Post-Harvest

Physiology and
Crop Preservation
NATO ADVANCED STUDY INSTITUTES SERIES

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_Post-Harvest
Physiology and
Crop Preservation
Edited by
Morris Lieberman
Late 0/ Beltsville Agricultural Research Center
Be/tsville, Maryiand

PLENUM PRESS • NEW YORK AND LONDON

Published in cooperation with NATO Scientific Affairs Division
 Library of Congress Cataloging in Publication Data
NATO Advanced Study Institute on Post-Harvest Physiology and Crop Preservation
(1981: Sounion, Greece)
Post-harvest physiology and crop preservation.
(NATO advanced study institutes series. Series A, Life sciences; v. 46)
"Proceedings of a NATO Advanced Study Institute on Post-Harvest Physiology and
Crop Preservation, held April 28-May 8, 1981, in Sounion, Greece" - T.p. verso.
Bibliogrpahy: p.
Includes index.
I. Food crops - Physiology - Congresses. 2. Food crops - Preservation - Congresses.
I. Lieberman, Morris, 1919- . II. North Atlantic Treaty Organization. III. Title.
IV. Series.
SB175.N37 1981 631.5'6 82-3645
AACR2
ISBN 978-1-4757-0096-1 ISBN 978-1-4757-0094-7 (eBook)
DOl 10.1007/978-1-4757-0094-7

Proceedings of a NATO Advanced Study Institute on Post-Harvest Physiology and

Crop Preservation, held April 28-May 8, 1981, in Sounion, Greece

© 1983 Plenum Press, New York

Softcover reprint of the hardcover 1st edition 1983
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N.Y. 10013
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
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 To
alI those who labored
and labor to preserve crops after
harvest for the benefit of mankind
MORRIS LIEBERMAN
 Morris Lieberman, the Director of the NATO Advanced Study
Institute on Post-Harvest Physiology and Crop Preservation, passed
away after completing the editing of the manuscripts of the present
book. The loss of this great scientist and fine personality is
deeply regretted by his colleagues and friends.

Lieberman contributed considerably to the knowledge of post-

harvest physiology, particularly to the understanding of the bio-
synthetic pathway leading to the hormone that regulates plant organ
senescence, ethylene. In the sixties, while working with Mapson at
Cambridge, he discovered methionine as a substrate for thissynthesis.
It was at the Post-Harvest Plant Physiology Laboratory at Beltsville,
which he directed, that rhizobitoxine analogs were found to inhibit
the main regulatory enzyme of the pathway, aminocyclopropanecarboxy-
lic-acid synthetase. Morris Lieberman died deeply involved in the
further analysis of the enzyme systems of this pathway.

Next to his enthusiasm for science, Morris had a vivid interest

in many aspects of life and was a very kind and friendly personwith
a beautiful sense of humor. Nothwithstanding his sharp insight his
judgment was mild. It was a privilege to be among his friends, only
few of whom knew that he was so ill that he nearly had to stay away
from the Institute that was his very conception and which he had
been fostering with such zeal. It gave hirn deep satisfaction to
direct the Institute and to edit this volume. We may accept thebook
with gratitude and respect as a memorial to a great man.

Johan Bruinsma
Co-Director
 PREFACE

Emphasis in agricultural research for many years has concen-

trated on crop production. This emphasis has become more important
in recent years with the realization that the population worldwide
is outstripping the food supply. There is, however, another side
to increasing the availability of the food supply. This simply
involves preservation of the harvested crop·for human consumption.
The losses incurred in harvesting, handling, transportation, storage
and marketing crops have become a greater problem as the distance
from the farm to the ultimate consumer increases. In the Western
world where modern transportation, storage facilities, and marketing
technology are widely used, post-harvest technology requires a
large input of energy which increases costs considerably. There-
fore, losses are more significant and the ability to provide fresh
fruits and vegetables, out of season, at reasonable costs will
depend on reduced post-harvest losses throughout the marketing
chain from the farm gate to the ultimate consumer.

The reduction in post-harvest losses depends on proper use of

current technology and further developments derived from a broad
spectrum of scientific disciplines. Biochemistry, plant physiology,
plant pathology, horticulture, agronomy, physics, engineering and
agricultural economics, all provide knowledge which has been useful
and will be useful in the future for improving post-harvest technol-
ogy and crop preservation. This volume records the Proceedings
of the NATO Advanced Study Institute on Post-Harvest Physiology and
Crop Preservation, held at Sounion, Greece, April 28 - May 8, 1981.
Lecturers at the Institute represented all of these scientific
areas, providing a broad spectrum view of current basic and practi-
cal information which impact on post harvest technology and crop
preservation.

Notice was also taken, at the Institute, of the special prob-

lems of crop preservation in developing countries wherein conditions
are quite different from those in the Western developed world. Quite
a different technology is applicable to developing world post-
harvest problems. Nevertheless, basic scientific information should
still be useful in establishing improvements which prevent losses

ix
x PREFACE

within the context of the primitive systems used. The economics of

some of these problems were also discussed in the course of the
Institute. Hopefu11y, the information in this vo1ume will provide
basic know1edge of current and emerging techno1ogy which will be
usefu1 in advancing post-harvest techno1ogy and crop preservation
wor1dwide.

The Institute was brought into being through the efforts of

many dedicated individua1s. I am especia11y indebted to my Co-
Director, Prof. Bruinsma, and the Organizing Committee members:
Prof. Bangerth, Prof. Come, Prof. Dekazos, Prof. Di11ey, Dr. Rhodes,
and Prof. V1itos. Their inva1uab1e he1p in arranging the program,
obtaining financia1 support in their own countries, and se1ecting
students made our Institute a success. A special thanks is due
Prof. Dekazos and Dr. Karaou1anis, members of the loca1 committee,
for se1ecting the site and making all the loca1 arrangements.

In addition to the generous support and sponsorship of NATO,

the Institute was co-sponsored by the Science and Education
Administration of the U.S. Department of Agricu1ture and the Greek
Ministry of Agricu1ture. Financia1 support was also provided by
the fo11owing organizations and companies: Air Liquide; BASF AG;
Bayer AG; Centre Technique Interprofessionne1 des Fruits et Legunle~;
Compagnie des Entreports et Gares Frigorifiques; Duphar; E.I. Dupont
de Nemours and Company; Froi1abo; Greek Ministry of Cu1ture and
Science; He11enic Organization of Tourism; Hoechst AG; Plenum Press;
Societe Francaise de Transports et Entreports Frigorifiques; Tate
and Ly1e Ltd; and Ve1sir:01 Chemica1 Corporation. Generous contri-
butions by these organizations provided support for additional
students and lectürers.

Finally I wish to acknow1edge my sincere appreciation to

Delores Sessions who carried the burden of extensive correspondence
and other matters re1ated to the Institute and retyped virtua11y
all the manuscripts in the required format. I accept fu11
responsibility for all editorial errors, which, despite my efforts,
probably exist.

Morris Lieberman
Be1tsvi1le, Mary1and
October 1981
 CONTENTS

I. BIOCHEMISTRY AND PHYSIOLOGY OF SENESCENCE

1. The General Biology of Plant Senescence and the

Role of Nucleic Acids in Pro tein Turnover
in the Control of Senescence Processes
which are Genetically Programrned . • . . 1
H. W. Woolhouse

2. Control of Ribonucleic Acid and Enzyme Synthesis

During Fruit Ripening 45
D. Grierson

3. Respiration and Energy Metabolism in Senescing

Plant Tissues . . . . . . . . . • . . . . 61
T. Solomos

4. Enzyme Activities and Post-Harvest Change . • . • . 99

M.J.C. Rhodes

5. Plant Membrane Lipids: Changes and Alterations

During Aging and Senescence . . . . . . . 123
P. Mazliak

6. Hormonal Regulation of Senescence, Ageing,

Fading and Ripening . . . . . . . . . 141
J. Bruinsma

11. CHARACTERISTICS OF SENESCENCE IN SPECIAL CROPS

7. Post Harvest Physiology of Seeds as Related

to Quality and Germinability • . • . 165
D. Came

xi
xii CONTENTS

8. Physiology and Storage of Bulbs: Coneepts

and Nature of Dormaney in Bulbs • . ,. , ... 191
M. Le Nard

9. The Formation of Enzymatie Produets in the

Fruits during Growth and Storage 231
G. Karaoulanis

111. PATHOLOGICAL ASPECTS - POST-HARVEST

10. Host-Pathogen Interaetions in Postharvest

Diseases . . . . . . . . . . • 247
J. W. Eekert and M. Ratnayake

111. Control of Postharvest Diseases with Antimierobial

Agents • . . . • . . . . . . . . . • . • . . 265
J. W. Eekert

12. Hydroxyproline - Rieh Glyeoproteins in the Cell

Wall of Diseased Plants as a Defense
Meehanism . . . • • . . . . . 287
M. T. Esquerre-Tugaye, D. Mazau,
and A. Toppan

13. Stress Metabolites 299

N. F. Haard

14. Myeotoxins as a Deteriorating Faetor in Stored

Crops • • • . • • . • . . . • . • . • . . 315
P. Krogh

IV. MANIPULATION OF THE PRE- AND POST-HARVEST ENVIRONMENT

TO INFLUENCE QUALITY

15. Hormonal and Chemieal Preharvest Treatments

whieh Influenee Postharvest Quality,
Maturity and Storeability of'Fruit 331
F. Bangerth

16. Effeet of Post Harvest Treatments of Growth

and Bioregulators on Quality and
Longevity of Fruits and Vegetables 355
E. D. Dekazos

17. Manipulation of the Postharvest Atmosphere for

Preservation of Food Crops • . . . . . . 383
D. R. Dilley
CONTENTS xiii

,18. Metabolism, Heat Transfer and Water Loss

under Hypobaric Conditions . . . . 399
S. P. Burg and R. Kosson

19. Maintaining Nutritional and Processing

Quality in Grain Crops During
Handling, Storage, and Transportation 425
P. C. Williams

20. New Post-Harvest Treatments of Horticultural

Produce and Developments to Maintain
Quality and to Prevent Damage in
Western Europe with Special Reference
to the Netherlands . . . . • . . . . . 445
W. S. Duvekot

/21. Postharvest Quality Maintenance of Fruits and

Vegetables in Developing Countries . . . 455
A. A. Kader

22. Instrumental Techniques for Measuring Quality

of Agricultural Crops . • . . . . . . . 471
K.H. Norris

V. POST-HARVEST LOSSES IN THE DEVELOPING WORLD:

ECONOMIC ASPECTS

23. Post Harvest Losses in Perishable Foods of

the Developing World . . . . . . . . 48"
D. G. Coursey

24. Solving Third World Food Problems: The Role

of Post-Harvest Planning . .
M. Greeley

25. Utilization of Agricultural Wastes: Same

Global Consideration . • • . . . . . . • . . 537
A. J. Vlitos

VI. PARTICIPANTS 547

VII. INDEX . . . . 553

THE GENERAL BIOLOGY OF PLANT SENESCENCE AND THE ROLE OF NUCLEIC
ACID IN PROTEIN TURNOVER IN THE CONTROL OF SENESCENCE PROCESSES
WHICH ARE GENETICALLY PROGRAMMED

Harold W. Woolhouse

John Innes Institute

Colney Lane
Norwich NR4 7UH, U.K.

PART I. SOME GENERAL PROBLEMS CONCERNING THE BIOLOGY OF SENESCENCE

Definitions

There are two terms "ageing" and "senescence" which are widely
used in reference to changes which impair the structure or function-
ing of living organisms. Medawar (1) defined ageing as referring to
all those changes which occur in time, without reference to death as
a consequence, indeed its use need not be confined to living organ-
isms. This is a convenient definition in that it allows of a clear
distinction of senescence as describing those changes which lead
sooner or later to the death of an organism or some part of it. As
Medewar puts it "It is a curious thing that there is no word in the
English language that stands for the mere increase in years; that is
for ageing silenced of its overtones of increasing deterioration
and decay. At present we are obliged to say that Dorian Gray did
not exactly 'age' though to admit that he certainly grew older. We
obviously need a word for mere ageing, and I propose to use 'ageing'
itself for just that purpose. 'Ageing' hereafter stands for mere
ageing, and has no other innuendo. I shall use the word 'senescence'
to mean ageing accompanied by that decline of bodily faculties and
sensibilities and energies which ageing colloquially entails. Dorian
Gray aged, but only his portrait disclosed the changes of senescence.
I hope that makes it clear."

Occurrence of Senescence

Figure 1 surnrnarizes the main senescence processes which occur

in seed plants arranged as they occur in successive stages of the
life cycle (2). The diagram emphasizes that we are dealing with a
2 H. W. WOOLHOUSE

wide range of phenomena, many but not all of which are integral parts
of the programme of development of the plant. If this is so then to
understand senescence one must understand development, and the im-
mensity of the task becomes apparent. It is a small step from
senescence as apart of the process of development and differentiation
to the view that "Natural" death is an inevitable consequence of
differentiation (3). Clearly there are certain forms of cellular
differentiation in both animals and plants in which the events which
lead to death are assured from the moment that the cell, or organ
of which it is apart, embarks upon its special differentiation.

It has long been recognized, for example, that the lignified

conducting elements of xylem tissues in plants and the formation
of enucleate erythrocytes in mammals are processes which have led
these cells to die or to astate where no resumption of synthetic
activity is possible (4). The operational difficulty in the study
of these forms of senescence is in deciding how far back in the
developmental sequence one should go; in the case ofaxylem element
it could be argued that once the crucial division takes place which
brings a particular daughter cell into the zone where the balance of
hormones or other determinent factors is appropriate for its dif-
ferentiation as a xylem element, then everything which follows
could, on the definition of senescence adopted here, be regarded
as part of the senescence process. In practice, however, one might
choose to focus attention more particularly on the later events in
the programme in which the disorganization of the cell was becoming
evident. Similarly, in the case of the erythrocyte, the point of
departure is the formation of the enucleate cell at erythropoeisis,
but again operationally, students of the senescence of erythrocytes
will often choose to concentrate on those particular changes in the
ce11 surface which occur after about 120 days of circulation in
the blood stream, whereby the red cell is no longer recognized as
"self" by the organism and is removed by phagocytosis. The impor-
tant point here is that when considering those forms of senescence
which are integral to development, it is weIl to bear in mind that
the moment of embarkation on a particular line of development, be
it as certain type of cell, or organ, is the point of departure in
the sense that the future of that cell or organ is now irretrievable,
nonetheless, it will often be a matter of operational choice in-
fluenced by the experience of the investigator, which will lead him
to single out more specifically terminal events in the differentia-
tion process for inclusion in the study of senescence.
Senescence and the Developmental Programme

The synchronous senescence of a whole population of plants, as

in the ripening of a field of corn, provides a spectacular example
of senescence manifested in the developmental programme. It is
important to recognize however that bland phrases such as "senescence
in the developmental programme," tell us little and tend if anything
GENERAL BIOLOGY OF PLANT SENESCENCE 3

,Free radical damage in ,1. Exhaustion of reserves. t1. Sene~nce of cotyledons.

: absence of repair :2. Failure of DNA repair. /2. Senescence in diflerentiation of
! processes. " 3. Loss of capacity for : tracheary elements, sieve lubes,
" I protein synthesis. : and some specialized cells.
: : : 3. Senescence in turnover 01 root
,: I I hairs and root cap cells.
: : I
DRY SEED IMBIBED SEED ' SEEDLING
I 11 111

1 J
FRUITING PLANT-- FLOWERING PLANT --VEGETATIVE PLANT
: VI \ V \ IV
l ~ t
1. Senescence of whole 1. Continuation of many 1. Senescence in cell diflerentiation
plant (monocarpism). senescence processes and tumover as in seedlings.
2. Senescence of aerial presenl in vegetative 2. Senescence in "self-pruning" of
shools (herbacious plant. branchlets in formation of tree
perennials). 2. Senescence and canopies.
3. Senescence and abscission of floral 3. Sequentialleaf senescence.
dehiscence of dry fruits. parts. 4. Seasonal, synchronous leaf
4. Senescence (ripening) senescence.
and abscission of fleshy 5. Senescence in developmenl of
fruils. lhorns and spines.
6. Abscission processes.

Fig. 1. A summary of the main senescence processes which are

found in seed plants, arranged as they occur in succesive
stages of the life cycle. Many but not all of the
phenomena shown are integral parts of the developmental
programme of the plant.

to obsure the multiplicity of mechanisms which may be involved.

For convenience I shall distinguish three types of senescence aris-
ing from the programme of development.

(1) Direct or positively programmed senescence. I have in

mind here those types of senescence in which there is released into
the cell a specific set of hydrolytic enzymes which will break down
its own macromolecules. If we consider some generalized form of
sequentially arranged operator-operon model as controlling the
production of the enzymic apparatus, then one could envisage a
"suicide squad" or "death clock," a bank of genes coding for hydro-
lytic enzymes, whose activation would lead to the death of the cello
As we shall see there are several instances, as in the autolysis
of the tail of tadpoles, the ripening of certain fruits and a
number of other senescence phenomena, in which something along
these lines seems to be happening.
4 H. W. WOOLHOUSE

(2) Indirect programming of senescence. There are a number of

examp1es in the senescence of plant organs, such as 1eaves and minor
branches, in which the senescence of the organ arises from unsuccess-
fu1 competition with other organs, because it comes to occupy an un-
favourab1e position relative to new organs which deve10p subsequent1y.
The sequentia1 senescence of the 10wer 1eaves when they come to be
shaded by newer 1eaves deve10ping on the stem above them, and the
10ss of minor branches within a tree canopy, are examp1es of this.
Now the detai1ed terminal events in the senescence of such organs
may invo1ve the positive action of a hydro1ytic enzyme "suicide
squad" of the type postu1ated under category 1. Equa11y it cou1d
arise through starvation or some other manifestation of inter-organ
competition; in which case the events in the senescence process may
not be direct1y-coded as genes for degradative processes in the ce11s
which die. They are, nonethe1ess, inimica1 to the deve10pment of
that plant simp1y because it puts its subsequent 1eaves and branches
at particu1ar ang1es in particu1ar p1aces. In doing this it has
sea1ed the fate of some of the 1eaves and branches which it used at
an ear1ier stage.

(3) A110metric effects, mechanica1 and physio10gica1 imba1ance.

A110metric growth, that is the unequal growth of different parts of
an organism, can lead to astate of mechanica1 or physio10gica1 in-
stabi1ity (5). It is frequent1y observed in various species of trees
that when a certain height is reached, the trunk may tend to snap
(as in p1antation-grown birches) or the plant becomes uprooted in
the event of strong ga1es as common1y occurs in ta11 e1ms. It may
be argued that in such cases the plant is contributing to its own
demise as a resu1t of its deve10pment to astate of mechanica1
instabi1ity. A more frequent1y canvassed view is that the senescence
of trees may result from their developing to astate of physiological
instability as a consequence of the gradual production of an increas-
ing proportion of non-photosynthetic tissues; the branches, trunk
and roots which comprise arespiratory burden which is greater than
the canopy can sustain (6). The concept is attractive but there is,
at the present time, no quantitative evidence to sustain this specu-
lation. Maggs (7) suggested that the distance between the shoot
apices andthe roots may be important in the senescence of trees,
possib1y as a resu1t of some constraint in the transport system of
the plant. This hypothesis also lacks experimental evidence but the
frequent1y observed "die-back" of distal branches in senescent trees,
al1ied to the finding that terminal twigs from such p1ants will grow
vigorously if rooted as cuttings, suggests that this hypothesis is
worthy of further investigation.

C10na1 Senescence

Many protozoa and unicellu1ar p1ants can be maintained as clones,

that is as specific cell lineages, for an apparently indefinite
GENERAL BIOLOGY OF PLANT SENESCENCE 5

period, provided that an appropriate regime of sub-culturing is

adopted.

Many perennial herbs, shrubs and trees can be propagated by

tillers, suckers or cuttings so that a particular genotype is con-
served over hundreds of years without an apparent decline in vigour.
The apple cultivar Winter Pearmain was known in England in 1200 AD
and was still in cultivation 600 years later (8). There are similar
examples of clonally propagated varieties of cassava (Manihot
usitatissima), date palms (Phoenix dactylifera) yams (Ipomea batatas),
plums (Prunus dornestica) and figs (Ficus dornestica) some of which
may have been grown from cuttings for 2,000 years (9). An early
reference to the possibility that clones might undergo senescence
is cited by the gardener Knight in 1795. In a letter to Sir Joseph
Banks, he describes difficulties relating to the propagation of the
older varieties of apples which make it clear that canker, now known
to be caused by Nectria galygena, accounted for much of the problem,
but continues "The wood of all the old fruits has long appeared to
me to possess less elasticity and hardness, and to feel more soft
and spongy under the knife, than that of the new varieties which I
have obtained from seed. This defect may I think be the immediate
cause of the canker and moss, though it is probably itself the effect
of old age and incurable." He goes on to list similar effects in
other species, the raspberry (Rubus idaeus) raised from seed is said
to live for 20 years, but the "common elm" (Ulmus ~.) a species
widely propagated vegetatively, is noted as an exception.

Arnong the many difficulties in most of the work from the time
of Knight to the present day, is that of deciding whether the supposed
decline in vigour of a given clone is due to cultural conditions, the
accurnulation of virus or other diseases or to genuinely endogenous
factors. The supposed greater rejuvenation following sexual repro-
duction, referred to so frequently in the work of Krenke and his
colleagues (10) could weIl be due to recombinatianof genes in the
offspring. Whilst there is clearly room for more work on this sub-
ject, the evidence at the present time is strongly in favour of the
potential immortality of meristemic cell lineages of many herbaceous
and woody perennials, provided that methods of vegetative propagation
exist. What is not disputed is that individual shrubs or trees will
have a finite existence, even though cuttings taken from old and
decrepit specimens can grow and develop to form normal healthy plants,
which resurne the growth rate of the parent tree when it was young.
This situation has been variously summarized: "The tree is in fact
not unlike a coral colony though a much more organized symbiotic
community (W.E. Baker) and apart from accident, disease and some
mechanical and nutritional considerations there seems no reason why
it should not live indefinitely," (11). A similar view is that of
Wynn-Edwards (12) "There is no universal life-span characteristic of
the species as a whole, indeed in some cases the durability and
resistance of the dead heartwood seem to be critical factors rather
than the true viability of living cells."
6 H. W. WOOLHOUSE

Senescence at the Gellular Level

We can distinguish three types of cell from the standpoint of

senescence and renewal, cells which are no longer capable of division,
cells capable of undergoing mitosis and cells of the germ line where
there exists the possibility of meiotic divisions.

(1) Post mitotic cells. The lifespan of post-mitotic cells

varies greatly according to the species and the tissue in which they
occur. Individual neurones in the brains of long-lived mammals may
continue in a functional state for a hundred years or more (13).
Similarly, cells in the xylem parenchyma of the wood of certain
trees may remain active, carrying out a seasonal starch storage
function for a hundred years or more.

Over the past forty years the so-called mutation hypo thesis
has held sway in much of the work on the origins of senescence at
the cellular level, particularly amongst zoologists. Gomfort (14)
offers three reasons for this, the fact that mutative changes in
cells would probably lead to eventual senescence if nothing else did;
the prevalence of theories based upon somatic mutation in cancer
and radiation research, and the readiness to which somatic mutation
lends itself to speculative exercises in higher mathematics (15).
The rich debate and often heated controversy which has surrounded
this somatic mutation hypo thesis have been admirably summarized
by Gomfort (16). It originated in the suggestion of King (17) that
cosmic rays could cause the accumulation of mutagenic changes in
the body. In successive refinements the possible nature of the hypo-
thetical lesions was elaborated and, und er the influence of physi-
cists, radiation biologists and cancer research workers. the mathe-
matical models gradually took leave of biological realities. The
hypo thesis was brought back into the ambit of experimental cel1
bio1ogy with the discovery that 1iving ce11s possess high1y specia1-
ized mechanisms for the repair of faults which arise in the genetic
material (18,19).

If the accumulation of genetic damage is involved in cel1ular

senescence it would be reasonable to expect that in organisms which
depend upon populations of post mitotic cells for their functioning.
there wou1d be some relationship between the capacity for DNA repair
and the lifespan and between the accumu1ation of damage in the DNA
and other cel1s and the efficiency of the repair system in such
ce11s.
Hart and Set10w (20) found a remarkable correlation between
1ifespan and the capacity for unscheduled DNA synthesis. a measure
of the amount of repair (21), for a range of species (Fig. 2). In
an attempt to standardize the comparison. equiva1ent cel1s of the
primary fibroblasts were taken from each species and the same range
of dos es of U.V. radiation was used to induce the DNA damage in the
GENERAL BIOLOGY OF PLANT SENESCENCE 7

cell samples for each species. Both the rate and the extent of
unscheduled DNA synthesis after U.V. radiation were found to in-
crease with the lifespan of the species (Fig. 3). The authors
emphasize ~hat such results must be interpreted with caution; the
extent of H-thymidine incorporation will be influenced by the
amount of DNA per cell, the specific activity of the internal thy-

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~ I /
..J
I I
/0 I. I:> HAMSTER
I I
o ./V RAT
I /1
O~ MOUSE
, 11
O~ SHREW

1
o 20 40 60 80
GRAINS/NUCLEUS

Fig. 2. The correlation between extent of DNA repair, measured in

terms of unscheduled DNA synthesis at 13 hours after dif-
ferent UV fluences, and the estimated life-span of the
species. (After Hart and Setlow (20».

midilate pools, the average size of the repaired regions and the
number of repaired sites per unit length of DNA. The authors are,
however, confident that it is the last of these factors which is
important for several reasons. (i) the fraction of the DNA dimerised
in comparable U.V. exposures is the same for mouse, hamster, cow
and man (22, 23, 24). (ii) Almost all mammalian cells contain about
8 H. W. WOOLHOUSE

50 0- o MAN
0/._ • ELEPHANT

/./
0//0 o COW

/JI
N 40
I
E

f
....,

-
0
a:
w
I-
11-
Cl 30

0 /6
fJ')
::> HAMSTER
w 6
...J
U
::>
Z
';;,
/6
~
Cl
a:
20
/6
Cl
w
~
6 ,
a: RAT
w
> MOUSE
Cl 10 0
6
SHREW

O~--~--~----r---~--~--~
o 10 20 30
TIME (h'r)

Fig. 3. The average amount of DNA repair, measured in terms of

unschedu1ed DNA synthesis, as a function of time after
exposure to a f1uence of 10 Jm- 2 of UV radiation, for
seven different species. (After Hart and Setlow (20».

tne same amount of DNA, 6pg per nuc1eus, (25) so that the total DNA
availab1e for repair is constant. (iii) Sma11 numbers of fibrob1asts
from the different species used in Set10w and Hart's experiments
underwent schedu1ed DNA synthesis in the course of the experiment
and all had simi1ar grain counts per nuc1eus suggesting that the
specific activities and the size of the thymidi1ate pools were not
significant1y different in the different species. (v) Measurements
of the photolysis of bromodeoxyuridine incorporated into DNA in p1ace
of thymidine during excission repair in ce11s of mouse, hamster and
man suggest that the average size of the repaired regions was simi1ar
in each species (26, 22).

If U.V. radiation is such a potent agent for inducing genetic

damage in ce11s. it is reasonab1e to suppose that many p1ants,
GENERAL BIOLOGY OF PLANT SENESCENCE 9

particularly those which grow at high altitude where the amounts of

U.V. received are greatest must have very efficient DNA repair
mechanisms. For example, in the Bristle Cone Pine, Pinus aristata,
which grows at an altitude of 4-5,000m in the White Mountains of
California, individual leaves can be retained in a functional state
for up to 30 years (27) during which time they must sustain an
enormous radiation load. There are numerous reports of visible
light stimulating recovery from damaging dos es of U. V. radiation
in leaves of higher plants (28,29) but it is not known at the pre-
sent time whether these effects are attributable to DNA repair.
Initial attempts to detect excission repair of DNA in plant cells
were unsuccessful (30,31), but with the development of more refined
techniques such repair has now been detected in Chlamydomonas
reinhardi (32) and in protoplasts of Daucus carota (33). In
C.reinhardi, U.V.-sensitive strains were deficient in excission re-
pair but there are, at the present time, no reports of comparative
studies which might enable one to say whether or not there are
correlations between the extent of the development of DNA repair
systems and the lifespan of plant cells or the magnitude of the
radiation load which they normally encounter.

(2) Cells having the potential for mitotic division. In some

relatively unspecialized organisms, mitosis appears to prevent
senescence. The head region of the flatworm Stenostomum consists of
non-dividing cells; the rest of the body includes cells that con-
trive to divide, giving rise to daughter animals by renewed growth
and differentiation of the posterior regions. If the head of the
animal is repeatedly severed and allowed to regenerate a new pos-
terior region, it soon becomes senescent and dies. If, on the other
hand, the newly developed posterior region is taken and allowed to
regenerate a new head then the organism can be clonally propagated
for an indefinite period (34). The possibility cannot be excluded
that selection may be taking place against cells which become senes-
cent, so that these are simply lost without trace: it is interesting
to speculate that an analogous selection against senescent cells may
account for the apparent immortality of the meristems of perennial
plants. An interesting parallel to the decline in regenerative
capacity of the post-mitotic cells of Stenostomum is the case of
frond development in the free-floating aquatic plant Lemna minor
(35,36,37,38,39). This group studied the regeneration capacity
of single parent fronds of L.minor when the successive daughter
fronds were removed as soon as they were expanded. It was found
that the successive daughter fronds became successively smaller as
the parent frond became progressively more senescent before the
final collapse of the parent frond. When small daughter fronds
arising from asenescent parent were cultured further, they gave
rise to fronds which were larger than themselves but took approxi-
mately six sub-culturings of this kind to regain the size of the
original parent frond.
10 H. W. WOOLHOUSE

Another interesting example of loss of regenerative capacity

in plants comes from work with cell suspension cultures (40). Clones
of cells fully established from the roots of carrot (Daucus carota)
can readily give rise to a new sporophytic plant when plated onto
an appropriate medium. When the cells in suspension culture are
sub-cultured through several generations and then plated, the capac-
ity to regenerate a new plant (totipotency) is greatly reduced even
though the growth rate of the cultured cells had not declined. How-
ever, in cases where a plant can be regenerated from an old suspen-
sion culture, other cells taken from the root formed on such a plant
are found to have regained their totipotent potential.

Steward (41) suggested that changes in the cytoplasm of the

cultured cells were the cause of the decline in capacity for re-
generation and these are apparently corrected during the formation
of a new plant.

Studies such as those on the senescence and rejuvenation of the

fronds of Lemna minor suggest that, as in some flatworms and protozoa,
there is an element of cell rejuvenation through the act of mitosis
per se. It is well known that in most, if not all, plants the cells
of the meristems are in symplastic contact with their neighbors by
means of plasmodesmata, indeed it has been estimated that even the
smallest meristemic cells have 1,000 to 10,000 plasmodesmatal con-
nections with the adjacent cells (42) the frequencies, length and
diameter of these connections shows wide variation according to
species (43) and there is evidence that these may function as major
channels for the transport of nutrients into the meristem (44).
Figure 4 is a diagrammatic representation of current views of the
structure and dimensions of a plasmodesma justaposed with a repre-
sentation of the dimensions of a range of plant viruses drawn to the
same scale (after Gibbs (45». The evidence suggests that the
genome of many virus es could pass readily through the plasmodesmata,
particularly if they lack a desmosome; in some cases the viruses are
small enough to pass directly through the plasmodesmata intact.
Figure 5 shows the particles of strawberry latent ringspot virus
passing through the plasmodesmata of Chenopodium amaranticolor.
Notwithstanding this evidence, considerable success has been achieved
in obtaining virus-free clonal stocks of a number of horticulturally
important species by culturing meristems isolated from the shoot
apices. This suggests that, if the virus es are able to enter the
apices, they are unable to survive in the cytoplasm during mitosis.
The detailed biochemical events in the mitotic cell are of enormous
complexity and are very imperfectly known, for review see Prescott
(46). The organelles and the ribosomes of mitotic cells remain in-
tact throughout division but their associated biosynthetic activity
shows large fluctuations. In the middle i f interphase (G 2) there
is a de novo synthesis and much increased activity of RNAase in cells
of Physa~ (47) but with the onset of the M phase, the capacity
of the cells for pro tein synthesis falls to a level which is only a
GENERAL BIOLOGY OF PLANT SENESCENCE 11

O.5p.-m

Fig. 4. Diagramatic representation of current views concerning

the structure and dimensions of a plasmodesma. For
purposes of comparison a range of plant virus es (a to h)
are drawn to the same scale. In some cases the viruses
are small enough to pass through the plasmodesmata intact.
(After Gibbs (45».

very small fraction of that during interphase. This decreased pro-

tein synthetic activity is associated with disaggregation of the
polysomes (48,49,50) and is apparently caused by the development and
persistence of an inhibitor of mRNA synthesis during this M phase
due to a transient incompetence of the ribosomes (51).
12 H. W. WOOLHOUSE

Fig. 5. Electron micrograph showing a chain of particles of

strawberry latent ringspot virus passing through a
plasmodesma of Chenopodium amaranticolor. W denotes the
cell wall. (After I.M. Roberts and B.n. Harrison (1980)
J.Gen.Virol. 7,47)

It is of interest that in dividing cells of pig kidney infected

with vaccinia virus, the cells lose the ability to support the rep-
lication of the virus at this stage, (52) either because the virus
is unable to recruit the inactivated cytoplasmic ribosomes needed
for its replication at this stage or it may be degraded as a
consequence of the increased ribonuclease activity in the cells.
The relationship of this sequence of events to the cell cycle 1S
shown in Figure 6. This scheme is of course conjectural but it
serves to emphasize the possibility of some limited "scrubbing"
or removal of unwanted components such as viruses, denatured nucleic
acids or other damaged cytoplasmic components, at this stage of
the cell cycle. In fairness it must be noted, however, that there
is good evidence from work with HeLa cells (53) and synchronized
Chinese hamster cells (49) that some long lived mRNAs synthesized
during G2 may persist through the M phase to be translated during
resurgence of pro tein synthesis which begins in late telophase and
proceeds into GI' It may well be of course that such mRNA mole-
cules ar.b protected against any general scrubbing process by complex-
ing with a protective protein or sequestration in a protected com-
partment of the cell in a manner not available to viral RNA or
damaged cytoplasmic components.

Another mechanism for the renewal of cell components which

might operate during mitosis is that certain genes could become
active only during cell division. If such genes were to code for
proteins which are required throughout the life of the cell, then
the life of the cell would be determined by the half-lives of the
GENERAL BIOLOGY OF PLANT SENESCENCE 13

(ci

Appearance of i nducer .........

factor for DNA synthes i s
Synthesis of thymidine
kinase.
Histone synthesis
I RNA synthesis
for mitosis
begins.

! Protein synthesis for

mitosis. Includes de
novo synthesis of-
rTliönuc 1ease

Arrest of RNA synthesis and turnover.

Period of ribosomal incompetence.
and dimi ni shed protei n synthesi s.
Period of potential cytoplasmic
Itscrubbingll process.

Fig. 6. Part-conjectural diagram describing events in the mitotic

cell cycle showing potential stages at which "scrubbing"
or removal of unwanted components such as viruses, denatured
or faulty nucleic acids or other cytoplasmic components may
occur. For discussion see text.

mRNA and pro tein molecules derived from them. There is no good
evidence that this is so. Certainly the levels of particular en-
zymes varies greatly at different stages in the cell cycle and in
relation to the stage of development of the tissue of which that
cell is apart. It is also weIl established that different enzymes
are turned over at very different rates, some having half-lives that
may be measured in hours, others in days or even weeks (see Part 11).
As far as the author is aware, however, there are few if any cases
from eukaryotic organisms of enzymes which are renewed only in the
dividing cell.

(3) Meiosis in relation to cell rejuvenation. It is evident

that whatever the cause of breakdown in the somatic cells of an
organism there must be far reaching events in the cells of the germ-
line which confer upon them the ability to sustain the continuity of
14 H. W. WOOLHOUSE

the species. There are, of course, the events of recombination in

the genome of the cell which may bring together new groupings o~
genes more favorable to a particular environment, but this is not
sufficient to explain the evident inunortality which graces the germ-
line, since there is no reason to suppose that the already existing
arrangement of genes is not entirely adequate for the progress of
the individual, but there is evidence of dtastic changes in the
cytoplasm of meiotic cells (54).

Autoradiographic studies in such widely different s.pecies as

Lilium, Rhoeo discolor, Zea mays and Paeonia show that there is
pronounced RNA synthesis in the pre-meiotic phase followed by a
per iod of declining RNA synthesis throughout the meiotic prophase
and a complete cessation of RNA synthesis from metaphase I until
the end of meiosis (55,56,57,58,59) which could be due to the fact
that during this per iod the pollen mother cells become sealed off
by callose walls which may prevent the passage of precursors into
the cells during this period. In Lilium and Trillium EM studies
reveal a decline in the ribosome population from zygotene to pachy~
tene (60). The ribosome population is restored during the meiotic
mitoses. Concomitant with the prophase elimination of ribosomes,
the endoplasmic reticulum changes from a plate-like appearance to
circular profiles of paired membranes often arranged in the manner
of concentric spheres, the membranes return to the normal form in
the last stages of breakdown of the ribosomes. Correlated with
these changes in the ribosomes and E.R. is a remarkable cycle of
dedifferentiation and re-assembly of the mitochondria and plastids.
In Tradescantia the mitocondria are elongate before meiosis and
become spherical by pachytene and may lose the capacity to divide
at this time (61). They subsequently lose internal structure until
by meiosis I the christae are lost and they appear as simple rounded
bodies 0.4nm in diameter. Redifferentiation of the mitochondria
follows a similar time course to the return to normal of the ER.

The plastids undergo a similar cycle of drastic dedifferentia-

ti on followed by re-assembly (62). At pre-meiosis in Lilium
longiflorum the proplastids of the pollen mother cells have a rudi-
mentary lamellar system containing starch, and, insofar as can be
judged from electron micrographs, are undergoing division. In
leptotene the starch is lost, the membrane system breaks down and
no more "apparently dividing" plastids are seen. In the transition
from zygotene to pachytene the plastids attain their simplest form
with scarcely any ribosomes present and a much reduced lamellar
system. From the late tetrad stage onwards there is a re-appearance
of starch, restoration of the plastid ribosomes and redevelopment of
a lamellar system. There are similar drastic alterations in the
cytoplasm of oocytes at meiosis and indeed recent work suggests that
during this period the nucleus may become relatively isolated
from the events occurring in the cytoplasm due to a transient clo-
sure of the nuclear pores.
GENERAL BIOLOGY OF PLANT SENESCENCE 15

The circumstantial evidence thus points very heavily towards a

wholesale eradication of cytoplasmic macromolecules in meiosis which
is at its height in the zygotene-pachytene transition. A basic rea-
son for this "scrubbing out" may weIl be the need to remove pre-
existing mRNAs in order to permit a re-programming of the cytoplasma
by the post meiotic nuclei (54). It is of interest that very few
plant viruses are seed born suggesting that if viruses were present
in the pre-meiotic cells they would also succumb to this scrubbing
process. It would therefore seem very probable that there is in
fact the opportunity for a very wide ranging renewal of damaged
components of the cytoplasm and its organelles at this stage, which
may be of crucial importance in counteracting cytoplasmic changes
which would otherwise give rise to senescence of the cello

Concluding Remarks

We have seen that in plants senescence processes may be linked

to the successive stages of the life cycle as the programme of
development unfolds. We have considered at some length the various
types of molecular damage which might be expected to cause senescence
in plant cells and described the processes of DNA repair, cell
selection and renewal at the time of cell division which prevent
such processes from becoming dominent factors in plant senescence.
It follows that in plants the majority of important senescence
changes incurred in the course of the life cycle arise from progranuned
rather than environmental insults of a stochastic nature. In the
second part of this paper I shall consider the turnover of nucleic
acids and pro teins as an example of the types of metabolic process
through which the developmentally programmed forms of senescence may
be initiated and controlled.

PART II.THE ROLE OF NUCLEIC ACID AND PROTEIN TURNOVER IN PLANT

SENESCENCE WITR PARTICULAR REFERENCE TO LEAVES

It is now over a hundred years since the suggestion was first

made that pro teins in plant tissues were continually being broken
down and resynthesized (63,64). This idea provided a framework for
measurements of the synthesis and breakdown of pro teins from tissues
which gave rise to the concept of a protein cycle (65,66). This
pro tein cycle involves continuous synthesis and breakdown of proteins,
even in circumstances where the total protein content of the tissue
may not be changing. Subsequent work has reinforced the concept of
a pro tein cycle in many plant tissues; for simplicity we shall con-
fine our attention to the case of leaves in this discussion. We are
confronted by three problems. Firstly there is the question of how
one shall measure the activity of this protein cycle - the problem
of pro tein turnover as it has come to be known. The two further
problems concern the mechanisms by which the synthetic and degrada-
tive aspects of this cycle are regulated. In the case of leaves it
16 H. W. WOOLHOUSE

is weIl known that there is a net decline in pro tein content in the
course of senescence (67,68,69,70). We shall therefore require to
consider the regulation of pro tein synthesis and degradation and
assess the extent to which changes in each of these processes
(Figure 7) are responsible for the net 10ss of protein (71,72,73).

Pro tein Turnover in Leaves

(1) Enzymes. Measurement of pro tein turnover in plant tissues

first became a practical proposition with the introduction of N15
and radioisotopes in the nineteen forties. There remain great
practical obstacles to the accurate measurement of pro tein turnover
in plants; I shall not dwe1l on the difficulties here, for arecent
review see Davies (74). Published values of protein turnover in
leaves are relatively few but suggest a half life of 3 - 5 days in
several species (74). This is of course an average value for the
leaf pro tein as whole and may derive from some pro teins which are
turning over rapidly and others which are turning over slowly or
not at all. It therefore becomes necessary to purify to homogeneity
specific pro teins in order that their several rates of turnovec
may be measured. In the past it has proved very difficult to purify
leaf pro teins except for RuBP carboxylase which is present in large
quantities. However, the recent introduction of immunological and
affinity column techniques should make it possible to purify and
measure the turnover of more leaf pro teins in the near future.

If the significance of pro tein turnover in the events of

senescence is to be understood it will be necessary to measure turn-
over of individual pro teins since averaged values of turnover for
unresolved pro tein mixtures can tell us relatively litt1e. Clearly
a protein carrying out an essential function, which is synthetized
at one phase of development but not renewed thereafter, could be-
come important in the declining functional capacity of a senescing
cello Such appears to be the case with the enzyme RuBP carboxylase
which, in the leaves of some species, declines both in activity
(75) and amount (76) in a manner which correlates approximately with
the declining photosynthetic capacity of the leaf (77). Turnover
studies on RuBP carboxylase have been carried out on leaves of
Perilla frutescens (71) barley (78,79) and maize (80). When con-
sidering these studies caution must be exercised in respect of the
methods used, but the general conclusion may be drawn that in Perilla
and barley RuBP carboxylase is not turned over whereas in maize
some turnover does occur. In Perilla and barley RuBP carboxylase is
synthesized during the development of the leaves but formation of
the enzyme ceases when expansion of the leaf lamina is completed,
thereafter the amount of activity of the enzyme declines gradually
in the mature leaf. In maize turnover of the RuBP carboxylase
GENERAL BIOLOGY OF PLANT SENESCENCE 17

Carbohydrate + NH 4 +

Kl
Ks K2
Protein ~ Amino acids
Kd
Kt
Precursor pool _~~~ Storage
pool
Fig. 7. A simple model for the turnover of proteins in the cell
incorporating an element of recycling of amino acids.
K denotes rate constants, Ks for synthesis of protein,
Kd for degradation of protein, Kl and K2 for synthesis
and degradation of amino acids and Kt for transfer to
a storage pool. (After Davies (74».

appears to continue in the mature leaf, albeit with degradation

proceeding faster than synthesis in the later stages (80).

The measurement of turnover is perhaps of greater significance

in the case of enzymes which are close to the threshold level at
which they are required in the cell and those which occupY key
positions in regulatory aspects of metabolism. Unfortunately the
small quantities of enzyme present and the difficulties in purify-
ing them have so far militated against measurements of turnover for
most of these enzymes, there are however a number of observations
which suggest that such measurements would be of interest. Enzymes
of the reductive pentose phosphate pathway in chloroplasts do not
all decline in activity at the same rate, which invites further
study of the rates at which these pro teins are synthesized and
broken down (81). Nitrate reductase is an enzyme which shows a
much more rapid turnover in leaf cells than the majority of the
soluble proteins. In leaves of Perilla frutescens the half-life
of the enzyme, based on activity measurements, was estimated to
vary from about 240 minutes in mature leaves to 120 minutes in
senescing leaves (Figure 8) (82). An analogous pattern of loss of
activity was observed in cell free extracts of barley leaves (83).
In the case of barley the nitrate reductase appeared to be con-
verted to a cytochrome C reductase, a modification which may re-
present the first step in breakdown of the enzyme.
18 H. W. WOOLHOUSE

100

90
,
\
80 \

,...
-=> 70 ,,
...o ,,
,,
:: 60 '~
o
\
u
:I \
\
~ 50 \
\

,,
\

\
::...
,,
,,
~-'
10
\
,
\
\

2 3 4 5 6
HOurs in darkness

Fig. 8. Decrease in nitrate reductase activity fo110wing darkening

in leaves of Perilla of different ages. 0---0, young leaves
(15 days from emergence); 0---0, mature 1eaves (45 days
from emergence);' " senescent leaves (70 days from
emergence. The data are expressed as percentages of the
activity at the time of placing in darkness. Subsequent
work has shown that 10ss of activity invo1ves breakdown
of the enzyme. (After Kannangara and Woo1house (82».

(2) Membrane and structura1 constituents. Changes in the

structure and properties of membranes are a universal feature of
senescence in plant ce11s: the evidence comes from electron micro-
scopy (84), x-ray-diffraction studies on isolated membranes (85,86),
and physio10gical measurements which show increasing permeabi1ity
in ce11s of senescing tissues manifested as 1eakage, that is to say
fai1ure to retain accumu1ated solutes (87,88). Indirect supporting
GENERAL BIOLOGY OF PLANT SENESCENCE 19

evidence for involvement of membrane changes in senescence comes

from a miscellany of sources, as for example the observation that
Ca 2+, a well-known factor in the stabilization of plant cell mem-
branes, will delay senescence in excised leaf discs of some species
(89).

Evidence that the constituent pro teins of membrane systems

may turnover at very different rates may be exemplified by the case
of the thylakoid membranes. One of the constituents of the thylakoid
is a protein encoded and synthesized within the chloroplast, having
a molecular weight of 32,000 (P-32,000) (90). The synthesis of
P-32,000 is light-dependent and involves the formation of a 33,500
M.W. precursor which is processed by proteolytic cleavage to the
P-32,000 form which is inserted into §he thylakoid. Studies in-
volving pulse-chase experiments with H-leucine labelled fronds
of Spirodela show P-32,000 to be turned over an order of magnitude
more rapidly than the other major chloroplast polypeptides (91).

Although accurate measurements of turnover of individual 1eaf

pro teins are so few, the ava1.lab1e evidence for turnover and age-
dependent differential loss of particular proteins is sufficiently
strong to suggest that we should look to the system regulating
pro tein turnover as an important site at which the senescence process
may be regulated. Underlying the turnover mechanism there are
undoubtedly hormonal controls which will be dealt with by Professor
Bruinsma elsewhere in this symposium but my present concern is the
dynamic system which is actua11y the subject of these controls.

The Regulation of Protein Turnover in Leaves

(1) General considerations. It will be evident from the

genera1ized scheme shown in Figure 9 that modulation of the level
of a particu1ar pro tein may be achieved by altering the balance of
synthetic and degradative sides of the process. Moreover, since it
is with relative rates of synthesis and degradation that we are
concerned, the balance may be altered in favor of synthesis or
degradation even when both processes are increasing or decreasing
in absolute terms.

A further general problem in considering pro tein turnover in

leaves arises from the fact that we have to consider three genomes
as sites of transcription, the nuc1eus, chloroplasts and mitochondria
and three sites of translation, the cytop1asm, chloroplasts and
mitochondria. As we sha1l see these different sites of protein
coding and synthesis may have important implications for the sequence
of events involved in 1eaf senescence.
 20 H. W. WOOLHOUSE
Structural and Functional
Macrornolecules

/
Biosynthetic reactions

Degradation reactions:
enhancernent
y inductior
activation
or de-
cornpartrnent-
ation

Degradation Products

1
Export to other parts of the plants

Fig. 9. Scheme for the turnover of pro teins and other macromolecular
constituents of the leaf in which a net loss of a component
may arise from depressed activity amongst elements oLthe
biosynthetic system or enhanced activity of enzymes on the
catabolic side of the cycle. Some breakdown products are
shown as being lost to the system by translocation whilst
some are re-cycled through the biosynthetic pathways. For
discussion of the implications of this process, see text.

(2) Control of Pro tein Synthesis. It seems to be a general

rule that in prokaryotic cells and in the organelles of higher
organisms the mRNAs are relatively shortlived and gene expression is
controlled primarily at the level of transcription. The mRNAs of
eukaryotes are longer-lived and control of pro tein synthesis occurs
at the levels of both transcription and translation (92). This
difference in extent of translational control in the 70s and 80s-
ribosomal system of prokaryotes and eukaryotes respectively may be
GENERAL BIOLOGY OF PLANT SENESCENCE 21

what occasions the much greater complexity of the 80s-ribosomal

based translation complex (Table 1).

a) Chloroplasts. The main evidence for changes in the bio-

synthetic side of pro tein turnover in relation to senescence comes
from studies of the photosynthetic apparatus. A consistent feature
associated with the declining photosynthetic capacity of the
senescing leaf is loss of RuBP carboxylase, the large sub-unit of
which is coded for and synthesized within the chloroplast. A de-
tailed study of the electron transport chain in senescing leaves
of Phaseolus vulgaris has revealed a declining activity associated
with alesion the PQ-Cyt-f section of the chain (93,73). Cyt-f is
a component of the electron transport chain which is also encoded
and synthesized within the chloroplast (94).

In leaves of Perilla there is a loss of capacity for chloroplast

pro tein synthesis in the course of leaf senescence, although cyto-
plasmic pro tein synthesis is maintained (95). Similar preferential
loss of capacity for synthesis of proteins in the chloroplasts has
been demonstrated in leaves of cucumber and f. vulgaris. Declining
chloroplastic pro tein synthesis associated with a loss of polysomes
and the cessation of chloroplast RNA synthesis. In f. vulgaris
arrest of chloroplast RNA synthesis coincides with the completion
of leaf expansion and has been traced to the loss of chloroplast
RNA polymerase activity. Chloroplast RNA polymerase is encoded in
the nucleus and synthesized in cytoplasmic ribosomes (96) suggesting
that the regulation of this enzyme may be a key to the shut-down of
pro tein synthesis in the chloroplasts (97,98). There is as yet no
evidence as to whether synthesis of the chloroplast RNA polymerase
is under transcriptional control in the nucleus or translational
control in the cytoplasm.

There is no evidence at present to suggest that the pattern

of events involving an early shut-down of chloroplast pro tein syn-
thesis is a feature of leaf maturation and early senescence in all
species; thus in wheat for example, Brady and Scott (99) have shown
that pro tein synthesis continues in the chloroplasts of senescing
leaves; chloroplast rRNA synthesis is also maintained in senescing
leaves of tobacco (134).

There is one set of circumstances in which arrest of chloroplast

pro tein synthesis becomes a major factor in leaf senescence; this
concerns the senescence which occurs when leaves are deprived of
light. It frequently happens in species growing in dense swards
that a particular value of the leaf area index is maintained be-
cause there is a sequential senescence of the lower leaves as they
become shaded by younger leaves developing above, (100). There are
probably a number of factors conspiring to promote this correlative
senescence of the older leaves. For example, in low light the
 N
N

Table 1. Factors involved in translational control in prokaryotes and eukaryotes

Soluble Protein Factors from Escherichia coli

Molecular Mole factor per

weight mole ribosome Function

1. Initiation
IF-l 9,000 0.18 Promotes IF-2 and IF-3 functions
IF-2a 115,000 0.15 Binds Met-tRNA: GTPase
IF-2b 90,000
IF-3 21,000 0.14 mRNA binding: dissociation

II. Elonga tion

EF-Tu 44,000 5-7 Binds AA-tRNA: GTPase

ET-Ts 30,000 1 GTP-GDP exchange on EF-Tu
EF-G 80,000 1 Translocation: GTPase

IH. Termination

RF-l 44,000 0.02 Recognizes UAA and UAG :J:

RF-2 47,000 0.02 Recognizes UAA and UGA
RF-3 46,000 Promotes RF-l and RF-2 GTPase :lE
:lE
o
o
r
:J:
o
C
cn
m
 G)
m
Table 1. Continued Z
m
::IJ
Soluble Protein Factors from Rabbit Reticulocytes :t>
r
tIJ
Molecule Number of 6r
weight polypeptid.es Function o
G)
-<
o"Tl
1. Initiation "1J
eIF-l 15,000 1 Promotes mRNA binding ~
eIF-2 150,000 3 Forms ternary complex with Met- Z
-i
t~A Cf)
m
eIF-3 -700,000 -9 Promotes Met-t~A and mRNA Z
binding dissociation m
Cf)
eIF-4A 49,000 Promotes mRNA binding ('")
1 m
eIF-4B 80,000 1 Promotes mRNA binding Z
('")
eIF-4C 17,500 1 Promotes Met-t~A binding m
eIF-4D 16,500 1 Stimu1ates Met-puromycin synthesis
eIF-5 150,000 1 Required for 80 S comp1ex formation
Co-EIF-2 22,000 1 Stimu1ates ternary comp1ex
formation
Cap binding 24,000 1 Binds to cap of m~A
11. Elongation
EF-l~ -55,000 1 Forms ternary comp1ex with AA-
t~A: binds to Rb: GTPase
EF-lß 30,000 1 Promotes exchange of GTP GDP on
EF-l y 55,000 1 EF-1~
EF-2 100,000 1 Trans1ocation: GTPase
111. Termination
RF 56,500 1 Promotes termina-tion. GTPase

w
'"
24 H. W. WOOLHOUSE

stomata will tend to close a common concomitant of leaf senescence,

(101). The transpiration stream through these leaves will thus be
much reduced leading to a depletion in the supply of cytokinins
from the roots, which appear to be essential for the maintenance of
pro tein synthesis in plant cells (102). There are other, more
direct effects of light, in regulating chloroplast pro tein synthesis.
Firstly light is necessary for photophosphorylation which provides
the energy for pro tein synthesis; in the absence of light chloro-
plast pro tein synthesis ceases and apparently cannot be sustained
by energy sour ces external to the plastid.

Evidence is accumulating to suggest "that chloroplast protein

synthesis may also be controlled at the transcriptional level in-
volving modulation of the activity of chloroplastic RNA polymerase.
In leaves of Perilla there is a loss of capacity for chloroplast
pro tein synthesis in the course of leaf senescence, although cyto-
plasmic protein synthesis is maintained (95). Similar preferential
loss of chloroplast activity has been noted in leaves of cucumber
and Phaseolus vulgaris. The decline of pro tein synthesis .in the
chloroplasts is associated with a loss of polysomes and the cessation
of chloroplast RNA synthesis (103). In~. vulgaris the arrest of
chloroplast rRNA synthesis at the time of completion of leaf ex-
pansion has been traced to the loss of chloroplast RNA polymer ase,
an enzyme which appears to be coded for in the nucleus and synthe-
sized on cytoplasmic ribosomes (96) suggesting that the regulation
of this enzyme may be a key to the initiation of shut-down in the
plastid metabolism,(97, 98). In this context it is of interest
that when leaves are caused to re-green by appropriate manipulation
of the plant, there is an early resumption of chloroplast RNA poly-
merase activity (98).

The chloroplast genome is made up of 45 ~m double circles of

DNA which codes for two proteins present in large amounts, the
large sub-unit of RuPCase and the 32,000 Dalton thylakoid pro tein
(104) and at least 100 other pro teins whicn are made in much smaller
quantities. Silverthorne and Ellis (105) have shown that the re-
lative proportions of the two major proteins synthesized by the
chloroplast, changes in the course of leaf development in spinach
due to changes in the amounts of translatable RNA for each. Bogorad
et al. (106) have isolated a protein of MW 26-29,000 which specifi-
cally stimulates transcription of maize chloroplast DNA by the
maize chloroplast RNA polymerase and have proceeded to show that
transcription of different parts of the chloroplast genome, iso-
lated by restriction endonucleases, is differentially influenced by
this "stimulatorH pro tein. The chloroplast genome also codes for
two sets of ribosomal genes per DNA molecule (107,108,109) and
for about 30 tRNA species (110). Figure 10 provides a working
hypothesis for a mechanism by which modulators like the stimulator
pro tein described above, may be produced by nucleus and cytoplasm
and passed into the plastid. The model is based on the envelope-
GENERAL BIOLOGY OF PLANT SENESCENCE 25

Ijucleus

~ nOljA

\
000000 80S ribosome system

1~ ''''''".i, "".i..
Chloroplast Protein precursors

Chloroplast
Envelope

CHLOROPLAS T

Fig. 10. Postulated mechanism for regulation of interactions between

nucleus and chloroplast. Proteins synthesized within the
cytoplasm which are destined for the plastid are identified
by aprecursor segment which is removed after entry into the
plastid stroma. SP indicates a stimulator protein modifying
the extent of transcription of particular genes in the
chl.DNA. ChlRNAp indicates chloroplast RNA polymerase, an
enzyme encoded in the nucleus.
26 H. W. WOOLHOUSE

carrier mechanism which appears to be involved in the transport of

the small sub unit of RubPCase into chloroplasts and may well serve
as a general mechanism. Through a mechanism of this type we may
envisage a ready means whereby the chloroplast RNA polymerase activ-
ity could be modified (105, 106) or totally arrested (98).

It is frequently observed both in gas-exchange studies (111)

and by electron microscopy that disorganization and loss of function
takes place much earlier in the chloroplasts than in the mitochondria.
The fact that the mitochondria can be self-sufficient for the energy
requirements of pro tein synthesis in darkness may account for this
difference in sequentially senescent leaves in which shading is a
factor. There are also other major differences in the pro tein
synthesizing machinery of the two organelles. For example both
organelles contain specific and immunologically distinct elongation
factors, EF - G and EF - Tu, as components of the protein synthesiz-
ing systems. Whereas EF - Gchl and EF - Tuchl are encoded and syn-
thesized within the plastids, EF - Gmit, and EF - Tumit are encoded
in the nucleus and synthesized on cytoplasmic ribosomes (112,113).
Thus in circumstances where there is preferential shut-down of
chloroplast metabolism at the chL-RNA pölymerase-transcription level
there will be attendant arrest of synthesis of the plastid-encoded
elongation factors. If cytoplasmic pro tein synthesis is continuing
at this time this could provide the necessary elongation factors and
al lied constituents of the mitochondria since these are encoded in
the nucleus and synthesized in the cytoplasm.

(3) Control of Protein Degradation. If we turn now to the

degradative side of the turnover process we find that the state of
knowledge is slight. The most obvious thing to look at first is
the activity of hydrolytic enzymes. I have culled from the litera-
ture a list of hydrolytic enzymes which have been reported to in-
crease in activity in senescing leaves and for purposes of compar-
ison a list of the enzymes which are active in the barley aleurone
on stimulation with gibberellin, which may be viewed as a somewhat
analogous senescing system (Table 2). The list of enzymes is
notably similar in the two organs although in the case of leaves the
list had to be compiled from widely scattered reports on a range of
different species. It is a matter of regret that there does not
appear to have been a systematic study of the spectrum of hydrolases
in the leaves of a single species.

As aprelude to examination of the regulation of hydrolytic

activity in senescing leaves it is well to ask whether the fre-
quently-observed increases in activity are in fact necessary at all
for senescence to occur. In many cases the hydrolase activity pre-
sent before the onset of senescence is more than adequate for the
observed rate of breakdown and the activity during senescence is
increased only by a factor of two or three (114), which may be of
much less significance than a change in compartmentation bringing
enzymes and substrates into contact.
 G)
Tab1e 2. Hydro1ytic enzymes reported to show increased activity in senescing m
Z
1eaves. Inc1uded for purposes of comparison (see text) is a list of m
:D
hydro1ytic enzymes re1eased from the a1eurone 1ayer of bar1ey. For »
r
references see Woo1house (131) OJ
or
Leaves o
G)
Protease -<
o
."
Tobacco Anderson & Rowan 1965 "'C
r
Mung bean (cotyledons) Baumgartner & Chrispee1s 1977 »z
Pea Storey & Beevers 1977 -t
Wheat Da11ing et a1. 1976 cn
m
Peop1es & Da11ing 1978 Z
m
Peop1es, Frith & Dal1ing 1979 cn
("")
Wa1ters et a1. 1980 m
Z
Fe11er 1978, 1979 ("")
m
Nair et a1. 1978
Wittenbach 1978
Maize Fe11er et al. 1977
Bar1ey Peterson & Huffaker 1975
Mi11er & Huffaker 1979
Oats Martin & Thimann 1972
Drivdah1 & Thimann 1977, 1978
Peri11a Kannangara & Woo1house 1968
Lo1ium temu1entum Thomas 1978

Ribonuclease

Rhoeo De Leo & Sacher 1970

Rhoeo Sacher & Davies 1974
Phaseo1us vu1garis Phi11ips & F1etcher 1969
Oat Udvardy et a1. 1967 N
--.J
 N
CD

Table 2. Continued

Acid Invertase

Lolium temulentum Pollack & Lloyd 1978

Chlorophyllase

Barley and oats Sabater & Rodriguez 1978

Esterase

Festuca pratensis Thomas & Bingham 1977

Acid Phosphatase

Perilla Kannagara & Woolhouse 1968

Aleurane layers of barley

a:-amylase Oe nova synthesis Varner 1975
protease De ~ synthesis Varner 1975
rOibonuclease Oe ~ synthesis (in part) Varner 1975
ß-glucosidase Oe ~ synthesis (in part) Varner 1975
phosphatase Release + de ~ synthesis Varner 1975 :J:
pentosanase Release + de ~synthesis Oashet & :E
Chrispeels
(1977) :E
0
peroxidase Release Varner 1975 0
r
esterase Release Varner 1975 :J:
0
C
cn
m
GENERAL BIOLOGY OF PLANT SENESCENCE 29

Hydrolase regulation. The possible levels of control of the

hydrolytic activity of senescing leaves, according to the present
state of knowledge, are summarized in Figure 11. The diagram offers
a vast array of possibilities but they may for convenience be
broadly divided under three headings (i) de novo synthesis of the
enzymes; reactions and factors a to k (ii~activation and in situ
regulation; reactions m and n, and (iii) compartmentation;-reactions
o to p. It is important to consider briefly the evidence for these
various levels of control.

De ~ synthesis: Reaction (a) Several workers have treated

excised leaves with actinomycin-D without affecting the rate of
senescence (115,116,117) from which the conclusion has been drawn
that a transcription contro1 is not involved in senescence. It is
not certain whether the inhibitor is ab1e to gain access to the
nuclei under the conditions of these experiments. Attempts to
demonstrate qualitative differences in the RNA produced in young and
old barley leaves by means of DNA:RNA hybridization have also proved
negative (118) though here again uncertainties surround the efficien-
cy of the technique employed.

Message processing: Reactions band c. Virtua11y nothing is

known concerning mRNAs for the hydro1ytic enzymes in leaves, of
whether they contain interrupted sequences and require processing
or of whether they become polyadenylated. Maize seedlings have been
shown to be an active source of RNA polydeny1ating enzyme and the
po1yadenylation inhibitor cordycepin has been shown to de1ay senes-
cence in leaf discs of Nicotiana (119) providing an interesting lead
worthy of further investigation.

Reactions and factors of the ribosome cycle: reactions d to

k. The ribosome cycle in eukaryotes is still imperfect1y und er-
stood but it seems reasonab1e to suppose that we are here concerned
with a vast array of possible controls on protein synthesis which
will include: availability of membrane surfaces upon which polysome
assembly can occur, availability of intact ribosomal sub units,
appropriate supplies of RNA and the corresponding aminoacy1 tRNA
synthetases and availability of active initiation, translocation
and te'rmination factars.

The cytoplasmic ribosome cycle in mature leaves has not been

ana1yzed at this level of detail and so we have no direct evidence
concerning rate-limiting reactions involving any of these factors.
Ca110w et al (95) obtained active 80s-based po1ysome fractions from
leaves of Peril1a throughout maturation and senescence up to the
time of abscission, long after the 70s-based plastid system had
become inactive. Most of the evidence on the involvement of protein
synthesis in senescence is however indirect and depends upon the
30 H. W. WOOLHOUSE

(a) (b) ( c)
Nucl eus - - Pro-message-- PI~~essed meS~POlyadenYlated message

~(e)
Translation system (f) the ribosome cycle

(g) membrane surface for

polysome assembly
(h) tRNA
(i) amino acyl tRNA synthetases
(j) initiation and elongation
Action on substrates factors
in the cell (k) supply of ribosomes and
energy (ATP, GTP)

(p)

I
subs tra tes from
the cell

'r
ActiV ".,..'~:::
~ymogens
/;;,'~fdrOlytic enzyme synthesis
(1 )
(n)

Process i ng and ~ Pro-enzymes

secondary modification

Fig. 11. Diagram indicating some of the multiple controls which may
be invo1ved in the regulation of the increased hydro1ytic
enzyme activity associated with the 1ater stages of 1eaf
senescence. Reactions and factors (a) to (1) are concerned
with aspects of de ~ synthesis of the enzymes; reactions
m and n denote in situ regulation of the enzymes by pro-
cessing, secondary modification or release from a zymogen;
reaction (0) indicates enzyme regulation by compartmenta-
tion; reaction (p) denotes enzyme regulation at the sub-
strate level.
GENERAL BIOLOGY OF PLANT SENESCENCE 31

credence which one is prepared to giye to the results of applying

inhibitors. It had been the general experience that inhibitors of
pro tein synthesis such as MDMP (120) and cycloheximide, (121, 122,
123, 79) arrest the progress of senescence in excised leaf tissue.
It is tempting to conclude that the key to this effect is the block-
ing of synthesis of degradative enzymes which mediate the autolysis
of cell constituents; it may be born in mind however that these
inhibitors will block the synthesis of all enzymes being made on
80s ribosomes and this may be sufficien~o arrest other aspects of
metabolism whether involved in senescence or not, rather than just
the synthesis of degradative enzymes.

The reader may cull from the literature numerous papers having
a possible bearing upon the ribosome cycle in leaves; I refer to
such matters as changing patterns of leucyl-tRNA and leucyl-tRNA
synthetases, changes in lability of rRNA under electrophoresis and
lowered protein: rRNA ratios in the ribosomes (124). In the opinion
of this reviewer there is insufficient evidence on which to accord
any of these observations a specific significance in the process of
senescence.

The only conclusive evidence for regulation by de ~~ synthe-

sis, of a hydro lase which increases during leaf senescence concerns
a ribonuclease. When leaf discs of Rheo discolor are incubated in
darkness there is a loss of RNA and protein accompanied by an in-
crease in ribonuclease and acid phosphatase activities (125). The
increase in activity of these enzymes is suppressed by IAA, kinetin
and inhibitors of RNA and protein synthesis and is promoted by
abscisic acid. Sacher and Davies (126) used a density labelling
technique to show that the increased ribonuclease activity is
accompanied by de novo synthesis of the enzyme. The precise level
at which this enzyme synthesis is being controlled is not known
and there is clearly a great need to extend this type of work to
the other enzymes which show increased activity in the course of
leaf senescence.

Hydrolase control by compartmentation. As may be seen from

Table 2, the reports of hydrolases associated with senescence in
leaves are becoming legion; likewise we have seen that the substrates
for these enzymes, the nucleic acids and proteins in particular, are
being lost at different rates within a given organelle and the time
course of losses is different for different organelles. Difficult
questions therefore arise as to the localization these hydrolytic
enzymes in the cello Do the enzymes move to the components which
are to be degraded or vice-versa? If the former then why is it that
the hydrolase having arrived in a particular organelle degrades one
protein relatively quickly but not its neighbour?
32 H. W. WOOLHOUSE

The currently popular view of leaf senescence urges that > 95%
of the proteases present in leaves are contained in the cell vacuoles
which function as lysosomes which degrade the pr 0 teins or indeed
whole organelles which pass into them (Figure 12) (127). At the
final dissolution of the cel1 the tonop1ast membrane will be ruptured
when these proteases pass from the vacuo1es a10ng with the rest of
the lysosoma1 hydro1ases bringing about the final dissolution of the
ce11 contents. For a detailed discussion of the vacuo1ar hydrolase
system see Woo1house (73). It is my purpose here 'however to suggest
that this represents far too crude a system to achieve the degree of
control imp1ied by the wide1y differing rates of turnover of the
various pro teins in the ce11. I wish to suggest that the vacuo1ar
system does not account for the ob'served facts of order1y events in
1eaf senescence and to propose in its stead an alternative hypothesis
invoking a precise1y regu1ated proteo1ytic system. Figure 13 shows
quasi-independent protein cyc1es within the cytop1asm and organelles.
In each of these cycles the numbers 1 and 2 refer to the generally
accepted view of protein synthesis, being the activation of an amino
acid (AA) to form aminoacyl tRNA and 2 the subsequent incorporation
via the ribosoma1 cyc1e. Steps 3 and 4 indicate a regu1ated de-
gradation of pro teins in which a pro tein to be removed is first
"recognized" and converted to an "activated" form which is suscep-
tib1e to proteo1ytic attack (step 3) and the activated protein is
then broken down (step 4).

Fig. 12. A simple llIodel to i1lustrate the autophagic vacuole concept

in relation to proteolysis in plant ce11s. CY (cytop1asm):
Chl (chloroplast) ; M (mitochondrion) ; N (nuc1eus) ; V
(Vacuo1e). The numbers 1 - 4 denote transport pathways
of pro teins from the organelles to the vacuo1e. Number 5
denotes the pathway of release of amino acids formed by
proteolysis in the vacuole. The difficulties in equating
a model of this kind with the variety of half-1ives of
pro teins in an organelle is evident (see text).
GENERAL BIOLOGY OF PLANT SENESCENCE 33
CYTOPLASM

?PROTEIN~
ACTIVATED ACT IVATED
A A PROTEIN

.
I
I
RltiC 1 R _t
I IRe_li ---------------
c

1 1
1 I
1+
YACUQlf

Fig. 13. Scheme representing the three pro tein cycles which co-
exist in leaf-cells and aspects of their interrelation-
ships. The numbered steps in each cycle indicate 1, amino
acid incorporation into protein, 3, energy-dependent pro-
tein activation prior to degradation (see test and Fig.
14) and 4. hydrolytic breakdown of protein.

The dotted lines bearing the symbol R represent possible

route along which amino acids or peptides may be trans-
ported, thereby linking the substrate pools of the 3
amino acid cycles as follows: Ro-c organelle to cytoplasm;
Rc-o cytoplasm to organelle; Rc-v cytoplasm to vacuole,
Rv-c vacuole to cytoplasm and Rc-t cytoplasm to the
phloem transport system mediating export from the leaf.

Regulation of degradation is envisaged as occurring particular-

ly at stage 3, which is an energy-dependent process. Step 3 is based
on the findings in animal and microbial cells, that there is a low
molecular weight protein APF-l (128,129) which combines with the
pro tein which is to be degraded, in an ATP-dependent reaction, to
form a covalently bonded APFl-protein ecomplex in which form the
protein moiety is now in a conformational state in which it is sus-
ceptible to proteolytic degradation. A more detai1ed picture of the
regulation of step 3 is presented in Figure 14.
34 H. W. WOOLHOUSE

2-
p"Ofe,n~

I'
+ 7 • (APF In P'ofe,n

nAPF ~
nAPF X'" AmIno .elds

Fig. 14. A suggested model for energy-dependent breakdown of

proteins. 1. APF-l-proteinamide synthetase (acting on
lysine E - NH 2 groups); 2, Amidase that allows correction
when n + 1 or 2; 3. Peptidases which act preferentially
on (APF-l)n derivatives, when n > 1 or 2; 4, Amidase for
APF-l-x. X is lysine or a small peptide. (From Hershko
et al. (130). For explanation see text.

In advancing this hypo thesis here I am seeking primarily to

point to new areas in which the study of plant senescence appears
to be leading. Considerations of space preclude the inclusion of
full discussion of compartmentation and energy-dependent protein
degradation which are required here, for details see references
(131,132) •

CONCLUSION

I have had in mind two main objectives in this introductory

review of plant senescence. Firstly, in Part I, I have surveyed the
various manifestations of senescence as they are to be found in all
phases of the life cycles of plants and have attempted to show how
the events ot senescence are embodied in the genetic programme of
development. 'Secondly in Part 11, I have considered just one facet
of the regulation of cell metabolism, nucleic acid and protein
turnover, in order to exemplify the deeper questions which must be
answered as we seek to explain how the senescence processes are
regulated. It is important to emphasize that I have made a deliber-
ate selection by way of an example.

An equally cogent case could have been made for considering

the regulation of liquid turnover and its role in altering membrane
functions in senescing tissues. Likewise I have said nothing of
hormonal controls which impinge upon many of the processes we have
mentioned (133). Thus we see that even in its basic aspects the
study of plant sßnescence is a vast and complex study; on these
grounds alone it is surely worthy of our serious attention but to
this may be added its enormouse economic importance in agriculture
andhorticulture with which we shall be much concerned in the later
stages of this symposium.
GENERAL BIOLOGY OF PLANT SENESCENCE 35

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CONTROL OF RIBONUCLEIC ACID AND ENZYME SYNTHESIS DURING FRUIT

RIPENING

Don Grierson

Department of Physiology and Environmental Studies

University of Nottingham, School of Agriculture
Sutton Bonington
Loughborough, LE12 5RD, U.K.

INTRODUCTION

When fruits ripen they undergo major changes in composition

which affect their attractiveness, storage life and nutritional
value. This is a consequence of alterations in the physiological
and biochemical processes occurring in the cells of the fruit.
A list of some of the major changes that occur in tomatoes is given
below. Similar changes occur in many other types of fruits.

1. Chlorophyll degradation
2. Synthesis of pigments such as lycopene and ß-carotene.
3. Changes in organic acids and amino acids.
4. Starch degradation and formation of sugars.
5. Synthesis of compounds contributing to flavour and aroma.
6. Cell wall degradation leading to softening.

Ripening usually takes place after a certain stage of maturity has

been reached. The timing is not directly related to chronological
age, however, but is determined by a developmental sequence which
is under genetic control. It is, therefore, misleading to consider
ripening as a type of "ageing" and it is probably more correct to
view it as aseries of coordinated metabolie events switched on at
a specific stage in development. In climacteric fruits, such as
tomatoes, apples and bananas, the initiation of these changes is
associated with increased synthesis of carbon dioxide and ethylene
by the fruit tissue. Ethylene is regarded as a ripening "hormone"
because unripe fruits can be stimulated to ripen by an exogenous
supply of the gas and natural ripening can sometimes be retarded
if ethylene produced by ripening fruits is removed from the sur-
rounding atmosphere.
45
46 D. GRIERSON

Early theories invoked alterations in membrane permeahility

and the release of degradative enzymes to explain the process of
ripening. However, although such events may contribute to the later
stages of ripening. there is now little doubt that the synthesis or
activation of specific enzymes is required for normal ripening to
occur. For example, the solubilization of cell~wall pectins that
take place during ripening of tomato is at least partly due to the
enzyme polygalacturonase. There is virtually no polygalacturonase
activity in mature-green tomatoes but enzyme activity appears as
fruits begin to ripen and continues to increase as ripening pro-
gresses (5,7,21). Invertase activity also increases dramatically
during ripening (5,10) and other enzymes, such as pectinmethyl-
esterase, may be present at the mature~green stage but subsequently
increase substantially in activity (5).

Although many aspects of ripening appear to involve changes in

enzyme synthesis or activity, including ethylene synthesis itseH,
which seems to depend on the appearance of enzyme ACC Cl-aminocyclo-
propane-l-carboxylic acid) synthase. this does not mean that all
enzyme changes occur by the same mechanism. Indeed this would be
surprising given the range of synthetic and degradative processes
which occur in such diverse cell compartments as the plastids, the
cytoplasm and the cell walls. Treatment of tomatoes with gibberel-
linst for example, permits the respiratory c1imacteric to proceed
while tomatoes remain green (3) and in the "greenflesh" mutant of
tomate the plastids tend not to lose their chlorophyll although
softening and lycopene synthesis appear to continue as normal
(Grierson, unpublished observations). Furthermore. under controlled-
atmosphere storage of green tomatoes, starch degradation occurs al-
though there is little or no softening or colour change (Coodenough.
Tucker, Grierson and Thomas, unpublished results). These observa~
tions suggest that some aspects of ripening can proceed independent-
ly of others and they do not all require exactly the same conditions
to be manifested. It therefore follows that there may be more than
one type of control mechanism operating during ripening although
there may be one common initiating event.

For those interested in understanding the control of ripening

the major challenge is, therefore, to learn how changes in enzyme
synthesis and activity are brought about and to determine whether,
and if so how, ethylene initiates these changes. In this article
I shall discuss the role of RNA synthesis and pro tein synthesis in
controlling enzyme changes during fruit ripening.

EVIDENCE THAT RIPENING INVOLVES CHANGES IN GENE EXPRESSION

A number of enzymes have been shown to change in activity dur-

ing ripening (18) including malic enzyme (9), polygalacturonase (7)
ahd invertase (10). Although such changes could be brought about
by a variety of mechanisms, as discussed below. there is good evi-
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 47

dence that specific stimulation of protein synthesis occurs during

ripening. Hulme (8) showed that there was a net accumulation of
pro tein during the ripening of apples and Hansen (6) showed ethylene
caused an increase in protein nitrogen in pears. Furthermore, a
stimulation of the incorporation of radioactive amino acids into
protein has been demonstrated during the ripening of avocado (15),
pears (4), apples (15), tomatoes (19) and bananas (17). In their
experiments with pears Frenkel et al. (4) showed that there was
enhanced incorporation of l4C-phenylalanine into malic enzyme and
other proteins during the early stages of ripening. These observa-
tions, and the finding that cycloheximide, which inhibits protein
synthesis on 80S ribosomes, retards or prevents ripening in pears (4),
citrus (16) and bananas (1), indicate that pro tein synthesis is an
important event in ripening fruit.

RNA synthesis also seems to be associated with ripening. There

is increased incorporation of radioactive uridine into RNA during the
ripening of tomatoes (13,19) and ethylene has been shown to stimulate
RNA synthesis in apples (9) and figs (11) and induce changes in the
relative amounts of certain iso-accepting tRNAs in tomatoes (12).

The involvement of both nucleic acid and protein synthesis in

the process of fruit ripening provides biochemical evidence that
there is a requirement for gene expression in order for ripening to
occur. There is also separate genetic evidence from the study of
various tomato mutants implicating specific nuclear genes in the
control of ripening (2·,20). For example, in the honozygous condition
the rin (ripening inhibitor) gene on chromosome 5 results in fruit
which do not exhibit arespiratory climacteric or produce a burst
of ethylene synthesis. Such fruit do not ripen normally but slowly
lose their chlorophyll and turn yellow. They produce no polygalac-
turonase (21) and do not soften to any great extent. In contrast,
fruit from plants homozygous for the Nr (Never ripe) gene on chromo-
some 9 show a reduced climacteric and ethylene evolution, turn
orange but not red, and soften slowly, probably due to the production
of low amounts of only one of two isoenzymes of polygalacturonase(21).
Both these mutations produce multipe phenotypic effects whereas the
ßf (green flesh) mutation on chromosome 8 results in fruit which
appear to ripen normally except for the fact that they do not lose
their chlorophyll.

POSSIBLE CONTROL POINTS IN GENE EXPRESSION

Figure 1 gives abrief outline of the steps involved in the

espression of genetic information in the form of proteins. The
number of active molecules of a given enzyme could be governed by
regulatory processes occurring at any one of the steps 1 - 7.
Although RNA and pro tein synthesis (steps 1-3) do seem to be
important for ripening, other regulatory mechanisms apart from
transcription and translation may also play a role (see article by
48 D. GRIERSON

(mony different genes)

ovoilobility of DNA for tronscription
(I) TRANSCRIPTION ./
" contral of RNA polyrnerose octivity.
\
(2) P.OST - TRANSCRIPTION processino ond
transport of mRNA'to the cytoplosm.
ImRNAI
(mony different types) mRNA moy be stored or mode
/ ovoiloble for binding to ribosmes

\ TRANSLATION ' " cofoClors such os tRNA moy be

" (3)
IimitinQ

moy require activator ~eins

DEGRADATION or cafoctors, or removal or
inoctivotion of inhibitors

'-1AC-,-TI-VE.........,..EN-Z-Y....
M--ES....I

Fig. 1. Possib1e contro1 points in gene expression. (From Grierson

et al. (5».

Rhodes in this volume). Figure 2 gives a general outline of the

steps invo1ved in the transcription, processing and translation of
mRNA and the production of active enzymes. After transcription by
RNA polymerase 11 many plant mRNAs are modified at the 5' and 3'
ends by specific enzymes. Modification at the 5' end may invo1ve
the addition of a special "cap" sequence starting with a 7-methy1
guanine 1inked via a 5'--5' triphosphate bridge to the second
nuc1eotide in the chain. Modification at the 3' end of some, but
not necessari1y all, mRNAs invo1ves the addition of a po1y(A)
sequence by po1y(A) polymerase. In addition to being modified at
each end the initial RNA transcript may be cut at specific points
by nuc1eases. Fo11owing this type of processing reaction, on1y
certain parts of the RNA sequence. are retained and sp1iced together
to produce functiona1 mRNA mo1ecu1es. Once formed, mRNAs may be
made avai1ab1e for polyribosome formation and protein synthesis
immediate1y or they may be stored unti1 such time as their transla-
tion is a110wed to proceed. It has been suggested that changes in
isoaccepting tRNAs may be important for trans1ationa1 contro1 (12).

The regu1atory imp1ications of the various RNA processing steps

are enormous. Insufficient information is avai1ab1e at the present
time, however, to allow a clear statement of the mo1ecu1ar mechanisms
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 49

DNA

I
I
Release of
compleled mRNA

mRNA 5'end
=====;:======5.
.':~:;r-'5-;:.
rtcOQllillon sequence
ond slorl signal
c:oding reQion I
~end

2
Processi"ll ond oddilion d
"cop· ond poly A loil

modiflld 5'end 3'end

;;=@. . ,.".
mRNA .t.ttNI 11
cop "I poly A toil
1 Translation

polyribosome
formalion
..000.
~

Release of newiy
synlhesized protein
ond foldi"ll 01
trimrninQ
Enzyme octivily may be modified ACTIVE
by chonges in cytoplosmic factors ENZYME

Fig. 2. The synthesis, processing and translation of messenger RNA.

(From Grierson et a. (5».

that deterrnine whether or how a specific rnRNA sequence is produced.

Despite the complexity of the situation this nettle will have to be
grasped if we are to understand how gene expression is controlled.

RNA SYNTHESIS DURING FRUIT GROWTH AND RIPENING

Figure 3 shows the change in nucleic acid content of two varie-

ties of tomate fruits during growth and ripening. Plants were grown
in a glasshouse and individual flowers tagged at anthesis. Fruits
were sampled at various stages of growth and fresh weight, nucleic
acid content and lycopene were measured. There is very little DNA
synthesis or cell division during fruit growth and the increase in
nucleic acid is due largely to RNA synthesis. The accurnulations of
RNA slows down or ceases at the mature-green stage (1-2 weeks before
ripening) and RNA content may actually decline just before or during
ripening (13) (measured by lycopene production).

Measurernents of RNA synthesis, based on the incorporation of

[5- 3H]-uridine into RNA by tomato segements, are shown in Figure 4.
The results indicate that there is a decline in the rate of RNA
synthesis as fruits approach maturity. Subsequently a burst of
incorporation into RNA occurs which reaches a peak at or just before
the initiation of ripening. This does not seem to be due to changes
50 D. GRIERSON

(0) Amberley cross

.60 90

1.0 5.0

40 60

05 2.5

1.5 7.5 (b) Mini popelld

1.0 5.0

t 60

05 25
! 2030

a-J
-..~
245678910
Aoe of tomoto fruit, weeks

Fig. 3. Changes in nuc1eic acid content during the growth and rip-
ening of tomatoes. Resu1ts are shown for a quick-ripening
(Amber1ey Cross, 3a) and a slow-ripening (Minipopella, 3b)
variety. Fresh weight of fruit (D); nuc1eic acid in mg
per fruit (e); nuc1eic acid in mg per g of fruit tissue
(0); 1ycopene content (.). There is virtua11y no ce11
division or DNA synthesis during fruit deve10pment and
ripening and the changes in nuc1eic acid are due 1arge1y
to RNA. Ripening is indicated by 1ycopene synthesis.
(From Rattanapanone et a1. (13».

in tissue permeabi1ity and uptake of [5- 3H]-uridine (13) and is

therefore interpreted as a genuine increase i~ RNA synthesis. Thus,
at the time when total RNA content is decreasing there is a 1arge
stimulation of RNA turnover as fruits approach ripening. It is not
c1ear whether this is an essential part of the ripening process but
evidence discussed be10w does indicate that the production of new
types of mRNA occurs as fruits ripen.

Polyacrylamide gel e1ectrophoresis has shown that during growth

of the fruit there is synthesis of 25S and 18S cytop1asmic rRNA and
po1y(A)-containing RNA (Figs. 5, 6 and reference 13). The po1y(A)~
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 51

1.2 6

2 3 4 5 6 7
Age of tomato fruit. weeks

Fig. 4. Rate of labelling of RNA in segments of tomate fruit at

different developmental stages. The Minipopella variety
was used. Nucleic acid per fruit (.); nucleic acid per g
(O)~ sp. radioactivity of RNA after 24 h incubation in
[5- H]-uridine (e); lycopene content (6). (From
Rattanapanone et al. (13».

containing RNA, purified by oligo(dT)-cellulose chromatography,

stimulates protein synthesis in vitro. It can therefore properly
be described as rnRNA (Figs. 7~nd references 13, 14). There is
also synthesis of plastid 23S and l6S rRNA during tomate fruit
growth but on a quantitative basis this is a relatively small part
of the total RNA(13). As fruits ripen the cells retain the capacity
for synthesis of high molecular weight 25S and l8S rRNA and soluble
RNA (Fig. 5). They also synthesis poly(A)-containing RNA, similar
to that shown in Figure 6. Thus the burst of RNA synthesis associa-
ted with ripening leads to the production of both stable rRNAs and
rnRNA. The ratio of total RNA to poly(A)-RNA does not change much
during the ripening period, suggesting that the production of mRNA
may actually be coupled to rRNA synthesis in some way (13).

Although in ripe fruit there is substantial breakdown of total

cell RNA, giving rise to a decrease in high molecular weight mole-
cules and an accurnulation of small products, this only occurs during
the later stages of ripening (13). This may be a consequence of the
general release of hydrolytic enzymes associated with the advanced
stages of senescence. In the early stages of ripening, although
there may be a controlled turnover of RNA molecules, the cells have
not lost their biosynthetic capacity and there is good reason to
believe that many RNA molecules are functioning in pro tein synthesis.
52 D. GRIERSON

lai 21 days
I.C 12

8
'"Q
)(
4
E
Co
v
E
e
on
CD
N
(bi
i
« 'Orange' :~
ü
075 :3 0
0
'Ö
~
050 2

025

2 4
Migration, cm

Fig. S. Incorporation of [S-3H]-urdine into different RNA fractions

in unripe and ripening fruits. Pericarp segments from un-
ripe (Sa) or ripening (Sb) fruit were 1abe11ed for 24 h
with 1 ~ Ci/~l [S-3H]-urdine. RNA was extracted, purified
and fractionated in polyacrylamide gels. E1ectrophoresis
was from 1eft to right. The optica1 density scan is
shown by the smooth curve. The peaks are, from 1eft to
rig4t; DNA, 25s and l8s ribosomal RNA and a mixture of 5s
and transfer RNAs. The histogram shows the distribution
of radioactivity in each 1 mm gel slice. (From Rattanapanone
et al. (13».

The most interesting question is whether new types of mRNA are

transcribed or accumulated at the onset of ripening. The answer to
this question seems to be yes. Purified po1y(A)- containing RNA
from green and ripening fruits directs the synthesis of a number of
major polypeptides in vitro. A comparison by gel e1ectrophoresis
of the pro teins synthesized in response to different tomato mRNA
samp1es has shown that during ripening at least two extra trans1a-
tab1e mRNAs appear (Figure 7 and reference 14). These experiments
provided the first direct evidence that there are changes in fruit
mRNA coding for specific proteins occurring during ripening. More
recent1y, simi1ar evidence has been obtained for avocado (23).
Experiments of this type most readi1y detect changes in those mRNAs
which accumu1ate in re1ative1y 1arge quantities. Furthermore, they
only relate to the poly(A)-containing mRNA fraction. Quantitatively
minor changes or those associated with the po1y(A)-minus mRNA would
have gone undetected. It therefore seems probable that further work
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 53

'" 6
Q
K
u
4

>-
:t::
~ 2
u
c
.2
"C
C
er 2 4 6
M igrofion. cm

Fig. 6. Gel electrophoresis of poly(A)-containing RNA from tomatoes.

Segments of pericarp from l-week-old fruit were labelled
with [5-~]-uridine for 24 h. Radioactive RNA was extract-
ed from the tissue and poly(A)-containing RNA was purified
by oligo(dT)-cellulose chromatography mixed with unlabel-
led carrier RNA (smooth curve) and fractionated by gel
electrophoresis as in Fig. 5. Unlike the radioactive RNA
in Fig. 5 the poly(A)-containing RNA is not present as
distinct peaks but has a broad size distribution, shown
by the histogram. Similar poly(A)-containing RNA fractions
were obtained from fruits at all stages of development and
ripening. (From Rattanapanone et al. (13».

may reveal other mRNAs that alter in amount during ripening. Never-
the less, the present indications are that those changes that do
occur involve only a small number of new mRNAs. We are not yet
certain which proteins they code for. Work which may help to solve
this question is discussed below.

POLYGALACTURONASE SYNTHESIS DURING RIPENING

As stated earlier there is a lot of evidence for a general

stimulation of protein synthesis during fruit ripening and there are
quite a few examples where an increase in activity of a specific
enzyme has been demonstrated. Generally speaking, however, there
is much less information about the mechanism of such enzyme in-
creases (whether by de novo synthesis or activation) and virtually
no evidence about mRNA coding for specific enzymes. Without such
information it is difficult to build up a proper picture of the
complexity of ripening-related changes and impossible to determine
where the regulatory factors, such as ethylene, operate. In order
to achieve this latter objective it is necessary to study specific
cases in much more detail than hitherto. We have chosen to in-
vestigate the problem of polygalacturonase production in tomato.
54 D. GRIERSON

IGreen
111 11 kI~II~III~ ~;~~ [:·:tfl{~l ~.
IRed 1IIIIII::tll::III{:~f~;;l::hl~~l t~.
e I I ®
~ )!
Fig. 7. Comparison of in vitro translation products of messenger
RNAs from green and ripening fruit. The radioactive
protein products produced by RNA translation in an in
vitro protein synthesis system from wheat germ were
fractionated in 15% polyacrylamide gels under denaturing
conditions and then detected by f1uorography. E1ectro-
phoresis was from 1eft to right. This is a drawing of the
original fluorograph. The approximate mo1ecu1ar weights
of the pro teins marked ß and y, estimated from the
mobi1ity of added marker pro teins fractionated in the
same gel, are 46,000 and 20,000. (From Rattanapanone
et al. (14».

Mature-green tomatoes contain extreme1y low amounts of extract-

ab1e po1yga1acturonase (PG). As fruits begin to change co1our
enzyme activity appears and there is a massive increase during the
1ater stages of ripening (21). There are at least two isoenzyme
forms of tomate po1yga1acturonase which have been purified and
studied. They have diff.erent physica1 properties (Le. mo1ecu1ar
weight. heat stabi1ity, density) but they both seem to be composed
of the same basic polypeptide (21). Isoenzyme 2 is the major form
of the enzyme which accumu1ated during ripening (Figure 8 and
reference 21). Purifie1d PG2 can be converted into PG1 in vitro,
confirming the belief that the enzymes are re1ated, and it is
probable that PG1 is a dimer of PG2 (22). The rin mutant produces
no po1yga1acturonase (Figure 10 and reference 2~and the Nr
mutant norma11y produces on1y PG1 (21).

There are severa1 1ines of evidence that suggest that po1y-

ga1acturonase is synthesized de ~ during ripening. First1y,
incubation of tomato fruit tissue with deuterium oxide leads to
the production of enzyme with an increase in density compared to
that of the water contro1 (Figure 9). Second1y, radioimmunoassay
(21) of tomato extracts, using an antibody raised against PG2, in-
dicates that there is a proportional relationship between enzyme
activity measured by conventiona1 ~ssay and the number of immun-
no1ogica11y active protein mo1ecu1es. Furthermore, no evidence
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 55

TOTAl PG
POTENTATE
100

50

.,""'e
~

C7I
Vl
t-
Z 200
::>

b:

100

PG1

G B 81 T T1 R R1

Fig. H. Total polygalacturonase activity and isoenzyme levels

extractable from individual fruit at various stages of
ripeness. A slow ripening (Potentate) and fast-ripening
(Ailsa Craig) variety were used. Isoenzyme activities
were determined by the heat stability method. After
heating for 5 min at 65°C the remaining activity can be
attributed to 90% of the original polygalacturonase 1.
Measurements were made on green fruit (G); breaker fruit
showing the appearance of colouration (B); la te breaker,
with half the fruit coloured (BI); fruit turning orange
(T): totally orange fruit (Tl); firm red fruit (R) and
slightly soft red fruit (Rl). Total polygalacturonase
activity (0); polygalacturonase 1 (e); polygalacturonase
2 (0). (From Tucker et al. (21».

of an i~nunologically active pro tein was obtained by assaying ex-

tracts from green tomatoes (Tucker, Robertson and Grierson, un-
published). Thirdly, electrophoresis of protein extracts from
green tomatoes in polyacrylamide-dodecyl sulphate gels shows no
56 D. GRIERSON

(A)

W
I/l
«
z
oCl:
::J
I-
~ (6)

~
~
o 0

7'00' 6'45' 6'30'

ANGLE OF REFRACTION

Fig. 9. Demonstration of de novo polygalacturonase synthesis by

density labelling. Individual fruits were taken 35 h
after the onset of ethylene production, the locule contents
removed and the hollow pericarp filled with an approximately
equal weight of either (A) 70% deuterium oxide or (B) water,
for 18 h. Total cell-wall-bound pro teins were then extract-
ed and fractionated on a CsCl gradient. Fractions were
assayed for polygalacturonase 1 activity after heating at
65°C for 5 min to remove any polygalacturonase 2 activity.
(Unpublished result of N.G. Robertson and D. Grierson).

detectable protein running at the position of purified PG. In

contrast, extracts from ripe tomatoes contain a very prominent
protein band at the position corresponding to PG (Figure 10). On
the basis of these three separate lines of evidence it seems
reasonable to conclude that the enzyme is synthesized during
ripening.

RELATIONSHIP BETWEEN THE SYNTHESIS 01" ETHYLENE AND POLYGALACTURONASE

It has been suggested that in tomatoes the synthesis of poly-

galacturonase precedes ethylene production and that the action of PG
 ......
RIBONUCLEIC ACID AND ENZYME SYNTHESIS 57

- - -- •
68
PG
--+
-
. '- 45
""0
I

~
)(

...
.....
3:

.-

"
17 ~
14

PG G R rin M

Fig. 10. Polyacrylamide gel profiles of cell-wall-bound protein .

Protein sampies were suspended in a buffer (40 mM Tris/
HCl pH 9.18 containing 50% w/v sucrose, 10% w/v sodium
dodecylsulphate and 5% v/v mercaptanoethanol) and boiled
for 5 min. Sampies containing 50 to 100 ~g were then
fractionated in a 10 to 15% gradient polyacrylamide slab
gel under denaturing conditions in 0.1% sodium dodecyl-
sulphate. The left hand track contains partially purified
polygalacturonase 2 (PG). The remaining tracks contain
total cell-wall-bound proteins from green normal (G),
ripe normal (R) and yellow (rin)fruit respectively. The
right-hand track contains marker pro teins Mr = 68,000,
45,000, 17,900 and 14,300. Gels were stained in 0.1%
Coomassie blue, 40% methanol, 7% acetic acid and destained
in 30% methanol, 7% acetic acid. (Unpublished result of
G.A. Tucker and D. Grierson).

on the cell walls of the fruit releases wall-bound enzymes important

for ethylene synthesis and other aspects of ripening (20). This
seems an improbable mechanism for the general control of ripening
since some fruits do not produce PG. Furthermore, in tomate the
kinetics of ethylene evolution and polygalacturonase synthesis do
not support this view. We have made careful measurements of the
timing of natural ethylene synthesis and polygalacturonase synthesis
in individual tomatoes. The results clearly show that the enhanced
ethylene production normally associated with ripening is initiated
some 20-30 h before polygalacturonase activity can be detected
58 D. GRIERSON

(Grierson, Robertson and Tucker, unpublished). We conclude that

ethylene synthesis precedes polygalacturonase synthesis. A similar
conclusion has recently been reached for cucumber (24). The
mechanism for switching on ethylene production is unknown.

IS THERE CONTROL AT THE LEVEL OF mRNA?

As shown in Figure 7, tomato mRNA stimulates the synthesis of

a number of polypeptides in ~ and there are specific mRNA
chariges occurring during ripening. One of the mRNAs which increases
greatly in quantity during ripening codes for a protein with a
molecular weight of 46,000 (Figure 7). This is only slightly larger
than the molecular weight of the polyga1acturonase protein in po1y-
acrylamidedodecyl sulphate gels (21). Furthermore, some of the
radioactive pro tein molecules synthesized in vitro in response to
mRNA from ripening tomatoes reacts with antibody raised against
po1ygalacturonase 2 (Figure 11). This provides an assay for
measuring the amount of mRNA for polyga1acturonase. Pre1iminary

Totat Protein
Synthesis
Precipitation of .in....xi.tm
translation products with TMV
polygalactu'onase antibody

Blank
o
TMV
O~====~======~_
o 5 10
pl Polygalacturonase Anti body
Fig. 11. Immunoprecipitation of pro tein synthesized in vitro in
response to tomato RNA, using an antibody raised against
polygalacturonase 2. The total incorporation into protein
in response to RNA from orange tomatoes or tobacco mosaic
virus is shown on the right. The resu1ts on the left show
the titration of the in vitro translation products with
increasing concentration of PG 2 antibody. (From Grierson
et al. in "Biochemistry of Fruits and Vegetables, 11 J.
Friend, ed., Academic Press, in press).
 RIBONUCLEIC ACID AND ENZYME SYNTHESIS 59

results suggest that there is an increase in this mRNA as tomatoes

ripen. Further measurements will be necessary before it can be
determined whether there is any mRNA for polygalacturonase before
ethylene synthesis begins and whether ethylene induces the accumu-
1ation of po1yga1acturonase mRNA.

ACKNOWLEDGEMENT This work was supported by a grant from the Agri-

cu1tura1 Research Counci1.

REFERENCES

1. C. J. Brady, J. K. Palmer, P.B.H. O'Conne1l and R. M. Smi1lie,

An increase in pro tein synthesis during ripening of the
banana fruit, Phytochemistry 9:1037 (1970).
2. L. A. Darby, Isogenic 1ines of tomato fruit colour mutants, Hort.
Res. 18:73 (1978).
3. H. C. Dostal and A. C. Leopo1d, Gibbere1lin delays ripening of
tomatoes, Science 158:1579 (1967).
4. c. Frenke1, I. Klein and D. R. Di11ey, Pro tein synthesis in
relation to ripening of pome fruits, Plant Physio1. 43:1146
(1968).
5. D. Grierson, G. Tucker and N. G. Robertson, The regulation of
gene expression during the ripening of tomato fruits, in
Long Ashton Symposium on 'Qua1ity in Stored and Processed
Vegetab1es and Fruit,' pp 179-191, P. W. Goodenough and R.K.
Atkin, eds., Academic Press, London (1981).
6. E. Hansen, Ethy1ene stimu1ated metabo1ism of immature Bart1ett
pears, Proc. Am. Soc. Hort. Sci. 91:863 (1967).
7. G. E. Hobson, Po1yga1acturonase in normal and abnormal tomato
fruit, Biochem J. 92:324 (1964).
8. A. C. Hu1me, Studies in the nitrogen metabo1ism of app1e fruits.
The c1imacteric rise in respiration in relation to changes in
equi1ibrium between protein synthesis and breakdown, J. Exptl.
Bot. 5:159 (1954).
9. A. C. Hu1me, M. J. C. Rhodes and L. S. C. Woodtorton, The re-
1ationship between ethy1ene and the synthesis of RNA and
pro tein in ripening app1es, Phytochemistry 10:749 (1971).
10. K. Iki, K. Sekiguchi, K. Kurata, T. Tada, H. Nakagawa, N. Ogura
and H. Takehana, Immuno1ogica1 properties of ß-fructofurano-
sidase from ripening tomato fruit, Phytochemistry 17:311
(1978).
11. N. Marei and R. Romani, Ethy1ene-stimu1ated synthesis of ribo-
somes, ribonuc1eic acid and protein in deve10ping fig fruits,
Plant Physio1. 48:806 (1971).
12. I. J. Mettier and R. J. Romani, Quantitative changes in tRNA
during ethy1ene-induced ripening (ageing) of tomato fruits,
Phytochemistry 15: 25 (1970).
60 D. GRIERSON

13. N. Ra t tanapanone , D. Grierson and M. Stein, Ribonuc1eic acid

metabo1ism during the deve10pment and ripening of tomato
fruits, Phytochemistry 16:629 (1977).
14. N. Rattanapanone, J. Speirs and D. Grierson, Evidence for changes
in messenger RNA content re1ated to tomate fruit ripening,
Phytochemistry 17:1485 (1978).
15. A. E. Richmond and J. Bia1e, Pro tein and nuc1eic acid metabo1ism
in fruits. 1. Studies of amino acid incorporation during the
c1imacteric rise in respiration of avocado, Plant Physio1 41:
1247 (1966).
16. J. Riov, S. P. Monse1ise and R. S. Kahan, Ethy1ene-contro11ed
induction of phenylalanine ammonia lyase in citrus fruit pee1,
Plant Physio1. 44:631 (1969).
17. J. A. Sacher, Permeabi1ity characteristics and amino acid incor-
poration during senescence of banana tissue, Plant Physio1.
41:701 (1966).
18. J. A. Sacher, Senescence and postharvest physio1ogy, Ann. Rev.
Plant Physio1. 24:197 (1973).
19. G. H. De Swardt, J. R. Swanepoe1 and A. J. Duvenage, Relations
between changes in ribosoma1 RNA and total pro tein synthesis
and the respiration c1imacteric in pericarp tissues of tomato,
Z. Pf1anzenphysio1. 70:358 (1973).
20. E. G. Tigche1aar, W. B. McG1asson and R. W. Buescher, Genetic
regulation of tomato fruit ripening, J. Hort. Sei. 13:508
(1978).
21. G. A. Tucker, N. G. Robertson and D. Grierson, Changes in po1y-
galacturonase isoenzymes during the 'ripening' or normal
and mutant tomato fruit, Europ. J. Biochem. 112:119 (1980).
22. G. A. Tucker, N. G. Robertson and D. Grierson, The conversion
of tomato fruit po1yga1acturonase isoenzyme 2 into isoenzyme
1 in vitro, Europ. J. Biochem. 115:87 (1981).
23. R. E.-Christoffersen, E. Warm and G. C. Laties, Gene expression
during fruit ripening, Plant Physio1. Supp1. 47:73 (1981).
24. M. E. Sa1tveit, Jr. and R. F. McFeeters, Po1yga1acturonase
activity and ethy1ene synthesis during cucumber fruit de-
ve10pment and maturation, Plant Physio1. 1019:66 (1980).
RESPIRATION AND ENERGY METABOLISM IN SENESCING PLANT TISSUES

Theophanes Solomos

Department of Hortieulture
University of Maryland
College Park, Md. 20742

INTRODUCTION

The seneseenee of detaehed plant organs is assoeiated with

eataelysmie physiologieal, histoehemieal and bioehemieal ehanges
(9,16,17,64,90,100). A1though the proeess is predominantly eatabolie,
anabolie reaetions are nevertheless neeessary for seneseenee to
proeeed (91). The prevention of seneseenee by inhibitors of anabolie
proeesses bears witness to the above (43,71,74). Furthermore, overt
eatabolie reaetions, sueh as the eonversion of stareh to suerose,
require at least one mole of ATP per mole of suerose formed (118).
The transport of inorganie and organie nutrients from seneseing
attaehed leaves to the parent plant must also be a sink of metabolie
energy. In detaehed seneseing organs respiration is the sole souree
of energy, since the photosynthetic capacity of eertain detaehed
organs is minimal and moreover seneseenee of detaehed leaves proeeeds
faster in the dark than in the light (69). In this presentation we
shall examine the rate of produetion, regulation, and physiologieal
signifieanee of metabolie energy in seneseing detaehed plant organs.

RESPIRATORY DRIFTS OF DETACHED PLANT ORGANS

Fruit

It was observed by Kidd and West (56) that the rate of respira-
tion of detaehed apple fruits deereased initially and after reaehing
aminimum, suddenly inereased rapidly, peaked and then deelined.
Kidd and West (56) named this sudden rise in respiration during
fruit ripening the elimaeterie. It was subsequently shown that this
phenomenon oeeurs during ripening of other fruits besides apples

61
62 T.SOLOMOS

(16,17,90). Fig. 1 shows the changes in the rates of 02 uptake and

C02 output of avocado fruits in the course of ripening. However
this burst of respiration during ripening is not a sine qua ~ for
all fruits. Thus the rate of respiration of detached citrus fruits
decreases gradua11y during storage (16,17), whereas the rate of
respiration of strawberries does not show pronounced changes during
ripening (58). Biale (16) divided fruits into c1imacteric and non-
climacteric groups, based on whether their respiration increased
or gradua11y dec1ined during ripening. Table 1 shows some fruits
representative of both groups.

Leaves

The changes in respiration during senescence have been studied

in both detached and attached 1eaves. Blackman was the first to
carry out extensive studies with senescing detached 1eaves (see
James, 53). The respiratory drift was divided into six phases as
shown in Table 2. Thus the respiratory drift of senescing leaves
shares many common features with that of climacteric fruits. The
most important is the rise in respiration during ye110wing (phase 4).

10 1'1.
"
OA'(56
Fig. 1. Respiratory drift of detached avocados.
RESPIRATION AND ENERGY METABOLlSM 63

Table 1. Classification of Fruits

Climacteric Non-climacteric

Apple Cherry
Apricot Citrus (grapefruit,lemon,orange)
Avocado Me10n
Banana Pineapp1e
Peach Strawberry
Pear
P1um
Tomato

A rise in respiration during the later stages of 1eaf senescence has

been observed with bar1ey 1eaves (53), tobacco 1eaves (4,67,66,103),
and oat 1eaves (69). On the other hand, work with pea 1eaves (104)
shows that respiration declines steadi1y during senescence. Woo1house
(124) studied the photosynthetic efficiency and rate of respiration
of attached 1eaves of Peri11a frutescens. It ean be seen in Fig. 2
that there is a e1imaeterie-1ike inerease in respiration prior to
senescenee.

F10wers

The respiratory behavior of detached f10wers has not been

studied as extensively as that of fruits and 1eaves. However, the
availab1e reports indieate that cut f10wers show both e1imacterie
and non-e1imacteric types of respiratory drifts (33,91,93). It has
been shown that cut carnations exhibit a typica1 climacterie
repiratory drift (39,80). Figure 3 shows that the rate of C02 out-
put of cut carnations decreases sharp1y after cutting, to increase
again during the wi1ting stage of the petals. The rate of respira-
tion of cut roses, on the other hand, dec1ines steadi1y during
seneseenee (55).

CAUSES OF TRE CLlMACTERIC RISE IN RESPIRATION

Sinee its discovery, the e1imaeterie rise in respiration

during fruit ripening has been attributed to a decrease in "organi-
zation" resistance of the cel1 (19). presenee of natural uncouplers
(77), enhancement of pro tein synthesis (50), and the synthesis of
specific "ripening" enzymes (90). The climacteric occurs in fruits
which differ widely with respect to growing conditions, composition
and physiology. The only common metabolie feature is their ability
to produce ethylene and to respond to exogenous ethylene with an
increase in respiration (16,17,90). Moreover, ethylene enhances
the rate of respiration of non-c1imaeteric fruits such as citrus
(17). The difference between eitrus and climacteric fruits lies
64 T. SOLOMOS

Table 2. Phases of Leaf Senescencea

Duration
Phase in days Slope Color of Leaves

I I high level green

2 6 rapid fall green
3 6 low level green-pale green
4 c.20 steady rise pale green yellow
5 c.20 steady fall yellow-brown
6 sharp rise brown

a James (53)

20 r
15

..:
.r:
;;-
E
-':!.N
0 10
U \
...:
\
\
5

Respiration
------x----------~xr_----

50 60 70
Time (days)

Fig. 2. Changes in the rates of respiration and photosynthesis

of attached Perilla leaves during the later stages of
development (Woolhouse, 124).
RESPIRATION AND ENERGY METABOUSM 65

1.00

19

,(00

.....
1 I~O
I-
~

.....
1 11..0
01
N
0
U 100

~
;:l

40

20

" ."
DAYS
10

Fig. 3. Rate of CO 2 output of cut carnation f10wers.

in the inability of the former to produce endogenous ethy1ene nat-

ura11y or in response to the application of exogenous ethy1ene.
Citrus fruits, however, can produce ethy1ene in response to stresses
(32,72) or if they are harvested at an immature stage (3). On the
other hand, ethylene is not produced by citrus fruits during normal
ripening. McMurchie et al. (75) treated bananas, a climacteric
fruit, and oranges, a non-climacteric fruit, with propylene, an
ethylene analog, and found that the rate of respiration was enhanced
in both fruits. Propylene failed to induce the production of ethy-
lene in oranges. To use their own termino10gy, oranges lack system
11, which the authors consider to be associated with the induction
of the autocata1ytic production of ethylene. The non-ripening
tomate mutants further demonstrate the c10se relationship between
the rise in respiration and ethy1ene. Thus, in the rin mutant,
which shows on1y a 1imited degree of ripening, there is no increase
in either the production of endogenous ethylene or respiration during
storage (48). These authors have shown that app1ication of exogenous
ethy1ene enhances the rate of respiration. Moreover, interrupted
app1ications of exogenous ethylene enhance respiration sequential1y.
66 T. SOlOMOS

In addition, ethy1ene enhances respiration in tissues other

than fruits where ripening is not at issue (89, 108-110). In
Tab1e 3 severa1 tissues are shown whose respiration is enhanced by
ethy1ene. In senescing 1eaves the association of the rise in res-
piration with ethy1ene production has not yet been estab1ished
unequivoca11y. It has been shown that senescing tobacco 1eaves
showed both an increase in respiration and ethy1ene production
(4, 103). Tet1ey and Thimann (113) c1aimed that there is no cor-
relation between senescence of oat 1eaves and ethy1ene. It has
recent1y been observed that the addition of 1-aminocyc1opropane-1-
carboxy1ic acid (ACC), the immediate precursor of ethy1ene (2), to
the bathing solution of senescing oat 1eaves enhances respiration
and senescence (Thimann, personal communication). We have found
recent1y that when ivy 1eaves (Hedera helix) senesce on the parent
plant, they produce about 2-3 n1 C2H4/g/h. The rate of respiration
of green ivy1eaves kept in the dark increases in response to ethy-
1ene treatment (Fig. 4). The data indicate that, as in the case of
fruits, the respiratory upsurge during 1eaf senescence may be
associated with an increase in the endogenous production of ethy1ene.

Certain cut f10wers produce ethy1ene during senescence (26).

Moreover the rise in respiration in carnations coincides with a
sharp increase in ethy1ene production (39, 73). We have found

Tab1e 3. The Effect of Ethy1ene on the Respiration Rate of

a Variety of Plant Tissues

Respiration Rate
Contro1 C2H4
Tissue
02 C02 02 C02
lJl/g/h
App1es 6 6 6 6
Avocados 35 36 150 140
Cherimoyas 35 35 160 150
Lemons 7 7 16 15
Grapefruit 11 11 30 29
Beetroot 11 11 22 22
Carrots 12 10 20 19
Potatoes 2.5 2.5 14 11
Sweet Potatoes 18 17 22 22
Rutabagas 9 9 18 15
RESPIRATION AND ENERGY METABOLlSM 67

Effect of C2H4 on Ivy Respiration

/"0

'!öO

/40

'30

,~ r (tll'1

IfIO

/".
L
..c
"-
rn
'0
"-N fO
0
u 70
c<
~ 100

st.
'10

'0

~o

'0 -
z 3 " $"" ,., 10 1\ ,~

(days)

Fig. 4. Rate of respiration of detached ivy 1eaves kept in the

dark in air and 10 p.p.m. ethylene

that, with cut carnations, the addition to the holding solution

of arninoethyoxyviny1 g1ycine, an inhibitor of ethy1ene production
(64), prevents both the rise in respiration and ethy1ene formation
(Fig. 5). In addition, the app1ication of exogenous ethylene en-
hances the rate of respiration of roses, which do not usually show
a climacteric type of respiratory drift (55).

In summary the evidence indicates that the rise in respiration

during senescence of fruits, cut flowers and possibly leaves is the
result of ethylene action. In this respect the division of fruits
68 T.SOLOMOS

.....
I
L..
~ 1(jI()

I
01
C\I 1'2.0
o
u
~
;l.80

':r
.,
a. I
.....

Go e 10 \-a.
DAYS
Fig. 5. Effect of AVG on the rates of respiration and ethylene
production of cut carnation flowers.

into climacteric and nonclimacteric should be based on their ability

to produce sufficient quantities of ethylene rather than on their
respiratory behavior.

MODE OF ETHYLENE ACTION ON PLANT RESPIRATION

Ethylene induces a wide variety of physiological responses in

higher plants (1.24,64,78,86). Theoretically the rise in respiration
can be the result of de novo synthesis of new respiratory enzymes
(coarse control), or the activation of pre-existing enzymes (fine
control) or a combination of the two. Several lines of evidence
indicate that with the possible exception of pome fruits (38,52),
the respiratory potential of preclimacteric fruits is sufficient
to sustain rates of respiration observed at the climacteric peak.
Thus Millerd et al. (77) reported that the rate of respiration of
preclimacteric avocado slices treated with uncouplers of oxidative
phosphorylation was similar to that at the climacteric peak. In
addition, the glycolytic potential of preclimacteric avocados is
adequate to handle the carbon flux required during the climacteric
(107). The initial rate of respiration of freshly cut carnations
equals or even exceeds the respiratory peak during senescence
(Fig. 3). Moreover the fact that inhibitors of protein synthesis
prevent ripening of intact pears and banana slices even though
they do not decrease the rise in respiration supports the above
proposition (43,74).
RESPIRATION AND ENERGY MET ABOLlSM 69

REGULATION OF PLANT RESPIRATION

In senescing fruits, carbohydrates are the principal source of

energy. Even in avocados, which may contain as much as 15% fat,
carbohydrates are the main respiratory substrates (18). On the one
hand, fat content does not change with ripening and avocados lack
the enzymes involved in the first steps of the mobilization of fats
(18). On the other hand, the stable carbon isotope composition of
respiratory COZ resembles that of cellular carbohydrates at all
stages of ripeness of avocado fruits (Table 4) (Solomos and Laties,
unpublished). In the case of leaves, proteins may contribute to
respiration at later stages of senescence (53).

The respiratory pathways of higher plants are the same as

those of other higher organisms. They include glycolysis, the pen-
tose pathway, tricarboxylic acid cycle and the electron transport
chain. The existence of a net flux and the necessity for a rapid
control in response to changes in the energy requirements necessi-
tate the displacement from equilfbrium of the overall reaction of
the respiratory pathways (5a, lZ, 79). It has been observed that
certain enzymatic steps of a metabolie sequence ar~ greatly dis-
placed from equilibrium and that these steps are usually regulatory
in nature. The attributes of regulatory enzymes are: (a) the re-
actions they catalyse are located at metabolie crossroads, (b) these
reactions are displaced from equilibrium, (c) the enzymes are highly
allosteric, and (d) there is an inverse relationship between the
changes in the overall flux and the level of substrate. Thus,
during a transition from a lower to a higher overall flux, the
concentration of substrate decreases. This pattern of changes
indicates that the activity of the enzyme under consideration has
been increased to a greater extent than that of both the immediately
preceding and the subsequent steps (5a,Z9,118). For areaction to
be considered non-equilibrium its observed mass action ratio must
differ by ZO-fold from the equilibrium constant (79). Although
the kinetic control of enzymatic reactions involved in energy pro-
duction is the most advantageous and prevalent (5a), in certain
cases equilibrium reactions contribute to metabolic regulation(60).

Glycolysis

üf the glycolytic enzymes, phosphofructokinase (PFK) and

pyruvate kinase (PK) are the key ones which fulfill the attributes
of regulatory enzymes. They are located at strategie metabolic
points (118), are highly allosteric, their activities being affected
by several metabolites (Table 5), their observed mass action ratios
are greatly displaced from equilibrium (Table 6), and crossover
changes occur under conditions where the rate of glycolysis changes
from one steady state to another (5,11,41,49,54,59,76,79).

The rate of glycolysis of pea seeds increases by two-fold with

transfer to NZ (11). This increase is associated with a character-
70 T. SOLOMOS

Tab1e 4. Carbon Isotope Va lues of Carbohydrate and Protein Enriched

Fractions, Total Fats and C02 in the Course of Ripening
of Avocado Fruits. a

13 12
o cl C(O/OO)
Stage of
ripeness Total
Carbohydrate Pro teins Fatty Acids

Prec1imacteric -23.31 -24.15 -31 -23

Climacteric -23.40 -24.08 -30.7 -23.2
Postc1imacteric -23.54 -24.29 -32.1 -23.5

a
Solomos and Laties, unpub1ished

Tab1e 5. Modulators of PFK and PKa

Modulators
Enzyme
Positive Negative

PFK PEP, 2PGA,

3PGA, ATP, citrate
gluconate-6-P
2+
PK Ca ,ATP.
citrate

a
Turner and Turner (118)

istic pattern of changes in the glycolytic intermediates. which re-

vea1 the existence of crossover changes at the levels of PFK and PK
(Table 7). It can be seen from the data of this table that the
changes in the known modulators of PFK and PK are in the expected
direction. Thus phosphoenolpyruvate (PEP). a strong inhibitor of
pea PFK (118). decreases sharply. Similarly the inhibitor ATP.
2-phosphoglycerate (2-PGA) and 3-phosphoglycerate (3-PGA) also
decrease. In the case of anaerobic castor bean endosperms there
is in addition a decrease in the total adeny1ate pool. which wou1d
further contribute to the activation of PFK (59). It is known that
citrate. an inhibitor of both PFK and PK. decreases in anaerobic pea
RESPIRATION AND ENERGY METABOLlSM 71

Table 6. Mass Action Ratios of Certain G1ycolytic Enzymes in

Air and NZ of Pea Seeds a

Mass Action
Ratio
Enzyme
Air NZ

Glucose phosphate isomerase 0.19 O.ZO 0.42

Phosphofructokinase 0.016 5.09 1.05 x 10 3
Phosphog1ycero1mutase 0.841 0.816 0.15
Eno1ase 0.Z88 0.Z16 3.7
Pyruvate kinase 6.90 13.60 11 x 10 3

a
Barker et a1. (11)

Tab1e 7. Changes in the sugar phosphates, adenosine

nuc1eotides and phosphoeno1 pyruvate of
green pea seeds in air and N2 .a

b Treatment
Compounds
Air 30 min in N2
f.I Ilffioles/100 gr
G6P 55.5 55.5
F6P 10.50 11
F,1,6diP 0.88 17.50
DHAP Z.10 10.57
3PGA 32.00 8.53
ZPGA 26.90 6.96
PEP 7.75 1.50
AMP 2.30 13.70
ADP 18.67 64.33
ATP 96.30 20

aBarker et a1. (11)

b
G6P (glucose 6-phosphate); F6P(fructose 6-phosphate);
F,1,6diP(Fructose 1,6di phosphate); DHAP (dihydroxy-
acetone phosphate); 3PGA (3 phosphoglyceric acid);
ZPGA(Z phosphoglyceric acid); PEP (phosphoenolpyruvate)
72 T. SOlOMOS

seeds (121). In addition, there is a l6-fold increase in the lactate

content of anaerobic pea seeds (121), which may decrease the pH of
the cytosol and hence enhance the activity of PFK (118). In short,
the changes in the levels of the modulators of PFK and PK are those
anticipated for the enhancement of glycolysis.

Crossover changes at the PFK and PK levels have been observed

during the climacteric with bananas (10,101), avocados (Fig. 6)
(107) and tomatoes (28). Because of the analytical difficulties
there are no data concerning the simultarteous changes of all factors
~nvolved in the regulation of glycolysis either during the climacteric
or during the rise in respiration of senescing detached leaves and
flowers. However, from the fragmented data it is clear first that
glycolysis is enhanced during the climacteric rise in bananas,
avocados and tomatoes, and second that this increase in gycolytic
activit~ is caused by an activation of PFK and probably PK. The
activation of these enzymes, however, cannot be completely explained
from the associated changes of the most important modulators. Thus,
apart from PEP, which decreases sharply,ATP increases in avocado,
cantaloupe, cherimoya and tomate fruits and in senescing leaves
(Table 8). In bananas, citrate either does not change (105) or in-
creases with the climacteric (82). It should be pointed out that
the concentration of citrate in bananas is such (ca. 15 mM) that it
is more than sufficient to completely inhibit PK in certain plant
tissues (118). It is thus obvious that in vacuolated plant cells,
cellular compartmentalization may alter the actual concentrations
of. the enzyme modulators, especially organic acids, at the site of
the enzyme (13,81). Chalmers and Rowan (28) proposed that the
activation of PFK during the climacteric of tomatoes is caused by
an increase in the cytosolic concentration of inorganic phosphate,
a positive modulator of PFK, in several plant tissues (117).

Avocado PFK is inhibited by ATP and stimulated by Pi (66).

Salminen and Young (101) have shown that the kinetic characteris-
tics of banana PFK change in the course of the climacteric. In
particular the enzyme exhibited negative co-operativity with re-
spect to the substrate, F6P. However, the negative cooperativity
was much more stringent with the enzyme isolated from prelcimac-
teric than from climacteric bananas. Furthermore the SO.5 changed
from 5.8 mM at the preclimacteric stage to 1.72 mM at the climac-
teric peak. Also, the inhibition by the adenine nucleotides changed
in the course of the climacteric. The most notable was that with
AMP, which lost its inhibitory effect at the climacteric. Pi and
NH 4 ions alone had no effect on the banana PFK. However, both ions
reduce the inhibition caused by ADP. On the basis of their electro-
phoretic data, Salminen and Young (101) concluded that first there
was no production of a new species of the PFK in the course of the
climacteric, and second the enzyme remained oligomeric at all stages
of ripeness.
RESPIRATION AND ENERGY MET ABOLlSM 73

c / 2 ~
ICl,maclefic l.L
I 6 ~
c Cl.
/ o
/ 12 l.L

cI
/ .,
V>

.8 ~
C
C
/ C C FDP c
/
A
--------~ Canlrol
~ ---.-------~---.----.- 0
lL. 320-L _______ ~ Conlrol ,

ißf 240i\ \'- G6P ~! ~~

Vl ''J c...
-"" 160~ .\:"-:'" (l,mocleflc 80 1;:'
E I- - - - - - 4 • conlrJol V>
c 80-1' PEP 40 -6
, ' E
I \.l' '"0 CI,mocleflc c
O~ 0

1:
:OO~
1
"-
~
w.. 80
I
~
"- 60 ~ C1 H.
0"-
E 40~ + ....-_""'-'0--0---C--O-.Q--~)-- Contral
......

20~

O~i
10 '4 18 2226
1
2 6
HOURS
Fig. 6. Course of respiration and the levels of PEP, G6P, and
FDP during the climacteric of intact avocado. Fruit
treated with 30 ~l/l ethylene where indicated

Isaac (1980) (reported by Rhodes (91», on the other hand,

found that PFK isolated from tomato fruits was oligomeric up to
the climacteric peak, while the enzyme extracted at the post-
climacteric stage was present in both oligomeric and low molecular-
weight species. Further, under certain conditions the oligomeric
form could be converted to smaller fractions. This form showed
negative cooperativity with respect to substrate, the degree of
which was determined by the Mg2~/ATP ratio, whereas the small mo-
lecular weight species exhibited classical Michaelis-Menten kine-
tics. Pi converted the oligomeric species to smaller molecular
weight species which in turn showed hyperbolic kinetics.

It has already been pointed out that PK shows all the charac-
teristics of a regulatory enzyme (118). However, no information
is yet available concerning the kinetic properties of fruit PK.
As in the case of PFK the changes of the negative modulators, ATP
74 T. SOlOMOS

Tab1e 8. Changes in the Levels of Adenine Nuc1eotides in Senescing

Fruits and Leaves

AMP ADP ATP

Tissue Stage nmo1es/gr

Avocado 1 Prec1imacteric 207 89

C1imacteric 124 140
Cherimoyas 2 Prec1imacteric 48
C1imacteric 115
3
Canta10upes Prec1imacteric 110
C1imacteric 350
Contro1s 40
Tobacco 1eaves 4 Senescing 90
5
Contro1 111
Bar1ey Senescing 218
Contro1 18 40.8 45
6
Ivy 1eaves Senescing 23.9 39.7 74

lyoung and Bia1e, (126)

2So1omos and Laties, (109, 110)
3
Rowan et a1., (95)
4Sis1er and Pian, (103)
5Malik and Thimann, (69)
6
Warman and Solomos, 1981, unpub1ished

and citrate, call for a decrease in PK activity in the course of

the climacteric, since ATP increases in most climacteric fruits,
and in the case of bananas, citrate may also+increase with the
climacteric rise. However the presence of K ions is a requirement
for PK and it also a1leviates the inhibition of n~gative modulators
(117). Thus, if the cytosolic concentration of K increases during
the climacteric, as has been reported for tomatoes (120), then PK
wou1d be expected to be activated. In short the present ana1ytica1
data indicate that the enhancement of glycolysis during the c1imac-
teric is caused by an activation of PFK and PK and that in bananas
and tomatoes a chaQge in the kinetic properties of PFK in the
course of the climacteric may be the most important factor in
bringing about the enhancement of the glycolytic f1ux.

Pentose Pathway

The measurements of Ashihara and Komamine (6) show that of

the enzymes of the pentose pathway, only the overall reaction of
RESPIRATION AND ENERGY METABOLlSM 75

G6P dehydrogenase is a non-equi1ibrium reaction. Further, this

enzyme is shown to be under both coarse and fine contio1s in
various plant tissues (118). The 1atter authors consider the
NADPH!NADP ratio the most important factor in the regulation of
G6P-dehydrogenase in vive in higher plants. With respect to fruits
there is no information available concerning the changes in reactants
and modulators of this enzyme in the course of the c1imacteric.

Tricarboxy1ic acid cycle

Mitochondria capable of 1inking the oxidation of Krebs cyc1e

intermediates to phosphorylation have been iso1ated from fruits at
various stages of ripeness (37,51,61,94). Further, there seems
to be no change in the mitochondria1 activities with ripening.
Lance et al. (61) stated: "In the presence of cofactors, taking
state 3 as the most reliab1e criterion, no major changes in the
oxidative abi1ities of mitochondria from avocados during the ripening
process have been found." With isolated mitochondria the avai1abi1ity
of ADP controls the rate of oxygen uptake. In vivo, a1though oxida-
tive phosphorylation exerts a strong control on respiration, since
the addition of uncoup1ers a1ways e1icits an increase in the rate of
respiration (see Beevers, 12), this contro1 cannot be thought of as
a state-4-to-state-3 transition. On the one hand, ADP is a1ways
present in sufficient quantities in plant tissues. On the other,
ADP decreases or shows marginal changes in the course of the
climacteric of avocados (126), and during the rise in respiration
of ivy leaves treated with ethylene (Table 8). Hence the control
on plant respiration of oxidative phosphorylation is not exerted by
the level of ADP per se, but rather by its feedback control on sub-
strate mobi1ization, name1y glycolysis and the pentose phosphate
pathways and possibly on some of the enzymatic steps of the Krebs
cycle (5a,117,123).

Several of the enzymes involved in the TCA cyc1e are located

at metabolic crossroads, and are highly allosteric, hence poten-
tial control points. Wiskich (123) has summarized the most important
regulatory metabolites of the enzymes of the Krebs cycle (Table 9).
The studies of the changes in the levels of the intermediates of the
Krebs cycle under different respiratory regimes and during the
transition from one steady state to another are limited and incon-
clusive. In fact the pattern of changes in the TCA cycle inter-
mediates in the air-to-N2 and N2-to-air transition of green peas led
Wager (121) to propose that in pea seeds the TCA cycle is not a
major pathway contributing to respiration. However, it shou1d be
kept in mind that measurements of overall concentrations may not
reflect the concentration present inside the mitochondria and also
that the availability of cofactors may distort the interpretation
of the results of the changes in the levels of the TCA cycle inter-
mediates. In addition, the metabolite transport systems, the pre-
sence of ma1ic enzyme, and the action of transaminase reactions
76 T. SOLOMOS

Tab1e 9. Metabo1ites Affecting the Activities of Enzymes of the

Krebs cyc1ea •

Modulators
Enzyme
Positive Negative

Pyruvate dehydrogenase NADH, CoA

Citrate synthetase ATP
Isocitrate dehydrogenase NADH
a-oxog1utarate dehydro-
genase AMP
Succinate dehydrogenase oxa10acetate
Ma1ic enzyme CoA NADH
Ma1ic acid dehydrogenase NADH, ATP

~iskich (123)

wou1d be expected to drastica11y alter the assumed cyc1ic uti1ization

of the TCA cyc1e intermediates. In banana fruits most of the TCA
cyc1e acids increase with ripening (Fig. 7) (10,82), and this in-
crease resu1ts in the decrease of the pH of the pulp (44). In the
case of app1es, however, ma1ic acid decreases during ripening due
to the synthesis of ma1ic enzyme (38, 52). It is obvious that on
the basis of ana1ytica1 data neither the activity of the TCA cyc1e
nor the possib1e regu1atory steps can be ascertained in senescing
plant tissues.

Metabo1ite Transport

The fo110wing metabo1ite transport systems have been identified

in plant mitochondria: pyruvate/OH-, Pi/OH-, dicarboxy1ate,
tricarboxy1ate, glutamate, a-oxog1utarate, adenine nuc1eotides and
adenosine diphosphate (122). Severa1 1ines of evidence indicate
that the metabo1ite transport system functions in vivo. For in-
stance in fresh potato slices, ma10nate inhibitS-the stimulation
of respiration by uncoup1ers, notwithstanding the absence of the
functioning of the TCA cyc1e (62). Further, the a-oxidation of
fatty acids, which are the respirab1e substrates of fresh potato
slices, is inhibited by rotenone (125), indicating that the e1ec-
tron transport chain is the terminal e1ectron carrier. Wu and
Laties (125) attributed the inhibition of the respiration of un-
coup1ed fresh potato slices by ma10nate to the impairment by the
acid of the ma1ate-oxa10acetate shuttle, which is considered to be
the vehic1e for the transport of reducing equiva1ents from the
NADH generated in the microsomes during the a-oxidation of fatty
acids to mitochondria. A1though the operation in vive of the meta-
bo1ite transporters is not in doubt, their regu1atory ro1e has not
RESPIRATION AND ENERGY METABOLlSM 77

. ,/:
.• 10
.S
! • •••
.-
10

I
'" 10
• f. "'.Iot •

• •
\,4
•
•
eo I
..• .0
~

-t.
~
ao

·
•. II!EJ!

..• ./
._._,J"
40

t ao
-f •
•:a.. d. GlCIloacetot.

..• aoo
300 ~
..
.I
~

k0
':"100
•"- ... .-. , ;I. c ketoglutarate

I
200

..• 150

·
-t
~ 100
2

50 , .-, ,J
• •
b. "",vate

f'-....
• •
/
..•11 11
I
-.
0
2 4
/
0
v •"- ____ e - e ./
E G. C02

o 8 12 111 20
0 ... .,.5

Fig. 7.a. The continuous line curve shows the changes in rate of CO 2
output of a single banana finger ripened at 18°C. The cir-
cles show the rates of respiration of other single fingers
taken for analysis at different stages of ripeness.
7.b,c,d,e,f. Changes in content of pyruvate, a-ketoglutarate,
oxalacetate, PEP, and malate, respectively, in banana in
which the rates of C02 output are shown in a.
78 T, SOLOMOS

been estab1ished. Wiskich (123) considers it un1ike1y to be of

major importance in the regulation of the TCA cyc1e.

E1ectron Transport

A1though the composition of the e1ectron transport chain in

senescing fruits has not been determined exp1icit1y, there is never-
the 1ess no reason to be1ieve either that it differs from other plant
tissues or that it changes in the course of ripening (61,94). In-
tact fruits and mitochondria iso1ated therefrom are resistant to
cyanide (94,109). It is we11 documented that cyanide resistant
oxidase branches from the main e1ectron transport chain at the
level of ubiquinone (14,36,47,106,111).

'It was observed that (a) HCN induces the c1imacteric rise in
respiration and eventual ripening of avocado fruits, (b) the changes
in the glyco1ytic intermediates of avocado and potato tubers treated
with HCN are simi1ar to those induced by ethy1ene,(c) ethy1ene and
HCN induce in potato tubers identica1 changes in respiration and
sugar content, (d) ethy1ene enhances respiration in tissues where
cyanide acts simi1ar1y, whi1e having no effect on tissues whose
respiration is inhibited by cyanide (45,107,108,109,110). On the
basis of the above observations it was proposed that for ethy1ene
to enhance plant respiration the presence of cyanide-resistant
respiration is necessary. Two crucia1 questions have to be examined
experimenta11y: first, is ethy1ene-enhanced respiration prevented by
inhibitors of the alternate oxidase? and second, is alternate oxidase
functioning in the absence of inhibitors of the cytochrome path?

To answer the first question, we treated with ethy1ene thick

banana slices which had first been infi1trated and were constant1y
supp1ied with a 3 mM solution of SRAM, an inhibitor of the alter-
nate oxidase (102). It can be seen in Figure 8 that the inhibition
of the alternate oxidase did not prevent the rise in respiration
in response to ethy1ene (Tucker and Solomos, unpub1ished). Simi1ar
data were obtained with ivy leaves (So10mos, unpub1ished observa-
tions). Thus ethy1ene enhances plant respiration independent of
the nature of the terminal oxidase.

To answer the second question, the mechanism which regu1ates

the apportioning of e1ectrons between the cytochrome and alternate
paths will first be examined. Bahr and Bonner (7,8) have shown
that the diversion of e1ectrons to the alternate path is independent
of the redox state of the cytochromes, since antimycin-A, which
inhibits the e1ectron transport at the level of the b cytochromes,
is as effective in diverting e1ectrons to the alternate path as is
cyanide. Also, the energy state of the mitochondria is not a con-
trolling factor, since e1ectrons are diverted to the alternate
oxidase in both coupled and uncoupled mitochondria. Bahr and Bonner
RESPIRATION AND ENERGY MET ABOLlSM 79

i
'tii
~
~
6
*•

f·
0
8N *•
2
rg .... )(.:~::~:~::2::$::~:~::Sr-:~----qe.,~

0 24 48 72 96 120
Time after slicing (hr5)

Fig. 8. Effects of SHAM on respiration of thick banana slices not

treated with C2H4 and treated continuously with 5 ppm
C2H4' Mannitol 0.3M was used as an osmotic stabilizer in
all samples. Each point is the mean of two composite
sampies each of four 1.5 cm thick slices. The symbols
indicate the following: 5 ppm C2H4(-); no C2H4(---);
C02 output (x,o); 02 uptake (*, 0); control (x, *); and
3mM SHAM (0, .).

(8) derived an equation by which the fraction of the alternate

oxidase which is engaged in the absence of inhibitors of the cyto-
chrome path can be calculated. For this, mitochondria were titrated
with inhibitors of the alternate oxidase in the presence of cyanide.
These values of oxygen uptake were called g(i). The authors carried
out similar titrations in the absence of cyanide. Assuming that the
degree of inhibition by the hydroxamic acid derivatives, inhibitors
of the alternate oxidase (102), is the same irrespective of the
presence of cyanide, then the total oxygen uptake VT= pg(i) + Vcyt,
where Vcyt is the flux through the cytochrome path and p is a number
between U and 1. If p = 1 then the full capacity of the alternate
path is realized, whereas if p = °
all electron transport is carried
out via the cytochrome oxidase. Bahr and Bonner (7) concluded that
the apportioning of electrons between the two paths is controlled
by an equilibrium mechanism. They proposed that both paths share a
common component Band the first component of the alternate path is
A which is in ready equilibrium with B. The standard redox poten-
tials of these two electron carriers differ, with B being more
positive, so that it allows A to be fully oxidized while B is
partially reduced.
RT AO X Bred
From the Nernst equation, E' = nF In ---d'
Are B ox
80 T. SOLOMOS

Bahr and Bonner calculated that the difference in the redox poten-
tials of the above carriers would be 35 mV. Storey (111) showed
that ubiquinone is the carrier share by the two paths (component
B) and FPma (Flavo-protein, mediumpotential, absorbing light) is
the first carrier of the alternate path (component A). The E
values of ubiquinone and FPma are +60 and +20 respectively (112)
which meets the requirement of a difference of 35 mV proposed by
Bahr and Bonner (8). The corollary of these observations is that
the flux through the cytochrome path alone determines the degree
of the diversion of electrons to the alternate path. In other
words as long as the capacity of the cytochrome path is sufficient
to handle the electron flux the alternate path will not be engaged.

Theologis and Laties (114-116) tested the above hypothesis

with a variety of tissue slices, including bananas and avocados at
various stages of ripeness. With the possible exception of avocado
slices, in all other tissues p was zero; i.e. the alternate path is
not engaged unless the cytochrome path is either inhibited or satu-
rated by the electron flux, as in coupled slices.

We have attempted to ascertain the contribution of the alter-

nate oxidase to the respiration of banana and sweet potato slices
by relying on the fact that the apparent Km for 02 of the alter-
nate oxidase is 5-8 fold higher than that of the cytochrome oxidase
(36,106,112). If the alternate oxidase is engaged and the mechan-
ism of controlling the apportionment of electrons is operative, the
concentration of oxygen below which the rate of oxygen uptake ceases
to be of zero order will differ in control slices and SHAM-treated
slices, being larger in the former case. It can be seen from
Figure 9 that the time versus rate of oxygen uptake of the oxygen
electron trace departs from linearity at the same point for both
control and SHAM-treated slices (13-19 ~M), while in slices treated
with KCN the rate of oxygen uptake becomes dependent on the oxygen
concentration at about 78-120 ~M of external oxygen concentration.

The data therefore indicate that, first, in the absence of

cyanide the alternate oxidase is not engaged. Secondly, the ratio
of the apparent Km for 02 of the alternate oxidase to that of the
cytochrome oxidase is about 6-8. It can be seen from Figure 9 that
when the external oxygen concentration decreases to 10.5 ~M the
rate of 02 uptake is reduced by 30%. If the slices are kept at
this concentration for 90 min, lactate accumulates (Table 10).
Hence when the concentration of oxygen becomes limited to the
cytochrome oxidase a Pasteur effect is induced.

It is a weIl established fact that reduction of external oxygen

tensions causes a decrease in the rate of respiration in several
plant tissues in general and in fruits in particular (42,53,57).
RESPIRATION AND ENERGY MET ABOLlSM 81

52

Fig. 9. Effects of 3 mM SHAM and .2 mM KCN compared to control,

on the rate of 02 uptake of 1 n~ thick sweet potato slices

Table 10. Effect of Oxygen Concentration on the Levels of Pyruvate

and Lactate in 1 mm Thick Sweet Potato Slices

nmoles/g FW
Treatment Pyruvate Lactate

Air 350 47
60 min in 10.5 ~M 02 522 356
90 min in 10.5 ~M 02 578 642.4

Mapson and Burton (70) proposed that in intact potato tubers two
terminaloxidases with a different affinity for oxygen are function-
ing. Chevillote (30) explained the decrease in respiration caused
by lowering the oxygen tension in terms of constraints of the oxygen
diffusion into the cells.
82 T. SOlOMOS

The beneficial effects of the low O2 concentrations on the

longevity of fruits and flowers preclude the possibility that the
decrease in the rate of respiration is the result of a diminished
activity of the cytochrome oxidase, since, as has been pointed out
earlier, this would be expected to lead to the accumulation of
glycolytic end products, such as lactate, ethanol and acetaldehyde,
which are known to be highly detrimental to stored fruits.

In order to avoid the complications of the interactions between

'0 2 and C2H4 action (27) we have studied the effect of low oxygen
concentrations on the rate of C02 output in sweet potato roots.
Figure 10 shows that the gradual decrease in external 02 concentra-
tion causes a drop in the rate of C02 output until the concentration
of the 02 drops to about 1.5 - 2%, where C02 output begins to in-
crease. The rate of C02 output continues to increase as the 02
concentration decreases to zero.

If the roots are kept for 12 h. at 1.4% 02' a concentration

at which the rate of C02 output stops falling and instead begins
to rise, lactate accumulated (Table 11). Hence the data indicate
that not until the 02 concentration is decreased below 2.2% does
the supply of 02 begin to limit cytochrome oxidase. In order to
further eliminate the possibility that the rate of 02 diffusion
was not the factor limiting respiration during the gradual decrease
of 02 concentration, we treated sweet potato roots with C2H4 at 10%
oxygen. Figure 11 shows that ethylene raises the rate of CO 2 output
by about 100% above that of the air level. Collectively the data
indicate that the decrease in the rate of respiration with decreas-
ing 02 concentrations is caused by the curtailment of an "oxidase"
with a lower affinity for 02 than cytochrome oxidase.. Furthermore,
the ratio of the apparent Km for 02 of the low affinity oxidase, ca.
8.5%, to that of cytochrome, ca. 0.90%, is about 9, which is close
to that, 6-8, found with the slices where the respective apparent
Km could be ascertained more accurately. Thus, in sweet potatoes
about 30-40% of the terminal electron transport is probably media ted
via the alternate oxidase.

It has been pointed out earlier than the diversion of electrons

to the alternate oxidase is regulated by the flux of electrons
through the cytochrome path. Unless the latter is inhibited or
saturated, the alternate oxidase is not expected to function. In
intact fruits and storage roots only a small fraction of the cyto-
chrome capacity is engaged. For instance, the rate of oxygen up-
take of intact sweet potato roots and uncoupled fresh slices at
25°C is 18-20 and 100-120 ~l/g/h respectively. Thus the contri-
bution of the alternate oxidase to tissue respiration should be
non-existent. However, the experimental evidence cited earlier
indicates that an "oxidase" with an affinity for oxygen lower than
that of the cytochrome oxidase, presumably alternate oxidase, con-
tributes substantially to the respiration of intact fruits and stor-
RESPIRATION AND ENERGY METABOLlSM 83

• ~o
s...
.c

10 I.. .1. 1&

DAYS

Fig. 10. Effects of Oz concentration lower than air on the rate

of COZ output of intact sweet potato roots.

Table 11. Effect of Oz Concentration on the Level of

Lactate in Intact Sweet Potato Roots
Lactate
runoles/g FW
Air < 10
10% Oz < 10
1.4% Oz lZ h Z60

age organs. It should be mentioned that (a) the low rates of oxy-
gen uptake of certain preclimacteric fruits and storage roots and
stems, (b) the high affinity for Oz of the cytochrome oxidase
(~~0.5 ~M), and (c) the absence of the accumulation of glycolytic
end products at Oz tensions lower than air all preclude the pos-
sibility that cytochrome oxidase is limited by the rate of oxygen
diffusion. These observations raise two crucial questions. If
84 T. SOLOMOS

\
;3'2.

'2.e>

..... 2q
I
L.
..c:::
..... 20 I~.,"'" 0,

1
I
01
N
0 .......-'---...~ .O,y.> "I. 0,

._1
"".-i IO~o.
U 1<0
cl
;::l

1'2.
&' 100 pp"" G,H.

'2 (0 e 10
DAYS

Fig. 11. Effect of 100 p.p.m. ethylene on the rate of C02 output
of intact sweet potato roots kept at 10% oxygen.

the availabi1ity of ADP is the main factor which restricts'cyto-

chrome oxidase in vivo, how then does site I of oxidative phospho-
rylation function. in preference to the other two sites, allowing
the operation of the alternate oxidase? Secondly, since there is
no accumulation of respiratory intermediates at oxygen tensions
lower than those in air, what is the feedback mechanism by which
the diminution of the activity of the alternate oxidase restricts
the initial steps of glucose oxidation?

Since it is very unlikely that preferential disengagement of

oxidative phosphorylation at site I is involved, then the elec-
trons from the oxidation of the NAD-1inked substrates mus't bypass
this step. Experimental evidence for this argument comes from
avocado slices, where amytal stimulated slice respiration by about
RESPIRATION AND ENERGY METABOLlSM 85

50% and this stimulation was sensitive to inhibitors of the cyto-

chrome path (see 106). Further work with isolated plant mitochondria
suggests that site I can be bypassed either by the action of malic
dehydrogenase (83,84) or through the transmembrane hydrogen trans-
port (34,123). Thus it appears that in plant mitochondria site I
of oxidative phosphorylation can be bypassed, thereby allowing
the operation of the alternate oxidase.

The mechanism by which the curtailment by low O2 of the low

affinity oxidases leads to the decrease in the mobilization of food
reserves is not clear.

Residual Oxidase

The combined application of cyanide and hydroxamic acid deri-

vatives to slices of storage organs and fruits does not completely
inhibit the rate of oxygen uptake (36,114). The nature of these
residualoxidases is completely unknown. We have recently observed
that the inhibition by the simultaneous application of inhibitors
of both the cytochrome and alternate paths decreases with increasing
oxygen tension (Figure 12). Thus while 63% 02 has no effect on the
rate of respiration of either control or cyanide-treated slices, it
enchances by more than three-fold the rate of 02 uptake of slices
treated both with cyanide and SHAM together (Table 12). In the case
of avocado slices, the increase in oxygen concentration to 100%
completely eliminates the inhibition caused by the simultaneous
application of cyanide and SHAM (Tucker and Laties, personal com-
munication). Since the rate of oxygen uptake of isolated plant and
fruit mitochondria is negligible when KCN and SHAM are added
simultaneously, the residual "oxidase(s)" must be cytosolic in
origin. The above observations raise the following crucial ques-
tions: What is the pathway of glucose oxidation in the presence of
both KCN and SHAM? Is the Krebs cycle partially functioning and
are the reducing equivalents then transferred to cytosolic enzymes
by the mechanism proposed by Day and Wiskich (34) for isolated
mitochondria? Or is glucose being oxidized exclusively by the
pentose phosphate shunt? The answers to these questions must re-
main unanswered until detailed analytical da ta become available
concerning the changes of respiratory intermediates in tissues in-
hibited by both KCN and SHAM. It should be noted that in short-
term experiments with tissue slices, like the present one, respira-
tory electrons are first channelled to the cytochrome path and
only the inhibition of the latter engages the alternate path.
Similarly since the increase in oxygen tension has no effect on
oxygen uptake when cyanide is present, and since the rate of oxygen
uptake in the presence of KCN is zero order up to concentrations of
02 equal to twice the apparent Km for oxygen of the alternate
oxidase, the alternate oxidase appears to be the main terminal
electron acceptor. Only when both paths are inhibited, are
respiratory-reducing equivalents transferred to 02 by cytosolic
86 T. SOLOMOS

182

ö
~ 130
~

78

.IOMIN·

Fig. 12. Effect of 02 concentrations on the respiration

of 1 mm thick sweet potato slices treated with
3 mM SHAM and 0.2 mM KCN simultaneous1y

Tab1e12. Rate of Oxygen and Uptake of Sweet Potato Slices Treated

with KCN, KCN and SHAM, and of the Contro1 at Different
02 Concentrations

jl1 02/g/min
jlM02
Contro1 3 mM SHAM 0.2 mM KCN 3mM SHAM
+ .2 mM KCN
260 0.92 0.92 1.13 0.20
780 0.91 0.92 1.15 0.69
RESPIRATION AND ENERGY METABOLlSM 87

oxidases whose affinity for 02 is much smaller than that of the

alternate path.

Cellular Organization

Blackman and Parija (19) speculated that the increase in the

rate of respiration during the climacteric was the result of the
IIdecrease in cellular organization resistance. 1I It was visualized
that with ~he onset OT senescence, the permeability of cellular
membranes increases, thus enhancing the availability of substrates
to the enzymes. There is little doubt that membrane permeability
increases in senescing tissues. Sacher (99) found that the free
space of banana slices increased prior to the climacteric rise in
respiration. Brady et al. (21) found an increase in membrane
permeability of ripening bananas before the increase in respiration.
However, when aseptically prepared slices were treated with ethylene,
arespiratory rise preceded the increase in amino acid leakage.
Hanson and Kende (46) also observed enhanced ion efflux in the rib
tissue of morning glory flower (Ipomoea tricolor). The authors
concluded that ethylene increases membrane permeability in selective
cells qf the rib tissue. However this increase is probably the
result rather than the cause of senescence. In the first place it
is difficult to measure changes in the membrane permeability of
bulky tissues like fruits. Secondly, it has been pointed out by
Burg (25) and Vickery and Bruinsma (120) that ion leakage, the
method used to determine changes in membrane permeability, may
reflect changes in the ions available for leakage. An increase in
the cytosolic ion concentration will be expected to show enhanced
efflux. The increase in the apparent free space observed during
ripening in bananas and avocados (15,99) may overestimate the
actual changes which occur in vivo (25).

It must be stressed that changes in the cellular distribution

of regulatory metabolites and ions need not require gross changes
in membrane permeability. It is to be expected that the compart-
mentalization of regulatory metabolites, particularly ions, are
under metabolie control (88). A case in point is the increase in
the cytosolic concentration of K+ in the course of the ripening
of tomato fruits (120). Since K+ is a requirement for PK and a
positive modulator of PFK, this increase in the cytosolic K+ will
be expected to contribute to the enhancement of respiration.

Hence the available experimental evidence indicates that the

rise in respiration during senescence of certain fruits, cut
flowers and detached leaves is caused by ethylene. Although ethy-
lene induces cyanide-resistant respiration in potato tubers (35,98),
it enhances respiration independent of the nature of the terminal
oxidase. The respiratory rise is associated with an increase in
glycolysis which in turn is caused by an activation of PFK and PK.
The activation of banana PFK appears to be due in part to changes
88 T. SOLOMOS

in its kinetic properties in the course of the climacteric and to

the decrease in the concentration of PEP. The probable increase
in the cytosolic concentration of ions, which activate both PFK and
PK, could also contribute to the enhancement of respiration.
Further, the results discussed above indicate that in tissue slices,
and probably intact tissues, the bulk of the respiratory electrons
is channelled via the cytochrome oxidase. However, in intact sweet
potato roots and probably other bulky organs, as weIl as preclimac-
teric fruits, a substantial portion of the terminal electron
acceptors (ca. 30-40% in sweet potatoes) is handled by an oxidase
other than cytochrome oxidase, presumably alternate oxidase.

A note of caution is in order. It is obvious that the cur-

tailment by relatively low 02 concentrations of the non-phosphory-
lating oxidation of glucose will be beneficial to the extension of
the storage life and quality of commodities where both of these
properties are critically dependent on sugars or other food reserves.
However the action of low 02 in delaying senescence cannot be
thought of as being exerted through the diminution of respiration.
In the first place, in fruits with large food reserves, such as
bananas, cherimoyas, avocados, etc, the shortage of respiratory
substrates is not at issue. The delaying effect of relatively
low 02 concentrations on senescence must be exerted through the
diminution of the activities of oxygen-utilizing enzyme(s) with re-
latively low affinity for oxygen and which are involved in the in-
duction, synthesis (2) and action of ethylene (25) and/or other
unknown processes (64). Alternatively, it has been proposed that
C2H4 and high 02 concentrations generate active forms of oxygen,
namely peroxides, which in turn enhance senescence (23,31,97).

PHYSIOLOGICAL SIGNIFICANCE OF TRE RISE IN RESPIRATION

It has already been pointed out that for senescence to pro-

ceed, metabolic energy is required. Teleologically an increase in
energy demand will be expected to decrease ATP concentration and
increase that of ADP. However, enhancement of anabolic activities
would require an increase in the energy charge (5a). The avail-
able analytical data concerning the changes in the energy charge
in the course of senescence of fruits and detached leaves are_
scanty. In avocados, energy charge increases (126), while in se-
nescing ivy leaves, the increase in the energy charge is not very
pronounced. It can be calculated from the data of Table 8 that
the energy charge in ivy leaves during senesc"ence increases from
0.51 to 0.6. The available analytical results show that the rise
in respiration in response to ethylene treatment is associated
with a large increase in ATP levels (Table 8). Further, in cases
where energy charge remains relatively constant. this increase in
ATP is associated with a net increase in the total pool of the ade-
nine nucleotides. In view of the inhibitory effects of these meta-
RESPIRATION AND ENERGY METABOLlSM 89

bolites on PFK, one would anticipate a decrease in the rate of res-

piration. Therefore, ethylene must decrease the regulatory re-
straints of the adenine nuc1eotides on respiration, allowing a
simultaneous enhancement in respiration, elevation of ATP and/or of
total adenine nucleotide concentration.

On the basis of the present state of know1edge regarding the

nature and magnitude of the sinks of metabolie energy in senescing
tissues, it is impossible to calculate the energy demand with any
degree of precision. At the c1imacteric peak of bananas at 20°C
the rate of respiration is 60-70 wl 02/g/h and the level of ATP is
90-100 nrno1es/g/h (Solomos, unpublished observation). If we assurne
that glucose is oxidized via glycolysis and the Krebs cycle, and
assuming tight coup1ing of glucose oxidation to phosphorylation,
about 18,750 nrno1es of ATP are synthesized per gram of tissue in
one hour (63). Hence the turnover of the ATP pool per hour is
approximately 200. Therefore, for respiration to proceed for any
length of time, ATP must be utilized very rapid1y.

In ripening bananas the conversion of starch to sucrose can be

a sizable sink for ATP. It can be calculated from the data presen~
ted by Gane (44) that at the climacteric peak the rate of sucrose
formation is about 1,900 nrno1es/g/h (Tab1e 13). If starch is broken
down by starch phosphory1ase, then one ATP is required for each
moleeule of sucrose synthesized (118). Hence, about 2,000 nrnoles
ATP/g/h are required for sucrose synthesis, a rather smal1 fraction
of the total ATP which cou1d be synthesized.

Brady and O'Conne1l (20) ca1culated that in ripening bananas

the rate of pro tein turnover represents the replacement of 25-50%
per day of the total proteins. If it is assurned that (a) the protein
content of bananas is 2%, (b) the average molecular weight of the
pro teins is 60,000, (c) the average weight of the individual amino-
acids is 135,. (d) four ATP are required for the synthesis of a
peptide bond (63), and (e) 50% of the total protein is replaced in
24 h, then 12,350 nmo1es ATP/g/h are required to sustain the above
rate of protein synthesis. Even this rather large overestimation

Tab1e 13. Calcu1ated rates of ATP Production and Uti1ization at

the C1imacteric Peak of Ripening Bananas
nrnoles sucrose nrno1es ATP/g/h
W1 C02/g nrnoles ATP synthesized required for
/h /g/h /g/h pro tein synthesis

70 18,750 1,900 12,350

 90 T. SOLOMOS

of protein synthesis together with the formation of sucrose cou1d

be satisfied from the point of view of energy with a sma11er incre-
ment of respiration than that extent at the climacteric peak (Tab1e
13). Moreover, ripening of bananas can proceed in the absence of
any perceptib1e rise in respiration (87). Further, in most fruits
the overt changes associated with ripening occur .after the c1imac-
teric peak.

It has been proposed that the upsurge of respiration in c1imac-

teric fruits ref1ects an increase in the energy requirements for the
ripening process, and further that the slow ripening process of
nonc1imacteric frutts does not require as much energy as do c1imac-
teric ones, hence the iack of arespiratory rise (86,90,92).

However, nonc1imacteric fruits such as strawberries ripen

rapid1y with no concomitant increase in respiration (58). Vendre11
(119) has shown that the infiltration of banana slices with auxin
solutions de1ays senescence caused by exogenous ethy1ene but does
not prevent the initial rise in respiration. Dostal and Leopo1d
(40) observed that app1ication of GA to green tomatoes de1ays the
breakdown of ch1orophy1 but has no effect on either respiration
or ethy1ene production. Further, the app1ication of exogenous
ethy1ene does not alter the effect of GA. Pratt and Goesch1 (85)
have found that in ripening canta1oupes, on the one hand 0.1 p.p.m.
ethy1ene induced softening and carotenoid synthesis and on the
other hand the enhancement of respiration required 3 p.p.m C2H4'
Moreover, the rate of pro tein and nuc1eic acid synthesis in severa1
fruits diminishes at the 1ater stages of the c1imacteric rise
(22,91,92).

In summary, the rise in respiration during senescence of fruits,

1eaves and cut flowers may not reflect the energy requirements of
• the senescence process but it is a facet of ethylene action and not
of senescence per~. To paraphrase Churchill, senescence is a
riddle wrapped in a mystery inside an enigma, but the parce1 is
mainly tied up with ethylene.

ACKNOWLEDGEMENTS

This is scientific artic1e no. A-3020, contribution no. 6083

of the Mary1and Agricultura1 Experimental Station and the Department
of Horticu1ture, University of Mary1and, College Park. The research
was supported in part by a grant from the National Science Founda-
tion No. PCM 76-21560.
RESPIRATION AND ENERGY MET ABOLlSM 91

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92 T. SOlOMOS

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RESPIRATION AND ENERGY METABOLlSM 93

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RESPIRATION AND ENERGY METABOLlSM 95

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96 T. SOlOMOS

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RESPIRATION AND ENERGY METABOLlSM 97

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ENZYME ACTIVITIES AND POST-HARVEST CHANGE

M.J.C. Rhodes

Agricultural Research Council Food Research Institute

Colney Lane
Norwich, England

INTRODUCTION

A complex series of metabolic adjustments occur in horticultural

crops after harvest which are influenced by the dislocation of supply
of nutrients, minerals and hormones from the parent plant to the
harvested plant organ. Basic respiratory processes are necessary to
supply the energy required to maintain the essential repair reactions
upon which the integrity and independent existence of the organ de-
pend. Over and above this maintenance metabolism, the harvested
fruit or vegetable is able to continue its normal development which
may lead to ripening and senescence in the case of fruits and leaves
or to dormancy and regrowth in tubers and seeds. In addition the
harvested organ has the ability to respond metabolically to the en-
vironment in which it is stored, to wounding and to microbial attack.
Such basic biochemical and physiological changes lead to major
changes in the chemical composition and physical structure of the
tissue and have an important effect on the quality of the horticul-
tural crop as food whether it is eaten directly or following some
food processing operation.

Underlying these biochemical changes are major changes in the

relative activities of metabolic pathways in the tissue. Figure 1
illustrates some of the visible changes that occur in ripening
fruits and the metabolic pathways which are likely to be responsible
for these changes. Changes in the flow of metabolites through the
pathways involved must be reflected in changes in the in viv~
activity of their constituent enzymes. Modern ideas on the regula-
tion of metabolic pathways stress the concept of control being a
function of the system as a whole with each of the constituent
enzymes of the pathway making some contribution to overall control

99
100 M. J. C. RHODES

COLOUR

1. Loss of green colour Lipolysis

Proteolysis
2. Formation of red Iblue colours Carotenoid and
phenylpropanoid synthesis

FLAVOUR

1. Loss of acidity TCAC and decarboxylation

2. Increase in sweetness Starch hydrolysis

3. Flavour volatiles Biosynthesis of alcohol,

esters etc.

TEXTURE

1. Softening Pectolytic activity

Cellulose breakdown

Fig. 1. Metabolie Pathways Associated with Fruit Ripening.

(13). However, the contributions of the individual enzymes are not

equal and it is frequently possible to identify steps in a pathway
catalysed by particular enzymes which make major contributions to
control. We need to concentrate on such enzymes in order to
understand how the metabolie adjustments which occur during the post
harvest period are achieved. The present paper will discuss the
mechanisms available for regulating enzyme activities in cells and
will then discuss the present state of our knowledge of the regula-
tion of two diverse areas of metabolism, both of which are of
importance in harvested organs.

Factors Affecting the Activity of Enzymes in vivo

The in vive activity of an enzyme is determined by factors

which affect either the number of active enzyme moleeules available
or the degree to which the potential c~talytic activity of these
available enzyme moleeules is expressed. The first group of factors
lead to a relatively slow acting gross control while the second
group involve fast acting, fine tuning responses of an existing
enzyme system. The actual number of active moleeules of a particular
enzyme in the cell is a function of the relative rates of its
synthesis and degradation and mayaiso be affected by mechanisms
which either activate or inactivate the enzyme reversibly. Increased
rate of synthesis or decreased rate of degradation provide a powerful
means of controlling the number of available enzyme molecules. There
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 101

is evidence in both plants and animals that different enzymes turn-

over at vastly different r,ates. For instance in rat liver the range
of half 1ives of individual proteins in the ce11 can vary from a few
minutes up to 15-25 days (20). Simi1ar variations occur among higher
plant proteins (Woo1house, Chapter 1).

In p1ants the nature of the proteases responsible for the pro-

teolytic breakdown of enzymes is unknown. In anima1s, degradation
is und er the contro1 of a range of proteases of varying specificity.
There are group-specific proteases which recognize the co-enzyme
binding sites of certain enzymes and sp1it the apoprotein at this
site. Group specific proteases for pyridoxa1 dependant, NAD
dependant and FAD containing pro teins have been described (31,34).
These enzymes appear to attack on1y a few peptide bonds near the
cofactor binding site and subsequent degradation is by other 1ess
specific proteolytic enzymes. In higher p1ants the presence of a
protease specific for nitrate reductase has been proposed (60) but
wider aspects of the spe~ificity of this protease were not studied
in detail. However, enzymes such as the insulin specific protease
of rat skeleta1 muscle, (15) specific for a particular protein do
exist in animal cells but these appear to be the exception rather
than the rule. Kahl (30) has discussed the factors which tend to
promote proteolysis and concluded that factors tending to promote
denaturation promote proteo1ysis whi1e those tending to preserve the
structural integrity of proteins protected against it. The binding
of ligands or attachment to ce11u1ar structures such as ribosomes or
membranes protect enzyme pro teins from degradation. It seems very
likely that conformational change is the first step in the break-
down of an enzyme molecule and it is suggested by Goldberg and St
John (20) that these initial conformationa1 changes in protein
structure are the rate 1imiting step in enzyme degradation rather
than the presence of specific proteases. However, the factors
responsib1e for these structura1 changes are unknown. In enzymes
attacked by the group-specific proteases the loss of the co-enzyme
must be an early event in these changes since only the apo-enzyme
is degraded and the presence of co-factor protects against proteoly-
sis. A possibly important but as yet unexplained feature of intra-
cellular protein degradation is its sensitivity to inhibitors of
energy metabolism such as cyanide or dinitrophenol. This is an
intriguing finding since it is unexpected on thermodynamic grounds,
the hydrolysis of the peptide bond being exergonic and none of the
proteases described require high energy intermediates (20). It
could be that the requirement for ATP is an indirect one but it
perhaps indicates that proteolysis is und er strict control.

In the synthesis of enzymes major structural modifications

occur following the release of nascent polypeptide chains from
polyribosomes leading to the formation of the complete enzymically
102 M.J.C.RHODES
active enzyme structurally located and operating i~ a pathway. The
rate of synthesis of polypeptide chains is sensitive to a number of
factors including the availability of the relevant mRNA species and
these factors will be discussed elsewhere in this symposium (Grierson,
Chapter 2). The development of secondary and tertiary structure
in the newly formed polypeptide chains is a function of the amino
acid sequence determined during translation. The folding of these
chains, the formation of disulphide bridges and other interactions
between chains determines the overall shape of subunits of the
enzyme molecule (11). The formation of quaternary structure in
more complex proteins involves interactions between subunits and
their assembly can be complex. The small and large subunits of
ribulose phosphate carboxylase:oxygenase are formed respectively
on cytoplasmic and chloroplast ribosomes. The small subunit is
transported across thechloroplast limiting membrane before the
complete molecule of the enzyme can be assembled within the chloro-
plast (6). The small subunit bears a polypeptide tail which is
removed by proteolysis during the process. Similarly, themito-
chondrial genome can code for only 8-10 polypeptides. These poly-
peptides are components of pro teins which contain polypeptide
constituents made in the cytoplasm and transported to the mitochon-
drion (38). Thus assembly of complex pro teins may involve diverse
sites of synthesis of subunits and their transport to the site of
assembly. Transport and assembly must be important with most enzymes
since their sites of action, which may be within organelles or on
cellular membranes, are distant from their sites of synthesis.
Possible mechanisms for incorporation of pro teins into a membrane
system have been reviewed by Wickner (61). Many enzymes require the
covalent attachment of a metal or prosthetic group to achieve
catalytic activity and enzyme synthesis may be coupled with the
synthesis of the prosthetic group and its incorporation into the
pro tein molecule may weIl itself be catalyzed by specific enzymes.
Many plant enzymes are glycoproteins and their synthesis involves
post translational transfer of sugar units to the protein. Plant
glycoproteins are generally glycosylated either by N-glycosidic
linkages to the amide N of asparagine frequently involving the C-l
of N acetyl-D-glucosamine to which D mannose residues are attached,
or by O-glycosidic linkages to hydroxyproline, serine or threonine
involving L-arabinose or D-galactose (51). In animals and plants
(27) groups to be linked N-glycosidically are first assembled, from
nucleotide sugar precursors, on a C80-l00 saturated isoprenyl
phosphate (dolichol phosphate) carrier and then transferred en bloc
to the protein acceptor. Oligosaccharides, to be linked by
O-glycosidic bonds, are assembled by direct transfer of sugars from
the nucleotide sugar precursor to the protein acceptor. Firestone
and Heath (19) studied the de novo synthesis in culture mouse cells
of a glycoprotein having alkaline phosphatase activity. They
showed that if pro tein glycosylation was inhibited using tunicamycin,
the formation of enzyme pro tein was also inhibited. It appeared
that glycosylation protected the nascent pro tein from proteolytic
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 103

attack. These findings indicate that glycosy1ation is an integrated

step in the synthesis of the enzyme. The function of the carbohydrate
in such enzymes is not fu11y understood but may p1ay a ro1e either
protecting against proteo1ysis or in the structura1 loca1ization of
the enzyme in the ce11 (51). It can be seen from the above discus-
sion that there are many steps in the synthesis of enzymes which
have to be integrated with other pathways such as those 1eading to
the formation of prosthetic groups or carbohydrate. These provide
possibi1ities for contro1 but their possib1e significance in vivo
is not known.

Another factor in addition to synthesis or degradation which

may affect the concentration of active enzyme protein in the ce11s
is the activation or inactivation of pre-formed enzyme pro tein. A
number of anima1 digestive enzymes are synthesized and stored as
inactive precursors (pro-enzymes) and these may be converted into
the active form by specific irreversible 1imited proteo1ysis. For
instance, the conversion of trypsinogen into the active trypsin
invo1ves the proteo1ytic c1eavage of a hexa peptide at the N-termina1
end and a change in conformation of the pro tein mo1ecu1e. In other
cases such as the conversions of procarboxypeptidase A to the active
enzyme in which near1y two-thirds of the mo1ecu1e is lost, the
proteo1ysis is 1ess 'limited' (63). In p1ants, there are a number
of cases reported of the presence of specific protein inhibitors
for particu1ar enzymes. These inc1ude invertase (40) and pheny1~
alanine ammonia lyase (8,12). Inactivation is thought to occur by
formation of stab1e protein:protein comp1exes between the inhibitor
and enzymes. Such a mechanism provides for a pool of inactive
enzymes which can rapid1y be activated in response to metabo1ic
demands. Such protein:protein interactions will be discussed further
in a 1ater section.

For a given amount of enzyme protein there is a maximum poten-

tial catalytic activity. There is scope for regulation of the
in vive activity of enzymes by the extent to which this potential
activity is actua1ly expressed. Many enzymes in the cell opera te
probably under conditions of limiting substrate and cofactors and
in the presence of potentially inhibitory products. Under these
conditions they are susceptible to regulation by factors which
either increase or decrease the supply of substrates or cofactors,
increase or decrease the rate of removal of products or which change
the effectiveness with which they are able to utilize a given level
of substrate or cofactor. The pR at which the enzyme operates in
vive is an important factor controlling activity. The supply of
substrate for a given enzyme may depend on previous steps in the
pathway or may in addition involve changes in cell membrane permea-
bility and the release of stored substrate from the vacuo1e or from
organelles. üf special significance in the overall regulation of
metabolie sequences is the action of small mo1ecular weight compounds
(a1losteric regulators) which, while not structurally related to
104 M.J.C.RHODES
either the substrates or products of the enzyme, may effect the
activity of regulatory enzymes (35). These regulatory enzymes are
characteristically complex both in quaternary structure and in
their kinetic behaviour towards their substrates. Allosteric regu-
lators bind to regulatory enzymes and change both the quaternary
structure of the pro tein and its catalytic activity. The effector
may either promote or inhibit the activity of the enzyme at a given
concentration of substrate by affecting the efficiency of binding
between substrate and enzyme. Such regulation may allow very rapid
reversible adjustments of the rate of flow of intermediates through
a regulatory step and appear to be of wide importance in cellular
regulation.

In addition to allosteric regulation, some enzymes typically

regulatory ones, are subject to reversible covalent modifications
which can have important effects on their catalytic and regulatory
properties. Figure 2 shows examples of the type of covalent
modifications occurring with animal enzymes which include carbamy-
lation, acylation, oxidation and reduction of thiol groups,
adenylation and phosphorylation. The most studied example of
covalent modification of proteins is the role of phosphorylation/
dephosphorylation in the control of glycogen phosphorylase which
catalyses the breakdown of glycogen in muscle. This reaction is
controlled by the combined action of non-specific protein kinases,
a highly specific phosphorylase kinase and phosphatase (36). The
protein kinases, which are activated by cAMP, phosphorylate pro teins
using ATP as phosphate donor. These activate an inactive form of
phosphorylase kinase by phosphorylating specific serine residues
in the molecule and causing a subsequent conformational change in
the enzyme protein. The activated kinase in turn phosphorylates
and activates an inactive dimeric form of the glycogen phosphorylase,
phosphorylase-b to form the fu11y functiona1 phosphorylase-a which
is tetrameric. The dephosphory1ation of phosphory1ase a and of the
activated form of phosphory1ase kinase are carried out by specific
protein phosphatases and the system of phosphory1ation/dephosphory-
1ation a110ws for very rapid activation or deactivation of the
glycogen degrading enzyme (44).

The importance of such reactions in higher p1ants is far from

c1ear and cova1ent modification of a regu1atory plant enzyme has
yet to be conclusive1y demonstrated. The plant enzyme equiva1ent
to glycogen phosphory1ase i.e. starch phosphory1ase does not appear
to have regulatory properties and there is no evidence that it can
undergo phosphory1ation (5). This lack of regu1atory mechanism may
be re1ated to the re1ative1y slow changes in the rate of uti1ization
of starch in p1ants compared with the rapid responses in glycogen
breakdown in anima1s during strenuous activity. Protein kinases
exist in p1ants (1,57) but these are not cAMP dependant and in fact
the presence of cAMP in plants is still open to doubt (4). The
phosphory1ation of proteins occurs in chloroplasts and the phosphory-
 m
Z
N
-<
s:
m
»('")
-I
<:
:::::j
m
cn
MODIFICATION DONOR ACCEPTOR ENZYME EFFECT ON ENZYME »z
o
""tl
o
Phosphorylation ATP Glycogen Phosphorylase Activation cn
-I
(Serine) :I:
»
:0
Carbamylation Carbamylphosphate Glutamic Dehydrogenase I nactivation <
m
cn
-I
('")
Adenylation ATP G lutamic Synthetase I nactivation I
( Tyrosine)
»
z
Cl
m
AD P- Ribosylation NAD+ Trans locase I nacti vation

S-H/S-S Pyruvate Lyase Activation

Fig. 2. Covalent Modification of some Animal and Bacterial Enzymes.

o(11
106 M. J. C. RHODES

lated products include the light haryesting chlorophyll:protein

complex. Phosphorylation of this complex has be~n proposed as a
regulatory mechanism by which photosynthetic systems adapt to
changing wavelengths of light (2). Covalent modifications allow for
relatively rapid responses in enzyme function to external factors
and can be seen as conserving metabolic energy as compared with
enzyme synthesis.

The foregoing discussion of the potential mechanisms for the

control of both enzyme formation and enzyme activity illustrate the
limited information available for plant as opposed to animal systems.
Yet even in animals, evidence for the functional significance of
such mechanisms in vivo is often incomplete. The second part of
this paper will discuss the state of our knowledge of the regulation
of two enzymes operating in fruit tissues. One is the role of
phosphofructokinase (PFK) in the control of glycolysis. a pathway
of primary metabolism and the other is the role of phenylalanine
ammonia-lyase (P.A.L.) in the control of the synthesis of a group
of secondary products. the phenylpropanoids.

The Role of Phosphofructokinase in the regulation of Glycolysis in

Plants

Glycolysis is a constituent metabolic pathway in all living

plant cells which can respond rapidly to respiratory and synthetic
demands made upon it. Davies (13) pointed out that, although the
control of glycolysis is a property of the whole integrated system.
the interaction between two of its constituents enzymes PFK and
pyruvate kinase was likely to be the major feature of thi.s control
in higher plants. PFK, which catalyses the essentially irrevers-
ible phosphorylation of fructose 6-phosphate (F6P) in the presence
of ATP to form fructose 1:6 diphosphate, is the first enzyme of the
sequence entirely committed to glycolysis. PFK is a high molecular
weight pro tein which exists as an oligomer. It shows non-linear
kinetics towards one of its substrates, F6P, and its activity is
affected by a wide range of compounds structurally unrelated to
either its substrates or products. It thus has the characteristics
expected of a regulatory enzyme (58). PFK is sensitive to allo-
steric effectors which are intermediates in glycolysis (2 phospho-
glyceric acid, 3-phosphoglyceric acid and phosphoenol pyruvate
(PEP», the pentose phosphate pathway (6-phosphogluconate). and
the tricarboxylic acid cycle (malate and citrate), as weIl as to
intermediates in energy exchange reactions (adenine nucleotides and
Pi). This coupled with its strategic position as the first step in
the pathway suggests an important role for PFK in the metabolic
control of glycolysis.

There is evidence from a number of different fruits including

the banana (64) and the tomato (10) that there is an increased f1ux
of carbon through the glyco1ytic pathway during the per iod of the
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 107

climacteric. A detailed discussion of the regulation of glycolysis

in plants has recently been published (13, 59) and it would not be
appropriate to discuss the overall regulation of the pathway here.
However, I would like to discuss some of our work on the properties
of PFK, in relation to its role in relation to the control of
glycolysis. In work in this laboratory PFK was isolated under care-
fully controlled conditions as we have shown that both the catalytic
and regulatory properties of PFK are sensitive to the molecular
state of the extracted enzyme (28). Table 1 shows estimates of the
maximum catalytic activity of PFK isolated from tomatoes harvested
at four stages of ripening and this shows that there are no signifi-
cant changes in the extractable activity of the enzyme. This
confirms other workers' findings that even though there is a major
increase in flux through glycolysis during ripening the maximum
catalytic activity of PFK remains constant (10,47). In studies of
avocado slices, Solomos and Biale (54) concluded that the glycolytic
potential of pre-climacteric fruit was sufficient to sustain the
rates of respiration found in fruit at the climacteric peak. These
findings suggest that there is potential of the maintenance of
widely different glycolytic fluxes by regulation of the activity of
pre-existing glycolytic enzymes without changes in the level of
available enzyme protein.

PFK was purified from the tomato (29) using affinity chroma-
tography and it was shown that the pH of isolation was critical in
retaining the structural and regulatory features of the enzyme.
Raising the pH during isolation promoted the breakdown of the oligo-
meric form of the enzyme (M.Wt. 180,000 daltons) giving progressively
more of a lower molecular weight form (35,000 daltons) which may be
a monomer. This loss of subunit structure was associated with a
loss of regulatory properties. The oligomer showed negative co-
operativity towards its substrate, F-6-P in the presence of MgATP
(28) while the dissociated form showed simple Michaelis-Menten
kinetics.

PEP was the most potent allosteric inhibitor of PFK found;

with the tomato enzyme 50% inhibition was caused by 10 ~M PEP
(see Fig. 3). The inhibition was sigmoidal in nature and if the
data was analyzed as a HilI Plot, the HilI coefficient was 1.5
showing marked deviation from hyperbolic kinetics (28). Such
inhibition by PEP could explain interactions between PFK and
pyruvate kinase since PEP is the substrate of the latter enzyme.
The presence of PEP reduced the binding of enzyme of both its sub-
strates, F6P and MgATP. However, the inhibiting effect of PEP
could be reversed by addition of Pi to the assay; 7-8 ruM Pi being
required to give 50% relief of inhibition in the presence of 20 ~M
PEP (see Fig. 4).

Chalmers and Rowan (10) proposed that an enhanced rate o~

leakage of Pi from the vacuole to the cytoplasm was important in
108 M. J. C. RHODES

Table 1. Activity of PFK in Tomatoes at

Various Stages of Ripening

PFK Activity Vmax.

Stage nKat/g nKat/ g nKat/ g
fresh wt protein protein

Mature-Green 191.8 0.085 0.2

Breaker 145.6 0.076 0.19
Orange 122.7 0.073 0.17
Red 118.2 0.073 0.17

(after 28)

100

c
o

.0
.c
c 50
~

0·01 0·02
mM PEP

Fig. 3. Effect of PEP on the activity of purified tomato PFK at

pR 7.5 (after 28).
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 109

100

c
o

..0

..c
c

4- 50
o
4-
Q)

Q)
er::.

10 20
mM Pi

Fig. 4 Effect of Pi in relieving the inhibition of activity of

purified tomato PFK at pH 7.5 by 20 ~M PEP (after 28).

controlling the rate of gl~colysis in tornatoes. They showed a

positive correlation between the rate of respiration of tomato fruits
.and their Pi contents. More recently, Woodrow and Rowan (62) have
studied the rise in respiration in slices cut from the pericarp of
mature-green tomatoes and incubated under conditions in which colour
changes occurred similar to those in the whole ripening fruit. They
measured changes in the fluxes of Pi and found that the flux from
vacuole to cytoplasm increased as the rate of respiration rose. The
effect of Pi could be due to its effect on PFK activity.

A number of workers (10,28,58) have shown that Pi stimulates

the activity of isolated PFK. Isaac (28) showed that Pi stimulated
PFK activity by up to 2 fold at concentrations up to 20 mM (see
Fig. 5). Pi caused a shift in the pH optimum of the enzyme from
8.0-8.25 to 7.5-7.8 and tended to promote degradation of the oligo-
meric structure with loss of the non linear kinetics. The oligomeric
form of PFK shows negative co-operativity towards its substrate, F6P
and Pi has the effect of both increasing the activity of the enzyme
and changing its kinetics towards its substrate to simple Michaelis-
Menten hyperbolic relationship. Such negative co-operative kinetics
towards substrate in the absence of Pi have been found in bananas
by other workers (46). Those authors considered that the effect of
110 M. J. C. RHODES

100

<Il

I'tl
~ 50
~
c

20 40 60 80
mM Pi
Fig. 5. Effect on Pi on the activity of purified tomato PFK at
pR 7.5 (after 28).

the negative co-operativity was for the binding of one substrate

moleeule to make the bindin'g of a second moleeule more difficult so
as to maintain the metabolism of carbohydrates at low levels in
spite of large changes in substrate level. The effect of Pi by
promoting deaggregation of the enzyme complex with resulting non~
interactive kinetics, could be to relieve the constraint imposed by
the negative co-operativity and thus stimulate the enzyme activity.
In the controlling interaction between PFK and pyruvate kinase an
additional effect of Pi would be to relieve the constraint on PFK
activity due to inhibition of the enzyme by PEP. Malate and
citrate, the major storage acids of the tomato can inhibit PFK
allosterically but less effectively than PEP and here again Pi can
relieve the inhibition. It is thus possible to propose a model
that increased permeability of vacuolar membranes leading to an
increased cytoplasmic level of Pi could stimulate glycolysis and
the climacteric by relieving the constraints on PFK imposed by sub-
strates and negative effectors.

Such a model is based on in vitro enzymological experiments.

It assumes that the isolated enzyme is both structurally and
kinetically identical to the enzyme in vivo. We know very little
of the substrate and product concentrations, and of structural
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 111

relations between enzymes which actually occur in the cello We know

average concentrations of metabolites but neither their actual con-
centrations in the organelles in which they act nor their rates of
change in concentration. In the case of the possible interactions
between PEP and Pi in controlling PFK, the concentrations required
to give 50% responses (10 ~M and 7-8 mM respectively) are at least
of the same order as the average concentrations of these compounds
in tomato tissues (10-20 ~M and 2-3 mM respectively (10). However,
it may be too simplistic to think of effectors acting singly and,
in all probability, complex interactions between groups of positive
and negative effectors may be important in the regulation of such a
complex enzyme as PFK. It has been calculated that for a single
enzyme with 8 reactants and effectors over 1.6 x 10 6 assays would
be required to establish the complete rate law (13) and many more
assays would be required for the complete analysis of a complex
enzyme such as PFK. There is clearly a gap between our relatively
detailed knowledge of the properties of high purified isolated
enzymes and our ignorance of the behaviour of enzymes in vivo.

Some aspects of the Regulation of Phenylpropanoid Metabolism in

Plants

The metabolism of phenylpropanoids is important in a number of

aspects of the post-harvest physiology of horticultural crops.
These compounds contribute to the colour, texture and flavour of
fruits and vegetables and are important in sorne responses to damage
and stress. Anthocyanins are responsible for the red and blue
colours of many fruits and vegetables while leucoanthocyanins are
important in the astringent taste of some fruits. Lignin formation
occurs in the endocarp of stone fruits and contributes to the tex-
ture such as pears by its presence in stone cells. In addition,
o-diphenols such as caffeic and chlorogenic acids and flavonoids
such as catechins are endogenous substrates for the browning reaction
of fruit and vegetable& following damage which depend on the enzyme,
o-diphenol oxidase. Frequently the synthesis of phenylpropanoids
is stimulated in response to stress in plants and some fruit and
vegetables respond to low temperature stress by increased metabolism
of chlorogenic acid (42).

All of these phenylpropanoid compounds are synthesized from

the aromatic amino acid, phenylalanine which in turn arises from
erythrose 4-phosphate and PEP by the shikimate pathway. Figure 6
shows that the route from phenylalanine to the various products is
highly branched and its regulation must involve control of the
total flow of carbon through the pathway and the distribution of
the flow to the particular branches. It is probably generally true
that not all branches operate in a given tissue and in some cases
the flow may be almost exclusively to a single product, as in ligni-
fying tissues. The pathway as outlined in Fig. 6 has a common core
consisting of the formation cinnamic acid and its ring substituted
 N

phenylalanine

1
cinnamic acid -+ o-coumaric acid -+ coumarins
1. cinnamic acid glycosides
p-coumaryl- p-coumaryl +- p-coumaryl ~ p-coumarlc
L alcohol aldehyde CoA acid C d
./ ~ 6- 1 compoun s
I tt!' caffeic acid ?

G
N ~ alcohol aldehyde 5 hydr~xy ~ ~
I funlic~d ~
N sinapyl .. sinapyl ~ sinapyl CoA ~ sinapi~ acid .
alcohol aldehyde c h a 1cones
ßavano~es C 6C l
/ ~ compounds
quinate
and isoflavonoids ßavonoids
shikimate
esters s:
c....
Fig. 6. The Pathway of Phenylpropanoid Biosynthesis.
n
:xl
:::I:
o
C
m
cn
ENZYME ACTIVITIES AND POST -HARVEST CHANGE 113

derivatives from phenylalanine (p-coumaric, caffeic, ferulic and

sinapic acids), and the activation of the cinnamic acid derivatives
to form their CoA thioester derivatives which are precursors for a
wide range of phenylpropanoid products (24). In the regulation of
this pathway it appears that the enzymes catalyzing the steps from
phenylalanine to cinnamic acid, phenylalanine ammonia-lyase (P.A.L.),
from t-cinnamic acid to p-coumarate, cinnamic acid 4-hydroxylase
(C.A.4H.) and from cinnamic acid derivatives to their CoA thioesters,
hydroxycinnamate CoA ligase (CL) are most important.

P.A.L., which catalyses the stereospecific deamination of

phenylalanine to form t-cinnamic acid, is a protein of molecular
weight 300-320,000 composed of two subunits each consisting of two
separate polypeptide chains (9,25). It displays non-linear kinetics
with apparent negative co-operativity towards its substrate (43)
and thus in its structural and kinetic complexity has the character
of a regulatory enzyme. The reaction it catalyses is reversible.
Havir and Hanson (26) demonstrated the formation of phenylalanine
from cinnamate but this required the presence of a very high concen-
tration of ammonium ions (0.374M) and even this was insufficient to
saturate the reverse reaction. Such ammonium ion concentrations
are not likely to occur under physiological conditions and since
neither free cinnamate nor its derivatives normally accumulate in
plant tissues it is likely that in vive the P.A.L. reaction is
effectively irreversible.

P.A.L. is mainly found in the 'soluble' fraction of tissue homo-

genates yet a small but significant fraction is bound to a membrane
fraction, consisting of fragments of the endoplasmic reticulum and
containing the second enzyme of the pathway, C.A.4H. C.A.4H is a
mixed function oxidase which is linked to a membrane bound electron
transport system in which the flow of electrons from NADPH2 to
cytochrome P450 is coupled to the hydroxylation of t-cinnamic acid
in the position 4 of the aromatic ring to form p-coumaric acid (45).
Kindl (32) has proposed that P.A.L. in vive exists as a membrane
bound complex associated with C.A.4H-.-and suggested that the complex
between P.A.L. and the membrane bearing C.A.4H involves a weak
interaction which is largely destroyed during homogenation under
aqueous conditions. Kindl (32) showed that in the complex phenyl-
alanine was a better precursor for p-coumarate formation than was
t-cinnqmic. The presence of such a complex in allowing restricted
exchange of intermediates, vectoral transport of intermediates and
to high local concentrations of substrates would offer major
advantages. Since the hydroxylation reaction is irreversible, the
channelling of cinnamate to the membrane bound system ensures that
the conversion of phenylalanine to p-coumaric acid by the complex
is irreversible. It is likely that the complex plays a major role
in controlling the pathway of phenylpropanoid biosynthesis. How-
ever, much of the work in the past has concentrated on P.A.L. since
C.A.4H. as a complex multienzyme system is much more difficult to
114 M. J. C. RHODES

handle experimenta11y. Another possib1e contro1 point i8 the 1igase

step, CL in which substituted cinnamic acids are activated in the
presence of ATP, CoA and Mg 2+ to form thioester derivatives. These
reactions are reversible and since they are inhibited by the ADP
and AMP (33,41) may be subject to contro1 depending on the avai1a-
bi1ity of high energy carriers.

In many tissues and in response to different environmental

and hormonal stimuli there is a rise in the production of pheny1-
propanoid products (9) and this is associated with co-ordinated
increases in the extractab1e activity of P.A.L., C.A.4R and CL. An
examp1e of such a response is shown in Fig. 7 in which increases in
the extractab1e activities of P.A.L., C.A.4R and CL in soya bean
suspension cu1tures fo11owing exposure to light (24a).

Measurement of levels of extractab1e enzyme activity invo1ve

a number of difficu1ties which inc1ude the quantitative extraction
of enzyme pro teins which are structura11y and cata1ytica11y intact
from tissues often containing high levels of aCidity and inter-
fering compounds, and the assay of the extracted enzyme. With
suitab1e precautions, the first of these two problems may frequent-
1y be overcome but measurements made of enzyme activities in vitro
may not be relevant to the conditions pertaining in vivo. To take
P.A.L. as an examp1e it is assayed in vitro at the-pR optimum (8.9)
in the presence of excess substrate-C13-14 mM, i.e. about x20 the
Km). It is un1ike1y that the physio1ogica1 pR is as high as 8.9
and it is interesting that all the other enzymes in this region of
the pathway have optima between 7.0-7.5 and this is probab1y nearer
the physio1ogica1 range found in p1ants (52). In this context it is
interesting that P.A.L. is inhibited by both its product, cinnamic
acid and by p-coumaric acid and this inhibition is more potent at
pR 7.0 than at the pR optimum (25). Margna (39) pointed out that
for a range of plant tissues, the average level of phenylalanine
was about 0.1-0.2 mM whi1e the Km va1ue for P.A.L. was in the range
0.2-0.7 mM. Thus it is un1ike1y that the maximum cata1ytic poten-
tial of the enzyme wou1d be fu11y expressed in vivo. Methods for
in vivo assay of enzymes inc1uding one for P:A.~3) have been
deve10ped but these have not been wide1y used and have their own
inherent problems. Margna (39) pointed out that since the maximum
cata1ytic potential of P.A.L. is not a1ways rea1ized in vive there
was considerab1e scope, at least in some cases, for the contro1 of
the pathway by increasing the avai1abi1ity of the substrate, phenyl-
alanine.

A1though it is not possib1e to genera1ize, there are now some

we11 documented cases in which it is c1ear that the observed rise
in P.A.L. activity invo1ves an increase in the level of avai1ab1e
P.A.L. protein rather than increased avai1abi1ity of substrate to
an existing enzyme system. The best examp1e does not re1ate to
post-harvest behaviour but to the effect of light on the formation
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 115

100

50

0
E
;:, 100
E
)(
CI
E
....0
~
0

...
>-
50

...
~~
u

..
CI

...
>

-a:..
CI
0
100
4CL
C

...................

...
50 \\ :
o
··.1
\, ,'p
'1

9,'''\. tn
("" .........
, I "',
· · . ·..·'tio
\ .....
000

o~--~--~----~~~--~
o 2 4 6 8 10
6rowth time of culture (days)

Fig. 7. Changes in P.A.L., C.A.4H and CL activities during the

growth of soybean cell suspension cultures (after 24a -
with permission).
116 M.J.C.RHODES

of two flavonoids, apiin and its methoxy derivative in cell

suspension cultures of parsley. Hahlbrock and his colleagues (22)
showed that the formation of apiin and methaxyapiin was preceded by
a co-ordinated incrßase in a number of enzymes including P.A.L.,
C.A.4H. and CL. The increases in these enzymes showed a similar
time course to a peak 15 hours after the start of illumination (21).
Hahlbrock (23) studied the rates of synthesis and degradation of
P.A.L. during the period of rising activity and proposed a model to
explain the observed pattern of changes in P.A.L. activity in terms
of large changes in the rate of synthesis coupled with a more or
less constant rate of degradation of the enzyme. The de novo
synthesis of the enzyme was indicated by its sensitivity to inhibi-
tors of both transcription and translation (49) but more definitive
evidence has come from experiments with antisera raised against
purified P.A.L. Schroder et al. (49) measured rates of formation of
P.A.L. in vitro using polysomes prepared from cultured cells after
varying per iods of illumination and measuring the incorporation of
35S-methionine into a pro tein product which was precipitable by the
antiserum to purified P.A.L. The immunoprecipitate which was
enzymically inactive (7) was analyzed by SDS-PAGE and among the
products were recognizable labelied fragments of P.A.L. In in vive
experiments in which 35S-methionine was fed to cells kept in~he--­
dark or after various periods following illumination, only in light
grown tissues was there significant incorporation into the immune
precipitate. Subsequent analysis by SDS-PAGE showed that the
radioactivity was associated with the 83,000 dalton subunit of P.A.L.
The time course experiments indicated that the maximum rate of P.A.L.
synthesis was reached 5-6 hours after the start of illumination and
corresponded with the time of maximum rate of increase of P.A.L.
activity. These findings confirmed the model proposed by Hahlbrock
(23) that the rise in P.A.L. activity observed was due to the effect
of light in promoting its synthesis. Subsequent work (50) showed
that the effect of light was mediated through an increase in mRNA
activity specific for P.A.L. It is, however, not clear whether
this involves de novo transcription of mRNA or the activation of
mRNA molecules-or-S-Combination of these two effects.

There are a number of other cases in which it is likely that

the observed rise in P.A.L. activity is due to an increased rate
of synthesis of the enzyme but the evidence is less complete than
that for parsely (14,37,48). The evidence is based on the use of
inhibitors of transcription and translation, density labelling
with heavy isotopes or on in vitro or in vive labelling of the
enzyme.

In other cases, however, mechanisms other than de novo syn-

thesis of the enzyme appear to operate. In a number of responses
of different plants to light, it has been shown that there is an
initial rise in P.A.L. activity to a peak value, which frequently
appears to bedue to enhanced enzyme synthesis, followed by a
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 117

period of decline in activity to a lower steady level. It has been

shown that application of inhibitors of pro tein synthesis to either
potato disks (65) or gherkin hypocotyls (16) at the point of maximum
P.A.L. activity inhibited subsequent decline in P.A.L. activity.
Similarly, if light stimulated gherkin hypocotyls, which had been
left at 4° for 24 hours during which time the P.A.L. activity fell
back to control levels, were subsequently transferred back to 25°
in darkness, there was a secondary rise in P.A.L. activity which
was insensitive even to a very high concentrations of inhibitors of
pro tein synthesis (17). Smith (53) showed a stimulation of P.A.L.
activity in cold treated gherkin hypocotyls which was insensitive
to cycloheximide. Subsequent density labelling experiments strongly
suggested that de novo synthesis was not responsible for this rise
in P.A.L. activity-csJ). Such experiments are taken as evidence
for the involvement of proteineous inhibitors playing a role in the
activation/inactivation of P.A.L. in vivo. In subsequent work an
inhibitor was isolated from gherki~(8) ~hich was present in both
the soluble and membrane fractions of aqueous extracts. It could
be 'solubilized' from the membrane fraction by treatment with
chaotropes. The inhibitor is apparently proteineous being both
thermolabile and sensitive to proteolytic attack. It has an apparent
molecular weight of 19,000 and inhibits P.A.L. in a reversible
manner. It is interesting that it also inhibits C.A.4H. but not a
range of other enzymes tested. Evidence for the presence of other
inhibitors of P.A.L. have been given by other workers (12,55,56)
but very little work has been done to purify these inhibitors and
to study their kinetic and mechanistic properties in any detail.
There is a limited amount of evidence at a physiological level
attempting to correlate changes in inhibitor level with observed
changes in P.A.L. activity.

There has been some work on the enzymic factors responsible

for the control of the accumulation of the red anthocyanin pigments
of the skin of apples which is known to be stimulated by exposure
to low temperatures and to light. Faragher and Chalmers (18) found
that in whole Johanthan apples, light stimulated anthocyanin for-
mation and a rise in P.A.L. to a peak followed by a secondary
decline. When cycloheximide was applied as a single drop to a whole
fruit, it stimulated P.A.L. activity and this may be interpreted as
an inhibition of the inactivation of P.A.L. Tan (5,56) proposed
that a P.A.L. inactivating system (P.A.L;-IS) plays a role in the
regulation of P.A.L. activity in vivo. Apples stored in constant
light accumulate high er levels of P.A.L. and more anthocyanin when
given alternating periods at 6° and 25° compared with fruit stored
continuously at 25°. The alternating temperature regime also led
to lower levels of P.A.L.-IS compared to 25° throughout. In general,
P.A.L. activity was found to be negatively correlated with the
level of P.A.L.-IS. The nature of the P.A.L.-IS was studied by
Creasy (12) who showed it to be associated with a heat labile high
molecular weight compound and that D-phenylalanine protected P.A.L.
118 M.J.C.RHODES
against P.A.L.-IS. P.A.L.-IS differs from the inhibitor of Billet
et al. (8) in being irreversible in its action. This prompted
Smith (53) to suggest it might be acting proteolytically on P,A.L.
but there is no clear evidence on how it functions.

In general, it appears that the responses of the pathway of

phenylpropanoid metabolism to external or internal stimuli are to a
large extent media ted by changes in the available level of P.A.L.
protein whether this be by de novo synthesis or by activation/
inactivation. This is the possibility of fine control of the acti-
vity of the individual enzyme since P~A.L. and C.A.4H. are sensitive
to both their immediate products (25,45) or to end products of the
pathway (53) and CL is susceptible to control by energy charge
(33,41). However, although it is likely that such control may be
important under some circumstances, the major control is on the
level of available enzyme pro tein.

It is important to return to the point made by Margna (39) who

questioned why it was necessary to invoke the production of new
enzyme pro teins when the existing system does not appear to be
fully saturated. It is possible to criticize this approach in
relating measured P.A.L. activities to production of particular
phenylpropanoid products. Since the pathway is highly branched it
is important to compare the catalytic capacity of P.A.L. with the
total throughput of the pathway by measuring all products (includ-
ing lignin which is frequently overlooked because of the difficulty
of its analysis but which is often quantitatively the most important
product of the pathway). While the estimate of the carbon flow
via P.A.L. may be too low it is almost certain that the in vitro
estimates of its activity are too high. The pH optimum is probably
weIl above the physiological pH of its cell eompartment and its
kinetie behaviour may weIl be very different at pHIS elose to 7.0
when it forms part of a membrane associated complex than in free
solution at pH 9.0. This points to a general problem that confronts
enzymologists and physiologists. The enzymologist carries out his
in vitro experiments measuring initial rates of reactions in the
pre~ of exeess substrate under conditions in which the concen-
tration of products is essentially zero. The activity of an enzyme
may be large when the concentration of products is low but may be
much lower when the substrate and products are present at close to
their equilibrium values. Thus the information the enzymologist
provides may not always be very useful to the physiologist who
wants information on the operation of enzymes in situ. It seems
likely that the reaction catalyzed by P.A.L. may und er physiological
conditions be limited by its catalytic potential in vive rather
than by the availability of substrate or cofactors.
In conclusion, this paper has set out some of the potential
mechanisms available for regulation of enzyme activity in vivo.
Dur knowledge of such regulation is at a very elementary level;
ENZYME ACTIVITIES AND POST-HARVEST CHANGE 119

many of the potential mechanisms have not yet been demonstrated

with plant enzymes and even with those that have been shwon to occur
it is very difficult to obtain evidence that they actually operate
in vivo. The two pathways we have considered represent reactions
or primary and secondary metabolism which play an important role in
the post-harvest physiology of fruits and vegetables. The present
state of knowledge suggests that very different mechanisms operate
in those two pathways. It is tempting to suggest the first type of
mechanism to be expected in obligatory systems such as glycolysis
while induction and inactivation may be appropriate to a pathway
which may only opera te in certain cells for limited periods of
their existence. At present, one can only speculate on the relative
importance of these two types of mechanism of control.

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PLANT MEMBRANE LIPIDS CHANGES AND ALTERATIONS DURING AGING AND

SENESCENCE

Paul Mazliak

University of Paris
4 place Jussieu, 75005 Paris

INTRODUCTION

Some types of lipid molecules (phospholipids or galactolipids),

because of their amphiphilic properties, are essential constituents
of all biomembranes. Changes occurring in the lipid composition of
these membranes will certainly modify their permeability, their
energy transduction capacity, the activities of their membrane-
bound enzymes, before and after harvest, when plants of economical
value are concerned. Furthermore, after harvest, many alterations
are likely to affect membrane lipids, thus playing a major role in
the senescence or the post-harvest physiology of plant crops. It
seems therefore reasonable to pay some attention to plant membrane
lipids when discussing the factors allowing good crop preservation.

LIPID COMPOSITION OF PLANT CELL MEMBRANES

11any articles have dealt recently with the lipid composition

of plant cell membranes (see for instance 1,2) and it will be suffi-
cient, for this short review, to present rapidly the lipid composi-
tion of some well-defined plant cell membranes (Tables 1 and 2).

Some general considerations can be drawn from the data ac cu-

mulated these last years. To summarize, one can say that:
(1) Polar lipids (glyco- and phospholipids) are the main lipid
components of all plant cell membranes. The amphipathic properties
of these molecules confer to membrane lipids a self-organization
(or auto-aggregation) tendency in aqueous mediums, due to the
thermodynamically favourable hydrophobic interactions occurring
between the non-polar parts of membrane lipids when they are
immersed in water.
123
 I'J
Table 1. Lipid compositio~ of some weIl defined plant cell membranes ~

Membranes % Total lipids

(origin) N.L* PC PA PI DGDG MGDG SL Refe-
PE PGP PS PG ren ces
Potato tuber (3)
32 12.6 17.5 1.1 7.2 12.5 17.1
plasmalemma
Potato tuber
mitochondria
outer membrane 52.6 25 12.1 10.3
------------
- inner membrane 19.5 24.5
(4)
27 29
Spinach chloroplast
- envelope 27 1.3 8.4 32 22 5.0
(5)
- thylakoids 3.2 1.4 9.1 26 51 7.1
Potato tuber microsomes 36.5 39 14.4 0.6 1.2 6.2 0.6 (6)
- Sunflower hypocotyl (7)
35.8 13.1 13.0 3.8 (6.1) 15.3 6.1 6.8
nuclear membranes
- Castor bean endosperm
. ~

49.0 31.4 2.4 11.4 6. 1 not measured (8)

glyoxysome
- Potato tuber (9)
22 16.3 41 10.9 10 traces -
peroxisomes

*Abbreviations : N.L = neutral lipids, PC = phosphatidylcholine, PE = phosphatidylethanolamine,

PGP = diphosphatidylglycerol, PA = phosphatidic acid, PI = phosphatidylinositol, PS = phosphati-
dylserine, PG = phosphatidylglycerol, DGDG = digalactosyldiacylglycerol, MGDG = monogalactosyl-
:-c
diacylglycerol, SL = sulfolipid.
s:
>
N
r
.. = lysophospholipids. »
A
 '1J
r
»
z
--l
Table 2. Main fatty acid composition of same plant cell membrane lipids s::m
s::
III
% Total fatty acids :D
Membranes
»
Z
m
Double Refe- r
(origin) 16: 1
16: 0 16: 1 18:0 18: 1 18:2 18:3 bond rences 3!
6 3 trans 0
index· Ul

Potato tuber plasmalemma 28.5 7.5 4.5 41 17.5 3.7 (3)

Potato tuber mitochondria
- outer membrane 24.0 3.5 1.8 58.0 12. 7 5.7
(4)
- inner membrane 12.5 2.3 0.8 67.4 16.8 12.6
Vicia faba chloroplasts
- envelope 13.3 1.8 0.0 4.3 5.8 11.5 63.3 12.5
( 10)
- thylakoids 5.9 0.0 1.2 1.6 3.2 5.2 82.9 33.8
Potato tuber microsomes 31 7.0 0.5 49.5 12.0 3.5 ( 1 1)
Sunflower hypocotyl
28.4 4.9 12.3 12. I 33.8 5.8" 2.3 ( 12)
nuclear membranes
Potato tuber peroxisome 22.0 2. 1 4.3 56 13.3 5.9 (9)

.Double bond index = % Monounsaturated + 2 x % Diunsaturated + 3 x % Triunsaturated fatty acids/

% saturated fatty acids.
"Eicosanoic acid (2.7 %) has also been found.

N
c.n
126 P. MAZLlAK

(2) Chloroplast thylakoid lipid composition is unique in several

aspects: i) galactolipids (MGDG, DGDG and SL) form the main part of
thylakoid lipids whereas phospholipids are predominating in other
cell endomembranes (the chloroplast envelope is somehow hybrid between
these two classes of biomembranes, although rich in galactolipids,
one can see that phospholipids (PC and PG) do represent important
percentages (more than 35%) of total envelope lipids, ii) a second
characteristic of chloroplast thylakoids, always observed in all types
of green tissues, is the high fluidity of these membranes due to very
high percentages of linolenic acid (C IB : 3 ) among total fatty acids,
which gives to thylakoid lipids a double-bond index far greater than
any other cell membrane lipid (Table 2).
(3) In cell membranes other than thylakoids, the major phospho-
lipids are always phosphatidylcholine and phosphatidylethanolamine,
phosphatidylinositol being often a third important lipid component.
Once again, the chloroplast envelope is in a curious position, phos-
phatidylcholine is predominating but phosphatidylglycerol (which is
important in thylakoids) is also important in the envelopes. All
other known phospholipids (PS, PA, PGP) are minor components,
envisioned most frequently as intermediary metabolites.

CURRENT SCHEMES FOR THE BIOGENESIS OF PLANT CELL MEMBRANE LIPIDS

Thanks to the joined efforts of many laboratories throughout

the world, the biosynthetic pathways of cell membrane lipids is
beginning to be known ($ee 13, 14). The striking point in this
research area is that the biosynthesis of all cell membrane lipids
necessitates an active cooperation between different cell compartments.
This necessary cooperation implies some transfer of acyl residues or
entire phospholipid moleeules within and between cell organelles.
Those transfers of mainly hydrophobie moleeules through the aqueous
cytosol cannot be realized without the participation of some kinds of
acyl carriers, for instance acyl carrier proteins within the plas-
tids (15), phospholipid transfer pro teins within the cytosol (16);
alternatively, membrane vesicles budding from the endoplasmic reti-
culum and membrane flow towards cell organelles can also be considered
following the conceptions of Morre et al (17). There is no doubt that
CO 2 photosynthetically fixed from the atmosphere, is the ultimate
precursor of all acyl residues of plant cell membrane lipids. However,
the biosynthetical process in chloroplasts stops with the production
of palmitic and oleic acids which are then exported towards the
cytosol in an unknown manner. The oleoyl residues are then desaturated
in the membranes of endoplasmic reticulum. Linoleoyl-phosphatidyl-
choline is the product of the desaturation. As linolenoyl-galacto-
lipids are the main lipid components of the photosynthetic apparatus
some transfer back to the chloroplast of linoleoyl-phosphatidyl-
choline must be envisioned to allow the last desaturation step to
occur in the plastidial compartment.
PLANT MEMBRANE LIPIDS 127

CHANGES INVOLVING CELL-MEMBRANE LIPIDS DURING AGING AND SENESCENCE

We have described some natural turn-oyer of membrane lipid

during the "aging" of adult plant cells of two quiescent organs;
abortive cauliflower inflorescence and dormant potato tubers
(waiting for some months the break of dormancy, in natural conser-
vation at 10-12 0 C). I must point out that this kind of research
upon the natural turn-over of plant membrane constituents is very
rare, as far as I know, although similar researches are rather
numerous in animal tissues (18).

Natural Turn-over of Membrane Lipid in Plant Organs

Half-lives of membrane lipids from potato or cauliflower cells

have been calculated, in our laboratory (19), by measuring, oyer
several days, the decrease in specific activities of phospholipids,
following a prior incubation of tissue slices in 14C-acetate solu-
tion. After a 3 hrs incubation period, the slices were transferred to
a medium without any radioactive precursor. In both tissues, the
phospholipid showed different turn-over rates in the different
cellular fractions. In cauliflower floret slices, where the inter~
pretation of the results was not complicated by a superimposed
lipid synthesis due to artificial aging, the measured half-lives
for phospholipids in the various subcellular fractions varied from
3 to 22 days which is very similar to the corresponding data obtained
with animal tissues (18). The greatest turn-over rates were found in
microsomal membranes and the lowest in mitochondria (although nuclear
membranes had not been examined). These resuits agree weIl with the
rates of direct labelling of the different PC or PE pools present in
the various subcellular fractions of caulifl~wer floret ti~sues. It
has been always found that 2 hrs pulses of C-acetate labelied the
phospholipids of the microsomal fractions more rapidly and more
intensively than those of mitochondria or any other fractions (11).

Similar re~ults have been obtained with onion stem slices (20)
labelled with 1 C-choline and the following order of decreasing
labelling has been found: microsomes > dictyosomes > smooth E.R. >
nuclei > mitochondria-proplastids.
14 3
Pulse labelling and dual substrate ( C-acetate and H-glycerol)
labelling were used by Wilson and Rinne (1976) (21) to evaluate lipid
turn-over in soybean cotyledons. These studies indicated first-·
order decay kinetics for phosphatidic acid, phosphatidylinositol,
phosphatidylcholine, phosphatidylethanolamine, N-acyl phosphatidyl-
ethanolamine and diacylglycerols. On the other hand, triacylglyce-
rols accumulated continuously in cotyledons, thirty days after
flowering. Phosphatidic acid and diacylg1ycerol turned over rapidly
(haIf-times of 6 min) , which simply resulted from their role of
intermediary metabolites in the biosynthetic pathway of triacylgly-
cerols. Half-times of 30-60 min were calculated for PC or PE and
128 P. MAZLlAK

were found simi1ar for the acy1 groups and the glycero1 parts of
these mo1ecu1es. On the contrary, in N-acy1-phosphatidy1ethano1amine~
acy1 groups were rep1aced independent1y of the glycero1 moiety
(ha1f-1ife: 3 hrs).

Moore (1977) (22) examined phospho1ipid turn-over in soybean

tissue cu1tures and found much 10nger ha1fI4imes than Wi1son and
Rinne. Phosphatidy1cho1ine, 1abe11ed from C-cho1ine, had 14ha1f-
1ife of 36 hrs and phosphatidy1ethano1amine, 1abe11ed from C-etha-
no1amine, decayed in three phases with ha1f-1ives of 12, 34 and 136
hrs. Those data, obtained without distinguishing the different pools
of a pecu1iar phospho1ipid, engaged for instance in severa1 organelle
membranes, point out the difficu1ty of measuring turn-over ha1f-1ives
without separating first, the different organelles or membranes of
the p1ant-ce11s under examination. Cumming and Osborne (23) recent1y
demonstrated a continuous turn-over of membrane phospho1ipids in
imbibed dormant e~bryos of the wild oat by carrying pu1se-chase
experiments with H-g1ycero1. The suggestion was made by the authors
that membrane turn-over was necessary to maintain the viabi1ity of
dormant imbibed seeds and that the capacity for continuous membrane
rep1acement may determine the 10ngevity of the wild oat-seed under
fie1d conditions.

Changes During Aging

It must be admitted, from the rare data co11ected in the litera-

ture, that, if the storage conditions chosen for various crops
(either seeds or fruits, or tubers) are adequate (re1ative1y 10w
temperatures, 10w relative humidity, darkness, slight anoxia), those
crops can survive severa1 months after harvest, in apparent1y good
physio10gica1 conditions, without disp1aying important changes in
the composition of their membrane lipids (24,25). That does not
mean, in any way, that metabolie activity in stored plant tissues
is nil: turn-over of membrane lipids can be measured with radio-
active precursors in those tissues, but this process does not lead
to any noticeab1e change in lipid composition of stored organs. It
must be pointed out here however, that arecent paper by Pearce and
Samad (26) showed a decrease in the total lipid content of peanuts
stored for 38 months at 5°C, without any change in the fatty acid
composition of the various neutral or polar lipids. If this aging
of the seeds was rea1ized in an environment of 38°C and 90% relative
humidity, the content of polar lipid fractions fell to almost 10%
of the original, in on1y 28 days.

It has been generally observed that a 10ng storage per iod at

10w temperature leads to an increase in the degree of unsaturation
of membrane fatty acids and this fact is usua11y interpreted as the
result of an adaptation of cells from chilling resistant plants to
low temperatures. It is assumed that such increase in lipid unsa-
turation would increase membrane fluidity and allow physiological
PLANT MEMBRANE LIPIDS 129

processes dependent on fluid lipid structure to be maintained at

lower temperatures.

Jerusalem artichoke tuber is a good example of such a chilling

tolerant organ (27). Artichoke tubers develop and mature during
spring and autumn but have to survive sub zero soil temperatures during
winter before sprouting next spring. Succinate-oxidase activity of
the mitochondria extracted from these tubers, at various times of
the year, has been studied, as weIl as the changes in the energy
activation of succinate-oxidation reaction at various temperatures.
The Arrhenius plots of succinate-oxidase activity thus obtained, in
various seasons, all displayed two breaks (T f and Ts ) corresponding
to two major changes in the molecular ordering of mitochondrial
membrane lipids; this was shown with coup~ed spin-label motion
studies. For instance at the end of autumn, the Tf and Ts for
mitochondrial membrane lipids of Jerusalem artichoke are 22 and 3 Q C,
respectively. Both Tt and T of tuber membranes decrease during
winter and during th~s timeSthere is a general increase in membrane
fluidity. This phenomenon is probably part of the mechanism adopted
by tubers to withstand freezing but cannot be correlated, in that
peculiar case, with any significant change in the unsaturation of
tha fatty acids of membrane lipids. Thus, many plants which survive
freezing temperatures, show negligible increases in fatty acid
unsaturation when exposed to low temperature(Siminovitch et al. 28),
Other mechanisms of the adaptation of plant cells to a low tempera-
ture stress can be envisioned: changes in the position of esteri-
fication of unsaturated fatty acids on the glycerol moiety of com-
plex lipids, changes in the relative proportion of membrane phos-
pholipids, etc •..

Lipid Breakdown During Senescence

Few studies are concerned with the changes occurring in mem-

brane lipids during senescence. However, all the data which can be
collected in the literature point out the same tendency: in every
senescent plant organ, whatever it is, there is a general lipid
breakdown. This phenomenon is generally accompanied by some disor-
ganization of membranes in senescent cells or organelles. Sometimes
the appearance of plastid globuli, probably built with membrane
lipid materials, is noticed, in chloroplasts for instance (29).

Newmann has thoroughly studied the evolution of chloroplast

lipids in the leaves of Cucurbita maxima, during development and
senescence: a noticeable reduction in percentage of linolenic acid
was observed in senescent leaves(30) (Table 3). The same author has
compared the lipid composition of the chloroplasts from leaves of
various trees, either green or yellow(senescent). He has found
again a noticeable reduction in the percentages of polyunsaturated
fattyacids (Table 4). In the same time, short chain fatty acids
tended to accumulate in senescent leaves.
130 P. MAZLlAK

Table 3. Evolution of the fatty acid composition of chloroplast

lipids during development and senescence of Cucurbita
maxima leaves (Newmann 30).

Percent of total fatty acid weight

Leaf age
Palmitic Linolenic
Oleic acid
acid acid

Cotyledons 10.3 2.0 66.9

First internode leaves 4.5 0.8 79.0
Second internode leaves 10.1 \.3 75.5
Sixth internode leaves 7.9 3.3 73.8
Tenth internode leaves 12.7 5.0 70.9

Table 4. Comparison between the fatty acid composition of the

chloroplastsof either green or yellow, senescent leaves
from two trees (Newmann 30).

Percent of total fatty acid weight

Species

Cladastris lutea
Green leaves 2.1 5.9 13.5 2.0 7.7 64.1
Yellow leaves 9.9 4.3 10.2 1.8 7.8 14.5 4.9 28.0
Gink~o biloba
Green leaves 10.6 9.1 23.9 4.6 4.3 8.9 4.0 34.7
Yellow leaves 12.8 19.2 17.2 \.3 6.4 9.5 4.6 7.0

These pioneering observatons were largely confirmed by

subsequent researches. In senescent leaves, rapid losses of those
lipids which are components of chloroplast thylakoids are seen (31):
the glycolipids are quickly deacylated (32) and the liberated lino-
lenic acid is particularly susceptible to lipoxygenase-catalyzed
attack. Accumulating unesterified fatty acids have also been.noted
to react with xanthophylls and various alcohols in senescing leaves
of Acer platanoides (33).

The degradation of linolenic acid has been observed in senes-

cent apple fruits (34), with the noticeable development of
PLANT MEMBRANE LIPIDS 131

osmiophilic granules in the yellowing peels of senescent fruits.

An unbalance between biosynthetic and degradative pathways of

lipid metabolism is probably the simplest explanation which can be
offered to this lipid breakdown during senescence. As it will be
indicated below, peroxidation of polyunsaturated fatty acids is
probably overtaking the biosynthesis of the same compound at the
onset of senescence, and this precise reversing of metabolic equi-
librium could mark the involvement of plant tissues towards an
irreversible disorganization.

Confirming this idea, changes in fatty acid metabolism were

studied in soybean cotyledons during senescence as well as in coty-
ledons which had been caused to regreen by removal of the epicotyl
from the seedling (35). The activities of the enzymes acetyl-CoA syn-
thetase and fatty-acid synthetase in plastids isolated from the
cotyledons decreased during senescence but increased in response to
regreening. These changes in enzyme activities followed the same
pattern as changes in the quantities of chlorophyll and polyunsatu-
rated fatty acids in these tissues. Similarly, the galactosyl trans-
ferase activity of chloroplast envelopes decreases in senescent
soybean cotyledons (36).

The loss of polyunsaturated fatty acids, during senescence has

obviously deep consequences on the fluidity of biomembranes in
senescing organs. This question has been studied by McKersie and
Thompson (37) in senescent cotyledons from bean. Lipid transition
temperatures for rough and smooth microsomal membranes isolated
from bean cotyledons at various stages of germination were determi-
ned by wide angle X-ray diffraction. For rough and smooth microsomes
from 2-day-old tissues, the transition occurred at 0 and 3°C, res-
pectively. By the 4th day of germination, the transition tempera-
tures had increased to 32°C for smooth microsomes and 35°C for rough
rnicrosornes. During the later stages of germination, the transition
temperatures of smooth microsorne lipids continued to rise to 56°C,
by day 9, at which time the cotyledons were extensively senescent and
beginning to abscise. The data indicate that substantial amounts of
membrane lipids are in a crystalline or pseudo-crystalline state in
senescing membranes. The onset of senescence in the cotyledons of
germinating beans is marked by a general metabolic decline (decrea-
sed ATPase activity, ATP-dependent cation transport, etc ... ) corre-
lating well with a structural disassembly of all membranes (38).

Finally, some relationship between phospholipid metabolism,

senescence and ethylene production has been described in senescing
tissues of the morning glory flowers (Ipomoea tricolor Cav.) (39).
Rib segments excised from flower buds passed through the same phases
of senescence as the respective tissues on the intact plant. It was
found that the phospholipid contents of rib segments had already
started to decline before rolling up of the ribs, a visible sign of
132 P. MAZLlAK

senescence. As the segments began to roll up and to produce ethylene,

the rate of phospholipid loss accelerated sharply.

During the same period, the level of fatty acids esterified to

phospholipids also fell by 40 per cent. No qualitative changes in
any lipid component could be detected during senescence. Exogenously
applied ethylene accelerated the loss of phospholipid and the senes-
cence of rib segments while benzyladenine retarded both of these
processes. Two metallic ions had also contrary effects: Ag+ inhibi-
ted the rolling up of flower segments but did not affect ethylene
generation or phospholipid loss; in contrast, C0 2+ reduced both
processes.

Those observations are interpreted in the following way: the

onset of senescence is triggered by some degradation of membrane
lipids resulting in increased membrane permeability. This loss in
cellular compartmentation is thought to cause mixing of previously
sequestered metabolites of the ethylene-generating system, thereby
stimulating ethylene evolution. Once formed, ethyleneenhanced its
own synthesis and accelerated aging by further increasing membrane
permeability and perhaps, by further reducing phospholipid biosyn-
thesis (40). As one can see from the preceding results andinter-
pretations, membrane lipid breakdown emerges as a crucial step in
the onset of senescence.

M&~BRANE LIPID PEROXIDATION: A STEP TOWARDS SENESCENCE

One of the good ideas which has been advanced concerning the
senescence of living tissues is that lipid peroxidation is greatly
involved in the phenomenon. The obvious advantage of this idea is
that experimental research can be done and has been done, indeed,
on the matter : the data collected so far strengthen the hypothesis.
On another hand, it cannot be masked that lipid peroxidation is
also of immense practical importance, for it leads to the deterio-
ration of tissues in vivo and so to the spoilage of foods.

The Mechanism of Lipid Peroxidation

Lipid peroxidation can result both from purely chemical or

photochemical processes as well as from biological reactions. In
the former case catalysts can be either organic or metallic; in
the latter case, the enzymes specialized in lipid peroxidation are
named lipoxidases or lipoxygenases (41).

Table 5 shows the detailed reaction mechanisms which have been

worked out for chemical or enzymatic lipid peroxidation. The first
products of such oxidations, involving the capture of atmospheric
oxygen, are always hydroperoxides which are very unstable products.
The best substrates of lipid peroxidation are common polyunsaturated
PLANT MEMBRANE LIPIDS 133

Table 5. Lipid peroxidation

AUTOXIDATION

~
50 % ••~----------~~~50 %
R1- HC=CH-CH=CH-CH- R2 R1-CH-CH= CH-CH=CH- R2
cis trans I I trans cis
OOH OOH

CHEMICAL CATALYSIS

- hematin comDounds
- hemoglobin
- myoglobin
- cytochromes
- trace metal catalysts (Cu, Fe, etc ... )

ENZYMIC CATALYSIS

R - HC = CH - CH - HC = CH -- R +
1 . 2 . 2
c~s c~s lipoxygenase

OH
o
os ~ t

II
R ~ HC~CH- HC=CH-C-R
position isomers, I 2
hydroperoxides
R1-C -HC=CH-HC . CH--R 2
I trans ~
o
I
OH
134 P. MAZLlAK

fattyacids (linoleic and linolenic), containing the reactive center

cis, cis, 1,4, pentadiene (-CH=HC-CH 2-CH=CH-). The hydroperoxides
formed constitute a great number of possible isomers and a major
difference between chemical lipid peroxidation (also called autoxi-
dation) and biological peroxidation is that, generally, the chemical
process leads to different isomers appearing roughly in equal amounts
while the biological process gives rise preferentially to one pecu-
liar isomer. Free radicals and chain reactions are characteristic of
lipid peroxidation.

Lipid peroxidation is indeed accelerated or inhibited by a

series of physical or chemical factors (Table 6); the lipid peroxi-
des, as indicated previously, are very unstable compounds and their
decomposition leads to many volatile end products (Table 7) (many of
which are responsible for the spoiling of foods). Lipid peroxidation
is often coupled to other compound oxidation, which, again contri-
butes to food deterioration.

Volatile products : the scavenging of lipid peroxides

Many of the flavours associated with vegetables (such as beans

and cucumbers (42»are formed by oxidative enzymic breakdown of un-
saturated tissue lipids. The sequence is begun by lipoxygenase. The
formed hydroperoxide cleaves to liberate volatile aldehydes that are
responsible for the aroma characteristics of many vegetables (Fig.l).

After cleavage of the hydroxyperoxides and departure of the

short volatile products, there remains always a portion of the acyl
chain which could well be at the origin of traumatic acid, the so
called "wound hormone," the structure of which is trans-lO-C 12 : l
dibasic acid.

A very recent finding deserves some attention : among the clea-

vage products of hydroperoxides many aldehydes are found. Some of
them could be reduced in vivo to short chain alcohols (butanol,
hexanol) : it appears that these short chain alcohols have senes-
cence retardant effects (43). This would allow considering the rela-
tively rare reductions of short chain aldehydes as feed-back re-
actions controlling negatively the onset of senescence (see beiow)

Table 6. Factors affecting lipid oxidation (41)

ACCELERATION BY INHIBITION BY
High temperature Refrigeration
Light (U.V. and blue) Opaque or colored
Ionizing radiation (a,ß,Y) Containers or wrappers
Peroxides Exclusion of oxygen
Lipoxygenases Blanching
Organic ir on catalysts Antioxidants
Trace metal catalysts Metal deactivators
PLANT MEMBRANE LIPIDS 135

Table 7. Hydroperoxides and volatile aldehydes formed In lipid

autoxidation(41)

Aldehydes formed by
Fatty acids Isomerie hydroperoxides
decomposition

Oleic 1 I-OOH-9-ene octanal

9-00H-IO-ene 2-decenal
8-00H-9-ene 2-undecenal
IO-OOH-8-ene nonanal

Linoleic 13-00H-9, I I-diene hexanal

II-OOH-9,12-diene 2-octenal
9-00H-IO,12,diene 2,4-decadienal

Linolenic 16-00H-9,12,14-triene propanal

14-00H-9,12,IS-triene 2-pentenal
12-00H-9,13-IS-triene 2,4-heptadienal
13-00H-9,11,IS-triene 3-hexenal
I I-OOH-9,12,IS-triene 2,S-octadienal
9-00H-IO,12,IS-triene 2,4,7-decatrienal

CUCUMBER
cleavage .
9-hydroperoxide ---'="~-cls-3-nonenal

...,,-
lipoxygenase ~
cleavage h exena 1
~13-hydroperoxide ---~..
_
linoleic acid

TOMATO
9-hydroperoxide - - -......-no cleavage

.. ~13-hydroperoxide - - -.....- hexenal

reaction
lipoxygenase /

linoleic acid

Fig. I. Comparison of the enzymic formation of volatile fragments

by hydroperoxide cleavage in cucumber and tomato fruits (42)
136 P. MAZLlAK

A Step Towards Senescence

Lipid peroxidation products on one side, hydroperoxides (lipo-

xygenase products) on the other side are susceptible to accumulate
in -
- vivo
- under certain circumstances. It must be stressed first that
lipoxygenase activity, for instance, is very different from one
species to another, as shown in Table 8. Secondly, the physiological
state of development of tissues is mostly important when considering
in vivo lipoxygenase activities. Accumulation of lipid peroxidation
products during senescence of fruit has been demonstrated (45). It
must be admitted that it is not easy, presently, to differentiate in
vivo enzymic and non-enzymic lipid peroxidation systems. Gardner (46)
has discussed important aspects of both enzymic and non enzymic for-
mation and breakdown of lipid hydroperoxides. But whatever the forma-
tion pathway of these compounds, both their biosynthesis and their
ultimate cleavage may produce free radicals which undoubtedly are
susceptible to greatly damage plant cell organelles and membranes.
These free radicals would react at random with other compounds
through hydrogen removal and a variety of addition reactions.
Through these reactions a wide variety of membrane lipid components
would be chemically damaged. The loss of some fatty acids, carotene,
pigments, etc ••• would appear of great significance for the physio-
logy of plant cell organelles and membrane bound-enzymes.

Table 8. Oxidation of linoleic acid by aqueous extracts of edible

plants (44)

Lipoxygenase activity·
Tissue (~l 02 consumed x min- 1 x g
fresh matter)

Lettuce leave 120

Spinach leaves 80
Strawberry 40
Pear o
Pumpkin 120
Avocado 720
Egg plant fruit 4320
Date o
Carrot root o
Beet root o
Turnip root o
Potato tuber 4560
Celery stalk 120
Artichoke heart 3360
Cauliflower florets 1440

·linoleic acid was used as a substrate.

PLANT MEMBRANE LIPIDS 137

For instance, damage to membranes may simply result in breaking the

diffusion barrier, which would allow cell contents to leak out. But
damage to membranes may be more complex as in the case of energy
transducing membranes where electron-transport systems or coupling
factors are greatly inhibited by lipid peroxidation, either in
chloroplast or mitochondria. Damages to lysosome membranes or tono-
plasts would be particularly devastating.

CONCLUSIONS

Let us propose in conclusion the following hypo thesis which is

not entirely original (47). Senescence could be the result of biomem-
brane damages within plant cells. At a certain period in the life
cycle of mature plant cells, there would cease to be the efficient
repression of the lipoxygenase peroxidation system by natural anti-
oxidants or superoxide dismutase. Free radicals would appear more and
more numerous formed from all membrane lipids, particularly those con-
taining polyunsaturated fatty acids which are so numerous. As a conse-
quence, tonoplast or lysosome membrane integrities, for instance,
could not be maintained and many transducing energy functions in
membranes would be hampered. Death, then, seems unavoidable.

In other words, one could say that lipid, in living cells, has
been an obligatory component since the origin of life, because of the
necessity to isolate the protoplasma from the external aqueous medium
or to divide the aqueous protoplasma into several distinct functional
compartments. The unfortunate and accompanying consequence of this
necessity is that membrane lipids, susceptible as they are to oxygen
attack, represent truely a potential danger for life itself. Membrane
lipid alteration could well be the first sign of senescence and the
leading pathway to death.

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HORMONAL REGULATION OF SENESCENCE, AGEING, FADING, AND RIPENING

Johan Bruinsma

Agricultural University
Wageningen
The Netherlands

INTRODUCTION

Senescence is generally looked upon as an irreversible process

of gradual degeneration and desintegration, eventually leading to
the death of the organism. Such a form of senescence, which may
proceed uncontrolled, occurs in most perennial plants, e.g. trees,
that after many years of flowering and fruiting (polycarpy) gradual-
ly die off.

However, senescence need not occur as a more or less patho-

logical event, but may proceed as a process under strict control
as is the case with any other stage of development. This can be
seen in monocarpic plants, mostly annuals or biennuals, in which
senescence starts in the course of the single reproductive state
of the plant. The lowermost plant parts, that were formed first,
may commence to senesce and the process moves acropetally.

Next to the senescence of organisms, such organs as leaves,

flowers and fruits also senesce. With polycarpic plants this may
often be to the benefit of the plant individual and species. For
instance, the dying off of leaves of woody and bulbous plants at
the approach of the unfavourable season is a means in the strategy
for survival of the perennial plant during the adverse period.

Detachment of plant organs usually advances the onset of

senescence. The artificially induced senescence of isolated plant
parts is called ageing. Without special precautions, cut leaves
will yellow sooner than when they remain attached. Similarly, cut
flowers will wilt, and picked fruit will ripen earlier than when
left on the plant. Insofar as the senescence of these organs
141
142 J. BRUINSMA

proceeds controlled, manipulation of the regulating mechanism(s)

may affect the longevity and shelf life of postharvest commodities.

It will be demonstrated that the senescence of monocarpic

plants is under the control of plant hormones. Subsequently, we
will consider how far the ageing of isolated plant parts, too, is
regulated by these hormones. The present state of our knowledge of
the course of hormone levels during yellowing, fading, and ripening,
and of the effects of treatments with exogenous plant growth regu-
lators during these processes, will also be surveyed. First of all,
however, the plant hormones themselves will be briefly introduced.

PHYTOHORMONES

Plant hormones are endogenously produced molecules that can

act at the site of their synthesis and also after translocation to
other plant parts. They exert their effects at low concentrations,
in the part per million (ppm) and part per billion (ppb) ranges,
mainly by interacting with the genetically controlled protein syn-
thesis, with the enzymatic activities of these proteins, and with
membrane-bound processes. In these respects, phytohormones are
similar to the hormones known from vertebrate physiology. They
differ from the animaiones in their chemical structures and in
that their actions are not confined to specific target organs. On
the contrary, phytohormones can be produced in different organs and
also exert their manifold effects in various plant parts. In this
way, locally synthesized hormones can interact with each other and
with those derived from elsewhere. This leads to shifting patterns
of agonistic and antagonistic components, which patterns specifi-
cally regulate the enzymatic composition and metabolic activity
of each organ at every state of development. With such a pattern
of promoting and inhibiting factors a much more precise regulation
can be obtained than with either one alone.

In order to be effective, the hormone molecules need to be

recognized by receptor molecules in the cello Insensitivity to a
hormone may mean a reduced amount or availability of its receptor
molecules. That the amount of receptor molecules may strongly vary
during development was shown for the cytoplasmic auxin receptor in
cultured tobacco-pith callus (Fig. 1). Hormones that antagonize
other hormones, might also do so by keeping receptor molecules at
a low level or unavailable for these other hormones.

At present, five groups of phytohormones have been established

with certainty. Moreover, other naturally occurring plant substances
may participate in the hormonal pattern and some of them may pos-
sibly be recognized as hormones before lang. Amang these are
HORMONAL REGULATION 143

phenolic, particularly steroid compounds (Grove et al. , 1979,

Hewitt et al., 1980). Polyamines are known to retard leaf senes-
cence (Kaur-Swahney and Galston, 1979), whereas a specifically
senescence-promoting substance, (-)-methyl jasmonate, has recently
been isolated from wormwood, Arternisia absinthurn L. (Ueda and Kato,
1980).

cr
CD
10 I--
r-'
r- ~
"0
C
8 r-
"..,...
)
:J
0
.0
r-
>..
"'iü 6 .h:
.S!
.....
'0
'-
r-
GI ,
a.
LIl
t.
«
«
i' -
,...., 2r- .
..,I
n
~
6.
......
.+
0
o 3 6 9 12 15 18 21
Days after transfer

Fig. 1. Course of amount of soluble auxin receptor during culture

in vitro of tobacco-pith callus (after Oostrom et al.,
1980).

The plant hormones are derived either from isoprene units or

from amino acids. The natural cytokinins are adenine compounds con-
taining an isoprene side chain (Fig. 2). Cytokinins are highly ef-
fective in delaying the onset of ageing in isolated plant parts
used as a bioassay to test senescence-promoting and -inhibiting
substances. The use of organ sections, e.g. leaf segments incubated
on test solutions in darkness, prevents interference by hormonal
substances from other organs of the plant. It is particularly the
deprivation of the roots that renders isolated plant parts liable
to advanced senescence because the roots are the main site of
cytokinin biosynthesis. On the contrary, enabling an overground
144 J. BRUINSMA

plant organ, e.g. a 1eaf, to form adventitious roots prevents the

1eaf to senesce because of re-establishment of its source of
cytokinin. This is the 'special precautions' referred to in the
previous section when discussing the advanced senescence of de-
tached organs.

The two other groups of hormones, derived from three and four
isoprene units, respective1y, are the abscisins and the gibbere11ins
(Fig. 2). The gibberel1ins are not very active in senescence bio-
assays, although they may retard ageing of leaf discs in specific
Rumex and Taraxacum species, and also counteract some ripening phe-
nomena. The abscisins, on the contrary, strongly promote the onset
and rate of senescence processes. The main representative, abscisic
acid (ABA), is known to enhance the synthesis and release of hydro-
1ytic enzymes that play a ro1e in senescence, and to decrease mem-
brane integrity. It specifica11y interferes with the biosynthesis
of cytokinin nuc1eotides (Miernyk, 1979). Gibbere11ins and abscisins
are formed both in roots and in 1eaves, especia11y in the chloro-
plasts of the leaf,parenchyma (Hartung and Abou-Mandour, 1980,
Heilmann et a1., 1980, Milborrow, 1979). They can be released from
the chloroplasts into the cytop1asm to exert their hormonal func-
tions. In the cytop1asm, ABA is gradua11y inactivated, part1y into
conjugate(s), part1y into (dihydro)phaseic acid (King, 1979).

The natural auxins are synthesized from amino acids (Fig. 3),
mainly in the young, overground plant parts. Also synthetic chemi-
ca1s with auxin activity have been deve10ped that can rep1ace the
natural auxins. When these growth regulators are app1ied at moder-
ate concentrations, they usua11y retatd senescence. High auxin
dosages, however, have the opposite effect, they promote senescence
by inducing the biosynthesis of the other amino acid-derived hormone,
ethylene (Fig. 3) (Aharoni et al., 1979). Ethylene is the most
potent accelerator of senescence phenomena and, according to its
ability to induce de novo enzyme synthesis, a true hormone
(Lieberman, 1979). It is the rate-limiting step in the production
of ethylene from methionine, vize the formation of l-aminocyclo-
propane-l-carboxylic acid (ACC) , which is strongly promoted by
auxins (Yu et a1., 1979). The same step can also be inhibited by
aminoethoxyviny1g1ycine (AVG) and by ~-aminooxyacetic acid (AOA).
These artificial regulators are thus potent inhibitors of senes-
cence phenomena (BoIler et al., 1979; Amrhein and Schneebeck, 1980).
The conversion of ACC into ethylene requires oxygen and can be
inhibited by ethanol (He ins and B1akely, 1980) and by coba1tous
ions (Yu and Yang, 1979), whereas si1ver ions prevent the action
of ethylene, probably by interfering with its receptor (Aharoni
and Lieberman, 1979; Veen et al., 1980). During its action,
ethy1ene may be oxidized to carbon dioxide or converted into
ethylene glycol and its glucoside (Beyer, 1979, BIomstrom and
Beyer, 1980). Ethylene can be replaced by the, less active,
propylene that is converted in a similar way.
HORMONAL REGULATION 145

CYTOKININS ABSCISINS GIBBERELLINS

A HN

N~N~
cc--~ (1 C~ ,.,".,,.,,".,-
~~_J
~)l~
t
pyrophosphate

I growth retardants

O~~O
Q91'"""'
absclslc aCid 'OH
(ABA)

~m
HO~H C
'OH
trans-zeatin gibberellic acid (GA 3 )

Fig. 2. Isoprenoid phytohormones.

AUXINS ETHYLENE

(O N
CH _~_C1'°
'I
NH,
'
OH
0- CH -c-c
,
H
1
NH,
~o
'OH
HO-CH -CH
, ,
_~_c1'°
1

NH,
'

OH
H
tryptophan phenylalanine
homoserine ---"?'"---A-TP9=:p +P
i i
--~ rransamlnallon

CH -S-CH -CH -~-c,,-o

(O
::=f::'" , ,.,
CH _c_d'° 3 1 ' ' I 'OH
adenosIne NH 2
, 11 'OH
N 0
H
indolepyruvic acid
,"r'-. .
phenylpyruvic acid
- - - oxidatlve decarboxylatlon- ----
o
c?

co-
CH,
I'c/, 'OH
/
CH, NH,
"p I-aminocyclopropane-
CH,-<
OH -I-carboxylic acid (ACCI

fH':'
N
H

CH, =CH 2 + H-cf + NH 3 +GO,

'OH
indoleacetic acid (lAA) Qhenylacetic acid (PAA)
ng. 3. Amino acid-derived phytohormones.
146 J. BRUINSMA

The physiological effects of plant growth regulators, both

artificial ones and the naturally occurring hormones, can be stud-
ied by their application and following the course of the develop-
mental process under study, e.g., senesce~ce. Also the level of
hormones during development can be followed by repeated samp1ing
and determination of hormone contents by bioassay or, preferably,
by physical methods (Hillman, 1978). It is by such methods that
phytohormones have been found to be involved in the regulation oft
e.g., monocarpic senescence.

MONOCARPIC SENESCENCE

The overall senescence of monocarpic plants, which occurs

during or after their single flowering phase, often coincides with
the approach of the adverse season. However, monocarpic p1ants do
not die because of unfavourable environmental conditions but rather
in response to internal changes. This is shown by individuals that
fail to flower and survive the adverse season (Leopold, 1975). The
coincidence i1lustrates the ecological adaption of the species.

Every gardener knows that removal of withered flowers prolongs

the longevity of his plants. This pro1onged viability is not so
much due to the removal of potential sinks for nutrients but rather
of producers of senescence-inducing substance(s) (Nood~n and Leopold,
1978). For examp1e, removal of developing soybean pods reduces the
abortion of younger fruit1ets and al10ws vegetative development to
continue; in fruitlets and shoot buds, where the ABA content is
lower than with intact p1ants, ABA app1ication inhibits growth of
fruits and shoots (Tarnes et a1., 1979 a, b). Therefore, the deve1op-
ing pods probably produce this inhibitor. However, because ABA
application fails to induce senescence in depodded plants, it is
probably not the 'death hormone' itself but an accompanying factor.
Very little research has been done yet to identify other hormonal
factors re1eased from developing fruit.

On the other hand, the application of cytokinin and auxin to

intact soybean plants largely prevents the yellowing and nutrient
export of the leaves without reducing the growth of the pods (Nood~n
et al., 1979). Apparently these regulators are able to counteract
the senescing factor(s) from the fruit. In cereal plants, the ABA
content of the ears increases during grain filling (King, 1979).
Here, too, leaf senescence during this period is retarded by cyto-
kinins from the roots (Biswas and Choudhuri, 1980).

When the basal, mature leaves of a tobacco plant start yellow-

ing, removal of the top of the plant will not only halt this but the
leaves will turn green again. This regreening demonstrates that
senescence, at least in its initial stage, is a reversible process.
That the reversion is brought about by root-produced cytokinins that
HORMONAL REGULATION 147

had otherwise moved to the top, is indicated by the regreening of

basal leaves at intact plants when sprayed with a cytokinin solution
(Dyer and Osborne, 1971). A similar reversibility of senescence is
shown by the recovery of wheat seedlings after two days of darkness.
After four days in the dark the plants are unable to recover com-
pletely, unless they had been treated with cytokinin (Wittenbach,
1977) .

Generally, the response of plants to environmental conditions

is mediated through hormonal messengers. As long as the soil condi-
tions are favourable, the roots produce cytokinins and gibberellins
that promote the growth and development of the overground parts.
However, when the roots perceive drought or lack of oxygen or of
mineral nutrients, they decrease the productiGn of these promoting
hormones and increase that of abscisins, so that the life of the
plant tends to be shortened by advancing senescence (Atkin et al.,
1973). For example, the leaf yellowing under nitrogen deficiency
may not directly be caused by shortage of this nutrient but rather
by lack of cytokinin from the root system (Wagner and Michael, 1969).

It can be concluded that plants, that cannot run away from

adyerse conditions, adapt their life cycle to their environment.
They regulate their physiological functions, including senescence
and death with monocarpic plants, by way of their hormonal patterns.
Developing fruits alter these patterns, either directly or by influ-
encing the hormonal production of the roots which is an important
factor in monocarpic senescence. In this form of senescence, a role
of ethylene has not yet been established.

LEAF AGEING

We now turn to the question of how plant organs react when they
are removed from the plant. To what extent is the inevitable senes-
cence of these isolated plant parts still under hormonal control?

Although the ageing of leaf sections is by far the most used

bioassay to test for senescence activity, yet the role of hormones
in the yellowing of leaf discs is still incompletely understood,
largely because the course of endogenous hormones during ageing has
hardly been studied. It is no doubt the removal from the source of
cytokinin supply which predisposes the leaf section to accelerated
ageing, but the mechanism by which cytokinins are able to prevent
the leaf tissue from senescing is not clear. Cytokinins are known
to activate anabolic pathways and perhaps they sustain the metabolic
state of the leaf cells to such an extent that it remains above some
threshold below which catabolic reactions become predominant (Thomas
and Stoddart, 1980). Ethylene and ABA, on the other hand, which
advance and accelerate leaf ageing (Hall, 1977; Nooden and Leopold,
1978), might do so by stimulation of catabolic pathways and reduction
148 J. BRUINSMA

of membrane stability (e.g. Collins and Morgan, 1980). Thimann and

Satler (1979) recently observed that treatments that cause stomatal
opening of oat leaf sections in darkness also retard ageing and,
conversely, treatments that induce stomata to close in the light
promote ageing. These treatm~nts in the light can be considered to
induce ABA as a response to stress, particularly in the absence of
root-supplied cytokinin. Exogenous cytokinin can, indeed, prevent
this ABA synthesis (Gepstein and Thimann, 1980). In prolonged dark-
ness, these leaf sections also accumulate ABA that may playa role
in accelerated ageing under these circumstances (Gepstein and
Thimann, 1980). This need not necessarily involve a direct causal
relationship with the stomatal closure in darkness. The ABA accumu-
lation may weIl result from release of the hormone fram the inactive,
cytokinin- deprived chloroplasts followed by further ABA synthesis in
these organelles. Radin (1981) showed ageing and stomatal closure to
be not directly related in cotton leaves, both processes responding
apparently independently to endogenous ABA levels.

Whatever the mode of action of hormones in leaf ageing may be,

the shelf life of leafy commodities can be enhanced by keeping the
levels of ABA and ethylene as low as possible; cytokinins are not
permitted to be applied to foodstuffs for toxicological reasons.

FADING OF CUT FLOWERS

The senescence phenomena of cut flowers are studied more thor-

oughly than those of leaf sections, on the one hand because of their
practical importance, on the other hand because of focusing upon the
changes in endogenous hormone levels in flowers. As early as 1970,
Mayak and Halevy reported that rose varieties have a longer vase
life when their petals have a higher cytokinin content. Immersion
of rose buds in cytokinin solution prolonged their longevity. Later
these authors reported that the endogenous cytokinin level in the
petals rapidly drops after flower opening and that subsequently
the ABA level rises. This occurs more rapidly in roses with a short...
er vaselife than in those with a better keepability. They also
state that the rise in ABA is provoked by ethylene. ABA should
accelerate senescence but suppress further ethylene biosynthesis
(Mayak et al., 1972; Mayak and Halevy, 1972). Although inhibition
of ethylene evolution by ABA has been occasionally reported
(Wright, 1980), it is more frequently observed that ABA promotes
ethylene that thereupon triggers the senescence process (e.g. Sagee
et al., 1980).

The crucial phenomenon in flower ageing, the fading process,

results from leakiness of petal-cell membranes, particularly of
the tonoplast surrounding the vacuoles. Such osmotically active
substances as sugars may preserve membrane stability and in the
spike of Gladiolus exogenously applied gibberellin (GA 3 ) may stim-
HORMONAL REGULATION 149

ulate starch breakdown and thus give a more full and prolonged
flowering by increased supply of soluble sugars (Ran~nuja Rao,
1979). Also the protecting effect of cytokinin seems to be exerted
through prolonged membrane integrity, and this effect can be mim-
icked by synthetic benzthiadiazoles (Apelbaum and Katchansky, 1977).
Cut flowers lack a cytokinin supply from the roots.

Ethylene, on the contrary, strongly promotes membrane perme-

ability (Asen and Lieberman, 1963; Kende and Baumgartner, 1974;
Hanson and Kende, 1975). This is the cause of the wilting of
ferti1ized f10wers: after pollination of, e.g., orchid blossoms,
ethylene is evolved from the pistil and reaches the petals that
subsequently start producing the hormone (Burg and Dijkman, 1967,
Nichols, 1977, Suttle and Kende, 1978). Consequently, inhibition
of either the biosynthesis of ethylene with AVG or ethanol (Suttle
and Kende, 1978, Heins and Blakely, 1980), or its action with sil-
ver ions (Beyer, 1976), effective1y extends the vase life of cut
flowers. If, in particu1ar, the silver is complexed in the thio-
sulphate ion (STS complex) it effectively enhances the flower
longevity (Veen and Van de Geijn, 1978). When added to the vase
water of cut carnations, the complex was found to be deposited on
the plasmalemma of the receptacle cells (Veen et al., 1980), which
membrane may be involved in ethylene biosynthesis (Lieberman, 1979).
lsolated protoplasts from morning glory flowers (lpomoea tricolor
Cav.) are still able to convert ACC into ethylene but the AVG-
sensitive synthesis of ACC from methionine is no longer possible,
one of the essential enzymes probab1y being located at the plasma-
lemma (Konze et al., 1980).

It must be questioned, however, as to how far ethylene can be

considered as the essential trigger for flower fading in general.
Although AVG completely suppresses the ethylene evolution from
Tradescantia hybrid petals, it on1y partially reduces the loss of
membrane semipermeability, indicating that other factors may be
involved in the initiation of membrane instability (Fig. 4). More-
over, in the petal cells of morning glory, the amounts of membrane
phospholipids decline, and fading starts, weIl before the increase
in ethylene production (Fig. 5). On the contrary, in carnation
flowers AOA suppresses not only ethylene synthesis but also com-
pletely removes the normal senescence of the flowers, characterized
by the rolling of the petals. Instead the flowers ultimately senesce
by desiccation and necrosis of the petals (Bufler et al.,1980).

Therefore, ethylene seems to exert two different functions. On

the one hand, in relatively long-living f10wers it may weIl be the
real trigger for senescence, particularly upon pollination. On the
other hand, in such ephemeral flowers as Tradescantia and morning
glory, which start fading adefinite number of hours after opening
al ready as determined by some endogenous clock, ethylene may act
as a regulator that accelerates the loss of compartmentation induced
150 J. BRUINSMA

0.3

,...
Itl
Itl
«0.2

01

0.5

30
111
Ö
~
~20
C\J

~
cJ' 1.0 RT+C 2H4

0.5 __~==_-_-_-:!:.=::a_.....- . .
::.::;;-O:_..._--_- -----t:::.-- _....
---------~Rt

12 16 18 21
Time of Day(hr)

Fig. 4. Differential effect of AVG (RT) on ethylene evolution and

pigment efflux, measured at 575 um, from Tradescantia pe-
tals (after Suttle and Kende, 1978).

by some other, clock-determined factor(s), thus speeding up rather

than triggering the terminal deterioration of the flower.

FRUIT RIPENING

The senescence of fruits starts with the ripening of the mature

fruit. Because fruit ripening involves the biosynthesis and activity
of several enzymes and requires large amounts of energy and the
prolonged integrity of membranes, some prefer to consider only the
deterioration of the fruit in its over-ripe stage as senescence.
However, since some of these enzymes are similar to those occurring
in other senescence phenomena such as abscission, and since the
hormonal regulation of ripening is also very similar, it is justi-
fiable to consider the ripening process itself as a senescence
phenomenon.
HORMONAL REGULATION 151

10r-------------------------------------------,

,~
I
8 p_,_;-O I
300
...
IJ)

" ~~II/
Z
loJ
...
IJ)

Z
~ loJ
CI ~
280
loJ CI
.,.,...
./ " ..
IJ) loJ
6 IJ)
CD
a: CD
er 260 ::
o
.,..
'0

..
o
0-
0.
I
4
4D

.,
0-
ROllinO_!
I 240
CI
z
...J
...J
o
,,I
o J:
0. N a:
U
...J
C 220
o
~ I... Ethy lene
2 I
& I
200

-
/
/ d
~,- ./ 0''''

16:00 2000 2400 400 8'00 12=00 1&00 2000

TIME OF DAY
Fig. 5. Time course of change in phospholipids, wilting (rolling),
and ethylene evolution of petal sections of Ipomoea tri-
color Cav. (after Beutelmann and Kende, 1977). ---

The onset of ripening is often advanced by the picking of the

fruit. For apple, this has been known since 1925 (Hulme et al.,
1969). It also occurs in tomate (Sawamura et al., 1978), while
such tropical fruits as mango and avocado hardly ripen pre-harvest
probably because of the presence of some hormonal, ripening-inhibi-
ting substance derived from the vegetative parts (Burg and Burg,
1964). On the one hand, roots may be involved in retardation of
fruit ripening since cytokinins have been reported to delay the
onset or decrease the rate of ripening (Bruinsma et al., 1974,
Varga and Bruinsma, 1974, Lieberman et al., 1977). On the other
hand, a mango fruit will ripen if a steam ring prevents supply to
it from the leaves (Burg and Burg, 1964). The nature of this foliar,
ripening-inhibiting hormone is unknown, but hormonal levels of
auxin are able to keep fruits from ripening (Tingwa and Young, 1975,
Inaba et al., 1976). In pear, endogenous auxin has to be removed
by oxidation before ripening can occur (Frenkel and Dyck, 1973).

Gibberellins can exert a pronounced, though limited, inhibition

01 ripening by retardation, or even reversion, of the degreening
process. They promote the re-assembly of functional chloroplasts
152 J. BRUINSMA

from plastids in various degrees of degradation into carotenoid-

filled chromoplasts, e.g. in citrus and banana peel (Bruinsma et
al., 1974, Goldschmidt et al., 1977). Other aspects of the ripening
process, however, are little affected by gibberellins.

Abscisic acid accumulates in maturing fruit and its exogenous

application may advance the onset of ripening (Bruinsma et al.,
1974; Inaba et al., 1976; Hale and Coombe, 1974). However, it may
well act by inducing ethylene biosynthesis. Because ripening can
be completely inhibited by preventing the biosynthesis or action
of ethylene, a direct role for ABA is doubtful (Bangerth, 1980).
Apparently, ethylene is the dominant hormone in fruit ripening
and the pre- and postharvest regulation of ripening largely con-
cerns the control of ethylene synthesis and action (e.g. Bangerth,
1978, Williams, 1980; Bramlage et al., 1980). The effect of the
other hormones in fruit ripening may well be exerted by affecting
either the biosynthesis of ethylene or the sensitivity of the tissue
to this hormone, for instance, by keeping the ethylene receptor sites
at a low level or inaccessible.

The sensitivity to ethylene gradually develops during matura-

tion and particularly after picking (Burg and Burg, 1962b; Sawamura
et al., 1978). Once the hormone becomes effective it also greatly
stimulates its own biosynthesis so that a burst of ethylene evolu-
tion, similar to the respiratory climacteric peak, is adefinite
indication of the onset of ripening. This autocatalytic ethylene
synthesis (Burg and Burg, 1965) can also be evoked by propylene,
although such an artificial induction may lead to a changed se-
quence of the ripening events, an artificial lack of synchroniza-
tion (Eaks, 1980). The inereased ethy1ene produetion results from
enhaneed activities of what are probab1y newly synthesized, ACC-
forming and -oxidizing enzymes. In over-ripe fruit ACC can accumu-
late considerably, possibly because of inactivation of the membrane-
bound ACC peroxidase during the gradual loss of membrane integrity.
This may contribute to the termination of ethylene evolution (Hoffman
and Yang, 1980).

Non-climacteric fruits show no peak, either in respiratory

activity or in ethylene evolution. Monogenie mutants of tomato
that fail to ripen behave as non-climacteric fruits: these rin- and
~-mutants form stress ethylene but have no autocatalytic ethylene
release. Because these mutants also lack the cell wa1l-softening
enzyme, polygalacturonase, and exogenous ethylene alone is unable
to induce its activity, Tigehelaar and MeGlasson (1977) hypothesized
that the biosynthesis or activation of this enzyme might be the pri-
mary, ripening-inducing process. By its action, alterations in com-
partmentation and/or release of cell wall-bound enzymes might occur,
amongst other phenomena leading to ethylene produetion. However,
HORMONAL REGULATION 153

there is no indication that these mutants would form ethylene upon

supply of polygalacturonase. In addition, these mutants do ripen,
and cell walls do undergo solubilization, in the presence of exo-
genous ethylene, provided the fruits either rernain attached to the
plants (Mizrahi et al., 1975) or are kept at a high partial oxygen
tension (Frenkel and Garrison, 1976). This indicates a genetic
block at the site of ethylene expression, including the stimulation
of its own biosynthesis, rather than at the biosynthetic locus of
one of the pectinolytic enzymes. Moreover, the appearance of poly-
galacturonase activity is not the first event in normal tomato-
fruit ripening.

The difficulty in determining time sequences of events in

fruit ripening is that because of lack of uniformity within fruit
sarnples the distinctness of the phenomena decreases. Fig. 6 shows
this for cucurnber fruit; if the four individual fruits were mea-
sured together, no clear peak of ethylene evolution would be detect-
ed. Since such a peak is correlated neither with fruit weight nor
with the nurnber of days since anthesis, the only solution to de-
termine the sequence of two events is to measure them in single
fruits and to plot thern, not against time, but against each other.
Because the dose-response curve of ethylene in fruit ripening is
logarithmic (Burg and Burg, 1962a), ethylene evolution should be
plotted logarithrnically. Such graphs show that in tomato, although
ethyleneand carbon dioxide evolutions may peak together, the endo-
genous ethylene level already increases when the carbon dioxide pro-
duction is still low and, thus, the rise in ethylene precedes the
climacteric rise in respiration (Fig. 7). Also the observation in
honeydew melon that the clirnacteric peak only occurs if the endo-
genous level of ethylene surpasses a threshold value (Pratt and
Goeschl, 1968) indicates that ethylene acts as the inducer of the
increase in respiration. In tomate this threshold value is high in
attached fruits, about 5 ppm on an average, but after picking it
drops to about 1 ppm, probably because of decay of a ripening
inhibitor (Sawarnura et a1., 1978).

Polygalacturonase activity rises in tomate at high ethylene

levels only, without any interference from the ripening inhibitor
(Fig. 8A). This renders it high1y improbable that this enzyme
plays a ro1e in the triggering of the ripening process. Water-
soluble pectin, the presumed product of cell wall-solubi1ization,
increases even before po1ygalacturonase activity enhances apprecia-
bly (Fig. 8B), so that the in vitro determination of enzyme activity
may be deceptive. What should be determined is the activity of the
polygalacturonase isoenzyme located within the cell walls and un-
der the conditions prevai1ing there which may be less than optimal.
However, the water-soluble pectin also increases only after an
ethylene threshold has been surpassed (Fig. 9).
 154 J. BRUINSMA

In sorne fruits, the development of ethylene sensitivity and

accumulation does not seem to be the primary event in ripening.
With avocado, for instance, the in vitro cellulase activity was
found to increase 1.5 days prior to ethylene evolution (Awad and
Young, 1979). However, particularly in avocado, both the sensitivi-
ty to ethylene and its production dramatically increase upon picking
(Burg and Burg, 1962 b), so that a role of ethylene as a trigger of
ripening is quite plausible in this fruit, too.

1eo

'\
140

p-
• . 120 / \

,•
~

.....
...•
D 100

J
I \
\ ,,
,,' ,\,,
80
...
Z V ~,' ~

,/\
.j I \
~ I \

......Z I \

,,
I

,,
\
I
,,
\
\
\
C
.,
,,
~ \
\
,.
'-..»c'.."..."'" '"
\
20 \ ",
.... _ _ _ 1

0
1 3 e 8 1e 21 25 30
DAYS AFTER HARVEST
Fig. 6. Non-synchronous ethylene evolution from four harvested
cucumber fruit (after Saltveit and McFeeters, 1980).

It can be concluded that ethylene is the primary hormone in-

volved in fruit ripening but whether it acts as the trigger, as it
does in the abscission process (Sagee et al., 1980), or only as
one of the key factors accelerating a multicausal process (aobson,
1979) remains to be established once the many methodological pit~
falls have been overcome. Whatever the outcome may be, the control
of ethylene sensitivity and biosynthesis is the main means of
regulating the shelf life of harvested fruits.
 :r:
o
'O~i-------------------------------------------------------------------' :D
s::
o
z
»r
:D
m
G)
0.8 C
r
»-I
oZ
.. 0.6
c
E
Cl
.Cl
:i. 0.4
('\j
o
()

0.2 •• .,

•• •
-., ••
e.:.
•••••
0' 0" -1 _, 10 H~O
ethylene pg. 9 . min
Fig. 7. Evolution of ethylene and carbon dioxide, measured together in the gas
from single 'Sonato' tomato fruits at different stages of ripening. t11
t11
 U1
(J)

401r-------------------------------------~ 0.4

A
O.
.....
, • .~ n,...r 0--30
CI CI
EO. 25
!!! 11-
·2 CI)
.2 B
~
~ 10 0.1
•
A

10· 8 A 0
~[]o..lD-cA.
o 0.1 1 5 10
0.5 50 100 0 10 20 30 40
ethylene (ppm) PG(unils.g-')

Fig. 8 (A), (B) Occurrence of po1yga1acturonase activity (PG) , water-so1ub1e pectin (WSP), and
ethy1ene in 'Moneymaker' tomate fruits, detached 25, 30 or 35 days after ferti1ization,
or 1eft on the vine unti1 analysis. (A) Relation between PG and interna1 ethy1ene. (B)
Relation between PG and WSP (after Sawamura et a1., 1978).

'--
OJ
:xl
C
Z
Cf)
s:::
»
 I
0
:D
25days 30days
s:
0
Z
03 0.3 »
r-
",/ :D
m
G)
0.2 C
'"'" A T ~ "'02 r-
»~
"'/ 0
0.1 '" ~ J')1 Z
'"
Cl
I
+D_D_D~D
'"
~O I I I 10
01 5 10 50 0.1 05 1 5 10 50
ä::
Cf)

:::
35days at1ached

03 ./ ed03
/0
02
° t
01
/02 01
e /e
7 e--

0 0
01 0.5 5 10 50 0.1 0.5 5 10 50
ethylene (ppm)

Fig. 9. Level of internal ethylene concentration and occurrence of water-soluble

pectin (WSP) in individual 'Sonato' tomate fruits, detached 25, 30 or
35 days after fertilization or left on the vine until analysis (after
Sawamura et al., 1978). (J1
-...J
158 J.BRUINSMA

CONCLUSlON

Senescence, also that of harvested plant parts, is regu1ated

by a pattern of phytohormones. In this pattern cytokinins, also
auxins, and to some extend gibbere11ins, postpone the occurrence
of senescence and retard the process, whereas abscisins and ethy-
1ene advance and acce1erate it. Pre- and postharvest treatments
with plant growth regulators, as we11 as physica1 storage conditions,
shou1d aim at the promotiön and maintenance of a hormonal pattern
that benefits the she1f 1ife of the harvested commodities. This
concerns main1y the prevention of accumu1ation of abscisins and,
particu1ar1y, the inhibition of the biosynthesis and action of
ethy1ene.

ACKNOWLEDGEMENT

The critica1 reading of the manuscript by Drs. E. Knegt, L. C.

van Loon, D. A. Priest1ey, A Varga, and H. Veen is high1y appreci-
ated.

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160 J. BRUINSMA

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HORMONAL REGULATION 161

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162 J. BRUINSMA

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HORMONAL REGULATION 163

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POST HARVEST PHYSIOLOGY OF SEEDS AS RELATED TO QUALITY AND

GERMINABILITY

Daniel Came

Laboratoire de Physiologie des Organes V~g~taux

apres Recolte, C.N.R.S.,
and Universite Pierre et Marie Curie (Paris 6)
4ter, route des Gardes, 92190 Meudon, France

INTRODUCTION

Seeds are remarkable organs in many ways and are of considerable

economic interest. Their role is essential in the everyday life of
men and animals, since they ensure plant reproduction. In this re-
pect they are used to produce nutritive and floral plants, as weIl
as fruit and forest trees. They are also used as direct sources of
food or in various industries such as oil manufacturing and malting.

Seeds are mostly highly dehydrated organs, naturally adapted

to long survival after their release by the mother plant until, in
an almost inert state, they find the necessary conditions for ger-
mination. The problems involved in their storage are therefore very
different from those which usually apply to other plant organs.

Some seeds germinate very weIl at harvesting but may lose this
property more or less quickly during storage. .Others are totally
unable to germinate or do so with great difficulty if they have not
been stored for sufficiently long or have not been given special
treatment.

BIOLOGICAL PECULIARITIES OF SEEDS AFTER HARVESTING

Seeds result from the development of the ovules after fertiliz-

ation. Despite their morphological diversity, their constitution is
fundamentally the same (1). The embryo is the essential seed struc-
turej it germinates and gives rise to a new plant. The reserves
are localized in the embryo cotyledons or in a particular tissue,
165
166 D. COME

the endosperm. The seed-coats protect the embryo and control its
germination. In some cases, like dry indehiscent fruit, the pericarp
and sometimes other parts of the flower adhere to the seed. In
certain Graminaceae, the caryopsis is covered by bracts called
glumellae.

Seeds are mostly organs of resistance living at a slowed rate

since their maturation is a very special phenomenon ending in extreme
dehydration. At maturity, they generally contain no more than 10-15%
water. Some seeds, however, contain more.

Consequences of Dehydration

The very great loss of water by seeds at the end of maturation

is accompanied by profound changes in their cell ultrastructure,
condensation of reserves and a significant decrease in metabolie
activity (2). During this dehydration period, the coats of certain
seeds become waterproof. This is known to be the case for Leguminous
plants (3), whose seed-coats have a very characteristic structure (4,
5,6). In addition, the hilum of these seeds plays an important part
in their dehydration, since it enables them to lose water without
taking it in again. In this connection, seeds have been shown to
lose water, first through all their surface and then, when the seed-
coat becomes waterproof towards the end of maturation, water escapes
through the hilum which remains open and acts as a valve (7). In a
dry atmosphere, the hilum opens in less than a minute, so the seed
can lose water, but in a humid atmosphere, it closes just as quickly
and prevents rehydration.

The very low metabolie activity of mature seeds due to dehydra-

tion slows down their life processes. However, except when seed-coats
are waterproof, this low activity ceases as soon as the seeds are
placed in the presence of water, since the metabolism of imbibing
seeds is quickly reactivated.

The ability of seeds to survive in adehydrated state enables

them to withstand conditions which in active life would lead to
their death and makes them highly resistant organs. As early as
1907, Becquerel,(8) showed that naturally dehydrated seeds could
survive exposure to the temperature of liquid air. However, such
resistance is only possible if seads are sufficiently dehydrated.
When imbibed, they resist no better than any other organ.

Seed Sensitive to Desiccation

Some seeds cannot withstand desiccation. Unlike most seed

types, they contain a good deal of water, and their embryo remains
active and dies of dehydration. This applies to various trees with
large seeds and to numerous tropical and subtropical species (9,10,
11,12,13,14,15). Under natural conditions, such seeds germinate
QUALITY AND GERMINABILITY OF SEEDS 167

as soon as they fall to the ground. These, however, constitute spe-

cial cases worth closer investigation. Although seed viability
greatly varies from one species to another, most seeds display an
ability to survive in the dehydrated state, which enables them to
remain alive for very long periods.

ORIGIN OF SEED DEATH AND ITS RELEVANCE TO SEED PRESERVATION

Seed Longevity

The extraordinary longevity of some seeds has fascinated

scientists for a long time. The earliest work on this subject dates
back to the middle of the last century (16).

Ewart, (17) in a treatise entitled "On the Longevity of Seeds,"

divided seeds into three classes, according to their longevity
under arnbient conditions:
a) Macrobiotic seeds, capable of surviving for more than 15
years. Exarnples of seeds with a longevity exceeding 50 to 100 years
are far from rare (18). Their seed-coats are often waterproof. In
1907 and 1934, Becquerel, (8,19) succeeded in making very old seeds
from the collections of the Natural History Museum in Paris germi-
nate (Table 1). Lotus seeds (Nelumbo nucifera) are also well-known
for their longevity of several hundred years (20,21). ~dum, (22)
obtained the germination of Chenopodium album and Spergula arvensis
seeds about 1,700 years old. The record is held by Lupinus arcticus
seeds which germinated perfectly even though they were more than
10,000 years old (23). However, it is quite untrue that wheat found
in Egyptian tombs was made to germinate (13).
b) Mesobiotic seeds with a life-span of 3 to 15 years. The
great majority of species are in this category, which includes wheat.
c) Microbiotic seeds which survive at most for 3 years. These
often die quickly, since they cannot withstand desiccation.
Harrington, (18) quotes many such cases. Under natural conditions,
some seeds only rernain viable for very short periods. Those of
Oxalis, for instance, die within a few days.

Viability Tests

Numerous tests have been proposed for evaluating seed quality

and vigour (13,24).

Loss of viability is chiefly shown by a decrease in germin-

ability (25), i.e. lower rates and percentages of germination, slow
growth of the seedlings obtained and production of abnormal plants.
The degree of germinability is the most cornrnonly applied criterion
of quality (26, 27).

Seed deterioration is also shown by diminished resistance to

168 D. COME

Tab1e 1. Longevity of some seeds (from Becquerel, (19))

Date Seeds Seeds Deter- Proba-

of growing growing mined b1e
Macrobiotic species co11ec- in in longe- longe-
tion 1906(X) 1934(X) vity vity
(years) (years)

Mimosa glomerata Försk. 1853 5 5 81 221

Me1i1otus 1utea Gue1d. 1851 3 o 55

Astragalus Massi1iensis Lam. 1848 o 1 86 100

Cytisus austriacus Linn. 1843 1 o 63

Lavatera Pseudo-o1bia Desf. 1842 2 o 64

Dioc1ea Paucif10ra Rusby 1841 1 2 93 121

Ervum Lens Linn. 1841 1 o 65

Trifolium arvense Linn. 1838 2 o 68

Leucaena 1eucocepha1a Linn. 1835 2 3 99 155

Stachys nepetifo1ia Desf. 1829 1 o 77

Cytisus bif10rus L'Herit. 1822 2 o 84

Cassia bicapsu1aris Linn. 1819 3 4 115 199

Cassia Mu1tijuga Rich. 1776 2 158

(X)10 seeds of each species were sown, except for Cassia

Mu1tijuga, of which on1y 2 seeds were sown.

different types of stress. To determine the vigour of some seeds,

a Cold Test is sometimes used, especia11y for maize, in which the
idea is to simu1ate bad natural germination conditions. The vigor-
ous seeds are those which resist this treatment best (28,29).

The tetrazolium test, very widely used to determine seed via-

bi1ity (30,31,32,33), is based on the reduction of co1our1ess tet-
razo1ium sa1ts and on the formation of red formazan by the
QUALITY AND GERMINABILITY OF SEEDS 169

dehydrogenases in imbibed embryos. Only living zones colour

intensely.

The measurernent of respiratory activity during imbibition has

been suggested as a viability test, but its validity is often lim-
ited (34).

Another way of judging seed viability and vigour is by the

arnount of inorganic salts or organic compounds which seeds release
into a waterbath, since darnaged seeds release more of these sub-
stances than vigorous ones (35). The electrolytes thus freed are
usually evaluated by measuring the electrical conductivity of the
waterbath (36,37).

X-ray analysis is used to determine the viability of seeds of

conifers and other trees (38,39), and can also be applied to other
species. Without any particular treatment, radiography permits
detection of seed malformations, but does not enable viable seeds to
be distinguished from non-viable ones. The method, based on the
impregnation of seed-coats and dead tissues with a barium chloride
solution followed by radiography, permits the dead parts, which are
more highly contrasted, to be distinguished from the nonimpregnated
living parts. Since neither the X-ray doses used nor the barium
chloride darnage the seeds, they can germinate after this treatment
and their germinability can be correlated with the damage observed
by X-ray.

Why do Seeds Die?

The numerous biochemical, cytological or genetic changes ob-

served during seed ageing have given rise to various theories
concerning their death (13,24,25,40,41,42,43,44). These are sum-
marized in Figure 1, reproduced from Roberts, (41).

Contrarily to what various authors have assumed, it does not

seem possible to impute the loss of viability to the degradation or
exhaustion of seed reserves, to the destruction or denaturation of
molecules essential to seed life such as vitamins, hormones and
enzymes, or to the production of toxic substances. Although fungi
accelerate the degradation of excessively humid seeds, they cannot
be involved when seeds are stored under favourable conditions (41).
The theories prevailing at present concern the accumulation of
damage in the nucleus and cytoplasmic organelles.

During dry storage of pea, broadbean and barley seeds, loss

of viability was found to be closely related to chromosome damage
(45,46). This chromosomal abberration was clearly visible during
the first divisions of the meristem cells at the end of the root
of the surviving seeds. Such darnage seems very general (41) and
apparently leads to the production of abnormal plants by the old
seeds. It sometimes persists for a very long time and is probably
170 D. COME

Iorüsing
radiations ------------------------,
Pathogen
Extrinsic attack
Growth
Fungi inhibitors

Mycotoxins

Mutagens

Theories to
account for
1055 of seed
viability

Gene
malfunction

Gene
derepression

ehydrogenases
(tetrazolum
test)

Intrinsic Other

Plastids,
lysosomes,
lipoprotein dictyosomes,
membranes etc.

Lipids

essential Proteins
metabolites Loss of
vitamins,
hormones,
etc.

Fig. 1. Classification of viability theories. (from Roberts,(41)

with permission).
QUALITY AND GERMINABILITY OF SEEDS 171

responsible for the abortive pollen formation in plants grown from

old seeds (47). Detailed studies of rye embryos have shown that DNA
undergoes fragmentation during dry storage (48,49).

Severe disorders in the cytoplasmic organelles have been observed

by electron microscopy in connection with loss of seed viability. Mi-
tochondrial degeneration was shown by Abu-Shakra and Ching,(50) in
Glycine max seedlings grown from 3-year-old seeds. The work of
Berjak and Villiers, (51,52) on maize has shown that disorders occur
in all cell organelles during ageing. Damage is reversible if ageing
is not too advanced, but increases with the loss of viability and
gradually becomes irreversible. The same types of degeneration were
observed by Hallam, (53) in non-viable embryos. Such cell disorders
are pcobably due to lipoprotein membrane deterioration and might
explain why the metabolite leak into the germination medium is
greater in old than fresh seeds (25,54,55).

Storage Problems

The maintainance of the nutritive value, viability and soundness

of seeds is obviously an economic problem and considerable loss may
result from germination, parasitic destruction or seed death during
storage. Storage problems therefore depend on the end in view and
the type of seed involved.

Numerous factors are likely to affect seed quality either be-

fore or during the harvest (18,56,57,58,59). We shall only consider
here those which are active during storage; they are first of all
temperature and moisture, and to a lesser degree, oxygen (60).

Prevention of germination - The problem of prevention germina-

tion only arises when seeds are stored in bulk in a very humid at-
mosphere or when they can only stand mild desiccation.

The nutritive seeds like cereals or leguminous seeds, germina-

tion and fungi growth may start if there is too much moisture in
the atmosphere or if seeds have been harvested too wet. Such condi-
tions cause appreciable heating of the seeds (61), thus favouring
these developments.

Seed water content increases with the relative humidity of the

atmosphere (15). For instance, wheat water content reaches 25% of
its fresh weight at a relative humidity of 95%. Temperatures within
the usual range affect the water vapour absorption rate of seeds
but do not greatly change their water content at equilibrium. Re-
frigeration of seeds does not reduce their water content signifi-
cantly but slows down germination appreciably. Below a given
temperature, which varies depending on the species, germination is
impossible. To prevent germination of seeds stored in silos or in
172 D. COME

1arge quantities, it, is therefore necessary to keep them in a dry

cold atmosphere.

Seeds which cannot stand much desiccation must be stored wet.

Tab1e 2 gives examp1es of such seeds. For some species 1ike certain
Citrus (11), essentia11y of tropica1 or subtropica1 origin, 10wering
of the temperature does not a110w the seeds to stand more intensive
dehydration. Such seeds can on1y be stored wet, at cold temperatures
(about +5°C), but in that case the risks of their being degraded by
fungi or bacteria are considerab1e. However, these difficu1ties can
be overcome by various chemica1 treatments (63). Other seeds which
cannot stand desiccation under natural conditions may, when desic-
cated and stored at cold temperatures, remain viab1e for 10ng peri-
ods. This is true of U1mus americana seeds, which die of dehyira-
tion within a year under usua1 conditions but can be stored for 15
years at -4°C if their water content is on1y 3% (12). The viabi1ity
of other seeds known to be very sensitive to desiccation can also be
prolonged by partial dehydration and storage at cold temperatures.
This applies to the seeds of oak (Quercus), beech (Fagus), horse-
chestnut (Aescu1us), walnut (Jug1ans), chestnut (Castanea), wi110w
(Sa1ix) and pop1ar (Popu1us) (10,64,65,66,67).

Anti-parasitic action - Microflora activity depends much more

on atmospheric humidity than on seed water content (60), but there
is of course a direct relationship between the relative humidity
of the atmosphere and the degree of seed hydration. The growth of
fungi is tota1ly inhibited when relative humidity is 1ess than
about 70% (68), and bacterial growth requires a relative humidity
of at least 90% (69).

Table 2. Minimum water content necessary

for survival, in several species of seed.

Minimal water
Species content (%) Reference

Acer saccharinum 30 34 JONES (62)

Quercus alba 25,6 KORSTIAN (9)
Quercus rubra 8,7 - 13,6 KORSTIAN (9)
Citrus sinensis 25 BARTON (11)
Citrus grandis 52 BARTON (11)
QUALITY AND GERMINABILITY OF SEEDS 173

A low temperature is just as important as a dry atmosphere in

combatting the growth of microflora (68). Seeds which can stand
desiccation should therefore be stored in a dry atmosphere at cold
temperatures.

It is much more difficult to prevent the often considerable

damage caused by insects, otherwise than by appropriate chemical
treatment. Seed storage in a dry cold medium can, however, kill
certain larvae such as those of weevils, which at aOc die within
two months (70).

In general, cold temperatures prevent germination and limit

seed deterioration, but they must be combined whenever possible
with dry storage conditions. It is also advisable to heat the seeds
with dry air immediately they are removed from cold storage, so as
to prevent water condensation on their surface.

Come and Tissaoui, (71) succeeded in destroying the fungi grow-

ing on Robinia pseudacacia seeds thanks to an unusual kind of treat-
ment. These seeds germinate with difficulty, since they are unable
to imbibe and are rapidly overgrown with fungi. Both satisfactory
germination and destruction of fungi were obtained as foliows: seeds
were plunged into water at room temperature for a day, allowed to
dry in the open air for another day and plunged into liquid nitrogen
for 15 minutes. As will be seen later, sudden freezing in liquid
nitrogen permits good germination of hard seeds, i.e. seeds whose
coats are waterproof. Although the immersion in water no doubt per-
mits germination of the fungus spores retained by the seed-coats,
these are subsequently killed by freezing.

Prolongation of viability - As the particular case of seeds

which cannot withstand desiccation has already been dealt with, we
shall consider here the great majority of seeds, which undergo normal
dehydration.

The main factors affeeting seed longevity during storage are

temperature and seed water eontent (13,18,60,72,73). In most eases,
the lower the seed water eontent and storage temperature, the longer
the viability. Results eoneerning the role of oxygen are sometimes
eonflicting (60,74,74). However, an inerease in partial oxygen
pressure is generally admitted to have an unfavourable effeet. In
addition, Barton, (13,40) emphasized that the hermetieal sealing of
containers improved the preservation of germinability.

The very elose relationship between seed longevity, water

eontent and temperature has enabled quite satisfaetory viability
equations to be established (60). Harrington, (73) reported that
seed longevity doubled when seed water content was reduced by 1% or
when temperature was lowered by 5°C. However, according to Roberts
and Ellis, (76) it would be more exact to say that longevity doubles
174 D.COME
when seed water content is reduced by 2.5% and temperature by 6D C.

At a given relative humidity, the water content of seeds in

equilibrium varies with the species considered. It also depends
on how seeds are brought to equilibrium. For the same relative
humidity, they contain more water when equilibrated by dehydration
than by rehydration (73). This phenomenon is known as hysteresis.
In general, small seeds can stand very fast artificial dehydration.
However, in large seeds, too sudden adehydration may fracture their
cotyledons or endosperm at germination (77).

Provided, of course, that seeds are not too greatly hydra ted,
very low temperatures (-20 D C or less) are often very helpful in
maintaining the viability of most species (13,60,78,79).

During the past 20 years, several countries have shown inter-

est in prolonging seed storage as long as possible, since this is
the best way of preserving plant genetic resources. The Interna-
tional Board for Plant Genetic Resources recommends storage at
-18 D C or less in hermetically sealed containers of seeds whose water
content has been reduced to 5 i 1% (76). Recent results suggest
that some seeds could be stored for a very long time in liquid nitro-
gen or by lyophilization. These methods might be very useful for
particularly fragile seeds. Table 3 gives the results obtained by
C6me (unpublished data) for certain floral and vegetable seeds treat-
ed for 4 years by lyophilization. They seem excellent compared to
results for storage under ambient conditions. However, it is still
too early for adefinite assessment of the lyophilization methode

Fina11y, it appears that natura1ly dehydrated seeds are not

totally inert organs. Although much reduced, their metabolism is
not nil, and in fact this residual metabolism seems to be at the
origin of seed death. Any treatment resulting in prolonged seed
survival reduces seed metabolism, as happens when the temperature
is lowered or seeds are artificially dehydrated. In the most extreme
case, total dehydration of the seeds by suitable methods should
completely suppress their metabolism and enable them to remain viable
almost indefinitely. Lyophilization might be one way of achieving
this.

ACQUISITION OF GERMINABILITY AFTER HARVESTING

Germination requires certain external conditions to be met.

The seed must first be able to become imbibed; next, the metabolie
activity started by imbibition requires oxygen and lastly, as in all
physiological processes, temperature is very important. Water,
oxygen and temperature are therefore the three decisive factors in
germination. Moreover, they are virtual1y indissociable (80, 81,82).
Light may also play an important part in germination (80,83,84).
QUALITY AND GERMINABILITY OF SEEDS 175

Table 3. Germinability of seeds stored by lyophilization

(from Ceme, unpublished data)

% germination
at harvesting after 2 years after 4 years
Species non
lyophi- open lyophi- open lyophi-
lyophi-
lized air lized air lized
lized

Lobelia sp. 89 79 50 75 30 80
Petunia hzbrida 84 90 37 64 14 70
Mimulus sp. 92 90 100 100 0 100
Helichrysum bractea-
turn 83 80 0 64 0 70
Cineraria sp. 80 95 58 80 10 84
Ageratum mexicanum 92 95 80 90 40 80
Alyssum sp. 96 100 100 100 80 100
Clarkia sp. 96 93 88 90 40 100
Reseda sp. 96 95 52 72 40 73
Papaver sp. 92 90 87 100 80 100
Linum sp. 80 89 74 80 8 66
Bellis perennis
(Daisy) 60 66 40 54 0 84
Rudbeckia sp. 84 66 7 65 0 59
Agrostis sp. 52 91 67 78 54 84
Polygonum fagopyrum
(Buckwheat) 96 96 100 100 32 100
Phalaris canariensis 96 1.00 92 92 84 100
Festuca pratensis 92 92 78 100 11 100
Dactzlis sp. 85 90 92 100 40 100
Nasturtium officinale
(Water-cress) 92 86 20 92 8 84
Milium sp. (Millet) 92 95 8b 100 72 100
Coriandrum sativum
(Coriander) 95 70 47 47 29 47
Ocimum basi1icum
(Basilica) 52 90 80 70 48 72
Cichorium endivia 96 90 84 100 4 100
Asparagus officinale
(Asparagus) 93 96 12 52 0 62
Foeniculum officinale
(Fennel) 100 83 80 100 4 72
A11ium cepa (Onion) 92 92 72 84 8 72
Valerianella olitoria
(Corn sa1ad) 88 96 68 97 4 100
176 D. COME

However, many seeds are unable to germinate or germinate with great

difficulty even under favourable conditions. This inability to
germinate is commonly called "dormancy." Some dormant seeds germi-
nate easily after storage, others need special treatment.

Inability to Germinate

When the term "dormancy" is used to designate the inability of

a seed to germinate properly, it does not characterize the physio-
logical mechanism involved. It may therefore be ambiguous, in which
case it is preferable to speak of "inability to germinate."

Inability to germinate can be of two types (1,80): in some cases

it is due to the embryo which cannot germinate properly even when
stripped of its various covering structures (seed-coats and some-
times endosperm). This phenomenon is called embryo dormancy. In
other cases, the naked embryo germinates perfectly, but the struc-
tures covering it inhibit its germination; this is known as inhibi-
tion of germination. Some seeds may display both types of inability.

Embryo dormancy is a very complex phenomenon and little is yet

known about its mechanisms (80,85,86,87,88). Inhibition of germina-
tion by seed-coats is characterized by the fact that seed-coat re-
moval or scarification leads to germination. Seed-coats often have
an inhibitory effect by depriving the embryo of oxygen (80,81).
They may also be waterproof, as in the well-known case of Leguminous
plants (3,80). In some species, such as Chenopodium amaranti-
color (89) and Fraxinus excelsior (90), they are permeable to water,
but their mechanical resistance is too great for the embryo to
break them. All these types of inability to germinate are very re-
lative phenomena whose manifestations depend on many factors. Thus
in some cases, such inability can be overcome by dry storage.

Effect of Dry Storage

Just after harvesting, the seeds of certain species germinate

with great difficulty at the usual moderate temperatures, but germi-
nate perfectly after various per iods of dry storage under ambient
conditions. This phenomenon is called after-ripening in dry
storage (80, 91). Crocker and Barton, (92) mention 41 species with
seeds capable of such after-ripening. Many photosensitive seeds
gradually lose their photosensitivity during dry storage (93), but
the most typical case is that of the Graminaceae (91,94), which we
shall consider here.

As an example, Figure 2 shows that wheat caryopses germinate

with difficulty at 20°C at harvest time, but germinate well after
storage at room temperature in the open air for 2 months or a year.
This particular dormancy of freshly harvested Graminaceae caryopses
is in fact only an inability to germinate at relatively high
 QUALITY AND GERMINABILITY OF SEEDS 177

temperatures, since germination occurs very easily at IO°C or

less (95,96). During dry storage, the caryopses become capable of
germinating at increasingly high temperatures (Fig. 3). The rate at
which they acquire this ability to germinate within a wide tempera-
ture range depends on the storage temperature. Storage at 35 or
40°C for a few days may have the same effect as prolonged storage at
20°C (96,97). On the other hand, storage at very low temperature
(-18°C) maintains caryopsis dormancy (95).
This phenomenon depends essentiallyon the external structures
of the caryopsis. In barley, for instance, the glumellae adhere to
the grain and are largely responsible for its bad germination at
harvesting. During dry storage, the inhibiting effect of the
glumellae gradually disappears (95).

Several explanations have been suggested to account for after-

ripening in dry storage. Some authors believe that volatile inhibi-
tors are eliminated during storage (97), but it is more likely
that the external structures of the caryopsis intervene by depriving
the embryo of oxygen and that their effectiveness in acting as
barriers to the passage of oxygen decreases during storage. This

100
~~~-"
0

75
c:

-
0

<':l
c:
E
.... 50
v
Cl

'*
25

o 2 3 4 5
Time (days)
Fig. 2. Germination of wheat caryopses at 20°C at harvesting (1), or
after dry storage at ambient conditions for 2 months (2) or
one year (3). (from Corbineau et al., (95».
178 D. COME

is proved by the fact that barley and wheat caryopses clearly require
less oxygen to germinate after dry storage than at harvesting (95,96)
and that, in the case of barley, the presence of the glumellae envel-
oping the caryopsis increases its need for oxygen (Fig. 4). Phenolic
compounds present in the seed-coats and glumellae might be respons i-
ble for oxygen absorption, as in many other seeds (81). However, it
is also possible that. at harvesting, caryopsis envelopes are made up
of living cells whose respiration might deprive the embryo of oxygen,
and the death of these cells during dry storage would render them
incapable of further oxygen absorption (98).

This inability of seeds to germinate easily as soon as they

ripen is of considerable economic importance for cereals, since it
prevents them from sprouting. However, it may be very inconvenient
when seed germination is desired just after harvesting. This is
especially the case of barley grown for malting.

Need for Special Treatment

The germination of seeds exhibiting embryo dormancy or whose

seed-coats are waterproof is only possible at harvest time or after

100

."
>-
(':I 75
\J

-
I"-
...
Q)
......
50
(':I

-
c:
0
( ':I
c:

...
E
Q) 25
Cl

'*
o 10 20 30 40
Temperature ("0
Fig. 3. Effect of temperature on the germination of wheat caryopses
(Champlein variety). (1) in freshly harvested caryopses; (2)
after 2 months of dry storage; (3) after one year of dry
storage. (from Corbineau et a1.~ (95».
QUALITY AND GERMINABILITY OF SEEDS 179

100

75

50

c:
25
0
....
C'Q
c:
...
E
(l)
Cl 0 2 3 4 2 3 4

*' 100 ~' ..5.4 ,....,03.5

......../0-0
~/

CD @
75

o 2 3 4 0 3 4
Ti me Cdays)

Fig. 4. Germination of barley caryopses (Sonja variety) at 20°C, in

atmospheres containing 0, 1.8, 3.5, 5.4, 9.6, 17 or 21% of
oxygen. (a) dormant caryopses with their glumellae; (b) non-
dormant caryopses with their glumellae; (c) dormant caryopses
stripped of their glumellae; (d) non-dormant caryopses
stripped of their glumellae. (from Corbineau and Come,(96)).
180 D.COME

dry storage if they have undergone special treatment.

Embryo dormancy. Embryo dormancy is characteristic of Rosaceae

seeds, but is also found in many other species. The subject has been
treated in a large number of investigations, of which certain authors
have made a synthesis (80,85,86,87,91,94,99).

Non-dormant embryos germinate easily at temperatures of the

order of 20°C. Dormant embryos, on the contrary, do not germinate
at these temperatures or do so very slowly. When they do, the seed-
lings they give - sometimes called dwarf seedlings - are abnormal,
and are generally not viable. Their growth anomalies are more or
less pronounced, depending on the state of dormancy of the embryos(80,
100).

Embryo dormancy is broken by placing the seeds at cold but

positive temperatures in a wet medium. In temperate climates, this
breaking process occurs naturally in winter. Cold temperatures only
break embryo dormancy if the embryo is imbibed. If not they maintain
its dormancy.

This property of cold temperatures has long been exploited by

nurserymen who put the seeds into wet sand or soil in cold rooms, or
simply plant them out in the open in winter. This method is known
as stratification. Other cold treatments are also possible (86, 88).

The breaking of embryo dormancy always requires prolonged cold

treatment. The evolution of this process is shown by the increasing-
ly fast germination of the embryos when sown at moderate temperatures
(Fig. 5) and by the regression of seedling growth anomalies. It is
also characterized by a widening of the range of temperatures per-
mitting germination (101) since, although dormant embryos can only
germinate at low temperatures, they acquire the ability to germinate
at higher temperatures during cold treatment (Fig. 6). Dormancy is
regarded as completely broken when the embryos germinate rapidly
within a wide temperature range, without producing abnormal seed-
lings.

There is a close relationship between temperatures and duration

of treatment. The higher the temperature, the longer the treatment
must last. For instance, the thermal optimum for breaking of apple
embryo dormancy is between 0 and 5°C; temperatures above l2-l5°C
have no effect (Fig. 7). The need for cold temperatures varies with
the species and variety considered, but in most cases a treatment
of one or several months at temperatures around 5°C enables embryo
dormancy to be broken.

Cold temperatures are not, however, absolutely necessary for

breaking embryo dormancy, since other treatments have the same
effect (86). For instance, apple embryo dormancy can be broken
QUALITY AND GERMINABILITY OF SEEDS 181

100
t::
0

<':l
t::
E
.... 50
Q.J
Cl

?J€-

0 5 10 15 20 25 30
Time Cdays)
Fig. 5. Germination at 20°C of Golden delicious apple embryos from
seeds treated at 5°C on wet cotton wool for 14, 21, 35 or
42 days. Curve C corresponds to control embryos which were
not given cold treatment. (from Thevenot, (88)).

100

U
<':l
Cl.
<':l
U
t:: 50
.2
....
<':l
t::
E
....
Q.J
\"j 2S

25 30
Temperature

Fig. 6. Effect of temperature on the gerimination capacity of Golden

Delicious apple embryos kept inside the fruit at aOc for
14, 42 or 105 days. Curve C corresponds to control embryos
which were not given cold treatment. (from Thevenot, (88)
modified) .
182 D.COME

100

'"'v'*
>-
U
<V
Co
<V
U
50
c::
..:::
0
<V
c::
·E...
CI.I
Q

o 234
Duration of treatment (months)"

Fig. 7. Variation in the germination capacity, at 18 D C, of

Reinette du Mans app1e embryos with duration of treatment
in a wet medium, at 0, 4, 7 or 12 DC (from C6me, (100).)

within 2 or 3 weeks when embryos are treated at 15 or 20 D C in a wet

medium without any oxygen (102). When apple seeds with dormant
embryos are placed in the presence of water at 30 or 35 D C, they do
not germinate, but the dormancy of their embryos is gradually elim-
inated (103), since at these temperatures no oxygen reaches them
under the seed-coats (100). Addition of gibberellins to the culture
medium can also more or less replace cold treatment -(104,105,106,
107,108).

Hard seeds. Some seeds are unable to germinate because their

seed-coats are totally impermeable to water and their embryo cannot
imbibe. Seeds which imbibe normal1y swell, soften and can easily
be crushed. Impermeable seeds remain dry and resist crushing, and
are therefore called hard seeds. This applies to 1eguminous seeds
and to those of many other species (3). The percentage of hard seeds
produced by a given species varies from one year to another. It
depends on the place of culture and climatic conditions. There
mayaiso be a connection between the proportion of hard seeds and
the colour of seed-coats (109).

Various treatments have been proposed to render hard seeds

permeable to water (3). Allof them partly destroy or crack the
seed-coats. Hardness in cultivated species is often eliminated by
QUALITY AND GERMINABILITY OF SEEDS 183

violently projecting seeds against a metallic wall in order to

break their seed-coats. However, the effectiveness of such treat-
ments is limited as the seeds too may be broken. Immersion of
seeds in concentrated sulfuric acid and their subsequent washing
with water can give good results. However, this treatment is
rather difficult to apply, since immersion must last long enough
to destroy the seed-coats, but not too long, to avoid killing the
embryos. In some cases brief immersion in boiling water is also
possible. An alternative method, which is very effective, easily
applied and does not damage the seeds consists of plunging them
into liquid nitrogen (Fig. 8). The process can be repeated until
satisfactory germination is obtained (71, 110). The enormous
temperature difference between liquid nitrogen and the ambient
air pro duces fine cracks in the seed-coats, which then become
permeable to water.

Trifolium ~pens ~ corniculatus

100 100 ...c- -- __ ___ o- _____ -c
~

2
,,
p
2
0,0------0-- - -- ----0 ,I
l
.-.- ....-.-.
I
50 50 I 1
----.-.-------.----
o
c::
"I

d ,...-/
1
,
~
T ----.
~
c::
E
.....
0 5 10 15 o 5 10 15
(1J
Cl

coronarium
100
p"'.o- - - - - .0---0---- 2- 0
,,
I

9
:'~._-.--

5 10 15
Time (days)
Fig. 8. Germination of Leguminous seeds at 20 o e. (1) untreated
seeds; (2) seeds frozen in liquid nitrogen for 15 minutes.
(from eÖme and Tissaoui, (71»
 184 D. COME

CONCLUSION
Seeds are very special organs whose postharvest physiology is
quite different from that of other plant organs. Their extraordinary
ability to survive in adehydrated state enables them to subsist for
a very long time until the conditions necessary to their germination
are present, and facilitates their storage. Their germination is
often delayed by numerous regulating systems. In wild species, in-
ability to germinate is very beneficial since it helps to preserve
these species when climatic or ecological conditions are difficult.
For cultivated species, however, dormancy often causes serious prob-
lems which have to be solved by suitable treatments.

REFERENCES

1. D. CÖme, Quelques problemes de terminologie concernant les

semences et leur germination, in: 11 La germination des
semences," R. Chaussat and Y. Le Deunff, ed., Gauthier-Villars,
Paris (1975).
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PHYSIOLOGY AND STORAGE OF BULBS: CONCEPTS AND NATURE OF DORMANCY

IN BULBS

M. Le Nard

Institut National de la Recherche Agronomique

Station d'Amelioration de la Pomme de terre et
des Plantes a bulbes, 29207 Landerneau, France

INTRODUCTION

Bulbous plants constitute a group of a very great diversity,

as much in their geographical origin as in their biological char-
acteristics. Moreover, the term "bulbous plants" is generally
used in a very broad sense and includes plants whose vegetative
multiplication organs are true bulbs, corms, rhizomes and even
tubers and tuberous roots.

Nevertheless, these "bulbs" have a point in common : after

being lifted they can withstand being stored for certain periods,
the duration depending on the species. Storage presents at least
two advantages. First of all, it allows the bulbs to be withdrawn
from adverse climatic conditions and thus to survive. Next it
enables them to be placed in such conditions that their behaviour
can be controlled and so to be prepared for a much more definite
use : bulb production, early or late flowering, etc •.•

Contrary to seeds, bulbs are organs generally rich in water

and show an active life after harvest, when they are sometimes
apparently at rest. Their physiological evolution continues there-
fore during storage and it will be easily understood that the envi-
ronmental factors will affect this evolution.

Another characteristic of bulbs is that it is generally possible

to lift them at different degrees of maturity, then to store them.
On the experimental level this is very interesting, for it is thus
possible to have at one's disposal organs which are physiologically
different and then to study the effects of environmental factors
on their evolution. It must however be pointed out that it is often
191
192 M. LE NARD

difficult to characterize adequately or define the degree of maturity

of a bulb. From a physiological point of view, the date of harvest
of a bulb is not therefore always a good reference.

There are at least three environmental factors which can

influence the evolution of bulbs during storage: humidity, composi-
tion of the atmosphere in the store, and temperature.

Control of humidity during storage can be a very important

factor in the case of bulbs, like lily bulbs, which are subject to
dessication. In other cases a too high humidity can be harmful for
it allows the growth of roots during storage. Similarly, by encour-
aging the development of certain parasites, chiefly fungi, humidity
can have an indirect influence on the physiology of bulbs.

The composition of the atmosphere in the store can also

strongly influence the physiological evolution of bulbs. The role
of agas like ethylene has especially been studied. Its effects can
be varied and sometimes very great on some species (1,2,3,4,5).

Temperature is certainly the factor whose effects have been

studied the most. This probably results from the fact that it very
soon appeared that its influence on the behaviour of bulbs could be
very great and that it was possible to benefit from this.

Extensive research concerning the influence of temperature on

the behaviour of a great number of bulbous species has been carried
out in the Netherlands under the direction of Blaauw. The results
of this work, which has largely contributed to the improvement of
our know1edge of the bio1ogy of bu1bous plants, has already been
sunnned up by numerous authors (6,7,8,9,10,11,12,13,14,15).

Blaauw and his co-workers have been mainly interested in the

influence of temperature on the differentiation of the f10wer bud
and on its elongation. The object was to determine the bases
necessary for the control of flowering and thus to produce flowers
of a good connnercial quality out of season and, if possible, all
the year round.

The results of Blaauw and his collaborators being henceforth

classic, the purpose of this review is not to make an additional
resume but, by providing complementary information, to try and
propose for a more global approach to the role of storage tempera-
ture in the physiology of bulbs. Indeed, it must be kept in mind
that bulbs are complex organs which, in addition to one or several
flower buds, possess vegetative buds, roots and storage tissues,
and that storage conditions affect the behaviour of this whole. To
i1lustrate this, the effects of storage temperature will be studied
in the case of two species possessing a true bulb with annual
replacement, the tulip and the bu1bous iris, and a species
PHYSIOLOGY AND STORAGE OF BULBS 193

possessing an annual corm, the gladiolus (Gladiolus grandiflorus

hort. )

The complexity of bulbs must also be taken into account when

tackling a subject like dormancy. This term, sometimes controverted,
is also sometimes used without precise definition and applied
indifferently to the bulbs or to the buds of bulbs. Abrief discus-
sion of the problem of dormancy in bulbous plants will be made,
referring more particularly to the behaviour of the tulip, bulbous
iris and gladiolus.

TULIP

The study of the development of the tulip bulb under temperate

climates, has shown that the initiation of the buds begins early,
at the end of winter beginning of spring, as the bulb en1arges,(16,
17). The most outer buds are initiated first, but their deve10pment
is then inhibited by the dominance exerted by the apica1 budo At
harvest, when the aerial organs of the mother-p1ant are mature, end
of spring beginning of summer, a bu1b of sufficient size possesses
an apica1 bud which has a1ready differentiated two or three 1eaf
primordia. This differentiation will continue during storage and
will end in the differentiation of a f1ower. The innermost vege-
tative bud, situated at the base of the flower bud, is the last to
be initiated, during the differentiation of the f10wer budo

Few observations have concerned the roots, but they seem to be

simi1ar1y initiated at the beginning of storage (18).

In the weeks that follow the end of its formation, a tu1ip

bu1b thus possesses all the organs which will give rise to the
daughter-plant : rooting in autumn-winter, growth of the aerial
parts, f10wering and en1argement of the daughter-bu1bs in spring.

The rate of bud and root differentiation and their subsequent

evolution are greatly influenced by the storage temperature of
bu1bs.

Effects of Storage Temperature on Bud and Root Differentiation

By exposing bulbs, immediately after lifting, to different

temperatures, from l,SoC to 3SoC, kept constant, B1aauw et a1.,(ll)
showed that f10wer bud differentiation occurred most rapid1y at
17°-20°C. The further the temperature moves away from this, above
or be10w, the more the differentiation is slowed down.

However, if instead of being stored at a constant temperature,

the bu1bs are subjected, immediate1y after lifting, to changing
temperatures, it is possib1e to show that a treatment of short
194 M. LE NARD

duration at high temperature leads simultaneously to a subsequent

more rapid flower bud differentiation and a more rapid root differ-
entiation which al10ws ear1ier rooting (19). The influence of a
high temperature treatment is quantitative, in the sense that extend-
ing it, within certain limits, a1lows a more and more rapid subsequent
flower bud differentiation and earlier and earlier rooting (Tab1e 1).
It has been noted elsewhere that extending the high-temperature
treatment led to an increase in the flower bud height at the end of
its differentiation (20).

As a direct effect of the high temperature is to delay consi-

derably the flower bud differentiation, a subsequent more rapid
differentiation can only be explained as a residual effect of the
high-temperature treatment. This induces the possibility of a more
rapid flower bud and root differentiation, but the induction can
only be expressed when the bulbs are then placed at mean tempera-
tures which a110w the actual differentiation.

Besides, when the duration of a high-temperature treatment is

extended beyond certain limits, the comp1etion of the flower bud
can be de1ayed because the acceleration caused by extending this
treatment no longer allows compensation for the delay caused by
moving the bulbs somewhat later into a temperature favorable to
the manifestation of the process induced (Table 1 and Table 2).
Similarly, it is obvious that if a high-temperature treatment is
applied when the flower bud differentiation is already well-advanced,
no beneficial effect on the earliness of flower bud differentiation
will then be observed.

If flower bud and root differentiation are both promoted by a

high-temperature treatment, it seems however that these two processes
could perhaps have slightly different requirements, and that rapid
rooting would require longer periods of high temperature than are
necessary to obtain rapid flower bud differentiation (20). Thus,
the results of Table 2 show that a treatment of several weeks at
30°C, which lead to a delayed flower bud differentiation neverthe-
less allows an earlier rooting even if it is applied fairly late.
On the contrary, a treatment of the same duration at 9°C, applied
at the beginning of storage, considerably delays rooting while it
allows earlier flower differentiation than the 30°C treatment.

The active differentiation of the axillary buds only starts

after completion of the flower budo By allowing an earlier flower
bud differentiation, a high-temperature treatment, applied immedi-
ately after lifting, also allows an earlier subsequent scale pri-
mordia differentiation in the axillary buds.

In summary, it appears that the high temperature induces the

possibility of a more rapid organogenesis, which however will not
take place unless the bulbs are subsequently placed in a temperature
PHYSIOLOGY AND STORAGE OF BULBS 195

Tab1e 1. Effects of duration of a 30°C storage of tu1ip bu1bs on

subsequent f10wer bud differentiation and rooting; cv.
"Pau1 Richter" (adapted from Le Nard, (19».
Contro1 : bu1bs p1aced, immediate1y after harvest, at
20 0 e for flower bud differentiation or planted at 15°e.
Stage G : completion of flower differentiation
(Beij er, (21».

Flower bud differentiation Rooting, at 15·e

at 20·e

Date of Days at Days after

stade G 20·e I Date 75 % rooting planUng

eontrol ••••••• 6 Aug. 49 12 Oct. 116

1 week 30·e ••• I 26 Jul. 31 6 Oct. 103

2 weeks 3o·e 26 Jul. 24 24 Sept. 84

3 weeks 30·e •• I 2 Aug. 24 14 Sept. 67

which allows the actual organogenesis to proceed.

The results reported in Table 2, confirming other observations

(20,22) show that there does not necessarily exist a corre1ation
between the earliness of flower bud differentiation and the earliness
of rooting. The observation of the degree of flower bud differenti-
ation on its own, therefore does not make it possible to characterize
the physiiological state of a bulb. in the sense that its subsequent
behaviour cannot be accurately predicted.

When small-sized bulbs are stored, immediately after lifting,

for several weeks at high temperature, there has sometimes been ob-
served an increase in the number of bulbs which show a floral initi-
ation (23). Therefore, the high temperature would not only affect
the rate of flower bud differentiation but would also influence
floral induction.

Storage Temperature and Induction of Growth Processes

Storage at high temperature, > 20 o e. At 20 o e, after its com-

pletion, the flower bud can immediately begin to grow. This growth
however is very slow and limited, the tip of the bud only growing
at maximum a few centimeters past the top of the bulb. When storage
196 M. LE NARD

Table 2. Effects of a 4-week storage at 30 oe, 20 0e or gOe, applied

at two different dates, on subsequent flower bud differ-
entiation and rooting at 15°e; cv. "Paul Richter."
Treatments applied on 2 July : bulbs previously stored
at 20 o e.
Stages of flower bud differentiation, see Beijer, (21).

4 w. 30·C! 4 w. 20·C I 4 w. 9·C

Stage of ·flower bud

differentiation at the I I P2 II
end of treatment •••••• I

Treatments applied
Date stage G .......... I 5 Aug. 23 Jul. 2 Aug.
on 14 Jun ••
Date 75 , rooting ••••• 27 Sept. I 14 Oct. I 10 Nov.
i.e. one week

after harvest
Days for rooting •••••• 77 94 121

Stage of flower bild

I differentiation at the II G+
1 end of treatment •••••• I
1
Treatments applied
1 Date stage G •••••••••• 1 19 Aug. I 21 Jul. 5 Aug.
on 2 Jul.
(Stage 11 of flower
I Date 75 , rooting ..... I 28 Sept. 6 Oct. 16 Oct.
bud differentiation) 1_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

Days for rooting •••••• 1 60 68 78

.1

continues beyond 6-7 months, dissections make it possible to estab-

lish that the flower is dry, although the exterior aspect of the
bulb is quite normal (See Fig. 3). The flower bud then progressively
dries up entirely.

The root primordia also lengthen during storage at züoe and

the rooting plate becomes more and more protuberant. However. if
the bulbs are planted at a temperature maintained above or at züoe,
rooting, i.e growth of roots out of the plate, does not take place
and elongation of the flower bud remains restricted.

During storage at züoe, the number of scale primordia increases

in the axillary buds. When storage is of long duration, some of
these scale primordia tend to change into leaves. Sometimes it is
even possible to observe a floral differentiation from an axillary
bud, usually the innermost one (Z4).
PHYSIOLOGY AND STORAGE OF BULBS 197

In the case of storage, even of very long duration, at a tem-

perature above or at 20°C, the axillary buds do not change into
daughter-bulbs, that is, bulbing does not take place.

If bulbs showing a completely differentiated flower bud are

then exposed to a high temperature, 30°C - 35°C, drying of the
flower can be rapidly observed (25,26).

Storage at low temperature. Blaauw et al.,(ll) have clearly

shown that a rapid extension growth of the flower bud can only be
observed after a bulb has been exposed to low temperatures. Other
results have shown that the low temperature also induces bulbing
(24,27), and that its influence varies according to the level of
the temperature, the length of its duration and the moment of its
application.

(1) Influence of the level of the low temperature. When

bulbs possessing a completely differentiated flower bud are stored
at different temperatures, it can be observed, after planting, that
plant growth is more rapid when the storage temperature is lower :
rooting, elongation of the aerial organs and daughter-bulb enlarge-
ment are more rapid (Fig. 1). The maturity of the plants is also
advanced and the duration of the growth per iod is shorter.

Thus, low temperature simultaneously induces a stimulation of

the elongation and bulbing. The induction is all the stronger, i.e
elongation and bulbing can subsequently proceed faster, when storage
temperature is lower, at least as far as 2°C. Other observations
suggest that a slightly negative temperature, - 1°C, would be as
effective as 5°C, (28).

Earlier flowering corresponds with more rapid elongation and

the production of longer flower shoots at anthesis and at the end
of growth if the bulbs are plan ted at a mean temperature and if the
environmental conditions then remain constant.

(2) Influence of the duration of application of low temperature.

Extending the duration of low-temperature storage leads, first, to
a more rapid growth, i.e. elongation plus bulbing, but shorter
lasting (Fig. 2).

Later, when the duration of storage reaches a certain length

which varies according to the level of the low temperature and the
cultivar, the subsequent plant growth is very rapid but reduced
(Fig. 2; sampie 25 w. IO°C). The plants are then short and slender,
and show blasted flowers. Finally, in the case of long duration
storage at low temperature, dissections of bulbs make it possible
to observe that bulbing has already begun during storage, that is
the enlargement of the daughter-bulbs has started (Fig. 3). If
such bulbs are then planted, neither rooting nor elongation of
198 M. LE NARD

Fig. 1. Tu1ip: effect of bu1b storage on subsequent elongation

and bu1bing; cv. "Pau1 Richter" (from Le Nard and Cohat,
(24»);left to right : bu1bs stored for 10 weeks at 2°C -
3°C (SlF2), 10 weeks at 10°C (SlM2) or 15 weeks at 20°C
(S3) before p1anting at 14°-16°C.

Plant length
cm.
_ _ 5 W 1000c.
50 _______ 10 -
.. _______ , 5 .....-.--.
40 .---.&---- 20
____ <>- ___ 25
~/ , / - 1 /
__ -------.
______ _
,'~, ,.-~,.--

~ Date of / / • //
30 flowering f
,','
/ ,,.
,/,/ /'
...t---... --.. - " /'
20
,
,~;
I ,.
/
/ / / /"
10
i':';/~ -//-'/ -",//
------------_... ...
~' ~ ......... "" ,.-,

20 25
after pl antlng

Fig. 2. Tu1ip : Growth of f10wer shoots produced by bu1bs stored

for 5,10,15,20 or 25 weeks at 10°C, before p1anting at
14°-16 Q C (from Le Nard and Cohat, (24)).
PHYSIOLOGY AND STORAGE OF BULBS 199

+- flower

d.b

Fig. 3. Tulip : Aspect of bulbs stored, immediately after lifting,

for 30 weeks at 18°C-20°C (5), 10°C or 2°_3°C (F); cv.
"Pax" (frorn Le Nard and Cohat, (24)). d.b = daughter-bulbs

aerial organs are observed, and the only growth process to take
place is bulbing.

Bulbing induction is all the stronger when the storage tempera-

ture is lower. However, bulbing during storage is noticeable earlier
at 10°C than at 2°C (Fig. 3). This can be explained by the fact
that a temperature of 10°C is more favorable than 2°C to the mani-
festation of growth processes.

These observations show that when storage at low temperature

is extended beyond a certain period, bulbing becomes the predominant
growth process and this results in a greater or lesser reduction in
elongation (roots and aerial organs).

If bulbs formed during storage at low temperature, 4°_5<1C, are

in turn maintained at low temperature for a long period, they also
produce daughter-bulbs during storage. In this case, the apical buds
do not differentiate any aerial organs and directly give rise to
200 M. LE NARD

daughter-bulbs which only possess one or two sca1es (fig. 4). Thus,
it appears that if the complete cycle of a tulip bulb proceeds at
10w temperature, organogenesis in the apical bud is very 1imited
but, even in this case, bulbing takes place.

(3) Influence of the moment of application of low temperature.

In natural conditions, because of the rhythm of the seasons, tulip
bulbs possess wel1-differentiated buds and roots when they are
subjected to low temperatures and subsequent growth is "normal,'"
On the contrary, if, experimentally, bulbs are placed at low tem~
perature at other times in their cycle, it is possible to show a
certain diversity in their behaviour (19,29).

By lifting bulbs periodically, then by storing them iIlUllediately

at low temperature, it can be observed that during a first phase,
which practically corresponds to the whole period of bulb enlargement,
the bulbs do not react to low temperature. A low-.temperature treat-·
ment applied during this phase does not induce any stimulation of
the subsequent growth : bulbs plan ted after this treatment give rise
to plants whose growth is very slow, comparable to that of plants
produced by bulbs planted at mean temperature immediately after
harvest (Fig. 5; bulbs placed at 2°C, on 14 March, 11 April and
9 May).

Fig. 4. Tulip : Apical bulbing in bulbs formed and maintained at low

temperature, 4°_5°C : grandmother-bulbs were placed at 40_
5°C on 16 Oct. 1976; the mother-bulbs, shown on this photo-
graph, were harvested and, in turn, kept at 4°-5°C; one or
two bulbs per cultivar have been dissected to show the api-
cal daughter-bulbs; photographed on 12 May 1978.
PHYSIOLOGY AND STORAGE OF BULBS 201

Fig. 5. Tulip: Effect of the date of treatment at 2°_3°C for 120

days on the subsequent behaviour of the bulbi cv, "Paul
Richter" (from Le Nard, (29»; F : date placed in 2°_JoC;
P : date planted at 14°-16°C; top: left to right; F
14 March, 11 April, 9 May; bottom : left to right; F :
6 June, 20 June, 18 July, 12 September.

From a certain stage, which we have called "physiological matu-

rity," (19), the bulb becomes sensitive to the action of low tem~
perature which then induces growth of the daughter-organs. The
growth process, or processes, which can then take place will depend
on the degree of bud and root differentiation at the time the low-
temperature treatment has been applied.

When the apical bud only possesses one or two leaf primordia,
these primordia change into scales and give rise to an apical
daughter-bulb (Fig. 5 : bulbs placed at 2°C one 6 June). When the
low-temperature treatment is applied a little later, the axillary
buds undergo bulbing, but the possibilities of elongation of the
flower bud depend on its degree of differentiation. If the differ-
entiation of the flower bud is incomplete, and although it can
later take place, the flower bua cannot elongate and dries up
(Fig. 5; bulbs placed on 2°C on 20 June) or it can show some elonga~
202 M. LE NARD

tion but the plants are slender and present blasted flowers (Fig. 5;
bulbs placed at 2°C on 18 July). Finally, if the low-temperature
treatment is applied even later to bulbs possessing a flower bud
and roots which are well differentiated, the subsequent growth is
"normal" and comprises elongation, roots and aer1.al organs, plus
bulbing (Fig. 5; bulbs placed at 2°C on 12 September). All the
transition stages exist between bulbing of the apical bud and
"normal" growth.

All these observations together lead to the conclusion that the

behaviour of a tulip bulb after a low-temperature storage depends on
the one hand on its physiological state and on the other on the
degree of bud and root differentiation. The physiological state
determines the possibility of induction of growth processes by the
low temperature, while the degree of bud and root differentiation
determines the growth process, or processes, which can take place;
bulbing alone or elongation and bulbing.

Bulb evolution towards "physiological maturity" is accelerated

by high temperatures and is very slow atlow temperature (19,29).
Having reached this stage, the bulb becomes progressively more and
more sensitive to the action of low temperatures, and a low-tempera-
ture treatment of fixed duration leads to a bulbing induction all the
stronger when it is applied later to bulbs previously stored at
high temperature (22) (Fig. 6)

The presence of buds which are sufficiently differentiated

constitutes a preliminary to all growth possibilities and it could
be supposed that before the "physiological maturity" stage a low-
temperature treatment would not induce any growth process solely be-
cause the bulb does not possess sufficiently differentiated buds.
It does not seem that this is the cause, for it has been possible
to show that the physiological evolution of a bulb towards a greater
sensitivity to low temperature can proceed in the absence of any
visible bud differentiation and that bulbing induction due to a
low-temperature treatment can be very strong even if the differenti-
ation of the apical bud is not far advanced at the time the treat-
ment is applied, (19,22). Besides, it has already been pointed
out that in the case of bulbs formed at low temperature, (see (2»
bulbing can take place even if the apical bud has only produced one
or two primordia. In fact, it appears, and this has been confirmed
by other results, (20), that the degree of apical bud differenti-
ation ~r se does not adequately define the physiological state of
a tulip bulb.

When a bulb has reached its "physiological maturity" and be-

comes sensitive to the action of low temperature, it is sometimes
possible to observe that bulbing of the apical bud stops shortly
after beginning. In this case, which we have called "incomplete
bulbing," the first primordium produced by the apical bud thickens
PHYSIOLOGY AND STORAGE OF BULBS 203

Fig. 6. Tulip: Importance of bulbing according to the treatment

applied to the bulbs before a 15-week storage at 2°_3°C;
cv. "Paul Richter" (from Le Nard,(19».
Bulbs lifted on 30 April and then placed for 1 (Cl' 2 (C2)
or 3 weeks at 30°C (C3) before cold treatment; Cl = incom-
plete bulbing (only one scale primordia has started to
thicken) and differentiation of aerial organs, a flower
bud (left) or one leaf; C2 = apical bulbing; C3 = bulbing
of different buds, apical and axillary (left), axillary
only, the flower bud having dried up (right).

and turns into a fleshy scale, but this bulbing stops and the apical
meristem of the bud produces aga in aerial organs, (19). This
organogenesis can go as far as the production of a flower if the
"incomplete bulbing" is not too great (Fig. 6).

Storage at High Temperature Following Storage at Low Temperature

The effect of a low-temperature storage, applied immediately

after bulb lifting, can sometimes be nullified if the bulbs are
then transferred at high temperature, (24,27,29). Recent observa-
tions, (22,30) have made it possible to determine more precisely the
effects of a high temperature applied after low-temperature storage.

Studies on bulbing have shown that in fact, a high temperature

has two opposed effects : it prornotes the manifestation of bulbing
induced by low temperature but at the same time it nullifies progres-
sively this induction. Thus, the behaviour of bulbs first stored at
low temperature then moved to high temperature, will depend on the
one hand on the strength of bulbing induction and on the other on
the duration of the high-temperature treatment. A weak induction
can be easily nullified : this is the case of bulbs exposed to low
temperature straight after lifting, that is, at a stage where they
are not very sensitive to the action of low temperature. On the
204 M. LE NARD

countrary, if bulbs are subjected later to low-temperature treat-

ments of fairly long durations, bulbing induction is strong and it
can no longer be completely nullified. Bulbing can then rapidly
proceed at high temperature. Between the two extreme situations,
weak induction completely nullified or rapid and complete bulbing,
all the transition stages can be observed. In these intermediate
situations, which we have called "incomplete bulbing," (19,29), the
primordia present in a bud begin to thicken, but bulbing stops and
new aerial organs are differentiated by the apical meristem of the
budo This differentiation can even go as far as flower differentia-
tion in the case where "incomplete bulbing" is not very great and
concerns the apical bud (27). When bulbing induction is completely
nullified, the bulb behaves practically as if it had never previous-
ly been exposed to low temperature and organogenesis can proceed in
the normal way.

The influence of low temperature on the stimu~ation of subse-

quent elongation can also be nullified by a high-temperature treat-
ment, insofar as the previous low-temperature treatment is applied
fairly soon after bulb lifting and it is of fairly short duration
(24). On the contrary, if a low-temperature treatment is applied
to bulbs possessing well-differentiated flower buds and roots,
subsequent storage of short duration at high temperature, 25°-27°C,
leads to a clear stimulation of elongation, that is, it leads to
the same final result as that produced by extending the low-tem-
perature treatment (30). However, looking at the plant growth
curves suggests that the mechanisms of action of the two tempera-
tures would not be the same.

When storage at 25°-27°C, or at a higher temperature, is ex-

tended, the flower is destroyed.

The effects of a high-temperature storage therefore vary con-

siderably according to the moment it is applied and/or according
to the previous storage conditions of bulbs.

Conclusions

The observations reported here, and summed up in Figure 7

show the importance of storage temperature on the behaviour of a
tulip bulb. They indicate that:

A high temperature applied at the beginning of storage, or

one which the bulbs have already been subjected to in the ground,
induces a more rapid subsequent organogenesis when the bulbs are
then exposed to a temperature which allows the manifestation of
this induction, that is, a mean temperature. Simultaneously, high
temperature speeds up the evolution of bulbs towards their "physio-
logical maturity" and a greater sensitivity to low temperatures.
The behaviour of bulbs formed at low-temperature and that of bulbs
early-stored at low temperature suggests that bud differentiation
PHYSIOLOGY AND STORAGE OF BULBS 205

would be restricted if the bulbs are not subjected to a high tempera-

ture for a sufficient duration.

The behaviour of a bulb following storage at low temperature

depends on the one hand on its physiological state which determines
the possibility of growth induction, and on the other it depends on
the degree of organogenesis which determines the growth process, or
processes, which can take place, bulbing only or elongation plus
bulbing, these two processes being induced simultaneously by a low-
temperature treatment.

It would seem necessary here to draw attention to the fact that

only the use of relatively extreme temperatures, 30 0 e and 2°e, allows
the induction of a process, organogenesis or growth, to separate from
its manifestation. Indeed, at a mean temperature the induction of a
process and its manifestation can both be expressed, as the cycle of
the tulip can proceed slowly, but completely, at a temperature in the
order of lSo-17°e for example.

A great deal of work has been devoted to the influence of low

temperature on the extension growth of the flower but while neglect-
ing its influence on bulbing. Now, the observations reported above
show that bulbing appears to be a fundamental growth process.

"Incomplete bulbing" of the apical bud observed following an

early storage at low temperature (see Fig. 6) and in the course of
study of the nullification of the influence of a low-temperature
treatment by a subsequent storage at high temperature, shows that in
a bud, the start of bulbing corresponds to an interruption of organo-
genesis (19,29). Where the apical bud itself is concerned this can
lead to an apical bulbing, while where the axillary buds are con-
cerned this results in the production of daughter-bulbs which will
possess fewer scales the earlier the bulbs have been exposed to low
temperature and/or the more rapidly bulbing has been able to start
(24). Inversely, if bulbs are stored at a temperature which does
not allow the induction of growth processes, > 20 oe, or after
bulbing induction has been nullified, organogenesis can continue
longer and the axillary buds can produce aerial organs and even
sometimes a flower.

These results lead us to distinguish two main phases in the

cycle of the tulip : a phase of organogenesis during which meriste-
matic activity is great, and a phase of growth during which meriste-
matic activity is considerably reduced. As far as the vegetative
buds are concerned, we have seen that bulbing corresponds to an
interruption of differentiation of new organs by their apical meri-
stems. Organogensis resumes during the daughter-bulb formation but
it only becomes active after the end of bulb enlargement. In the
case of the flower bud, cell divisions are especially active before
the extension growth, this chiefly resulting from the elongation of
pre-existing ce11s (18,31).
206 M. LE NARD

BULBING
(bulb formation) HIGH

~ ~HrTU~

1
deter.ines response to
induction
of • more r.pid
org.n ogenesis

I
low temper.tur.

differentiation
LOW prollote. d
TE"PERA TURE .e.n temper.h"s

\ induction
!
", ,"owth proeessll ORGANOGENESIS

... 1 '''uds Ind roots)

I
o • IniflS t Ition
.'" of ....wth pro .ot.d
.t ...11 t • .,,,.tII'lI
o
.
.~

d ...." of DrqIll04enesis
...
z:
• ..ter.inlS growth proeess (es I
!! which ean t.ke place

!..
u

!
induetion is Welk
CI/I b.
~ •
nullifitd
hitll t ••per.tur.
11. ,rowtll
1
buds buds
suffiei.ntl~ insuffieien tl~
difftrtntilt.d differentiahd

induction is .tron:
"nnot bl IIUIlifi.
1 l
b~
• hiCJh t,.plr.tur. GROWlH GROWlH
:
CJrowth Elongation Bulbinq
•
8ulbing
.lone

8ULBING
=
lilterrupti.. 01 ORGANOGENESIS

Fig. 7. Tulip: Role of storage temperature in the cycle of the

tulip.
PHYSIOLOGY AND STORAGE OF BULBS 207

The importance of the physiological state of a bulb has been

noted above, for that determines its reaction to low temperature.
As long as the bulb has not reached its I1 physiological maturity,11
a low temperature only delays the physiological evolution of the
bulb and does not induce any growth process. It can be supposed
that the bulb is then in astate such that at the end of a low-
temperature treatment its reserves could not be mobilized and, con-
sequently, no growth would be possible. On the contrary, when the
stage of " physiological maturity" is reached, low temperatures induce
growth processes and the bulb reserve mobilization, necessary to
ensure daughter-organ growth, is then possible. Mobilization of the
reserves is all the more rapid the later low-temperature treatment
is applied to bulbs previously stored at high temperature (20,22).

It has been shown repeatedly that the physiological state of a

bulb cannot be characterized by the degree of flower bud differentia-
tion, and that the physiological evolution of a bulb can even pro-
ceed in the absence of any visible organogenesis in the apical budo
This has an important consequence in practice where the degree of
flower bud differentiation is used as morphological point of refer-
ence during the preparation of bulbs which will be forced. It
appears in fact that bulbs showing flower buds at the same degree
of differentiation will not necessarily be in the same physiological
state and, consequently, their subsequent behaviour can be different
(20).

On the practical level, the observations reported here, show

that all storage of tulip bulbs at a positive temperature, and even
slightly below O°C (unpublished results), is limited in time. A
fairly long storage at low temperature, lO-2°C, can only be planned
in the ease of bulbs whieh have not reached their I1 physiologieal
maturity,11 (29).

A great deal of work has been carried out to determine the

temperature treatments which should be applied in order to obtain
winter flowering, and a very rieh literature exists on this subjeet
(see de Hertogh (32». It must always be kept in mind that flower-
ing of good quality can only be obtained if the two growth proeesses,
elongation and bulbing, can proeeed harmoniously. If bulbing be-
comes predominant (the case of bulbs plaeed too early or stored for
too long at low temperature, and bulbs whose planting or rooting
is delayed after the end of low-temperature treatment). the plants
are poor and, very often. show blas ted flowers (33).

Bulbing in the absence of elongation, obtained in the case of a

long storage at low temperature, cannot usually be observed ~n nat-
ural eonditions where the environmental faetors allow rooting and
growth of the aerial organs.
208 M. LE NARD

BULBOUS IRIS

Temperature Requirements for Flower Differentiation

Bulbous iris of the dutch iris group (I. x"hollandica) have a

cycle very similar to that of the tulip. In temperate climates of
Western Europe, bulbs plan ted in autumn root rapidly, the growth of
the aerial organs begins in winter and continues into spring, and
daughter-bulb enlargement ends at the beginning of summer.

However, there is a great difference between tulip and bulbous

1r1S. In the case of the tulip, flower differentiation occurs in
the weeks following bulb lifting, at rather high temperatures. In
the case of bulbous iris, flower differentiation takes place after
bulb planting, during autumn and winter, that is, at a rather low
temperature (34).

At lifting time, the apical bud of an iris bulb, usually pos-

sesses 3 leaf primordia. The subsequent evolution of this bud de-
pends on several factors. It depends first of all on the size of
the bulb, that is, on its weight. There is a critical size, variable
according to the cultivars, below which flower initiation cannot
take place. The apical bud in that case, will only produce, besides
the leaf primordia, scale primordia and will give rise to an apical
daughter-bulb.

Temperature also greatly affects apical bud evolution. A high

temperature, above 20°C, favors subsequent flower initiation.
However, the actual flower differentiation will only proceed if the
bulbs are subsequently moved to a mean or low temperature, the most
favorable temperature for flower differentiation being between 9°
and 15°C (11).

A high temperature therefore allows iris bulbs, of adequate

size, to acquire the capacity to differentiate a flower, capacity
which can only be expressed when temperature becomes favorable. The
duration of high-temperature storage necessary for the acquisition
of this capacity is all the longer the nearer the bulb is to the
critical size.

Iris bulbs can be stored for long per iods at high temperature,
25°C-30°C. In these conditions, flower differentiation cannot pro-
ceed, but the bulb retains its flowering capacity and it is possible
to produce flowers all the year round (10,35,36). After planting,
the development of the flower shoot will depend on the environmental
factors (11,37.38,39,40,41).

The different temperature treatments applied to iris bulbs after

lifting therefore affect greatly apical bud differentiation. How-
ever, storage temperatures do not only affect the possibilities of
PHYSIOLOGY AND STORAGE OF BULBS 209

differentiation of this bud but also influence the whole bulb evolu-
tion.

Storage Temperature and Subsequent Bulb Behaviour

Influence of low temperature. A low temperature treatment,

applied to bulbs having acquired the capacity to initiate a flower,
induces simultaneously a stimulation of the elongation of the aerial
organs and bulbing (42,43). However manifestation of these effects
of low temperature depends on the one hand on the length of its
application and on the other on the time it is applied.

(1) Influence of the duration of low-temperature treatment.

In the case of bulbs possessing the capacity to initiate a flower,
extending the low-temperature storage leads first to a more and
more rapid elongation of the flower shoot (earlier flowering) and
a more and more rapid daughter-bulb enlargement (Fig. 8; C7,C6Ml'
C5M2, C4M3 and some plants of C3M4 ).

Later, when low-temperature storage is extended beyond aperiod

which varies according to the cultivar and the temperature level,
the extension growth of the flower bud is still very rapid but very
reduced and the flower, although present, sometimes aborts and
dries up (Fig. 8; C3M4)' Bulbing proceeds still more and more
rapidly.

Finally, if storage at low temperature lasts very long, bulbing

can begin during storage. If the bulbs are then planted, elongation
of the daughter-plants is reduced or nil (Fig. 8; C2M5)'

In the case of the bulbous iris, as in that of the tulip, it

therefore appears that elongation will be limited or nil if bulbing
becomes the predominant growth process.

When elongation of the aerial organs is rapid, as a result of

an extension of the duration of low-temperature storage, a decrease
in the leaf length, and sometimes also in the flower shoot length,
can be observed (37,42,44,45,46).

(2) Influence of the time at which low temperature is applied.

Kamerbeek (37), has shown that early application of low temperature
limits the differentiation of the apical bud which then produces
only 2 or 3 leaves, and scales. By applying low-temperature treat-
ments earlier, or of longer duration, it is even possible to obtain
an apical daughter-bulb in the absence of production of any aerial
organ (42,43). In this case the leaf primordia of the apical bud
change into fleshy scales (Fig. 8; M7)' An early apical bulbing
then results in a limited organogenesis.

The time at which low temperature treatment is applied does not

210 M.LENARD

Fig. 8. Bulbous iris : Flower shoot elongation and bulbing follow-

ing storage at 10°C applied after, or in the absence oft
a previous treatment at 30°C; cv. "Wedgwood" (from Le Nard,
(42» .
C = 30°C, M = 10°C; left to right : C7 = 28 weeks at 30°C;
C6Ml = 24 w. 30°C + 4 w. 10°C ; CSMZ= 20 w. 30 DC + 8 w.
10DC; C4M3= 16 w. 30°C + lZ w. 10°C ; C3M4= lZ w. 30°C
+ 16 w. 10DC (two types of plants) ; C2MS= 8 w. 30° C +
20 w. 20°C; M7 = 28 w. 10°C.

only influence the possibilities of apical bud differentiation but

has other consequences on the subsequent bulb behaviour.

Thus, bulbing begins earlier and is then completed more rapidly

when the bulbs have been stored at higher temperature before exposure
to low temperature (47). This result suggests that a high tempera-
ture allows a more rapid subsequent bulb reserve mobilization after
a low-temperature treatment.

When a similar low-temperature treatment is applied to bulbs

previously stored for fairly short periods at different temperatures,
the plants issued from the bulbs stored for the longest time at high
temperature produce fewer leaves, flower earlier and better, i.e.
flowering percentage is higher (47). These observations, which agree
with those of Kimura and Stuart (4S) and Uhring (48) suggest that
apical bud differentiation at low or mean temperature proceeds more
quickly when previous storage at high temperature is extended.
Corresponding on the one hand to an earlier flower differentiation
would be a decrease in the leaf number and on the other an earlier
shoot elongation. A better flowering could be explained, at least
PHYSIOLOGY AND STORAGE OF BULBS 211

in part, by the favorable influence exerted by the high temperature

on subsequent bulb reserve mobilization.

All these observations show that the behaviour of an iris bulb,

after a low-temperature treatment depends on its physiological state
which determines the bulb's response to low temperature, and on the
possibilities of differentiation of the apical bud which remains
vegetative or becomes floral.

Storage at high-temperature following storage at low tempera-

ture. In certain conditions a high-temperature treatment can nullify
the influence of a previous storage at low temperature (42,43).

When a low temperature is applied immediately after bulb lift-

ing and for a fairly short period, its influence can then be com-
pletely nullified by a subsequent storage at high temperature. In
this case bulbing does not take place and organogenesis in the
apical bud can then continue normally if the bulb is exposed at
favorable temperatures. When low-temperature storage is of long
duration, a subsequent high-temperature treatment can no longer
nullify its influence, bulbing occurs and the mother-bulb sc ales
shrivel.

Between these two situations, bulbing induction completely

nullified or rapid bulbing at high temperature, there are intermedi-
ate situations. In these cases, bulbing induced by low temperature
can start but stops before complete shrivelling of mother-bulb
scales, and, later, a new differentiation of aerial organs is observ-
ed in the bud which had begun to form a bulb (42).

Conclusions

In an iris bulb, high temperatures anq low temperatures induce

processes which occur successively in the buds in the course of the
bulb cycle. High temperatures induce an organogenesis and simulta-
neously promote the bulb's physiological evolution towards a greater
sensitivity to low temperature. Organogenesis induced by a high
temperature only proceeds rapidly if the bulbs are subsequently ex-
posed to a medium to low temperature, 9° to lSoC.

Low temperatures, necessary to the stimulation of elongation,

also induce bulbing. The start of bulbing seems to lead to an in-
terruption of organogenesis in the buds. Thus, following the degree
of apical bud differentiation at the time bulbing starts, so bulbing
will be apical or axillary. The importance of the physiological
state of a bulb on its behaviour at the end of a low-temperature
treatment has been demonstrated. High temperatures. which allow a
more rapid subsequent bulb reserve mobilization, accelerate bulb
evolution towards a greater sensitivity to low temperature. This
evolution, as in the case of a tulip bulb, can take place in the
absence of any visible organogenesis. It has appeared besides,
212 M. LE NARD

that a high-temperature treatment of sufficient duration to allow

subsequent flower initiation, does not Qecessarily allow satisfactory
growth of the flower shoot (47).

All these results indicate that the effects of storage tempera-

ture on the behaviour of an iris bulb appear to be very similar to
those observed in the case of a tulip bulb.

Study of the effects of temperature on the behaviour of iris

bulbs furnishes the basis for storage methods used in practice. Use
of the sequence high temperature-low temperature makes it possible
to control flower initiation and the choice of the length and level
of temperatures to be applied, depends on what result is desired,
early or late flowering, and on the type of plant looked for (33,36,
37,49,50,51).

The sequence high temperature-low temperature can also be used

to prevent flower initiation. This objective, especially desired in
the case of seed bulbs, can sometimes be attained if the low tempera-
ture applied is considerably lower than that which promotes flower
differentiation (23,33). This result could be explained by the fact
that a direct effect of low temperature is to slow down organogensis
and, simultaneously, to induce bulbing. This could then start
rapidly after planting and would limit the differentiation of the
apical budo However, the low-temperature treatment must not be
applied too early and/or for a too long period, for if bulbing be-
comes predominant, plant growth will be poor.

One of the greatest impediments for a rational application of

these different temperature treatments, is the absence of criteria
enabling the bulb's physiological state to be defined. In the
course of storage there is no morphological point of reference com-
parable to that which is used in the case of a tulip bulb, for in
the iris bulb, flower differentiation generally occurs after plant-
ing. It is therefore not always easy to predict accurately the
behaviour of iris bulbs nor to reproduce certain results.

GLADIOLUS

In the temperate climates, the cycle of the gladiolus differs

from that of the tulip and the iris. Corms are planted in spring.
Flower differentiation occurs after planting and growth of the
aerial organs proceeds in spring and summer. The new corms are
lifted in autumn and are at rest in winter.

The buds which will produce the aerial organs of the daughter-
plant are initiated early, during corm formation, and at lifting
time the apical bud has already produced several leaf primorida (52).
PHYSIOLOGY AND STORAGE OF BULBS 213

The rate of subsequent apical bud differentiation and the

daughter-plant growth can be greatly affected by corm storage tem-
perature.

Influence of Storage Temperature on Sprouting, Growth of the Aerial

Organs and Formation of New Corms

Immediately after lifting, the corms and cormels cannot usually

show any growth, even if they are placed in favorable conditions.
Earliness of bud and root growth depends first of all on the cultivar
and on the environmental conditions during the formation of the
corms : corms produced at high temperature and/or at high light
intensity sprout later than corms produced at me an temperature and/
or at weak light intensity (53,54,55,56,57).

Storage temperature, and sometimes moisture conditions, also

greatly affect sprouting of corms and cormels (53,54,58,59,60,61,
62,63,64). These different studies indicate that sprouting results
from the successive realization of two processes : first the acquisi-
tion of growth capacity (dormancy release), then the differentiation
and growth of buds and roots.

The acquisition of growth capacity is favored by low temperature,

5° to 10°C. The length of the low-temperature treatment needed to
acquire this capacity depends on the cultivar and on the physiologi-
cal state of the corms at lifting. Cormels require langer low-
temperature storage than corms.

At low temperature, organogenesis is practically stopped. It

occurs later when the corms or cormels are placed at high tempera-
ture, 20°-25°C. Ta obtain homogeneous early sprouting it is there-
fore necessary to store the corms first at low temperature then at
high temperature.

High temperature storage does not only promote organogenesis

but also affects subsequent plant growth : leaf growth, flowering,
cormel formation and new corm enlargement are speeded up (58,63,64,
65,66,67,68,69). Extending the high-temperature storage beyond a
certain duration can result in the production of less vigoraus
plants : leaf and flower number per plant and the final plant height
are decreased (67).

In the temperate climates of Western Europe, storage at high

temperature has a favorable effect not only on the earliness of
new corm formation but also on their final size. Daughter-corms
produced by corms stored at high temperature have the peculiarity
of possessing more tuberized interna des and, for a given size of
corm, therefore have a more globular appearance (64,67). (Fig. 9).

As storage at high temperature leads to earlier sprouting, it

214 M. LE NARD

could be supposed that this would explain a more rapid subsequent

plant growth. It has been possible to show that this is not the case
and that differences between growth in plants produced by corms
stored at different ternperatures could originate from the fact that
the corms show different physiological stat'es at planting.

Influence of Storage Ternperature on the Physiological Evolution of

eorms

The physiological evolution of corms has been studied by stor-

ing them for increasing per iods at different ternperatures, then by
planting thern in the dark, at 20 0 e (67,68). Following a high-
ternperature storage applied to corms which have already acquired
growth capacity, the weight of sprouts produced in the darkness
within a certain time increases, rapidly reaches a maximum, holds
its own for a while, then progressively decreases (Fig. 10). Simul-
taneously, the new corms are formed more and more rapidly after
planting. When high-ternperature storage is extended beyond a certain
period, daughter-corm formation can even begin during storage. In
this case, after planting, the growth of leaves and roots is very
poor or nil.

After storage at low temperature, the growth rate of the sprouts

increases progressively to a maximum, then stays constant for a long
period (Fig. 10). In the case of corms stored for a very long time
at 2°_4°e, up to three years, daughter-corm formation has not been
able to be observed during storage.

It seerns therefore that high ternperatures induce a possibility

of a more rapid growth when the corms are subsequently placed in
conditions favorable to the expression of this induction. When
corms are stored for a longer period at a higher ternperature, at
least up to 30 o e, the subsequent growth is more rapid (roots,
sprouts, corm formation). This indicates that growth induction is
stronger after a 30 0 e storage than after a 20 0 e storage for example.
However, in the case of a long storage, daughter-corm formation
occurs earlier at 20 0 e than at 30 o e. This result can be explained
by the fact that 20 0 e is more favorable than 30 0 e to corm formation
(68,70). Manifestation of tuberization, in a given environment,
is therefore the result of the double action of the temperature of
this environment on the induction of tuberization and on the rate of
its expression.

eonclusions

Having acquired the capacity for growth (ernergence from dorman-

cy), a gladiolus corm evolves constantly during storage, rapidly at
high temperature and slowly at low ternperature. The physiological
state of a corm at planting, all things being equal everywhere else,
influences the behaviour of the daughter-plant. Corms physiologically
PHYSIOLOGY AND STORAGE OF BULBS 215

Fig. 9. Gladiolus: Effects of corm storage on the earliness of

growth, flower shoot elongation and new corm formation;
cv. "Happy End"; plants produced by corms stored at low
temperature, SO-l2°C (left), or placed at 20°C, 79 days
before planting (right).

little-evolved, that is, stored at low temperature, produce vigorous

plants whose sprouting, flowering, tuberization and maturity are
delayed. Corms physiologically evolved produce less vigorous plants
whose growth is accelerated. Only a low temperature, which slows
down the physiological evolution of corms, allows a long-term
storage.

Daugher-corm production during storage at high temperature,

> 20°C, can be compared to what has been observed in the case of
tulip and iris bulbs stored for a long time at low temperature.
In these last two species, it has been possible to show that
organogenesis and bulbing are mutually exclusive in a budo In the
case of gladiolus corms, a similar relation does not seem to have
216 M. LE NARD

-'-'- 5·(
SPROUT LENGTH ---- 10·C
_________ 20·C
80
1 (H.
_ _ 300C

60

40

20

o~__~--~--~--~--~~~~~~--~--~--~--~~~~
4 8 12 16 20 24 28 32 36 40 44 48
STORAGE WEEK S

Fig. 10. G1adio1us Evolution of the 1ength of sprouts produced

by corms stored for increasing periods at 5°, 10°, 20°
or 30°C (adapted from Cohat (68»; sprouts measured 28
days after p1anting at 20°C, in darkness.

been observed. Some observations neverthe1ess show that when the

formation of daughter-corms starts off rapid1y, the formation of
new organs by a bud wou1d be 1imited. Thus, fo11owing a quite long
storage at high temperature, 1arge corms produce p1ants with fewer
flowers (67), while small corms produce a higher percentage of
vegetative plants, that is, organogenesis in their apica1 buds
wou1d be 1imited (64, 71). Final1y, when daughter-corm formation
begins during storage, after p1anting no 1eaf differentiation can
be seen.

CONCEPTS AND NATURE OF DORMANCY IN BULBS

Concepts of Dormancy

The notion of dormancy in bu1bs has not a1ways been we11-defined

and is often rather subject to discussion. A review of dormancy
in bu1bs and corms has been made by Kamerbeek et a1., (72). Refer-
ring to the definition of dormancy proposed by Amen, (73) these
authors have c1assed the bu1bous species in three groups.

The first group inc1udes species 1ike gladio1us, 1i1ium, a11ium,

etc ••• , which show a relatively long per iod during which differentia-
tion of new organs as we1l as their elongation are tota11y interrupt-
ed. This period, which occurs after the bu1b or corm has been form-
ed, would correspond to the state of dormancy. Dormancy would dis-
PHYSIOLOGY AND STORAGE OF BULBS 217

appear slowly, and would sometimes last for several months as in the
case of gladiolus cormels (54).

The second group includes bulbs in which hardly any true physio-
logical dormancy can be observed : tulipa, hyacinthus, narcissus,
etc ••• In these species, dormancy would be very slight as can be
noted in the case of the tulip where bulb formation is immediately
followed by flower bud differentiation. The leaves and the flower
can grow even if the bulb is not subjected to low temperature. Only
the flower shoot does not show practically any growth in the absence
of low-temperature treatment. Dormancy would therefore be restricted
to the stern. In opposition to the dormancy of the first group
(gladiolus, etc ..• ), which begins before flower formation, dormancy
in the tulip will become manifest after flower formation.

The third group includes species like bulbous iris, in which

apparently no physiological dormancy can be observed. At bulb
lifting, initiation of leaf primordia has already started, and their
growth can be observed if bulbs are exposed to a mean ternperature.
On the other hand, if bulbs are stored at high ternperature, > 25°C,
the growth of leaf primordia is arrested. This would be a typical
case of imposed dormancy.

The concept proposed by Kamerbeek et al., is based first of all

on "the fact that many bulbs and corms only develop weIl after having
been subjected to aperiod of cold. Therefore, it seern justifiable
to suppose that during certain developmental periods, these plants
are in dormancy that can be broken by cold." Besides, Kamerbeek
et al. only take into account the ability of the floral bud to
develop new organs arid to elongate.

When comparing the basis of this classification with the be-

haviour of tulip bulbs, iris bulbs and gladiolus corms during and
after storage, two remarks have to be made. First, it is not always
low ternperatures which accelerate the development of a bulb. Second,
it has been shown that the development of buds depends very greatly
on the physiological state of a bulb. The importance of the physio-
logical state of the gladiolus corm on the subsequent bud develop-
ment has also been demonstrated in in vitro culture (74).

It appears difficult therefore to dissociate a bud's behaviour

from a bulb's or corm's general evolution, and it seerns to us more
logical to consider the bulb in its entirety. The results reported
above for tulip bulbs, iris bulbs and gladiolus corms, show that it
is possible to discern a certain unity in the behaviour of these
species. In taking the period of bulb or corm formation as a start-
ing point, two important phases in their cycle can be seen.

During a first phase, more or less long according to the species,

no growth of daughter-organs is possible, even if the bulbs or corms
are placed in conditions which are favorable to the induction and/or
218 M. LENARD

the manifestation of growth processes. At the end of enlargement,

then in the course of storage or in the ground in natural conditions,
a physiological evolution takes place which leads the bulbs or corms
to acquire a growth capacity. According to whether the species grows
in spring and summer, gladiolus, or in winter and spring, tulip and
iris, this physiological evolution is accelerated, respectively,
either by low temperatures or by high temperatures.

Once the growth capacity has been acquired, the second phase,
which corresponds to growth, will only be able to come about rapidly
if the temperatur es change, high and mean temperatures for gladiolus,
low and mean temperatures for tulip and iris.

In the case of the gladiolus corm, the first phase coincides

with the period of dormancy proposed by Kamerbeek et al (72) a term
used moreover by many otherauthors.

In the case of tulip, we have shown (19,29) that as long as the

bulb has not reached its "physiological maturity," it cannot react
to external factors favorable to induction and manifestation of
growth processes. This situation seems close to astate of dormancy
which for Chouard (9) is "a state of rest for which the actual cause
resides in the dormant organ itself." This type of dormancy is
also called "true dormancy," "internal dormancy" or "rest" (75).

The behaviour of iris bulbs is very similar to that of tulip

bulbs, as has been shown above (see section BULBOUS IRIS).

If it can be accepted that we apply the term dormancy to a

bulb in its entirety, it then appears that the per iod of bulb dor-
mancy could be defined as being the period during which a bulb is
not able to react to environmental factors which induce and/or
allow the manifestation of growth processes. In the case of the
tulip, iris and gladiolus, this period of dormancy would begin during
bulb or corm formation and would end all the more rapidly when the
bulbs or corms are then subjected to favorable temperatures in the
ground or during storage: low temperatures for gladiolus, high
temperatures for tulip and iris.

It can be noted here that temperatures which promote bulbs'

emergence from dormancy are also those which, later, slow down
their physiological evolution, in this sense they are unfavorable
to the induction of growth processes. After emergence from dorman-
cy, bulbs and corms would then stay quiescent if the temperatures
do not change. Temperatures wh ich accelerate dormancy release
would also be those which allow long-term bulb storage.
Two major characteristics of the bulb dormancy per iod appear
PHYSIOLOGY AND STORAGE OF BULBS 219

to be the following. First, the observations made in the case of

tulip and iris bulbs show that during the per iod of dormancy, bulbs
are in such a physiological state that their reserves would not be
available for the daughter-organ growth (19,22). High temperatures,
which lead to an earlier and a more rapid reserve mobilization when
the bulbs are subsequently placed in environmental conditions which
allow the actual mobilization, that is, low and mean temperatures,
therefore accelerate bulbs' physiological evolution towardsemer-
gence from dormancy. Dormancy release would be gradual, the bulbs
becoming more and more sensitive to the action of low temperatures.

The second characteristic of bulb dormancy per iod concerns

organogenesis. It has been pointed out that organogenesis begins
during bulb or corm formation, but it is not however very advanced
at the end of bulb or corm enlargement. It can only become active
after the bulbs or corms have been subjected either to high tempera-
tures for tulip and iris, or to low temperatures for gladiolus, that
is, temperatures which promote dormancy release. The period of bulb
dormancy would correspond then also to aperiod of low meristematic
activity. However, as already mentioned above, the physiological
evolution of bulbs or corms towards emergence from dormancy, can
occur in the absence of any visible organogenesis. Thus, it appears
that the physiological evolution of storage tissues, and eventually
that of the basal plate tissues, could proceed independently of
that of the buds. These would express growth potentialities condi-
tioned by the physiological state of other tissues (storage tissues,
basal plate). The type of growth would also depend on the state of
the bud, its degree of differentiation and the correlations it is
subj ec ted to.

The term of dormancy, although it is not weIl defined has al-

ready been applied to the iris bulb by different authors (48,76,77).
Tsukamoto (77) considers that high temperature breaks dormancy in
winter-growing bulbs and that low temperature breaks dormancy in
summer-growing bulbs. This agrees with the concept proposed here.

The behaviour pattern which has just been presented for tulip
and iris bulbs, and gladiolus corms, seems to be relevant to other
species. For example, the results of Aoba, (78,79) show that freesia
corms behave very similarly to tulip or iris bulbs. Moreover, it
has long been admitted that high temperatures break dormancy in
freesia corms (10). In the case of narcissus bulbs, whose tempera-
ture requirements for development are very similar to those of tulip
bulbs (11,15), we have observed (unpublished result) that a low-
temperature treatment applied before the end of bulb enlargement,
did not lead to any stimulation of the subsequent daughter-organ
growth, but, on the contrary, had a retarding effect as in the case
of the tulip.
220 M. LE NARD

The concept of bulb dormancy as it has just been proposed for

the tulip, iris and gladiolus, seems applicable to a larger number
of bulbous species whose behaviour patterns appear then more homo-
geneous. The length of the per iod of bulb dormancy can however vary
from one species to another : it is relatively stort in iris while
it can be long in gladiolus. For a species, according to the growing
conditions (year, area, etc ••• ), the length and the degree of dorman-
cy would also vary.

In conclusion, this short discussion of the problem of dormancy

in bulbs brings to the fore differences of conception in the approach
of the phenomenon and shows, once again, the difficulties encountered
on the level of terminology.

It can be noted here, that, apparently, a similar situation

exists in the case of another storage organ, the potato tuber. In
this plant, according to Burton (80,81) dormancy would begin at tuber
initiation, but the concept of dormancy raises the same problems as
in the case of bulbs, for if many authors (see, for ex. Rappaport
and Wolf, (82) Burton, (83»talk of bud dormancy, some results (Madec
and Perennec (84), Bailey et al.(85» suggest that both the buds and
the storage tissues exhibit dormancy, and that breaking of this dur-
ing after ripening operates independently.

Finally, perhaps, it ought to be considered as with de Hertogh

(32) that "the question of dormancy in bulbous plants is highly
academic." while recognizing that "the fact is that bulb species
have evolved mechanisms to survive adverse climatic conditions and
man has attempted to describe these processes." What is sure is that
our understanding of these processes is very limited, especially in
some species.

Breaking of Dormancy and Biochemical Bases of Bulb Dormancy

In horticulture, the existence of bulb dormancy, in the sense

we have defined it, constitutes an obstacle when early flowering is
desired. For this reason a great deal of work has been carried out
to determine the factors which can be used to reduce the length of
the dormancy period.

The role of temperatur es has already been pointed out, so only

a short review of the chemical substances which break dormancy will
be made. The major observations concerning the biochemical bases of
dormancy will also be reported.

Extensive research on these subjects has especially been done

in the case of a species like gladiolus whose corms and cormels
sometimes show long periods of dormancy. In most other species
knowledge is much more limited.
PHYSIOLOGY AND STORAGE OF BULBS 221

Gladiolus. Corm and cormel sprouting can be accelerated by a

certain number of chemical substances among which are : ethylene
chlorhydrin, methanol, 2-chloroethylphosphonic acid (2-CEPA), aqueous
solution of calcium cyanamide, cytokinins (55,59,60,86,87,88,89,90,91
92). The action of these substances is more or less pronounced and
can vary according to the conditions in which they are used. Thus,
2-CEPA which accelerates sprouting of dormant corms and cormels,
tends to inhibit or delay sprouting of corms or cormels previously
stored at low temperature, that is, which are no longer dormant.

Proof of the efficacity of cytokinins in breaking dormancy of

corms (91) and cormels (55) is at the origin of work which has
furnished some information on the mechanisms of dormancy regulation
(55,56,74,77,92,93). These different authors have proved in the
case of corms and cormels the presence of inhibitors, including
abscisic acid, fatty acids (linoleic, linolenic, stearic, palmitic)
and phenolic compounds. Tsukamoto (77) has shown synergism in the
inhibitory activities of these substances.

The inhibitor content is related to the degree of dormancy and

corms or cormels of slightly dormant cultivars contain less than
those of very dormant cultivars. The abscisic acid level in dor-
mant cormels is 5 to 10 times that of non-dormant ones. The in-
hibitor content decreases during a low-temperature storage which
allows dormancy release. On the other hand, applications of
abscisic acid, fatty acids and phenolic compounds inhibit sprouting
of non-dormant cormels.

Abscisic acid and benzyladenine exert antagonist actions, but a

treatment with benzyladenine which breaks dormancy, does not lead to
a decrease in the content of inhibitors. It would prevent or counter-
act their activities.

Word done by Yazawa (74) shows that during a low-temperature

storage, which eliminates dormancy, it is difficult to detect any
cytokinin activity. However, if the corms are subsequently placed
at high temperature, cytokinin activity rapidly increases. Low
temperature would therefore induce cytokinin synthesis, and the
actual synthesis takes place later, at high temperature. This syn-
thesis would be necessary to allow sprouting, the decrease in inhib-
itor content not on its own being able to explain this. Antagonism
between cytokinin and inhibitors in breaking dormancy would be re-
lated to RNA metabolism.

Gibberellin treatments do not break dormancy of gladiolus corms.

However, Halevy et al. (94) have shown an evolution in the relative
importance of three forms of gibberellin during dormancy release,
an evolution which could affect synthesis of enzymes influencing the
breakdown of reserves in the storage tissues.
222 M. LE NARD

All these resu1ts together show that dormancy regulation in

gladio1us corms and corme1s wou1d resu1t from a balance between
inhibiting and promoting substances.

Other species. Most of the other bu1bous species do not seem

to have been the object of such detai1ed study as the gladio1us.

In the bu1bous iris, sprouting can be acce1erated by soaking

bu1bs in a solution of 2-CEPA (89). Dur own observations (unpub-
1ished resu1ts) confirm this resu1t and show that the inf1uence
of 2-CEPA is very comparab1e to that of high temperature in that it
promotes simu1taneous1y subsequent f10wer initiation and a more
rapid growth of f10wer shoot. The effects of high temperature and
2-CEPA seem moreover to add to each other. These resu1ts have to
be compared to the observations of Stuart et a1. (3) and Uhring (48)
concerning the inf1uence of bu1b exposure to ethy1ene, and those
of Durieux and Kamerbeek (95) and Kamerbeek et a1. (96) concerning
the inf1uence of plant spraying with 2-CEPA. The mechanisms of
action of ethy1ene are not known however. Tsukamoto and Ando (76)
and Tsukamoto (77) have shown the presence of inhibitors, abscisic
acid and fatty acids (capric acid), in iris bu1bs. Storage at high
temperature decreases the inhibitor content and wou1d thus a110w
dormancy release. It must also be pointed out that Rodrigues
Pereira (97,98,99,100) has shown that iris bu1bs contain a certain
quantity of free gibbere11in.

In the case of tu1ip bu1bs, very few studies have borne on what
we have defined as being the dormancy period, that is the per iod
which precedes the "physio1ogica1 maturity." During bu1b formation,
the presence of abscisic acid and the existence of a gibbere11in
activity have been proved (101,102,103,104,105). These substances
also exist in the bu1b after lifting (106,107). Ito et a1. (108)
have shown that during storage, inhibiting substances decrease whi1e
auxin content increases in sca1es. But, in the tu1ip, the inf1uence
of growth regulators and their eventual ro1e have especia11y been
studied in relation to the possibi1ities of f10wer shoot elongation.

In the case of 1i1y bu1bs, gibbere11in treatments wou1d promote

sprouting (72). Wang and Roberts (109) and Tsukamoto (77,110) have
proved the presence of inhibitors and auxin, A.I.A., in the bu1b
sca1es. The young sca1es contain more inhibitors than the old ones
and their removal favors sprouting. Removing old sca1es can, on
the contrary, de1ay sprouting. The inf1uence of sca1e removing
seems however to vary in time.

The observations made in the case of 1i1y bu1bs show some

ana10gies with those made on the onion by Kato (111,112,113,114).
This author distinguishes between a rest period and a dormant per iod
in the onion bu1b.
PHYSIOLOGY AND STORAGE OF BULBS 223

In the hyacinth, a study of the possible role of growth regula-

tors in the development of the plant has been made and a conception
of the hormonal control of growth and development has been suggested,
(115). From this study, where dormancy applies to flower stem elonga-
tion, it appears that knowledge relative to bulb formation is still
quite limited.

CONCLUSION

This review of the physiology and storage of bulbs, although

limited to three species, has nevertheless made it possible to prove
on the one hand the importance of storage temperature in bulbs' phys-
iological evolution, and on the other a certain unity in behaviour
of species which at first glance seem quite different.

Certainly, according to the species, a same temperature does

not have the same effects, but this must be related to the cycle of
the species in its original environment. Thus, tulips which come
mainly from central Asia and Asia Minor, grow in winter and spring,
and are apparently at rest during the hot dry summer. Gladiolus,
which come from the more or less mountainous areas of tropical
Africa, grow during the hot damp summer and are, also, apparently at
rest during the dry and cool season. It must be kept in mind that
during the apparent rest period or during storage, bulbs and corms
are continuously evolving, more or less rapidly according to the
temperature. In natural conditions, the temperatures encountered
during this period slow down the development of bulbs or corms in
that they are unfavorable to induction and/or manifestation of the
growth processes.

The influence of storage temperature on the behaviour of a

fairly large number or bulbs is at present weIl known and used in
horticulture to manipulate the bulbs in order to act on their flower-
ing. However, it must be recognized that our knowledge of the
fundamental processes which determine the behaviour of bulbs is
very limited, especially in some species. This is clearly shown
when it is a question of tackling a problem like dormancy. In this
field, and abstracting problem of terminology, it appears that in
species like tulip and bulbous iris very little is known about the
mechanisms which intervene in the formation of the storage organ
which enables these plants to survive.

This situation results perhaps in part, from the fact that most
research work, often done for practical purposes, has been aimed at
studying flowering while relatively little work has borne on bulb
formation and its physiological evolution. Yet, it appears obvious
that basic research on this subject is necessary if we want to
understand how a bulb works. Such research could have practical
applications in two areas: the characterization of the physiological
 224 M. LE NARD

state of a bulb, and the use of growth regulators to replace, at

least partially, some long and costly temperature treatments. The
possibility of characterizing the physiological state of bulbs would
also allow a more rational application of temperature treatments at
present in practice.

Fundamental research would also furnish some information on

floral induction and perhaps would make i t possible to determine why
small bulbs do not initiate flowers.

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PHYSIOLOGY AND STORAGE OF BULBS 227

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(1966).
113. T, Kato, Physio1ogica1 studies on the bu1bing and dormancy of
onion p1ants. IX. The relationship between bu1b dormancy
and the components of the juice. J. Jap. Soc. Hort. Sei.,
35, 297 (1966).
114. T. Kato, Physio1ogica1 studies on the bu1bing and dormancy of
onion p1ants. X. A germination inhibitor in onion juice.
J. Jap. Soc. Hort. Sei., 35, 395 (1966).
115. R. M. Rudnicki, Hormonal contro1 of growth and deve10pment of
hyacinth. Acta Hortic., 91, 185 (1979).
THE FORMATION OF ENZYMATIC PRODUCTS IN THE FRUITS DURING GROWTH

AND STORAGE

George Karaoulanis

Food Technology Institute

Lykovrissi-Amaroussion
Athens, Greece

INTRODUCTION

It is well-known that research on fruits is important not only

for their commercial and nutritive value, but also for the realiza-
tion that biological processes that are common to all living organ-
isms can be studied in fruits.

It is also known that there are several reasons for using

fruits for the study of the general biological problems of ageing.

The fruits are capable of prolonged independent existence even

after removal from the parent plant and as long as they are kept
under suitable conditions (humidity and temperature) they do not
require external supply of water to maintain the cells in a turgid
condition.

Most fruits are amply supplied with respiratory substrates,

in the ordinary sense, although the depletion of certain specific
substances required in small amounts and not synthesized in detached
fruits may playa role in the metabolie pattern. On the other hand,
the study of fruits avoid complexities resulting from the interplay
of several processes, such as happens with photosynthesis and
respiration in leaves.

It is well-known, that the central reactions in ripening are

biological oxidation. This term is used advisedly to include gly-
colic as weIl as respiratory activities.

The former refers to the conversion of carbohydrates to pyru-

vic acid in the presence or absence of oxygen. The pyruvic acid is

231
232 G. KARAOULANIS

converted in the absence of oxygen to fermentative end-products

such as alcohol, lactic acid, and propionic acid. The term respira-
tion is reserved then for the reactions requiring oxygen for the
enzymatic oxidation of carbon compounds to CO 2 , water and energy.
Anaerobic respiration of higher plants was first noted (as evolu-
tion of CO 2 in absence of air) by William Cruikshann in 1797. In
1872 Pasteur and also Lechartier and Balley, reported formation of
alcohol by apples and pears in absence of oxygen and Pasteur said
this was due to the'chemical and physical life of the cells" and
linked it to "that of the cells of a ferment" (yeast).

Argument followed amongst plant physiologists, some (including

de Saussure) holding that aerobic and anaerobic respiration were
distinct and separate, while others (Pfeffer, Palladin 1900-1903)
thought they were basically similar, and that fermentation was the
first stage in respiration, the second stage being oxidation of
alcohol.

In 1913 Kostytschew, on the basis of experiments in wh ich he

measured the CO 2 : Alcohol ratios, concluded that anaerobic respi-
ration was not Identical with respiration. Boysen-Jensen (1932)
was rather more cautious, concluding that there is not necessarily
any connection.

Then work on the intermediate metabolites notably by Newberg,

Meyrhof, Embden and Parnas, built up the modern theory, in which
aerobic and anaerobic respiration are linked by common intermediates.

In subsequent works Thomas (1925) and Fidler (1933) observed

that the anaerobic respiration of carbohydrates by apple fruits
is qualitatively and quantitatively similar to alcohol fermentation
of yeast.

This presentation is an attempt to review the research works

on the formation of the anaerobic enzymatic products (ethyl alcohol
and acetaldehyde) in the fruits,the causes of their concentration,
and to discuss whether the concentration of ethyl alcohol can be
used as an additional criterion for the determination of a) the
stage of maturity of the fruits, b) the duration of their preserva-
tion under different storage conditions,and c) the optimum storage
condi tions.

Formation of Enzymatic Products in the Fruits During Their Growth

G. Karaoulanis (1968,1978 and 1979) and P. Davis (1970) observed
that during the growing of citrus fruits, mainly oranges, the in-
crease of alcohol (ethanol) concentration is higher than the ratio
Brix/acidity, Index which is used for the determination of their
maturity.
THE FORMATION OF ENZYMATIC PRODUCTS 233

In experiments with oranges of W. Mariel, Valencia and Marsh

and Ruby Red variety, picked at different steps of maturity (during
their ripening) the concentration of ethyl alcohol in the peel and
in the flesh during ripening was observed. (Table 1.). Similar ob-
servations were reported for apples (several years ago) by other
researchers (Fidler. 19S1).

Formation of Enzymatic Products in the Fruits During Their Storage

in Air and Under Different Temperatures

Apples of the varieties Newton Wonder and Bramley's Seedlings

stored at 3°C showed low concentrations of alcohol and acetaldehyde.
However, when exposed to high temperatures even for a short period
of time, i.e. at 18°C, the concentration of the above substances
increased in general and more markedly during their senescence
(Fidler, 1933) (Table 2.). The same was observed when the oranges
W. Navel were kept at 2.SoC, SoC and 10°C in air (Karaoulanis, 1979).

Formation of Enzymatic Products in the Fruits During Storage Under

Low 02 Concentration

High quantities of ethyl alcohol and acetaldehyde accumulate

in apples of different varieties (Thomas and Fidler, 1933) and
oranges W. Navel and Valencia (Karaoulanis, 1969) and in most
vegetable tissues, when preserved under low oxygen concentrations.
This was verified by the work of Furlong (1961) for apples of the
Coxes variety which showed very high concentrations of enzymatic
products when preserved under anerobic conditions, compared to
those developed und er normal conditions. He also observed that the
Coxes apples produce more alcohol than the Bramley's Edward or
Cox's Orange Pippin apples in a low oxygen concentration environment.
Work at Ditton Laboratory has shown that ethanol accumulation in
Cox's Orange Pippin, Barnack and "Lambourne" apples stored at 02
levels below 2% at 3.SoC increases. Further experiments with Cox's
Orange Pippin and Bramley's Seedling held for 7 days in 0% 02 at
3.SoC and then in 3% 02 at the same temperature yielded the follow-
ing results: After 7 days at 0% 02' the ethanol content of Cox's
Orange Pippin was 2S0 mg/100 gr. and Bramley's Seedling 140 mg/100 ge
of flesh (Furlong, 1961b).

Fidler (19Sl) also studied the effect of air and N2 atmospheres

at various temperatures on the loss of carbohydrate and acid and
the production of C02 and ethanol in Sturmer Pippins and Bramley's
Seedling apples. The experimental results indicated that the rate
of acid loss was not affected by the presence or absence of 02;
the presence of 02 had a converse effect on the loss of carbohydrate
and acid, accounted quantitatively for the production of C02 and
ethanol in air and in N2 atmospheres.

It was also observed that during anaerobiosis the apples

234 G. KARAOULANIS

Tab1e 1. Month1y averages of ethanol, solids, acid and pH of juice

of Ham1in and Valencia oranges

Ethanol Solids Acid Solids/

Variety Season Month (mg/100 m1) (%) (%) acids pH

Hamlin on C1eo-
patra mandarin 1
rootstocks 1968-69 October 4.0 9.3 1.05 8.9
Do 1968-69 November 20.0 9.5 0.89 10.6
Do 1968-69 December 25.4 10.4 0.87 12.0

Do 1969-70 September 0.2 8.9 1.46 6.3 3.044

Do 1969-70 October 1.7 9.4 1.04 9.1 3.243
Do 1969-70 November 9.3 10.1 0.94 10.7 3.371
Do 1969-70 December 24.5 10.8 0.91 12.0 3.407

Hamlin on sour
orange root-
stock 1969-70 September 0.2 9.2 1.53 6.1 3.057
Do 1969-70 October 0.8 9.4 1.21 7.9 3.043
Do 1969-70 November 9.1 10.0 0.97 10.3 3.333
Do 1969-70 December 38.1 10.6 0.94 11. 2 3.417

Valencia on
rough 1emon
rootstock 1968-69 October 0.5 8.5 2.83 3.0
Do 1968-69 November 4.7 9.1 2.28 4.0
Do 1968-69 December 9.5 10.6 1.88 5.8
Do 1968-69 January 28.9 11.3 1.67 6.7
Do 1968-69 February 40.0 11. 7 1.46 8.0
Do 1968-69 March 45.7 12.4 1. 29 9.7
Do 1968-69 April 48.0 11.8 1.04 11.5

1Figures represent an average of four week1y samp1ings of 10 fruits

each. From Davis 1970

showed an increase in the rate of ethyl a1coho1 accumu1ation, with

a subsequent increase in acetaldehyde. When app1es are fed with
ethanol, the ratio acetaldehyde to ethanol remained the same as in
the absence of ethanol feeding.

Formation of Enzymatic Products in the Fruits During Storage under

High CO 2 Concentrations

The high concentrations of ethanol and aldehyde observed in

fruit tissues, main1y app1es, oranges etc. during their preservation
 -i
::c
m
"o:0
s:
»
-i
Tab1e 2. Inf1uence of the harvest, the temperature and the duration of the storage on the concen- o
trat ion of ethyl a1coho1 in the flesh of oranges of the W. Nave1 variety (mg. of ethyl Z
a1coho1 per 100 g of f1esh weight) o
"m
Z
Days CONDITIONS OF PRESERVATION N
-<
in 1st Harvest 2nd Harvest 3rd Harvest s:
storage O°C 2.5°C 5.0°C 10°C Mean O°C 2.5°C 5°C 10°C Mean O°C 2.5°C 5°C 10°C Mean »
-i
()
0 8.60 8.60 8.60 8.60 8.60 8.80 8.80 8.80 8.80 8.80 17.57 17.57 17.57 17.57 17.57 -0
60 27.20 13.44 26.50 23.50 22.66 19.60 8.97 21.50 22.50 18.13 11.12 15.52 33.95 39.26 24.96 :0
o
120 29.19 28.80 29.92 50.12 34.51 27.15 14.84 24.94 27.80 23.64 37.42 20.83 38.36 81.12 44.43 o
38.45 33.64 36.94 37.72 36.68 53.00 35.24 50.29 84.41 55.73 c
160 77.40 41.46 48.70~6.61 81.09 ()
-i
Cf)

Mean 34.09 23.07 28.43 59.68 23.51 16.50 22.83 24.20 29.78 22.29 35.04 55.59

L.S.D. 5% 1% 5% 1% 5% 1%
Btn Cond.Pr.* +1.620 +2.240 +0.250 +0.344 +0.215 +0.296
11
Days 11 +1.620 +2.240 +0.250 +0.344 +0.215 +0.296
Interaction +3.240 +2.240 +0.501 +0.690 +0.430 +0.593

*Between Conditions of Preservation

From Karaou1anis 1979

w
'"
U1
236 G. KARAOULANIS

und er high concentrations of CO 2 are due either to the anaerobic

conditions or to damage in the fruit tissues or to both reasons.

Thomas (1925) attributed the severe damages of the fruit cells

to the "C02 zymosis" and to the high concentration of aldehyde
(poisoning of their internal atmosphere).

Also Fidler (1951) studying fruits preserved und er high C02

concentration observed the following:
a) The rate of increase of acetaldehyde concentration is higher
than that of ethanol.
b) The appearance of injuries is speeded as the C02 concentration
increases.
c) When the C02 concentration was higher than 30% the fruits showed
damages similar to Brown Heart.
d) The different varieties of apples have different sensitivity to
the high CO 2 concentration, i.e. the Newton Wonder variety is
more sensitive to injuries than other commercial varieties.
e) The CO 2 zymosis can be induced in a wide variety of plant tis-
sues. Already in 1932 it was observed that when the C02 concen-
tration in the storage room is higher than 80% the ratio of
ethyl alcohol to acetaldehyde concentration produced in the
plant tissues can have the following values:

a. 30:1 to 100:1 in swede, beet, horseradish, potato, barley

seedlings, pea seedlings.
b. 3:1 to 10:1 in pears, oranges, bananas.

Formation of Enzymatic Products in the Fruits with Internal or

External Physiological Disorders

As to whether zymosis, precedes 1nJury, or injury leads to zy-

mosis, Thomas (1929) came down firmlyon the side of disorganization
of state of the tissue being the precursor of ~ymosis. Part of the
evidence on which this conclusion was based is given in effect of
injury of surface tissue of Newton and Wonder apples by tapping
with a pestle, in which the external tissue was mechanically damaged.
This damage leads to increase of alcohol number (i.e. aldehyde+
alcohol, no separate figures are given).

Effect of injury of surface tissue by tapping with a pestle

gave the following results:
Newton) 1 .. (Whoie apple 100 mg Alcohol/lOO gr.
Wonder) app e var1et1es (Untreated apple 6 mg 11 / \1

Further, Thomas (1929) showed that subjecting apples to -5°C

for 3 days led to an increased "alcohol number" when kept 3 days
subsequently at ordinary temperatures. Complete killing of the
tissues at -10°C for 7 days has less effect. A similar autolytic
THE FORMAnON OF ENZYMATIC PRODUCTS 237

effect was found in work by Fidler when apples were treated with
chloroform vapour.

Anaerobic zymosis is characterized by a high proportion of al-

cohol related to aldehyde; in low temperature injury, senescent
breakdown and brownheart, the quotient alcohol/aldehyde is lower
and in freshly damaged "brownheart" tissue can be as low as unity.

a. Low temperature injuries, freezing injuries etc. It i8

known that the injury becomes more severe at ordinary temperatures,
subsequent to storage at very low temperature. It is observed that
in Coxes apples the enzymatic products increase with increased in-
juries.

On the other hand, Wills and McGlasson (1968, 1971) observed

that the appearance of low temperature breakdown is related to the
presence in the apple tissue of esters of Oxalic Acid and Alcohols,
compounds which appear in high concentrations at low temperatures.
Also, the hardening of the skin, because of low temperatures, seems
to prevent the diffusion of gases leading to accumulation of CO 2 and
the appearance of anaerobic environment in the fruits.

b. Brownheart: It has been stated previously that a feature of

brownheart, when the injuries are of recent occurrence, is the high
concentration of acetaldehyde. If, however, the injury took place
some considerable time before the trouble was evident, and the in-
jured zones dried out to form the characteristic cork cavities then
it has been found that both acetaldehyde and ethyl alcohol have to
some extent disappeared.

c. Breakdown: Neal and Hulme (1958) showed that the peel of

ßramley's Seedlings apples decarboxylated malic acid to acetalde-
hyde. The NADP-specific malic enzyme was isolated from McIntosh
and Cox's Orange Pippin apples (Dilley et a1. 1964, Hu1me et a1.
1962). Also C1ijsters (1965) has found that a) The malic acid con-
tent of diseased "Jonathan app1es" decreased and that severa1 vo1-
ati1e products accumu1ated, especia11y ethanol (l963 a and band
1965 b). "Jonathan" app1e tissue decarboxy1ates malic acid to
acetaldehyde and ethanol. Malic acid decarboxylation occurs as weIl
in healthy apples as in diseased fruits affected by "Jonathan"
Internal Breakdown.

Formation of Enzymatic Products in the Fruits During their Storage

Work with apples, pears (Furlong 1961 and Karaoulanis 1968) and
oranges (Karaoulanis 1978) showed that the concentration of anaerobic
zymosis products increases with time of preservation (Table 2)even
at the suitable conditions.

In the case where the concentration of ethanol was higher than

238 G. KARAOULANIS

150-1500 mg/100 gr. of fresh weight, the fruits were unsuitable for
consumption.

Changes in the Internal Atmosphere of Fruits During Senescence and

Their Influence on Metabolism

Changes in the internal atmosphere of the fruit during ripening.

The relationship between respiratory activity, its course and the
composition of the internal atmosphere has been studied in several
kinds and varieties of fruits.

In the case of banana (Musa Sapientum) and papaya (Carica

papaya) Wardlaw and Leonard (1939) observed that the climacteric
phase coincides with the maximum concentration of CO 2 inside these
fruits. This concentration drops as the respiratory activity in-
creases and reaches the lowest value in the last stages of senescence.
These remarkable changes in the composition of gases inside the
fruits led the above reserarchers to conclude that the concentration
of 02 in their internal atmosphere regulates the mechanism of the
climacteric phase.

Trout and his colleagues (1960) reported also that the lack of
02 depresses the respiratory activity and delays the ripening of
mature apples. They also showed that Granny Smith apples stored at
7°C and 29°C in air had 17% 02 and 2% respectively in their internal
atmosphere, while the CO 2 concentrations were 2% and 17% respectively.

Ulrich (1958) and Ulrich and Marcellin (1955) showed thatthe

highest concentration of CO 2 in the intercellular space occurs dur-
ing the climacteric peak. They also investigated the percentages
of gases in the internal atmosphere and made observations on the
epidermal layers as the tissue that offers the major resistance
to gaseous diffusion for the apples.

Smith (1954) reported that the maximum concentration of 02 is

found near the epidermis and drops as we proceed to the center of
the fruit, while the CO concentration increases. Biale et al.
(1962) observed that after the climacteric phase the concentration
of C02 in the intercellular spaces increases while that of 02 de-
creases due probably to their filling with fluids from the surround-
ing cells. The rate of fluid leakage from the cells increases with
ripening and this is a factor leading to the senescence of the
fruits.

Henze(1969) in aseries of experiments showed that the decrease

of the percentage of the intercellular space combined with the high
respiratory activity leads quicker to the maximum tolerable CO 2 and
02 concentration level, above which anomalous metabolism takes place
in the fruit.
THE FORMATION OF ENZYMATIC PRODUCTS 239

Ben-Yehoshua et al. (1963) observed for the Avocado fruits

that there is a correlation between internal atmosphere and respi-
ration rate for the lag per iod and respiratory rise; upon softening
this correlation changed. Despite the decline in respiratory activ-
ity the oxygen percentage dropped to as low as 1-2% and CO 2 per-
centage rose to 15%.

The cross-sectiona1 area of air-space of firm fruit was ca1cu-

1ated to be at least four times 1arger than the cross-section of
air space of ripe fruit.

Lyons et a1. (1961) experimenting with Cantaloupe fruits found

simi1arity to the changes in the internal concentrations of CO 2 and
02 in canta10upe to those reported ear1ier by Ward1aw and Leonard
(1939) for bananas.

To conc1ude from all the above observations one can say that
during senescence the atmosphere within the fruits changes, result-
ing in an increase of C02 and decrease of 02 concentration. The
new environmental composition depresses the oxidative processes and
favours the accumulation of enzymatie produets.

Changes in the interna1 atmosphere of oranges and app1es.

Henze (1969) experimenting with app1es of the A1exander Lueas and
Coxes Orange varieties observed that as the temperature inereases
the eoneentration of C02 inereases and that of 02 decreases within
the fruits with an inerease of ethyl a1eohol aceumu1ation in the
same time.

Eaks and Ludi (1960) have shown that the interna1 atmosphere
of eitrus fruits is affeeted by temperature, washing and waxing.

Vines and Ober1aeher (1961) found that eertain paeking house

treatment inereased C02 eoneentrations within eitrus fruits.

Also Davis et al. (1967) found for the Temple oranges, that
the internal 02 and C02 of fruits held at O°C were usually within
1% of those of the external eoneentrations. Fruits held at 4.5°C
varied somewhat more in their internal 02 and C02 levels. This
is probably due to the greater metabolie aetivity at 4.5°C, where-
in 02 eoneentration tended to be lower and CO 2 higher. Waxed fruits
eonsistently had lower 02 and higher C02 in the internal gases than
in the atmosphere.

Aeeording to Ben-Yehoshua (1969) the eommereial degradation

of oranges is eonneeted with transpiration, the respiratory aetivity
and the eoneentrations of CO 2 , 02 and ethylene within oranges. He
also observed that during storage of oranges their respiratory
aetivity deereases, the interna1 eoneentration of CO 2 inereases
240 G. KARAOULANIS

from 2-4% to 5-10%, and that of 02 decreases from 17-19% to 10%

and even lower.

The epidermis mainly the flavedo hardens resulting in a reduc-

tion of gaseous diffusion and a change in the composition of the in-
ternal atmosphere.

Acetaldehyde and Ethanol Metabolism' in Plant (fruit) Tissues

As it 'is already shown many plant tissues are known to produce

ethanol when they are subjected to experimental stresses such as
low oxygen supply or a variety of respiratory inhibitors. Ethanol
production has also been observed und er natural conditions in such
tissues as germinating seeds, fruits and root tips (Cossins et al.
1963). There is one question whether ethanol, particularly when it
has accumulated in periods of natural or imposed anaerobiosis, can
be metabolized when the tissues subsequently gain better access to
°2'
The question has two directions: a) Whether plant tissues
which have become alcoholic as a r~sult of accidental deprivation
of oxygen during storage, can be rendered palatable and b) what is
the fate of any alcohol which may be lost. On the first point,
that of disappearance of alcohol formed within the tissues in the
absence of oxygen, Furlong (1961) gives some figures in the D. L.
Annual Reports for 1960 and 1961. Typical data are given in Table 3.

Two further sets of data exist, one relating to a sample from

Norfolk fruit growers, from a store which accidentally went anaero-
bic, and tue other obtained by Karaoulanis (Table 4).

From Tables 4 and 5 it will be seen that alcohol disappears

from Coxes apples in air at 38°F only very slowly, the rate of loss
in 3% 02 at 38°F is of the same order as in air. At 65F the rate
of loss is more rapid, but if the initial concentration is of the
order of 150 mg/100 gr. or higher, the apples lose quality before
they are acceptable to the taste. A low oxygen store can probably
be anaerobic for a maximum of about 6 days, after which the apples
would slowly recover in 3% 02 at 38°F or could be marketed after
10-14 days at 65°F.

Turning now to the question of the fate of alcohol which is

lost, Fidler (1951), gives data on the disappearance of alcohol
fed as vapour to apples in air (Table 5). Analyses of the data
show that in the first periods of aeration, for both Sturmers and
Dunns, the alcohol could have been oxidized. But in periods (ii)
and (iii) for Sturmers and (ii) for Dunns, the C02 and oxygen
THE FORMATION OF ENZYMATIC PRODUCTS 241

Table 3. Changes in concentration of alcohol and aldehyde in Coxes

Apples

mg/lOO g Alc./ Loss of Loss per

Sample ald. alc. ald. alcohol day

Initial 152
After 7 days, AIR,65°F 62 90 13
Initial 302
After 26 days,AIR,65°F 52 250 10
Initial 4 207 70
After 9 days,AIR,65°F 3 123 41 84 9
" 11 " 59 148 13
Initial 3 169 57
After 9 days ,AIR, 65°F 98 71 8
Initial 4 207 70
After 28 days,3% 02,38°F 3 134 41 73 2.5

From Furlong 1961b

equivalents of the alcohol lost exceed the observed total gaseous

exchange, and therefore the alcohol must have been lost by some
process other than oxidation.

Cossins and Beevers (1963) experimented with pears, beans and

corn on metabolizing of ethanol, observed clearly that all the tissues
investigated were 4able to convert the supplied ethanol labelled with
C14 (ethanol-I-Cl) into organic acids, aminoacids, lipids and sugars.

These observations are consistent with the conversion of ethanol

to acetyl coenzyme A, which is then involved in the reactions of
established pathways. Based on what has been already discussed, one
can say the following:
a) The presence of ethyl alcohol in the fruits is connected to their
maturity and accompanies them until their death.
b) The storage of fruits under different conditions leads to increase
in the concentration of ethyl alcohol. The presence of ethanol
in some cases results in such a change of their quality that
they become unsuitable for consumption.
c) Since there is a relation of concentration of ethanol to length
and temperature of storage and to the degree of maturity at the
time of storage, we would say that the content of ethanol, may be
used as an additional criterion for determination of 1) The
maturity of the fruits;2) the suitable storage conditions;and,
3) the length of their storage life.
 Tab1e 4. Changes in eoneentration of aldehyde and a1eoho1 in Cox's Orange Pippins, in air
~
'"
Subsequent history mg/lOOg. Loss of
'"
Sampie TOF days a1d. ale. ale. Notes

N.F.G. reed.22/11/65) initial sampies 4.5 312

N.F.G. " 24/11/65)
38 8 6.4 296 18
38 15 5.4 296 18
38 22
38 36 5.2 242 70
65 8 6.0 250 62
65 15 5.1 172 140
65 22 3.7 69 243 A1eoholie taste
65 36 2.2 23 289 Seneseent

Karaou1anis
After 6 days N2 ,38°F Initial 3.2 125
38 7 2.7 109 16
" 14 2.5 111 14
65 7 2.7 51 74
" 14 1.8 21 104 Yel1owing,norma1
After 12 days N2 , 38°F Initial 5.4 215
38 7 3.7 210 5
" 14 3.2 187 28
65 7 4.4 131 84 G')
" 14 2.2 64 151 Aleoho1ie
After 18 days, N2 ,38°F Initial 5.9 274 '"»
4.0 256 18 :rI
38 7 »
" 14 3.8 238 36 0
65 7 4.3 163 111 cr-
" 14 3.1 113 161 »
z
From Karaou1anis 1968 cn
Tab1e 5. Changes in concentration of aldehyde and a1coho1 fo110wing a1coho1 feeding (as vapour) -I
:J:
in air m
"Tl
mg/lOO g o
A1coho1 A1coho1 Lost ::c
Change in Transpired by Processes
s:~
-I
Days in concen.of into Air Other Than Ö
Date Variety TOF Air Aldehyde A1coho1 A1coho1 Steam Transpiration Z
o"Tl
X1.33 Bram1ey 68 Initial 3 148 m
2 4 156 +8 14 None, gain of 22 Z
11 " 11 N
9 4 143 -5 52 47 -<
15* 4 230 +80 84 " " " 164 s:
~
22 5 290 +140 115 " , " " 255 -I
*on this date, an untreated samp1e in air had formed 3 aldehyde + 100 a1coho1 (i
'"'\J
II.34 Sturmer 54 ::c
(i) After 1st period of feeding: o
C
Initial 0.7 64 c
("")
6 0.8 51 13 8 5 -I
(ii) After 2nd period of feeding:
cn
Initial 1.1 200
6 0.8 136 64 13 51
(iii) After 3rd period of feeding:
Initial 2 525
4 2 413 110 15 95
The R.Q. fell progressive1y from 1.35 initia11y to 1.00 at the end.
IV.34 Dunn's 54
Seed1ing (i) After 1st period of feeding:
Initial 1.6 163
4 2 144 19 9 10
(ii) After 2nd period of feeding: 6
Initial 4 602 6
4 4 430 170 17 153 6
The R.Q. fell progressive1y from 1.8 initia11y, to 0.95 at end. .j:>.
'"
w
From Fid1er (unpub1ished).
244 G. KARAOULANIS

REFERENCES

Ben-Yehoshua, S., Robertson, R. N. and Bia1e, J. 1963. Respiration

interna1 atmosphere of avocado fruits. Plant Physio1. 38: 194.
Ben-Yehoshua, S., 1969. Gas exchange transpiration and the commercia1
deterioration in storage of orange fruit. J. Amer. Soc. Hort.
Sci. 94:52L
Boysen-Jensen P. 1932. The gentic connection between normal and
intramo1ecu1ar respiration of p1ants. Kg1. Danske. Vidensk.
Se1sk. Bio1. Medde1. 4,1.
C1ijsters, H. 1963a. Omzetting van appe1zuurinetano1 bijverschi11ende
appe1varieteiten. Arch. Int. Physio1. Bioch. 71:127
C1ijsters, H., 1963b, Que1ques aspects biochimiques de 1a pourriture
de 1a pomme Jonathan, en frigo. Rept. XVI, 4th Int. Hort. Congress,
Brusse1s 1962 (Ed. Dueu10t S.A. Gemb1ous, 3:449.
C1ijsters, H. 1965. Ma1ic acid metabo1ism and initiation of the
interna1 breakdown in "Jonathan" app1es. PhysioL P1ant.18:85.
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THE FORMATION OF ENZYMATIC PRODUCTS 245

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HOST-PATHOGEN INTERACTIONS IN POSTHARVEST DISEASES

Joseph W. Eckert and Malinie Ratnayake

Department of Plant Pathology

University of California
Riverside, CA 92521

INTRODUCTION

Every fruit, vegetable and root crop is exposed to hundreds

of species of microorganisms during its lifetime yet the vast
majority of these are incapable of extensively invading the plant
tissues as long as the cells are living and the tissues are co-
herent. Approximately 25 species of fungi and bacteria are re-
sponsible for the major decays of plant products after harvest.
Most of these microorganisms have the potential for attacking only
a few different products from related species of plants. For ex-
ample, Penicillium digitatum causes green mold on citrus fruits
but does not cause disease in apples and pears. ~. expansum on the
other hand, attacks apples and pears, but not citrus fruits.
Successful pathogens must have the eapability to overcome the host
defenses, must be able to initiate growth under conditions of water
potential, pH and nutrients present in the host tissues, and must
elaborate enzymes that macerate host tissue and cause arelease of
nutrients required to sustain the indeterminate parasitic develop-
ment of the pathogen in the host tissues.

As living entities, the host and pathogen possess the potential

to interact with each other as well as with the physicochemical
environment. Their individual reaction to a stimulus may be vastly
different, however, because of physiological differences of the cells
of higher plants and microorganisms. For example, ethylene acceler-
ates senescence of the tissues of many fruits and increases their
susceptibility to attack by pathogenic microorganisms. Yet, this
plant hormone has no measurable effect upon most pathogenic micro-
organisms. Fungal infection of certain fruits is stimulated by
certain chemie al constituents of that fruit, whereas other chemieals

247
248 J. W. ECKERT AND M. RATNAYAKE

inhibit the growth of the pathogen in the host. The attacking patho-
gen may elicit the production of fungitoxic substances by the host
to ward off invasion. Finally, constituents of the host often in-
duce the pathogen to elaborate enzymes that destroy host tissues,
whereas other plant constituents inhibit the action of these enzymes.

Some host-pathogen interactions promote the development of

pathogenesis, whereas other interactions retard or prevent the
process. Since living fruits and vegetables are usually inhospitable
substrates for extensive invasion by most microorganisms, the un-
favorable interactions eVidently outweight the favorable interactions
in most plant-microorganism combination. The nature of these inter-
actions and their influence upon the development of postharvest
diseases is the subject of this chapter.

DEVELOPMENT OF POSTHARVEST DISEASES

Diseases of fruits and vegetables observed in the marketplace

and in the horne may be traced chronologically to: 1) infection or
infestation of the product during the per iod of its development in
the field, or 2) infection through wounds created during the har-
vesting operation and through physiological injuries resulting from
an unfavorable storage environment (Table 1). The subsequent prog-
ress of an infection depends upon the growth requirements and
enzymatic capabilities of the pathogen and upon the physiological
resistance of the plant tissue to invasion.

Table 1. Time and Site of Infection in some Representative Post-

harvest Diseases

Disease Pathogen Infection Site

1. Field Infection
Apple lenticel rot GloeosEorium lenticel
Banana anthracnose Colletotrichum epidermis
Peach brown rot Monilinia epidermis
Orange stem-end rot DiElodia calyx
Strawberry gray mold Botrytis flower
H. Postharvest Infection
Banana crown rot Colletotrichum crown cushion
Pineapple black rot ThielavioEsis fruit stem
Peach RhizoEus rot RhizoEuS surface injuries
Citrus blue-green mold Penicillium surface injuries
Tomato sour rot Geotrichum surface injuries
Potato soft rot Erwinia surface injuries
HOST-PATHOGEN INTERACTIONS 249

Infections of several species of fungi, especially if initiated

early in the growing season, enter a latent or quiescent state soon
after the initial stages are completed because the host tissues
resist further invasion. After harvest the fruit gradually loses
this resistance as it ripens, and the pathogen resurnes growth, giving
rise to an active decay lesion on the host. Diplodia, Phomopsis,
and Alternaria establish latent infections in the stern ends of citrus
fruits because this part is vulnerable to attack when the fruits are
very small. Alternaria mayaiso cause a latent infection in the
stylar end of the navelorange. Gloeosporium rots of apples arise
from quiescent infections in the lenticels of the fruits which are
initiated months before the rot is apparent in storage. Anthracnose
diseases of bananas, papayas, and mangoes arise from latent infec-
tions initiated randomly over the entire surfaces of these fruits.

Gray mold (Botrytis) on grapes and brown rot (Phytophthora)

on citrus fruits come from infections of fruits in the field that
begin shortly before harvest. These infections may not be truly
quiescent, but grow rather slowly in mature fruits, depending upon
the disease resistance of the host tissues and the ambient temper-
atures.

The second major class of postharvest diseases originates by

infection through wounds which occur during the harvest and prep-
aration of the product for market. These infection sites may be
localized--the severed stern of the pineapple, the avocado, and the
pear--or more or less randomly distributed on the entire surface as
abrasions or cuts caused by rough handling. Several examples of
disease caused by preharvest and postharvest (wound) infection are
given in Table 1. The etiology of the postharvest diseases of
tropical and temperate crops has been considered in detail in other
reviews (24,25,26).

ROLE OF PATHOGENS' ENZYMES IN DISEASE DEVELOPMENT

Plant cell walls are held together by intercellular material

composed mainly of pectic polysaccharides. Development of a post-
harvest disease depends upon the capability of the pathogen to
secrete enzymes that depolymerize these insoluble pectic polymers,
leading to loss of tissue coherence and separation of the individual
cells, a process referred to as tissue "rnaceration." The cells of
the macerated tissues increase in permeability and die, allowing
outward diffusion of host metabolites which may be used as substrates
for growth by the pathogen (52).

Pathogens involved in postharvest diseases produce endopoly-

galacturonases, endo pectin lyases, and endo pectate lyases (7).
Although the random hydrolysis of a-l,4 glycosidic linkages in
polygalacturonans is sufficient to cause tissue maceration and cell
250 J. W. ECKERT AND M. RATNAYAKE

death, cellulases and hemicellulases may also be involved in patho~

genesis. Recently, a proteinaceous factor has been isolated which
stimulates tissue maceration by fungal enzymes, but the factor is
essentially devoid of activity alone or in in vitro assays with pure
enzymes (42). - ---

The endopolygalacturonases are enzymes which hydrolyze the

a-l, 4 galacturonide linkages of pectic acid with different degrees
of randomness to produce aseries of oligogalacturonides. These
enzymes have been associated with tissue maceration by several
microorganisms that cause postharvest disease, e.g., Penicillium,
Rhizopus, Geotrichum, Sclerotinia and the bacterium Erwinia
carotovora, as well as many non-pathogenic microorganisms growing
in culture (61). Maceration of citrus peel by Geotrichum (73),
sweet potatoes by Rhizopus (65), and persimmons' hY Gloeosporium
(71) can be accounted for entirely by the action of extracellular
endopolygalacturonases produced by the pathogens.

The endopectin lyases split the a-l, 4 galacturonide linkages

of pectin to different degrees of randomness by a transelimination
mechanism so that aseries of C~-C5 unsaturated methylated oligo-
galacturonides are produced. Tne pectin lyases which have been
characterized are all of fungal origin. Pectin lyases have been
implicated in the maceration of citrus fruit peel attacked by
Penicillium digitatum and Penicillium italicum (19), but this has
been questioned recently by Barmore and Brown (3,4) who identified
endopolygalacturonase and exopolygalacturonase as the principal
enzymes responsible for the maceration of orange peel by Penicil-
lium italicum and~. digitatum, respectively. These investigators
did not find pectin lyases in extracts of fruit invaded by either
of these pathogens.

The third group of enzymes associated with postharvest diseases,

the endopectate lyases, are similar to the pectin lyases except that
their substrate is pectic acid and their product, oligogalacturonides.
This group of enzymes is commonly produced by pectolytic bacteria
and the pectate lyases are the major cause of softening of potatoes
attached by soft rot bacteria (53), although endopolygalacturonases
may also be involved (8).

The production of extracellular endopolygalacturonases and

pectin lyases by fungi is generally induced by pectate or pectin
in their environment. Cooper and Wood (21) reported that these
enzymes in the fungi Verticillium and Fusarium were induced by low
concentrations of galacturonic acid rather than pectin or pectic
acid and that the production of the enzymes was repressed when the
inducer was in slight excess over that required for growth of the
fungi. The extracellular pectate lyases produced by Erwinia
caratovora are usually induced by pectin and, may or may not, be
subject to catabolite repression, depending upon the individual
HaST-PATHOGEN INTERACTIONS 251

isolate of the bacterium and the environment (56, 79).

Although microorganisms which cause postharvest disease must

produce extracellular pectolytic enzymes, it does not follow that
microorganisms that produce pectolytic enzymes, can cause post-
harvest diseases. A number of nonpathogenic fungi and bacteria
are capable of producing large amounts of enzymes which macerate
slices of potatoes and other plant products (42). Both virulent
and avirulent isolates of Penicillium spp. and Erwinia carotovora
produce pectolytic enzymes in culture, indicating that the ability
to attack native pectin in the host is not measured by pectolytic
activity in vitro. However, avirulent strains of Erwinia ~
tovora showed lower activity of pectate lyase and endopolygalacturo-
nase than did virulent strains (8,31).

SUSCEPTIBILITY OF HOST TISSUE TO DISEASE DEVELOPMENT

The susceptibility of fruits and vegetables to postharvest

decay is influenced dramatically by the maturity of the crop at
the time of harvest and by subsequent physiological changes,
collectively termed "ripening." The susceptibility of apples to
blue mold (Penicillium expansum) increases with both maturity and
ripeness, and appears to result from an increase in susceptibility
of the flesh to bruise damage. Likewise, navel oranges become
more susceptible to invasion by Penicillium molds with advance
in maturity during the season. The resumption in activity of
quiescent infections during ripening of fruits is a clear example
of increasing disease susceptibility, rather than increased
susceptibility of the host tissue to mechanical injury. Lenticel
rotting of apples is virtually unknown at the time of harvest but
develops in storage as the fruit ripens. Different pathogens
appear at different stages of ripening of apples in storage. A
striking increase in Alternaria-stem-end-rot occurs when lemons
pass a certain threshold of ripeness during storage. The lemon
peel is highly resistant to invasion by Alternaria citri prior to
this stage of development. Latent infections of Colletotrichum on
banana, papaya and mango fruits seldom become a serious problem
until the fruit approaches ripeness. A number of investigators
have observed that potatoes become more susceptible to Fusarium
dry rot during storage; the susceptibility of carrots to Botrytis
and Centrospora at SOC also increases with duration of the storage
period (22, 23, 32).

A few examples of disease susceptibility decreasing with

maturity or storage of the crop are known. Several investigators
have recorded that green tomatoes are more susceptible than red
tomatoes to bacterial soft rot. White potatoes become less
susceptible to bacterial soft rot during the first few weeks of
storage because the periderm layer matures during this period.
252 J. W. ECKERT AND M. RATNAYAKE

Since susceptibility to disease increases with ripeness, it

is not surprising that the application of ethylene gas to acceler-
ate ripening has been observed to increase Alternaria rot on
tomatoes (63), and stern end rot and anthracnose on citrus fruits
(17,50). McCornack (50) dernonstrated that ethylene degreening of
oranges increased Diplodia stern end rot, but not Penicillium green
mold, and that there was a direct relationship between the ethylene
concentration (4-50 ppm) and stem end rot (Table 2). Five to ten
parts per million ethylene is the minimum required for satisfactory
coloring of the fruit. The ethylene treatment accelerates senescence
and abscission of the fruit button, thereby stimulating the develop-
ment of quiescent infections of Diplodia in this organ and providing
an avenue for the fungus to enter the fruit (18).

Ethylene treatment of green tangerines stimulates appressoria

of Colletotrichum present on the surface of the fruit to form in-
fection hyphae which then penetrate the epidermal cells of the
fruit (14, 17). Apparently the ethylene treatment simultaneously
triggers an unknown resistance mechanism since the fruit become
resistant to infection if they are treated with ethylene three
days before inoculation with Colletotrichum spores.

The intrinsic and variable resistance of fruits and vege-

tables to postharvest disease, especially with aging, might be
associated with one or a combination of several properties of the
host (Table 3).

STIMULATION QF PATHOGEN BY HOST CONSTITUENTS

The development of a pathogen on the surface of its host may

be influenced by chernicals exuded from the interior of the host
plant. There are many reports that simple sugars and nitrogenous
compounds are released to the surface of fruits and leaves where
they stimulate spore germination and germ tube growth of plant
pathogens. However, these substances often delay the formation of
infection structures such as appressoria.

Anthranilic acid, which has been identified in the leachate

from banana fruit, is metabolized to 2,3-dihydroxybenzoic acid by
the fungus Colletotrichum musae, the pathogen of banana anthrac-
nose (36, 70). The latter compound stimulates conidial germination
and appressorium formation by the pathogen. Similarly, leachates
of apple fruits (cv. Bramley's Seedling) contained chlorogenic
acid and ~-coumarylquinic acid, both of which stimulated spore
germination and formation of appressoria of the apple-rotting
pathogen Diaporthe perniciosa (11). The leachates showed most
stimulatory activity in mid-August when the concentration of
E-coumarylquinic acid was highest in the leachate.
HOST -PA THOGEN INTERACTIONS 253

Table 2. Effect of Ethylene on Anthracnose, Stem End Rot, and

Green Mold of Citrus Fruits

Ethylene Percent fruit decayed by

(j.ll/liter air) Anthracnose l Stem end rot 2 Green mold 2

o o o 6
5 l3 3
10 17
20 57
50 86 23 2

~ata of Brown and Barmore 1977. Anthracnose incited by Colletotri-

chum gloeosporioides. Robinson tangerines exposed to ethylene for
2 days and then stored for 1 wk at 26°C and 100% relative humidity.
2Data of McCornack 1972. Stern end rot incited mostly by Diplodia
natalensis and green mold by Penicillium digitatum. Valencia
oranges exposed to ethylene for 2 days and then stored at 21°C for
4 wk.

Table 3. Intrinsic Factors Influencing Resistance and Susceptibility

of Plant Products to Attack by Microorganisms

1. Stimulators of pathogen growth and infection

2. Inhibitors of pathogen growth or enzyme action
a. Preformed
b. Elicited by pathogen attack
3. Hypersensitivity of invaded tissue
4. Water status
5. pH and nutritional level
6. Morphological barriers to pathogen spread
7. Resistance of host tissue to pathogen's enzymes

INHIBITION OF PATHOGEN GROWTH AND ENZYME ACTIVITY BY HOST CONSTIT-

UENTS

Two types of microbial growth inhibitors have been recognized

in plant tissues: 1) preformed compounds and 2) inhibitors that are
synthesized by the host in response to attempted infection by a
pathogen. The latter are referred to as "phytoalexins." Examples
of preformed inhibitors that may offer resistance in fruits to
infection by pathogens are tannins and 3,4-dihydroxybenzaldehyde
in bananas (5,34,54) and phenolic compounds in apples (20,41,55).
254 J. W. ECKERT AND M. RATNAYAKE

The biosynthesis of antimicrobial compounds by plant tissue

in response to injury is a rather common phenomenon (45). Several
investigators have reported that the production of 6-methoxymellein
by the carrot is stimulated by the application of spores of Rhizopus
stolonifer, Botrytis cinerea and other storage pathogens to the
injured surface of the roots (33,35,37,38,5l). The loss of bio-
synthetic ability of the carrot to produce this compound during
long-term storage is believed to be an important factor in the in-
crease in susceptibility of carrot roots during this period (32,37).
The carrot also synthesizes ~-hydroxybenzoic acid and the poly-
acetylene falcarinol which are fungitoxic and may play a role in
the resistance of the carrot to postharvest disease (35).

The pulp of green tomatoes contains substantial amounts of

tomatin (an alkaloid saponin) but the concentration decreases with
fruit maturity until only traces are detectable in yellow and red
fruit. Tomatin is a weak base and possesses maximum fungitoxic
activity in its non-ionized form which exists in neutral to alkaline
solutions. Although the tissue of green tomatoes is acidic,
Schlosser (62) reported that the fungus Gloeosporium fructigena in-
creased the pR of the tomato tissue at the infection site to pR 6.4
at which pR the amount of tomatin present was inhibitory to the
pathogen. Rence, Q. fructigena is capable of colonizing mature
tomato fruits only and not green fruits. Verhoeff and Liem (75, 76)
believed that latency of the pathogen Botrytis in tomato fruit could
also be explained in part by tomatin.

The antimicrobial terpenoid, rishitin, formed in potato tubers

inoculated with Erwinia carotovora and stored in air may be a factor
in restricting the expansion of lesions of bacterial soft rot which
develops under these conditions (48).
Nectria galligena infects apple fruits in the late summer but
resistance of the fruit tissue prevents further growth of the fungus.
Attack by the pathogen causes the host tissue to synthesize benzoic
acid around the site of infection. At the pR of the immature apple
flesh (pR 3), benzoic acid is in its most fungitoxic form (undis-
sociated) and prevents extensive growth of the fungus in the apple
(9,10). During storage, the pR of the fruit tissue increases,
benzoic acid dissociates to the anionic form and the fungus resumes
growth producing disease lesions in the fruit. Synthesis of benzoic
acid is e1icited by an extrace1lu1ar protease produced by Nectria
galligena; other app1e pathogens such as Penici11ium expansum that
do not elaborate the protease do not e1icit the production of
benzoic acid by the app1e tissue (68, 69). •

Brown and Swinburne (12) demonstrated recent1y that phytoa1exins

probab1y p1ay a ro1e in the 1atency of Co11etotrichum ~ on
banana fruit. Drops of water containing spores of this fungus p1aced
on green banana fruit cause necrosis of ce11s in the tissues direct1y
HOST-PATHOGEN INTERACTIONS 255

beneath the drops. Five compounds that inhibited the growth of ~.

musae were isolated from the necrotic tissue on green fruit, but the
fungitoxic compounds disappeared as the fruit ripened. Brown and
Swinburne (12) isolated a glycan containing polysaccharide from the
cell wall of ~. musae which elicited necrotic lesions, as well as
the two major fungitoxic compounds elicited by living conidia of
~. musae. It seems likely that the fungitoxic compounds elicited
by this pathogen play a role in the latent behavior of the fungus
on green fruit and the fungus resumes growth when the compound
disappear as the fruit ripens.

The distribution of substances in plant tissues that may in-

hibit enzymes of the pathogen is probably more general than is
usually appreciated. General enzyme inhibitors such as polyphenols
are known to be present in the flesh of apples (20, 41, 55) and
the level of these compounds generally decreases as the fruit
mature. Dicotyledonous plants have pro teins associated with their
cell walls which are capable of inhibiting the action of endopoly-
galacturonase secreted by plant pathogens. Albersheim and Anderson-
Prouty (2) suggest that a pathogen is incapable of attacking a plant
unless the pathogen can secrete sufficient endopolygalacturonase to
overcome the inhibitor present in the wall of the plant.

HYPERSENSITIVITY OF HOST TISSUE CHALLENGED BY THE PATHOGEN

Appressoria of Colletotrichum gloeosporioides are commonly

present on the surface of Robinson tangerines during the per iod of
fruit development in the grove but they do not germinate and infect
green fruit. Treatment of the fruit after harvest with ethylene
caused a loss of chlorophyll and an increase in carotenoids in the
fruit, as well as stimulating the appressoria to send out infection
hyphae into the fruit (14,15,16,17). However, if the appressoria
are removed from the surface of green fruit before treatment with
ethylene, the ethylene-treated fruit become orange in color and
simultaneously develop resistance to invasion by Colletotrichum
spores applied subsequently. Brown (16) has shown that penetration
of an epidermal cell of orange-colored fruit by an infection hypha
from an appressorium of C. gloeosporioides results in the death of
four layers of cells surrounding the infection hyphae. The necrotic
cells become filled with phenolic compounds. Apparently, the dead
phenol-filled cells of the host form a barrier to further spread of
the fungus hyphae, localizing the pathogen to the area penetrated
by the infection hyphae.

WATER STATUS OF THE HOST

Many fruits and vegetables are more susceptible to invasion by

pathogens when their tissues are turgid. This is usually attributed
256 J. W. ECKERT AND M. RATNAYAKE

to the presence of a water film associated with injuries which sup-

ports the growth of the pathogen during the infection process. Thus,
slight desiccation reduces the susceptibility of citrus fruits to
Penicillium digitatum (27), potatoes to bacterial soft rot (58) and
carrots to Centrospora acerini (22,23). In contrast, both Botrytis
and Rhizopus preferentially infect wilted carrots (23,32). The
susceptibility of carrots to infection by these fungi increases
when the tissues exceed 8% water loss; more turgid roots are resis-
tant. The increase in susceptibility of slightly flaccid carrots
has been attributed to an increasein the intercellular spaces as
the cells begin to separate at approximately 7% water (72). Cabbage
also seems to be more resistant to Botrytis and other pathogens
when stored at very high humidity which delays the onset of senes-
cence of the outer leaves (74,77,78). These effects of host water
status upon the disease reflect the physiological status of the
host rather than a direct effect upon the pathogen, since most
microorganisms involved in postharvest diseases grow weIl at water
potential deficits larger than those that are likely to be en-
countered in the tissues of palatable fruits and vegetables.

pR AND NUTRITIONAL LEVEL OF ROST TISSUES

Acidity of the tissue of many fruits may be one of the most

important reasons for their general resistance to bacteria that
cause soft rot of many vegetable crops (47). Tomatoes, peppers
cucumbers, and pears are the few fruits known to be seriously
affected by bacterial soft rot. Fruit tissues are usually below pR 5
which inhibits the growth of most bacteria capable of degrading
plant tissues; vegetable tissues are generally less acid (23, 47).
A decrease in the acidity of the flesh of Bramley's Seedling apple
during storage may be responsible for the decrease in resistance to
Nectria galligena (68). The increase in pR of the flesh associated
with ripening in storage favors the dissociation of benzoic acid in
the apple to the less fungitoxic dissociated form.

Several examples of plant constituents that stimulate preinfec-

tion development of the pathogen on the host surface have been
described. Clear-cut examples of unique nutrients or growth stimu-
lants that determine susceptibility in fruits and vegetables are rare,
but the possibility of their existence must none-the-less be con-
sidered. Penicillium digitatum, a unique pathogen on citrus fruits,
requires a complex medium for rapid germination and vigorous hyphal
growth. Citrus fruits contain both ascorbic acid and certain
terpenes that are known to stimulate germination of f. digitatum
spores (30, 57) as weIl as a complement of organic nutrients that
sustain vigorous growth during the infection process. Otazu and
Secor (56) found a highly significant correlation between reducing
sugar content and susceptibility to bacterial soft rot in Norgold
Russet potato tubers of various ages held in different storage
HOST-PATHOGEN INTERACTIONS 257

temperatures, and Bartz et al. (6) reported that bacterial soft rot
susceptibility of tomato fruit was influenced by nitrogen fertiliza-
tion of the plant in the field.

RESTRICTION OF PATHOGEN GROWTH BY MORPHOLOGICAL BARRIERS OF THE

HOST

In response to injury, plant tissue may form protective barriers

of tightly packed cells, provided that the tissue is still capable
of cell division, or the cells surrounding the injury can deposit
lignin and suberin in their walls to protect against the action of
macerating enzymes of microorganisms. Lenticels of potatoes are
normally blocked with a layer of suberized periderm wh ich prevents
the entry of soft rot bacteria into the cortex of the tuber (1,29).
Rupture of this periderm layer, either mechanically or by prolifera-
tion of underlying cells of the tuber in response to free water,
makes the lenticels susceptible to invasion by soft rot bacteria
(29,58). A similar situation exists in the case of lenticels of
immature apples (28).

If potato tubers are superficially wounded and then placed at

high humidity and moderate temperatures, suberin begins to form in
the walls and intercellular spaces of the living cells surrounding
the injury and, within several days, a suberized periderm forms
beneath these suberized surface cells (29). These structure offer
substantial resistance to the invasion of pathogens at wounded areas
on the tuber. The surface cells of carrots also become suberized in
response to wounding and this suberized surface layer is a barrier
to infection by Botrytis (32, 37, 39).

Suberization of cells around 1nJuries is probably the most

important defense of the potato against infection by microorganisms,
since this reaction takes place within 24 hours under optimal
environmental conditions (29). The suberized periderm which follows
in about four days is highly resistant to infection by microorganisms
because it is composed of tightly packed cells, the walls of which
are impregnated with suberin and have only a small amount of pectin
(29). Periderm formation is most rapid at 2l-27°C at high humidity,
but storage temperatur es of l6-2loC are usually recommended to re-
duce the rate of growth of Erwinia carotovora and Fusarium. Smith
and Smart (64) found that the periderm formed on potato slices at
lOoC in four days was adequate to protect against bacterial soft rot
whereas slices held at 4.4°C formed only a slight amount of suberin,
no periderm, and were highly susceptible to decay after transfer to
2l DC. Low temperatures favor the development of gangrene (Phoma)
on potatoes in storage because the pathogen continues to develop
while the low temperature prevents the formation of barriers by the
host. Carrots held at 22-26°C and high humidity for two days to
permit wound healing before storage had less decay after long-term
258 J. W. ECKERT AND M. RATNAYAKE

storage at SOC than an uncured lot (22). Curing of sweet PQtatoes

and yams for 4-7 days at 26-32°C and 8S~90% relative humidity a110ws
for the'tubers to form a suberized periderm to protect harvest in-
juries against invasion by Rhizopus and Endoconidiophora during
subsequent storage of the crop (67). Wo und hea1ing proceeds most
rapid1y at 30°C whereasRhizopus grows most rapid1y at 24°C (49),

The ability of tissues of some p1ants to form a barrier of

lignin around an injury is apparent1y an important mechanism of
defense against invasion by a pathogen. Holding oranges at 30°C
and 90-96° RH for severa1 days after harvest reduces Penici11ium
decay (13,43) because this environment is high1y favorable to the
synthesis of lignin and the temperature is too high for the deve1op~
ment of Penici11ium (Tab1e 2). The lignin barrier is not attacked
by enzymes of the pathogen and probab1y prevents pectic enzymes of
the pathogen from depo1ymerizing the midd1e 1ame11a of the host
ce11s (60). Resistance of immature papaya fruit to Co11etotrichum
gloeosporioides has been co~re1ated with the ability of the wounded
immature fruit to ward off the pathogen by the formation of a
1ignified periderm (66).

RESISTANCE OF HOST TISSUE TO PATHOGEN'S ENZYMES

The coherence of the f1esh of fresh fruits and vegetab1es is

dependent, to a 1arge degree, upon the pectic substances in the
midd1e 1ame11a which functions as an adhesive, binding the ce11s
together. The pectic materials are made up of linear chains of
D-ga1acturonic acid, methy1ated to varying degrees (pectin), and
with side chains of neutral sugars. As initially laid down by the
cell, the pectic substances in the middle lamella are in an in-
soluble form known as protopectin. The insolubility of protopectin
is a consequence of high mo1ecular weight and an anchoring of the
linear chains of pectin and pectic acid to the cellulose of the
cell wall through bridges of neutral polysaccharide and perhaps
pro teins (59). In this insoluble state, the midd1e lamella is
resistant to hydrolysis by pectolytic enzymes of plant pathogens(7).
Kuc et a1. (44) conc1uded that this cross linking of pectin to the
ce11 walls was a major reason for the resistance of immature app1e
fruits to the rot caused.by Botryosphaeria ribis.

As fruits and vegetables ripen, the bonds anchoring the pectic

materials to the cel1 wall are broken with the result that the
pectic materials become soluble and the tissue begins to soften.
The increased solubility of the pectic substances and perhaps, in~
creased openness of the midd1e lamella makes the tissue more vu1~
nerable to maceration by the pectolytic enzymes of pathogens. The
susceptibility of strawberries to attack by Botrytis cinerea is
highly corre1ated with the soluble pectin content of the fruit (40).
Barmore and Brown (3) found that pectin was demethy1ated by pectin
HaST-PATHOGEN INTERACTIONS 259

methylesterase that leaked from the cells of orange peel attacked by

Penicillium digitatum and that the resulting polygalacturonic acid
was more readily depolymerized by the exopolygalacturonse of the
pathogen than was native citrus pectin.

CONCLUSION

The fact that relatively few microorganisms are capable of

parasitizing fresh plant products, i.e., living fruits, tubers
and leafy parts, reveals that a unique relationship exists between
host and pathogen in postharvest pathology. This situation suggests
that opportunities may exist to reduce postharvest deterioration by
increasing the resistance of the host to infection or at least by
delaying the onset of senescence that is invariably accompanied by
an increase in disease susceptibility.

Harvested fruits and vegetables possess the ability, to varying

degrees, to reduce the vulnerability of surface injuries to pathogen
invasion by the deposition of biologically-resistant barriers of
lignin, suberin, callose and layers of tightly packed cells (wound
periderm). These anabolic reactions are favored by high relative
humidity and usually the temperature-rate relationship is distinct
from that of the pathogen growth curve. Thus environmental condi-
tions can often be discovered that encourage the development of a
barrier in wounds before the pathogen can invade the tissue.

Another interesting possibility for disease control involves

the intensification of the natural defense mechanisms of the plant,
pathogen-inhibiting compounds or structural barriers, by the appli-
cation of chemical or physical agents to the plant product after
harvest. Langcake (46) recently reviewed the status of research
in this field as applied to the treatment of growing plants.

Numerous investigations on the optimum environment for storage

of plant products have clearly demonstrated the benefits of de-
laying senescence upon resistance to disease arising from pathogens
that are suppressed in the immature host (latent infections) and
those that invade through wounds. Temperature management, relative
humidity, inert atmospheres, ethylene removal and hypobaric condi-
tions can all have a beneficial role in reducing disease lossess.
Senescence-inhibiting compounds such as 2,4-D and gibberellin are
used in practice to reduce susceptibility of citrus fruits and this
approach may develop greater applicability as more versatile agents
of this type are discovered.

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260 J. W. ECKERT AND M. RATNAYAKE

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HOST-PATHOGEN INTERACTIONS 261

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31. Friedman, B. A. (1962). Physio10gica1 differences between
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262 J. W. ECKERT AND M. RATNAYAKE

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HOST-PATHOGEN INTERACTIONS 263

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CONTROL OF POSTHARVEST DISEASES WITH ANTIMICROBIAL AGENTS

Joseph W. Eckert

Department of Plant Pathology

University of California
Riverside, CA 92521

INTRODUCTION

Pathological diseases of fruits and vegetables can be reduced

to a certain extent by maintaining the natural resistance of the
host, lo~ temperature storage, low oxygen atmospheres, and treat-
ment with growth regulators that delay senescence. However, these
beneficial practices may not adequately protect the crop from micro-
bial attack, especially during prolonged storage or movement of the
crop through export market channels. This is particularly true for
crops of tropical origin such as bananas, sweet potatoes and lemons,
that suffer physiological injury at the near-freezing temperatures
that are required to inhibit growth of fungi over an extended per iod
of time. The maximum storage life of many fresh fruits and vege-
tables can be realized only by treating the product with an anti-
fungal agent before storage in an optimal environment. The anti-
microbial treatment is not a substitute for a satisfactory storage
environment, since these agents rarely influence the rate of phys-
iological deterioration of the fresh product. However, the anti-
microbial agent is most efficient when the host possesses intrinsic
resistance to infection and the environmental conditions are least
favorable for the growth of the pathogen.

STRATEGIES AND METHODS OF DISEASE CONTROL

Several strategies for controlling postharvest diseases are

outlined in Table 1. The application of a protective fungicide to
the developing crop in the field was formerly the principal means
for controlling postharvest diseases initiated through preharvest
infection. This approach has been used extensively in the tropics

265
266 J. W. ECKERT

Table 1. Strategies for the Control of Pathogenic Microorganisms

on Fresh Fruits and Vegetables

Strategy Agents
1. Spray developing crop with fungicides Protective fungicides-
to prevent preharvest infection. captan, maneb, etc.

2. Inactivate pathogens in postharvest Broad spectrum sanitizing

environment. agents - formaldehyde,
hypochlorite, quaternary
ammonium compounds.

3. Inactivate latent infections in Systemic fungicides

harvested crops. benomyl, thiabendazole.

4. Eradicate spores in harvest- o-phenylphenate, sec-

related injuries. bu ty lamine.

5. Protect surface of product Benomyl

against infection through
wounds created after application
of fungicide.

6. Inhibit fungus sporulation and the Biphenyl, benomyl, S02

spread of hyphae to adjacent fruits. dicloran, metalaxyl.

to control anthracnose on mangoes and papayas and in Europe to

control lenticel rots of apples (1,2,3,4). The necessity for re-
peated applications of fungicides in the field has been reduced
considerably in recent years due to the development of systemic
fungicides that can be applied after harvest to inactivate the
pathogen situated within the host. However, even today preharvest
fungicide treatment is the only satisfactory means for control of
brown rot (Phytophthora) of citrus fruits, a disease that is initi-
ated by zoospore infection of fruit on the tree (5). The destruc-
tion of spores in packing houses is an essential practice to reduce
the inoculum that may find its way into harvest-related injuries
and to suppress the build-up of fungicide-resistant mutants that
accumulate under the selection pressure created by the fungicide
treatment. Formaldehyde and quaternary ammonium compounds are
used as broad spectrum sanitizing agents for applications that do
not involve contact with the food product. Solutions of hypochlorite
and sodium ~-phenylphenate are used to surface sterilize the prod-
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 267

uct itself or to sterilize water that is recycled to wash, transport

or cool the product.

Over the past thirty years, about twelve postharvest fungicides

have been developed that are highly effective against latent infec-
tions, wound-pathogens, and the spread of pathogens between adjacent
fruits. Table 2 lists the antimicrobial compounds that are in
practical use today, the crops treated and the diseases controlled.
The chemical structures of these compounds are shown in Figure 1.

The selection of an antimicrobial compound for a specific post-

harvest application depends upon: 1) the sensitivity of the pathogens
to the chemical agent; 2) the ability of the agent to penetrate
through the boundary layer of the host to the infection site; 3)
the tolerance of the plant product to the chemical agent, both from

I [
OH

6O
I#
f~
-

y
NH2

c'AC'
y
N02
Fig. 1. Fungicides used in practice to control postharvest diseases.
I - biphenyl, II - ~-phenylphenol, III - thiabendazole,
IV - benomyl, V - sec-butylamine, VI - dicloran.
268 J. W. ECKERT

Table 2. Practical Treatments for Control of Postharvest Diseases

of Fruits and Vegetables

Crop Disease pathogens Antimicrobial agents

Apple Penicillium E..-Phenylphenate

Pear Botrytis Thiabendazole
Pezicula Benomyl

Banana Colletotrichum Thiabendazole

Fusarium Benomyl
Ceratocystis Thiophanate methyl
Imazalil

Citrus Penicillium E..-Phenylphenate

Diplodia Thiabendazole
Phomopsis Benomyl
sec-Butylamine
Biphenyl
2,4-D
Imazalil

Grape Botrytis Sulfur dioxide

Mango Colletotrichum Thiabendazole

Papaya Diplodia Benomyl

Melons Alternaria Dimethyldithio-

Cladosporium carbamate

Peach Monilinia Dicloran

Nectarine Rhizopus Benomyl
Cherry E..-Phenylphenate

Pineapple Ceratocystis E..-Phenylphenate

Benomyl

Potato Fusarium Thiabendazole

Phoma Benomyl
Oospora ~-Butylamine
Helminthosporium

Sweet potato Ceratocystis Dicloran

Rhizopus E..-Phenylphenate

Tomato Botrytis Discloran

Rhizopus E..-Phenylphenate
Geotrichum
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 269

the standpoint of phytotoxicity and the effect of the treatment upon

the sensory properties of the product. No specific bactericidal
compounds have been developed for postharvest use and, until recently,
there were few compounds available that were effective against phy-
comycetes, Alternaria or Geotrichum and diseases incited by these
pathogens were very difficult to control. Even those compounds that
exhibited in vitro activity against these pathogens did not prevent
their development in the host, apparently because these compounds
were unable to penetrate to the sites of infection, e.g. latent
infections. Postharvest fungieides of the first generation, e.g.
sodium ~-phenylphenate, dicloran and sec-butylamine are effective
in preventing decay by the wound-invading pathogens, e.g. Penicillium
and Rhizopus, but have little effect on the development of latent
and other deepseated infections (2). Several fungicides developed
since 1965 have shown a high degree of efficiency against such latent
infections as weIl as protective and antisporulant action in control-
ling certain post-harvest diseases (4,5).

Fungieides may be applied to harvested products either in a

gaseous state or mixed with water or wax formulations, depending
upon the physical and chemical properties of the fungicide and the
convenience of each method of application. A fumigation treatment
.is advantageous for treatment of strawberries and other highly
perishable commodities that are not normally washed after harvest
because they might be adversely affected by hydration. Sulfur
dioxide is extensively used to fumigate grapes for control of Botrytis
and seems to be effective on raspberries as weIl. Ethylene oxide
is an effective fumigant for control of yeast on dried dates, but was
discontinued in the late 1960's when residues of toxic ethylene
chlorohydrin were found in treated fruit (4). Acetaldehyde has re-
ceived considerable attention as a fumigant for apples and straw-
berries in recent years because it is effective in controlling mold
growth and also is a natural metabolite in fruits.

Today, most fungieide treatments are applied in water-base

formulations that are sprayed or flooded onto the plant product as
it is conveyed mechanically through the packing houses. Also, formu-
lations of fungieides in water-base waxes and in hydrocarbon-solvent-
base waxes are in popular use. All of the organic fungieides listed
in Table 2 have been applied in these ways. Fungieides applied in
water-base formulations to prevent infection at injury sites may be
divided into two categories according to their ~istribution on the
surface of the treated product: 1) Non-ionic water insoluble com-
pounds (e.g., thiabendazole) that are applied as a uniform deposit
over the entire surface of the product; 2) water soluble salts such
as sodium o-phenylphenate, sec-butylamine, and sodium carbonate that
are applied in relatively concentrated solutions (0.5-3.0%) in water.
Aqueous solutions of these salts are absorbed by fruit injuries but
the intact cuticle is not permeable to such ionic compounds. After
treatment, the product is rinsed lightly with fresh water to remove
270 J.VV.ECKERT
most of the fungicide from the surface of the fruit except for a
significant residue that remains in the injury site to inhibit growth
of the pathogen there (Fig. 2). The pR of injured tissue of most
fruits is considerab1y be10w the pKa of o-pheny1pheno1 (10.01),
resu1ting in the precipitation of o-pheny1pheno1 from the solution
of sodium o-pheny1phenate (6). An-advantage of uniform non-se1ective
coverage of the product with a fungicide is that the entire surface
is protected to some degree against infections at injuries incurred
after the treatment is app1ied. For examp1e, benomy1-treated citrus
fruits are difficu1t to inocu1ate through superficia1 injuries made
after the benomy1 treatment is app1ied. Apparent1y, benomy1 enters
the surface ce11s of the host, making the tissue an unsatisfactory
substrate for the growth of the pathogen (7). Fungicides that carry
some risk of phytotoxicity are app1ied se1ective1y to the potential
infection sites and the excess compound must be removed by rinsing
the fruit with water to prevent injury to the fruit surface. sec-
Butylamine and thiabendazo1e are not phytotoxic; therefore, sub-
stantia1 deposits may be 1eft on the fruit without causing injury.
Rinsing with water after treatment significant1y reduces the fungi-
cida1 residue on the product, but also reduces the antimicrobia1
effectiveness of the treatment as we11.

CONTEMPORARY FUNGICIDE TREATMENTS (Fig. 1)

~-Pheny1pheno1 (11) is a broad spectrum antimicrobia1 agent

that is toxic to fungi, bacteria and p1ants. The undissociated
phenol is 1etha1 to microorganisms and is injurious to most plant
products at concentrations of 200-400 mg/L, depending upon the
temperature of the solution and the per iod of contact. The dis-
sociated form (o-pheny1phenate anion) is not phytotoxic and a
solution of sodfum ~-pheny1phenate (SOPP), containing excess alkali
to suppress hydrolysis of the sa1t, is quite safe for treatment of
severa1 fruits and vegetab1es. The intact waxy surface of citrus
fruits, apples and other fruits is impermeable to the ~-pheny1phenate
anion as indicated by the low residue levels of fruit treated at
pR 12 (6). Rowever, the ~-phenylphenate anion diffuses selectively
into injuries where the surface ce11s of the fruit have been rup-
tured and is hydrolyzed to o-pheny1pheno1 in the presence of fruit
acids and metabolic carbon dioxide. Rinsing the treated fruit with
water removes most of the sodium o-pheny1phenate residues from the
surface of the fruit, but a subst~ntia1 amount remains associated
with injuries and prevents the deve10pment of pathogens at these
sites. The se1ective accumu1ation of o-pheny1pheno1 in injuries
and its ro1e in the prevention of fruit decay is shown in Fig. 2.

The benefits of treating fruits and vegetab1es with sodium

~-phenylphenate are two-fold; 1) Fungus spores and bacteria on the
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 271

4 HOPP CONSTANT AT 60
I

,........15
\\ 32mg/LlTER
o 50
n. \
n.
o \ % DECAY o
:x: \ 40~
~,
<3:

--
W
[10
CI.
\
\
\
30 0
~
I-
5
e:
:::l \
o
,
.
20
~5 ;;e
""
.
Cl::
o RESIDUE-Sound Fruit
• '~
.....---------ll
10

o • o
o 2 3 4 5
9 SOPP I LITER

Fig. 2. Effect of concentration of SOPP (sodium o-phenylphenate

tetrahydrate) upon decay of oranges inoc~lated with
Penicillium digitatum and upon the residues of HOPP (0-
phenylphenol) in sound and injured tissues of the frults
peel(6).

surface of the fruit or in cleaning solutions are killed; 2) a

residue of ~-phenylphenol is deposited in wounds on the fruit sur-
face, preventing infections at these sites during marketing and
storage of the crop. Solutions of sodium ~-phenylphenate have
been used extensively for the control of postharvest diseases of
citrus fruits, apples, pears, peaches, sweet potatoes and other
perishable fruits and vegetables. The treatment is applied in
one of several ways. The fruit may be soaked in a bath of SOPP
for 1-3 minutes; l:he fruit may be flooded with a recirculated
solution of SOPP; the fruit may be brushed in a foam of SOPP for
15 seconds. In all cases, the fruit are rinsed with a spray of
fresh water after treatment. Krochta et al. (8) demonstrated that
a foam prepared from a solution of 0.5% SOPP and 0.3% sodium lauryl
sulfate was highly effective as a cushion for mechanically harvested
tomatoes as they dropped into a bin and also prevented decay of
fruit caused by Geotrichum and Rhizopus.

Biphenyl (I) has been added to the packaging material for

citrus fruits for the past forty years to provide vapor phase
inhibition of sporulation of Penicillium digitatum and P. italicum
272 J. W. ECKERT

during long distance shipment and marketing of the crop. Despite

these many years of successful use, biphenyl has three recognized
problems that limit its value as a postharvest treatment. 1) Treated
fruit may have temporary chemical odor. Although this problem is
minimal with citrus fruits, biphenyl has never been used on other
fruits and vegetables because of this odor problem. 2) Residues of
biphenyl on treated fruits may exceed the 70 mg/kg tolerance in
Europe and Japan. This problem is greatest with fruit that tend to
absorb substantial quantities of biphenyl, especially if they are
not adequately cooled during shipment. 3) Strains of Penicillium
that are resistant to biphenyl and cross-resistant to ~-phenylphenol
are commonplace in citrus packing houses.

~-Butylamine (V) may be applied to harvested crops either

as a fumigation treatment or as a solution of a salt of sec-butyl-
amine. The fumigation treatment controls Penicillium on citrus
fruits. The injuries on the fruit surface become alkaline after
absorption of the amine vapor and fungistatic residues of neutral
sec-butylamine salts persist in injuries after the fumigation treat-
~t is terminated (9). The sec-butylammonium cation is uniquely
fungistatic to several species of fungi (10,11). The optical isomer
(-) sec-butylamine is substantially more active than the (+) isomer,
although the racemic mixture is used in practice to control post-
harvest diseases. Fumigation of seed and warehouse potatoes in the
U.K. with sec-butylamine controls Oospora and Phoma during storage
(12). Gaseous sec-butylamine penetrates deeply into the potatoes
so that the fumigation can be delayed for 14 days after harvest
without diminishing the effectiveness of the treatment. However,
~-butylamine is not effective against Fusarium dry rot.

Oranges in California are drenched with a solution of 1% sec-

butylamine (phosphate or HCl salt, pH 9) to protect superficia~
injuries against infection by Penicillium species during the ethylene
degreening period and during storage (10). The degreening operation
is highly conducive to the development of decay since it involves
exposure of the fruit to a low concentration of ethylene gas (5 ppm)
for several days in a warm humid environment. se~Butylamine is
also added to wax formulations applied to lemons before storage to
control Penicillium, but the treatment has no effect upon diseases
caused by Alternaria or Geotrichum which are resistant to ~-butyl­
amine. Penicillium expansum and Pezicula spp. have been controlled
on apples treated with solutions of sec-butylamine salts (13,14).
The effectiveness against Penicillium was diminished, however, when
treated fruit were stored for several months at low temperatures.
The major problems that have beset the use of sec-butylamine are
injury to Navel oranges if the peel absorbs the solution, and the
build-up of sec-butylamine resistant strains of Penicillium. In
California, sec-butylamine is used mostlyon lemons in a schedule
with other fungieides which do not select strains of Penicillium
that are cross-resistant to ~-butylamine.
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 273

Dic10ram (VI) is the most effective fungicide for contro1 of

Rhizopus stolonifer, a wound invading pathogen of many fruits and
vegetables (15). Cherries, peaches and other stone fruits are
usua11y treated with this fungicide before shipment to distant
markets. Dicloran is much less active against Monilinia fructicola,
another major pathogen of stone fruits, and is only weakly active
against other Rhizopus species and Mucor species (16). Nectarines,
peaches and cherries are usually treated with a combination of
dicloran and benomyl after harvest to control the major postharvest
pathogens, Monilinia fructicola and Rhizopus stolonifer (17).

Dicloran is the standard postharvest treatment for control of

Rhizopus rot of sweet potatoes, but the treatment is ineffective
against Ceratocystis black rot or Penicillium rots (18). Recent
trials in California have demonstrated that dicloran app1ied to
tomatoes after harvest is highly effective in controlling Botrytis
and Rhizopus rots (19). However, the dicloran treatment has no
influence upon Geotrichum rot.

The principle postharvest fungicides prior to the mid 1960's,

borax, sodium carbonate, SOPP and sec-butylamine controlled decay
by preventing the invasion of pathogenic microorganisms through
harvest-related injuries on the surface of the fruit. Biphenyl and
dicloran function in this fashion; but in addition, inhibit the
sporu1ation of the pathogen and prevent its spread to adjacent fruits.
The introduction of the benzimidazole fungicides (Fig. 3) thiabenda-
zole, benomyl, carbendazim, and thiophanate-methy1 in the 1ate
1960's made it possible for the first time to prevent the development
of latent infections of such fungi as Dip10dia, Phomopsis and
Colletotrichum on certain tropical fruits. These fungicides also
are highly effective in preventing infection of fruit injuries
incurred during harvest and in handling after application of the
fungicide treatment. Finally, at high dosage rates, thiabendazole
(4-6 g/L) and benomyl (2-3 g/L provide a protective barrier on the
fruit surface that inhibits the sporulation of Penicillium and other
fungi on diseased fruits. The significance of this latter property
is that the benzimidazole fungicides potentially offer an acceptable
alternative to the biphenyl treatment for the control of spoilage of
citrus fruits.

All of the benzimidazole fungieides and their derivatives share

a common antifungal spectrum although they differ quantitatively
in activity (20). In general, the benzimidazole fungieides have
been used interchangeably for certain postharvest applications while,
in other cases, a specific compound is preferred because of its
biological or chernical properties. The benzimidazole fungicides
have been used world-wide to control Penici1lium decay and stern end
rots of citrus fruits, Penicillium blue mold and Gloeosporium
(Pezicula) lentice1 rots of app1es (1,21), Colletotrichum and
Ceratocystis on several tropical fruits (22,23,24,25,26) and
274 J. W. ECKERT

Moni1inia rot of stone fruits (peaches, cherries, p1ums). Benomy1

is usua11y combined with dic10ran for treatment of stone fruits in.
order to contro1 both Moni1inia and Rhizopus, the major postharvest
diseases of these fruits (17). The success of the benzimidazo1e
fungicides in the contro1 of postharvest diseases can be attributed
to two factors: 1) high intrinsic activity of these compounds against
many of the fungi responsib1e for postharvest diseases and 2) the
ability of the benzimidazo1e fungicides to penetrate at least some
degree through the waxy surface of the host to reach the site of
infection (7,27,28). Benomy1 appears to penetrate the host's sur-
face more efficient1y than thiabendazo1e, carbendazim or thiophanate
methyl and may be more effective in disease situations in which the
pathogen is situated be10w the surface of the host.

Numerous trials conducted in the U.S. and in Europe have

demonstrated that the benzimidazo1e fungicides can contro1 severa1
diseases of potato tubers during storage--dry rot (Fusarium),
gangrene (rhoma), skin spot (Oospora) and si1ver- scurf (Helmintho-
sporium (29). Thiabendazo1e appears to be somewhat more effective
than benomy1 for the contro1 of Fusarium and Phoma (30,31).
Thiabendazo1e is app1ied at 42 g/metric ton by means of an ultra
low volume mist app1icator as the potatoes are transported into
storage (31). High volume app1ication may be more effective, but
the residual water on the potatoes in storage may increase the
risk of bacteria1 soft rot. A simi1ar treatment of sugar beets
has been effective in reducing decay by Penici11ium and other fungi
that cause a sugar 10ss in the beets during storage in outdoor piles.

Thiabendazo1e and carbendazim are stab1e under most conditions

that might be encountered in the treatment of fruits after harvest.
Thiabendazo1e can even be app1ied as "smoke" in fruit and vegetab1e
storage rooms. Benomy1 and thiophanate methyl, on the other hand,
are unstab1e in aqueous solution or in organic solvents. Benomy1
suspensiqns in pure water appear to be stab1e because of the very
low solubility of benomy1 in water (ca. 4 mg/L); however, benomy1
is not stab1e in very di1ute aqueous solutions or in formu1ations
of Wax coating app1ied to fruits and vegetab1es after harvest.
Under neutral conditions, benomy1 decomposes significant1y within
a few hours to methyl 2-benzimidazo1ecarbamate (carbendazim); under
a1ka1ine conditions, benomy1 is converted to 1,2,3,4-tetrahydro-3-
butyl-2, 4-dioxo-s-triazino (a1pha)-benzimidazo1e(STB) within a
few hours (7,32,33; Fig. 3A). Carbendazim is almost as active as
benomy1 in preventing infection of citrus fruits by Penici11ium,
but STB is essentia11y devoid of antifunga1 properties. Carbendazim
is more polar than benomy1 and is significant1y 1ess effective in
controlling sporu1ation of Penici11ium on citrus fruits because
it does not penetrate into the pee1 of the fruit as efficient1y as
benomy1 (7). C,!Jrbendazim and thiophanate methyl, a progenitor of
carbendazim (Fig. 3B), have been used as substitutes for benomy1
(24,34). For some applications these compounds are equiva1ent
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 275

A 0
11 1:1 5 0

(XI
C- NHC 4 H9
I
N
0
11
}-NHCOCH 3
(XI 111 11
NCNHCOCH3

"... N "... NCNHCOCH3

'\.
benomyl ' \
/ ~~ g
H thiophonote-methyl

(XI
I
N ~
}-NHCOCH 3
::--.. N

corbendozim

Fig. 3A. Reactions of benomyl in aqueous formulations resulting

in the formation of carbendazim (MBC) and STB.
B. Spontaneous conversion of benomyl and thiophanate-
methyl to carbendazim (MBC).
276 J.VV.ECKERT
in effectiveness to benomy1, but for uses that require significant
penetration into the host~ MBC and thiophanate methyl appear to be
inferior.

All benzimidazo1e fungicides have two serious bio1ogica1

1imitations: 1) All compounds in this group are inactive against
severa1 microorganisms responsib1e for important postharvest diseases
(20)--Rhizopus and other muc~rs, A1ternaria, Geotrichum, Phytophthora
and all bacteria, inc1uding Erwinia spp. which causes soft rot of
potatoes and vegetab1e crops. 2) Pathogens that are usua11y contro1-
1ed by treatment with benzimidazo1e compounds have bui1t up ben-
zimidazo1e-resistant strains fo11owing intensive use of these
fungicides.

Diseases of secondary importance caused by A1ternaria, Geotrichum

and other pathogens of citrus fruits that are resistant to ben-
zimidazo1e fungicides have become a 1imiting factor in the storage
and handling of this crop after treatment with a benzimidazo1e fungi-
cide. The resistance of strains of Penici11ium to benzimidazo1e
fungicides has serious1y diminished the usefu1ness of these compounds
as postharvest treatments for citrus fruits and it now appears that
this problem may also arise on other crops.

RESISTANCE OF PATHOGENS TO POSTHARVEST FUNGICIDES

Strains of Penici1lium resistant to biphenyl were recognized

in Israel in 1940. These strains were not regarded as a practica1
problem unti1 the 1ate 1960's when severa1 shipments of Ca1ifornia
lemons packed with biphenyl-impregnated papers arrived in distant
markets with high levels of Penicil1ium decay and sporu1ation.
Harding (35) observed that many of these problem shipments origin-
ated in packing houses where the 1emons were treated with sodium
o-phenylphenate before they were stored for several months prior
to shipment. Harding found that strains of Penicil1ium digitatum
that were resistant to biphenyl were also resistant to SOPP, a
relationship that would be anticipated from the structural similarity
of the two fungicides (Fig. 1). He demonstrated that residues of
o-pheny1pheno1 on SOPP-treated 1emons were sufficient to suppress
the growth of biphenyl sensitive strains, thereby a1lowing the
biphenyl resistant mutants in the population to bui1d up on the
fruit. In a survey of Ca1ifornia citrus growing areas, Harding
observed biphenyl resistant variants of K. digitatum at a frequency
of about 0.15% of the iso1ates co1lected in citrus groves and
packing houses that had never been exposed to SOPP. Packing houses
that had a his tory of SOPP use had a resistant strain frequency of
14-18% of the Penici11ium digitatum population and 60% of the
Penici1lium isolates were resistant to biphenyl in packing houses
that had used SOPP continuously over aperiod of years.
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 277

The origin of the SOPP/biphenyl resistance problem can be

explained by the preexistence of mutants of ~. digitatum that are
cross-resistant to both of these fungieides (36). These strains
are apparently less well adapted in the natural population of
Penicillium digitatum in the citrus grove, but under selection
pressure of SOPP/biphenyl treatment they selectively proliferate in
the packing house to become the dominant component of the ~.
digitatum population. Mutants of several fungi that are cross-
resistant to biphenyl, SOPP and dicloran have been reported, but
dicloran resistance does not appear to be a real problem at this
time in the crops that are treated with this fungieide after harvest.
Dicloran is not used on citrus fruits.

sec-Butylamine may be applied to oranges and lemons before

they are place in storage. These applications create a situation
that is highly favorable for the selection and proliferation of
sec-butylamine resistant strains of ~. digitatum and P. italicum
in the natural population. Thus, the continuous use of sec-
butylamine over aperiod of months may lead to a failure of the
treatment to control Penicillium decay of citrus fruits.

The most serious problem of fungieide ·resistance arose in

connection with the continuous use of benomyl and thiabendazole on
citrus fruits (36,37). Mutants resistant to one benzimidazole
fungieide are also resistant to every other member of this group.
Benzimidazole resistance in Penicillium spp. was observed in
California citrus packing houses about fifteen months after thia-
bendzole was incorporated into the wax formulation applied to lemons
before storage. These resistant isolates occurred at a high fre-
quency in the atmosphere of packing houses that used thiabendazole
intensively as aprestorage treatment for lemons and they were not
controlled by treating the fruit with the highest dosage of thia-
bendazole recommended for commercial treatment. Benzimidazole re-
sistant strains of R. digitatum and P. italicum have been isolated
also in Florida, Israel, Australia, and Japan. A survey of citrus
fruits arriving in the port of Rotterdam revealed that approximately
one-half of the iso1ates of Penicillium digitatum and~. italicum
recovered from fruit from twenty countries were resistant to ben-
zimidazole fungieides (38). Benzimidazole resistant strains of
P. expansum have been reported on harvested apples and pears in
Oregon, New York and Austra1ia (14, 39, 40). Most observations of
benzimidazole resistant strains of Penicillium spp. have followed
intensive use of thiabendazole or benomyl over aperiod of some
months but Kuramoto (41) reported a severe resistance problem on
satsuma mandarins in Japan following application of a single annual
spray of thiophanate methyl immediately before harvest. Several
investigators have recorded the isolation of benzimidazole resistance
strains from citrus groves and packing houses where benzimidazole
fungieides had never been used (36).
278 J.VV.ECKERT
CURRENT"DEVELOPMENTS IN POSTHARVEST FUNGICIDES

Several new fungicides (Fig. 4) now at various stages of com-

mercial development could correct most of the deficiencies of post-
harvest treatments in commercia1 use tqday. Guazatine (VII), a
diguanadine, is a broad spectrum fungicide that is effective against
wound infection of citrus fruit by Penici11ium spp., inc1uding
strains that are resistant to the benzimidazo1es and other fungicides
now in popu1ar use. Guazatine also contro1s Geotrichum sour rot on
citrus fruits, but not the sporu1ation of Penici11ium since it is
not a systemic fungicide (42). Guazatine is used on a commercia1
basis in Austra1ia, but the treated fruit are not acceptab1e in
most importing countries at this time.

The imidazo1e fungicides (Fig. 4), imaza1i1 (VIII), phenaproni1

(IX) and prochloraz (X) are more effective than benomy1 and thia-
bendazo1e in controlling Penici11ium decay and sporu1ation on citrus
fruits, inc1uding strains of this pathogen that are high1y resistant
to the benzimidazo1e fungicides (43,44). On the other hand, the
imidazo1e fungicides are 1ess effective than benomy1 and thia-
bendazo1e against Diplodia stem end rot, the most important cause
of early-season decay in citrus fruit produced in the humid sub-
tropics. lmaza1i1, phenaproni1 and proch1oraz (45) in common with
the benzimidazole fungicides, do not contro1 Geotrichum sour rot or
Phytophthora brown rot diseases. Imaza1i1 does contro1 A1ternaria
rot on tomatoes (46) and pre1iminary observations indicate some
benefit against this disease on citrus fruits as we11.

The effectiveness of imaza1i1 in controlling Penici11ium decay

has been demonstrated in 1arge sca1e trials conducted throughout the
world (44). lmaza1il offers outstanding potential for the contro1
of Penici1lium decay in citrus fruits due to its effectiveness
against strains of Penicillium that are resistant to thiabendazo1e
and benomyl (47). Despite the obvious advantages, imaza1il is not
wide1y used at present because several countries, including the
U.S. and Japan, have not estab1ished permanent tolerances for this
fungicide on citrus fruits. lmazalil was very effective in control-
ling benzimidazo1e-resistant strains of Penici11ium expansum on
app1es during long-term storage in a modified atmosphere (14, 48).
Our tests showed that imaza1i1 was more effective than benomy1 in
controlling Moni1inia fructico1a (benzimidazo1e sensitive strains)
on sweet cherries. Others have reported good contro1 of this patho-
gen on peaches (49). The triazo1e fungicides, CGA 64251 and its
propy1 homolog, CGA 64250 (50) are equiva1ent to the imidazo1e
fungicides in controlling Penici11ium decay and sporu1ation. Re-
markab1y, these fungicides are high1y effective against Geotrichum
sour rot (51), but like all other organic fungieides currently in
use, the triazole fungieides have no effect against Phytophthora
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 279

XII

Fig. 4. New fungicides currently being evaluated for control of

postharvest diseases. VII-guazatine, VllI-imazalil,
IX-phenapronil, X-prochloraz, XI-CGA 64251, XlI-metalaxyl.

brown rot. Experiments conducted in California, Greece and Israel

have demonstrated the effectiveness of metalaxyl (Fig. 4, XII) in
controlling Phytophthora rot on citrus fruits (52) and apples (53).
If applied early in the incubation period, metalaxyl can penetrate
the waxy surface layer of the fruit to eradicate incipient infec-
tions of Phytophthora beneath. Short range systemic action is
essential for control of these diseases because Phytophthora in-
fection is initiated in the orchard, but a postharvest fungicide
treatment cannot be applied until several days later. Current
practical control measures for Phytophthora rots consist of pro-
tective fungicides (copper or captan) applied to the citrus grove
before the onset of the rainy season.
280 J. W. ECKERT

SUCCESSES AND CHALLENGES IN POSTHARVEST DISEASE CONTROL

Substantial progress has been made over the past decade in the
chemical control of postharvest diseases. The benzimidazole fungi-
eides provided a conceptual breakthrough in that they demonstrated,
for the first time, that latent or incipient infections located
within host tissues could be inactivated by chemical treatment applied
to the surface of the product after harvest. Stern-end rots of citrus
fruits, lenticel rots of apples, latent infections of Colletotrichum
on bananas and other tropical fruits, and late season infections of
Monilinia on peaches have all been controlled by postharvest fungi-
eide treatment to an extent thought impossible in earlier times.
Surface deposits of thiabendazole, benomyl and imazalil on citrus
fruits, within legal residue tolerances, can suppress the sporulation
of Penicillium mold on the surface of citrus fruits more effectively
than biphenyl, the standard fungieide for export shipments of citrus
fruits for four. decades. Thiabendazole has equalled or surpassed the
organomercurial fungieides in controlling certain important diseases
of seed potatoes and this technology has been extended to the re-
duction of postharvest losses due to fungal pathogens in warehouse
and fresh-packed potatoes as weil. The outstanding successes of
the benzimidazole fungieides in reducing storage los ses in these
crops has emphasized, by comparison, the inadequate control of
diseases incited by microorganism species that are tolerant to this
c1ass of fungicides, Le., Alternaria. Geotrichum, Phytophthora"
Mucors, bacteria and strains of fungal pathogens that have been
selected for benzimidazole resistance.

The new imidazole and triazole fungieides discovered over the

past few years potentially can correct the deficiencies of the
benzimidazole fungieides. As a group, they are able to control
Penicillium mutants that are resistant to the benzimidazoles and
several compounds can control Alternaria and Geotrichum sour rot
as weil. Furthermore, genetic studies with this class of compounds
suggest that they are less vulnerable to the problem of fungieide
resistance than the benzimidazole fungicides. The benzimidazole
compounds bind to fungal tubulin causing mitotic arrest (54, 55),
whereas the imidazole compounds are inhibitors of sterol biosynthesis
(56). Large increases in resistance to benzimidazole fungieides can
result from a simple mutation involving the benzimidazole binding
sites, whereas laboratory-induced resistance to the imidazole fungi-
eides involves only a small increase in resistance to this class of
compounds. Speculation regarding the ease of resistance develop-
ment has not been tested since these new fungieides have not yet
been subjected to the enormous selection pressure that prevails in
citrus packing houses. However~ strains of Penicillium expansum
(an apple fruit pathogen) that are resistant to imazalil were isolated
recently in Israel (57). The fact that imazalil and related compounds
are less effective against Diplodia stern end rot than the ben-
zimidazole compounds could result in a use pattern of the two fungi-
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 281

cides that could delay the emergence of strains of Penicillium that

are resistant to both classes of fungicides. Benomyl or thia-
bendazole are preferred early in the season when Diplodia is the
major problem, but they should be replaced by an imidazole fungicide
at mid-season when Penicillium decay and sour rot become the major
problems. Benzimidazole resistant strains of Penicillium that are
selected by the early season use of benomyl and thiabendzole could
be controlled by an imidazole compound later in the season.

Recent tests with metalaxyl have shown that incipient infections

of Phytophthora can be eradicated by postharvest treatments dictated
when environmental conditions before harvest forecast the probability
of an impending disease problem in storage. Although a few diseases
of fresh fruits and vegetables (e.g. bacterial diseases) cannot be
satisfactorily controlled by existing postharvest treatments, cur-
rently available fungieides have demonstrated the potential for
controlling the major fungal pathogens responsible for postharvest
losses. The major thrust of current research in postharvest pathology
should be directed towards the development of existing fungieides
into practical and economical treatments for the reduction of crop
losses during storage and distribution to the consumers.

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some new treatments for the control of rotting of stored
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282 J.VV.ECKERT
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(1973). Use of 2-aminobutane as a fumigant for contro1
of gangrene, skin spot and si1ver scurf diseases of potato
tubers. Potato Res. 16: 109.
13. Bompeix, G. and Morgat, F. (1977). Cires, anti-echaudures,
fongicides et conservation des pommes. Fruits 32: 189.
14. Koffman, W., Penrose, L. J., Menzies, A. R., Davis, K. C. and
Ka1dor, J. (1978). Contro1 of benzimidazo1e tolerant
Penici11ium expansum in pome fruit. Scientia Hortic. 9:
31.
15. Luepschen, N. W., Rohrbach, K. G., Jones, A. C., and Peters,
C. L. (1971). Methods of controlling Rhizopus decay and
maintaining Co10rado peach qua1ity. Co10rado State Univ.
Expt. Sta. Bu11. 547S.
16. Ogawa, J. M., Mathre, J. H. Weber, D. J., and Lyda, S. D. (1963).
Effects of 2,6-dich10ro-4-nitroani1ine on Rhizopus species
and comparison with other fungieides on contro1 of Rhizopus
rot of peaches. Phytopatho1ogy 53: 950.
17. Wade, N. L. and Gipps, P. G. (1973). Postharvest contro1 of
brown rot and Rhizopus in peaches with benomyl and dicloran.
Austri. J. Expt1. Agr. Animal Husbandry 13: 600.
18. Steinbauer, C. E. and Kushman, L. J. (1971). Sweetpotato cu1ture
and diseases. U. S. Dept. Agr. Handbook 388: 35.
19. Chastagner, G. A. and Ogawa, J. M. (1979). A fungicide-wax
treatment to suppress Botrytis cinerea and protect fresh-
market tomatoes. Phytopatho10gy 69: 59.
20. Eckert, J. W. and Rahm, M. L. (1979). The antifunga1 activity
of alkyl benzimidazo1e-2-y1-carbamates and re1ated compounds.
Pestic. Sei. 10: 473.
21. Edney, K. L. (1970). Some experiments with thiabendazo1e and
benomy1 as postharvest treatments for the contro1 of storage
rots of app1es. Plant Patho1. 19: 189.
22. Cho, J. J., Rohrbach, K. G. and Apt, W. G. (1977). Induction
and chemica1 contro1 of rot caused by Ceratocystis paradoxa
on pineapp1es. Phytopatho10gy 67: 700.
23. Couey, H. M. and Farias, G. (1979). Contro1 of postharvest
decay of papaya. Hort. Sei. 14: 719.
24. LBcher, P. and Hampe1, M. (1973). Contro1 of postharvest diseases
of bananas, pineapp1es and citrus with carbendazim. Proc.
7th British Insecticide Fungieide Conf. 1:301.
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 283

25. Meredith, D. S. (1977). Recent advances in contral of post~

harvest deterioration using thiabendazole with special
reference to tropical and subtropical crops. Proc. 1977
British Crop Protection Conf.
26. Muirhead, I. F. (1976). Postharvest control of mango anthrac-
nose with benomyl and not water. Austral. J. Exptl. Agr.
Animal Husbandry 16: 600.
27. Ben-Aire, R. (1975). Benzimidazole penetration, distribution
and persistence in postharvest treated pears. Phytopathology
65: 1187.
28. Phillips, D. J. (1975). Detection and translocation of benomyl
in postharvest-treated peaches and nectarines. Phytopathology
65: 255.
29. Meredith, D. S. (1975). Control of fungal diseases of seed
potatoes with thiabendazole. Proc. 8th British Insectic.
Fungic. Conf. 2: 581.
30. Boyd, A. E. W. (1975). Fungieides for potato tubers. Proc. 8th
British Insectic. Fungic. Conf. 3: 1035.
31. Logan, C., Copeland, R. B. and Little, G. (1975). Potato
gangrene control by ultra low volume sprays of thiabendazole.
Ann. Appl. Biol. 80: 199.
32. Chiba, M. and Doornbos, F. (1974). Instability of benomyl in
various conditions. Bull. Envir. Contam. Toxicol. 11: 273.
33. White, E. R., Bose, E. A., Ogawa, J. M., Manji, B. T. and
Kilgore, W. W. (1973). Thermal and base-catalyzed hydrolysis
products of the systemic fungieide, benomyl. J. Agr. Food
Chem. 21: 616.
34. Vonk, J. W. and Kaars-Sijpesteijn, A. (1971). Methyl ben-
zimidazole-2-yl-carbamate, the fungitoxic principle of
thiophanate methyl. Pestic. Sei. 2:160.
35. Harding, P. R. Jr. (1962). Differential sensitivity to sodium
o-phenylphenate by biphenyl-sensitive and biphenyl-resistant
strains of Penicillium digitatum. Plant Disease Reptr. 46:
100.
36. Eckert, J. W. and Wild, B. L. (1981). Problems of fungieide
resistance in Penicillium rot of citrus fruits. in Pest
Resistance to Pesticides: Challenges and Prospects, C. P.
Georghiou and T. Saito, eds., Plenum Press (in Press).
37. Harding, P. R. Jr. (1972). Differential sensitivity to thia-
bendazole by strains of Penicillium italicum and P. digita-
tum. Plant Disease Reptr. 56: 256.
38. McDonald, R. E., Risse, L. A., and Hillebrand, B. M. (1979).
Resistance to thiabendazole and benomyl of Penicillium
digitatum and P. italicum isolated from citrus fruit
from several countries. J. Amer. Soc. Hort. Sei. 104:333.
39. Bertrand, P. F. and Saulie-Carter, J. L. (1978). The occurrence
of benomyl-tolerant strains of Penicillium expansum and
Botrytis cinerea in the mid-Columbia region of Oregon and
Washington. Plant Disease Reptr. 62:302.
284 J. W. ECKERT

40. Rosenberger, D. A. and Meyer, F •. W. (1979). Benomy1-to1erant

Penici11ium expansum in app1e packing houses in Eastern
New York. Plant Disease Reptr. 63: 37.
41. Kuramoto, T. (1976). Resistance to benomy1 and thiophanate-
methyl in strains of Penici11ium digitatum and P. ita1icum
in Japan. Plant Disease Reptr. 60: 168.
42. Kuramoto, T. and Yamada, S. (1976). DF-125, a new experi-
mental fungicide for contro1 of satsuma mandarin postharvest
decays. Plant Disease Reptr. 60: 809.
43. Kaplan, H. J. and Dave, B. A. (1979). The current status of
imaza1i1: a p.ostharvest fungicide for citrus. Proc. F1a.
State Hort. Soc. 92: 37.
44. Lavi11e, E. Y., Harding, P. R. Dagan, Y. Rahat, M., Kraght,
A. J., and Rippon, L. E. (1977). Studies on imaza1i1 as a
potential treatment for contro1 of citrus fruit decay.
Proc. Int. Soc. Citricu1ture 1: 259.
45. Birchmore, R. J., Brookes, R. F., Copping, L. G., and We11s,
W. H. (1977). BTS 40 542 - A new broad spectrum fungicide.
Ninth British Insect. Fungicide Conf. 2: 593.
46. Spa1ding, D. H. (1980). Contro1 of A1ternaria rot of tomatoes
by postharvest app1ication of imaza1i1. Plant Disease
Reptr. 64: 169.
47. Anonymous (1980). lmaza1i1: a new weapon in the fruit decay
batt1e. Citrograph 65: 95.
48. Rosenberger, D. A., Meyer, F. A. and Ceci1ia, C. V. (1979).
Fungicide strategies for contro1 of benomy1-to1erant
Penci11ium expansum in app1e storages. Plant Disease
Reptr. 63: 1033.
49. Bompeix, G., Coeffic, M., and Greiffier,
I
P. (1979). Lutte
contre 1es pourritures des peches a Moni1ia sp., Botrytis
sp. et Rhizopus sp. Fruits 34: 423.
50. Van Geste1, J., Heeres, J., Janssen, M. and Van Reet, G.
(1980). Synthesis and screening of a new group of fungi-
cides: 1-(2-pheny1-1, 3-dioxo1an-2-y1-methy1)-1,2,4-triazo1es.
Pestic. Sci. 11: 95.
51. Brown, G. E. (1979). Bio1ogy and contro1 of Geotrichum candidum,
the cause of citrus sour rot. Proc. F1a. State Hort. Soc.
92: 186.
52. Chitzanidis, A. and Laskaris, D. (1980). Effects of postharvest
dips of oranges in meta1axy1 against brown rot. Proc. 5th
Congress of the Mediterranean Phytopatho1ogica1 Union.
Patras, Greece, Sept. 1980. pp 151.
53. Edney, K. L. and Chambers, D. A. (1981). Postharvest treatments
for the contro1 of Phytophthora syringae storage rot of
app1es. Ann. App1. Bio1. 97: 237.
54. Davidse, L. C. and Flach, W. (1977). Differential binding of
methyl benzimidazole-2-y1-carbamate to funga1 tubu1in
as a mechanism of resistance to this antimitotic agent
in mutant strains of Aspergillus nidu1ans. J. Ce11
Biol. 72: 174.
CONTROL OF DISEASES WITH ANTIMICROBIAL AGENTS 285

55. Davidse, L. C. and Flach, W. (1978). Interaction of thiabenda-

zole with funga1 tubu1in. Biochem. Biophys. Acta 543: 82.
56. ,
Leroux, P. and Gredt, M. (1978). Effets de que1ques fongicides
systemiques sur 1a biosyntheses de l'Ergostero1 chez Botrytis
cinerea Pers., Penici11ium expansum Link et Usti1ago maydis
(DC) Cda. Ann. Phytopatho1ogy 10: 45.
57. Prusky, D., Bazak, M., and Bendrie, R. (1980). To1erance of
Penici11ium expansum in Israel to postharvest fungicide
treatments. Proc. 5th Congress Mediterranean Phytopath.
Union, Patras, Greece, Sept. 21-27. Published by the
He11enic Phytopath Soc.
HYDROXYPROLINE-RICH GLYCOPROTEINS IN THE CELL WALL OF DISEASED

PLANTS AS A DEFENSE MECHANISM

Marie Therese Esquerre-Tugay~, Dominique Mazau and

Alain Toppan

Universite Paul Sabatier

Centre de Physiologie V~g~tale - L.A. 241 CNRS
118, route de Narbonne
31062 Toulouse, France

INTRODUCTION

The plant cell wall may be considered as a barrier which sepa-

rates a protoplast from the surrounding medium. Yet it is not a
passive barrier, as indicated by active modifications under natural
and stress physiology processes such as growth, maturation, ripening,
abscission, senescence, and plant-microorganism interactions. This
activity results, in part, from the presence of pro teins and glyco-
pro teins inside the cell wall. Although already mentioned sixty
years ago(l) , cell wall pro teins were mainly studied after the dis-
cQvery in the 1960's (2), that some of these pro teins contain high
amounts of hydroxyproline (Hyp). This paper is concerned with the
study of these Hyp-containing pro teins that we will abbreviate,
according to their chemical nature, i.e. HRGP (Hydroxyproline Rich
Glycoproteins). The following points will be successively consider-
ed:

1 - Hydroxyproline-containing components in plants: their occurrence

and structural features;
2 - Hydroxyproline-rich glycoproteins in plant-pathogen interactions;
3 - Significance of the accumulation of hydroxyproline-rich glyco-
proteins in diseased plants;
4 - Role of cell surface interaction in the elicitation, via ethylene,
of hydroxyproline-rich glycoprotein biosynthesis.

287
288 M. T. ESQUERRE-TUGAYE ET AL.

1 - HYDROXYPROLINE-CONTAINING COMPONENTS IN PLANTS

Hyp-containing components are present in varying amounts in all

plant parts: roots (3), stems and leaves (4), tubers (5), seeds (6,7),
sap, and in the growth medium of cells in culture (8,9). Theyare
either located within the cell wall, or found inside or outside the
cello The main features of the chemical composition of these molecu-
les are summarized in Table I. They all have in common a high level
of Hyp, of the ß-hydroxyaminoacids serine and threonine, of alanine
or glycine, and they all contain sugars, mainly arabinose and galac-
tose. Accordingly these molecules are glycoproteins. In addition
to their sugar to protein ratio which varies from about SO to 90%,
they differ by their solubility. Intra- and extracellu1ar Hyp-
glycoproteins are quite soluble, whereas cel1 wall Hyp-glycoproteins
(HRGP) are highly insoluble in the native state once they are in-
corpora ted into the wall. Despite this limitation, ce11 wall HRGP
are better characterized because chemica1 and radiochemica1 tech-
niques were originally devised at the aim of understanding the
structure and biosynthesis of these glycoproteins.

Chemical (HC1(10), Ba(OH)2(11), N H40H (12), NaOC1-CH COOH (13»

and enzymic (14,15) hydro1yses and oxi~ation (Tab1e 11) a1tow recov-
ery of either Hyp alone, or glycoaminoacids and glycopeptides rich
in hydroxyproline. The resu1ts indicate that most Hyp residues in
isolated peptides are found in aseries of consecutive or distant
pentapeptides in which 4 Hyp residues are linked to serine at the
N-terminus (14) : -Ser-HYP4-' In these pentapeptides, the
1 D-galactopyranosyl residue is O-glycosidically linked to serine(14),
and 1 to 4 L-arabinofuranosy1 residues are O-glycosidica11y linked
to Hyp (11,16), a1though varying levels of non-g1ycosy1ated serine
and hydroxypro1ine are also found, depending on the group (Dicots
as compared to Monocots) (17,18), or on the species and on the age
of a particular plant (19). Altogether, the resu1ts allow predic-
tion of a left-handed helix with 3 Hyp residues per turn for the
secondary structure of the Hyp-rich portions of the wall HRGP (20).

In addition to structural investigations, one can also measure

the biosynthesis of ce1l wall HRGP using radio1abe1ed techniques (21,
22,23). The rate of 14C_Hyp and thus of HRGP deposition into the
cell wall can be measured in tissues or ce11s incubated in the pre-
sence of l4C-proline, since peptidyl Hyp originates from peptidy1
proline (21,24).

A combination of these techniques was retained to study ce1l

wall HRGP in plant microorganism interactions.
 ::I:
-<
0
:D
Table I. The major amino acids and sugars of Hydroxyproline-Rich Glycoproteins (HRGP) in 0
plants X
-<
"'tI
:D
Amino NELON TOMATO POI'ATO CORN TOBACCO WHEAT LOLIUM mu1tif1orum 0
r
acida, stems 1eaves tubers pericarp ce11s endosperm r--ce11s - - ,
sugar ce11 wa11s intrace11. intrace11. extrace11. Z
intrace11. extrace1~. m
( 10) (It)b (5) (6) (8) ( 7) ( 9) :D
n
::I:
HYP 11.7 23.1t 20.1t 12.2 23.8 16.7 9.9 11t.8 G)
r
ASP 10.9 S S S S 6.3 6.3 s
THR 6.2 11.1t 6.1 6.0
n-<
9.1 5.7 9.3 6.5 0
"'tI
SER 8.6 11t.3 12.6 7.7 11t.8 9.6 9.5 9.8 :D
GLU S 6.9 S S 6.6 0
7.5 12.2 -l
GLY 12.2
m
11.5 19.6 15.5 S S 7.8 6.7
Z
ALA SC S S S 21.3 21t.3 22.5 Cf)
17.3
PRO S S 6.9 10.3 S S S S

ARA + + ++ + ++ + + +
GAL + + + + ++ + ++ ++
other
HEXOSES ++ +
G1cN + + + +

aMo1ar " of tota1 amino acids residues.

b(4) Reca1cu1ated from Lamport 1970.A.R.P1.Phys 21:235 (1970) ; (5) A11en et a1. Biochem J. 171:665
(1978) ; (6) Boundy et a1. J. Bio1. Chem. 21t2:21t10 (1967) ; (7) Fincher et a1. Biochem J. 139:535
(1974) ; (8) Hori et 81. Phytochem. 16:1485 (1977) ; (9) Anderson et a1. Austr. J. P1. Physio1.
4:11t3 (1977) ;(10) Esquerre-Tugaye C.R. Acad. Sci. Paris Sero D 276:525 (1973).
Numbers correspond to reference 1ist.
cSma11 amounts.

I'.J
CD
c.o
290 M. T. ESQUERRE-TUGAYE ET AL.

Table II. A typical hydrQxyproHne~rich glycopeptide of the wall

HRGP and a sunnnary of A~ chelnical and B;~ radiochemical
methods used to study the major structural features and
the biosynthesis of this glycoprotein.

Ara,

~I~
Ara,

:I~
Ara,

~I ~
Gal (Ara')oa4 Ara,
la P
I~ I
.... _Ser_Hy p_Hyp_Hyp_Hyp_Lys_ ....
~I~
I
Method Result Recovery of

A - CHEMICAL .and BIOCHEMICAL METHODS(a)

Acid hydrolYSiS 15 Cleavage of peptide Hyp, o:ther

6 N HCI, 100'C, 18 h and glycosidic bonds amino acids
in glycoproteins

Partial acid hYdrolysis 14 Cleavage of furanosi- Deglycosylated

0.lNHCI,100' C, 1 h dic linkages Hyp-containing
material

Partial alkaline hYdrolysis 26 Cleavage of peptide Hyp-arabinosides

0.44 N Ba(OH 2 ), 90' C, 20 h linkages

Hydrazinolysis 15 Elimination of glyco- Non glycosylated

N2 H4 OH, 110' C, 18 h sylated -Ser and-Thr -Ser and-Thr
in HRGP
Oxidation by 1 % NaOCl_ 13 Cleavage of phenolic Hyp-rich
1 % CH 3 COOH, 75' C, 30 min crosslinks glycopeptides

Enzymic hydrolysis 14 Cleavage of peptide Hyp-rich

Trypsine pH 8.0, 2)' C, 18 h bonds involving basic glycopeptides
amino acids residues

B - RADIOCHEMICAL METHOD

14C_Proline incorporation 21 Synthesis of 14 C_pep _ Cell wall 14 C _

tidyl proline, leading peptidyl Hyp
to 14C-peptidyl Hyp
after hydro~~lation

a_ Numbers correspond to reference list.

HYDROXYPROLlNE-RICH GL YCOPROTEINS 291

2 - HYDROXYPROLINE-RICH GLYCOPROTEINS IN PLANT-PATHOGEN INTERACTIONS

Working on the hypothesis that host proteins could serve as

substrates for proteolytic degradation by fungal pathogens we dis-
covered, in 1973, that, instead of being lowered, the level of cell
wall protein and of Hyp is higher upon infection (10). This was
originally found in melon seedlings (cv. Cantaloup charentais) in-
oculated with Colletotrichum lagenarium, a fungal pathogen which
develops on the aerial part of this plant, notably on the fruits,
but the roots are left symptomless. Under greenhouse conditions,
melon seedlings are killed 7 to 9 days after inoculation.

Infected seedlings exhibit a general increase in both cell wall

pro tein and Hyp, whatever the organ considered, the roots included.
As shown in the sterns, this increase begins 3 days after inocula-
tion (19), at the time of symptom appearance, and then proceeds
throughout the disease to a level of Hyp which can be ten times as
high as that in healthy plants.

The structural features which normally characterize cell wall

HRGP, namely : a, their glycosylation pattern and, b, the presence
of extractable Hyp-rich glycopeptides were studied upon infection.
a. Hyp-arabinosides were isolated after hydrolysis of the wall with
Ba(OH)2 and ion exchange chromatography (26). Galactosyl serine was
measured in the presence of hydrazine (N 2H40H) which cleaves this
O-glycosidic linkage by aß-elimination process (12,22). It was
found that the two glycosylated amino acids were present in much
higher amounts in the cell wall of diseased plants but that the
tatios of glycosylated serine to Hyp were very similar in healthy
and infected plants.
b. Serine and Hyp were also found together in glycopeptides obtain-
ed by trypsinolysis of the wall and, upon sequencing, were recovered
under the form of the pentapeptide -Ser-HYP4-which typifies the wall
HRGP (15).

From these data, it was concluded that infected melon seedlings

have an increased ability to accumulate HRGP of the kind already pre-
sent in the wall of healthy plants, but in minor quantities.

This response was later shown to occur in several plants chal-

lenged by different microorganisms, as indicated by Hyp and Hyp-
arabinoside analyses of the cell walls of plants inoculated with
either fungi (37) or bacteria (unpublished data). However, it was
noticed that increases in Monocots were very low as compared to in-
creases found in Dicots.

Because of its widespread occurrence, the significance of cell

wall enrichment in HRGP upon infection was investigated.
292 M. T. ESQUERRE-TUGAYE ET AL.

3 - SIGNIFICANCE OF HYDROXYPROLINE-RICH GLYCOPROTEIN ACCUMULATION

IN DISEASED PLANTS

Melon seedlings were retained throughout this study because of

their ability to accumulate large amounts of cell wall HRGP in a
relatively short time upon infection by C. lagenarium. Experiments
were designed in order to control the extent at which this accumula-
tion proceeds, and to measure the amount of pathogen present in the
seedlings under these conditions.

Plants with higher than normal amounts of HRGP were obtained by

treatment with ethylene, 6 days prior to inoculation. As formerly
reported (27), this hormone promotes a Hyp enrichment of the cell
wall, artd this increase corresponds to the deposition of HRGP, as
demonstrated by Hyp-arabinosides analysis (22). Amounts of this
glycoprotein, lower than in control infected plants, were also
obtained by feeding plants with L-trans Hyp for 6 days during the
course of the disease. This amino acid specifically irthibits the
synthesis of the wall HRGP, which results in lower amounts of hyp-
arabinosides (22).

The amount of pathogen present in both situations was assessed

by measuring glucosamine, a component which characterizes the cell
wall of many fungi, but is present in only trace amounts in
plants (28). An inverse relationship was found between the amount
of HRGP and the extent of pathogen spread inside the plants (22).
Plants pretreated with ethylene contained much less glucosamine,
i.e. pathogen, and showed restricted symptoms as compared to untreat-
ed, infected controls. As a consequence, a large number of them
recovered, instead of being killed as usual. However, plants whose
HRGP accumulation was irthibited by free Hyp showed an accelerated
susceptibility to the disease : they contained more pathogen and,
as a result, were killed earlier (Fig. la, lb).

These experiments allowed us to conclude that the accumulation

of HRGP in melon seedlings is closely associated to their defense
against C. lagenarium. This defense becomes efficient provided it
starts early, before appearance of symptoms. The fact that ethylene
or 1 mM Hyp bring about a two to three fold increase, or a 30% in-
hibition in ItRGP deposition, only, suggests that even moderate
changes can be significantly associated to resistance. Such moderate
differences were later found in cultivar specific resistance and
also in Legume-Rhizobium symbiosis under conditions where the roots
were resistant to nodulation either constitutively or in the presence
of irthibitory concentrations of nitrates (unpublished data).

Because of the high insolubility of HRGP once they are incorpor-

ated into the wall, one cannot explain yet the biochemistry of
their association with resistance. One may hypothesize that they
are structural pro teins which strengthen the cell wall, or that they
HYDROXYPROLlNE-RICH GL YCOPROTEINS 293

" Hyp AND

" GIeN OVER
" Hyp and TIIE CONTROL
" GleN over
the control
+)0

+100

+10

------i----i . . . . .
..........
..........
............. GleN
-50 '" .........
......
-)

INOCULATION

l
-100

DAYS + C2H4 DAYS AFTER INOC.

I ,

15 19 22 27 )1 o 0.5 1 1.5 2 4
AGE OF THE SEEDLINGS (DAYS) (HYP] IN THE GROliTH MEDIUM (mM)

Fig.la. Cell wall hydroxyproline (I) and glucosamine (~) in

plants treated with ethylene before inoculation in a % of the same
components in control plants. Melon seedlings treated with ethylene
received this hormone for 7 days. These plants and non-ethylene
treated controls were then inoculated with Colletotrichum lagenarium
and grown in the air of the growth room without added ethylene. The
stems of both kinds of plants were harvested from day 0, before
starting ethylene treatment, to day 9 after inoculation, and their
cell wall extracted. Data represent the mean + SE of three
replicates (From M. T. Esquerre-Tugaye et al. i979 Plant Physiol.
64:320).

Fig.lb. Cell wall hydroxyproline (I) and glucosamine (.) in

diseased plants grown in the presence of L-trans hydroxyproline, as
a % of the same components in control plants. The addition of hy-
droxyproline at different concentrations to the culture medium start-
ed one day after inoculation of melon seedlings with Colletotrichum
lagenarium, and lasted 6 days. Control plants were simultaneously
inoculated and grown without hydroxyproline. The stems of both kinds
of plant were harvested 7 days after inoculation and their cell wall
extracted. Data represent the me an + SE of three replicates (From
M,T Esquerre-Tugaye et al. 1979 Plant Physiol. 64:320).
294 M. T. ESQUERRE-TUGAYE ET AL.

have a lectin-like activity. A Hyp-containing lectin which bears

structural similarities with cell wall HRGP has been isolated from
potato tubers (5,29). This Hyp-lectin isthought to be associated
with the resistance of potatoes against Pseudomonas solanacearum
through its agglutinating activity towards avirulent, but not to-
wards virulent, bacteria (30). This agglutination involves the LPS
fraction of the bacterial cell wall and is inhibited by oligosac-
charides that contain N-acetyl glucosamine residues.

Probably the most recent evidence that HRGP accumulation is a

novel parameter, which must be taken into account in plant-micro-
organism interactions, comes from the study of its elicitation.

4 - ROLE OF CELL SURFACE INTERACTION IN THE ELICITATION, VIA ETHYLENE,

OF HYDROXYPROLINE-RICH GLYCOPROTEIN BIOSYNTHESIS

The fact that HRGP begins to accumulate in infected seedlings

at a time when the pathogen is still restricted in the host (31)
suggests that cell surface interaction only, between both partners,
could be sufficient to elicit this accumulation. The role of ethy-
lene as a media tor in the elicitation of this phenomenon was in-
vestigated since ethylene alone can promote an enrichment of the
cell wall in HRGP (22), and that infected seedlings accumulate large
amounts of this hormone early on (32). Using several inhibitors of
the ethylene pathway in plants (33), namely L-Canaline, Benzyl iso-
thiocyanate and Aminoethoxyvinylglycine (AVG), as weIl as l-Amino-
cyclopropane-l-carboxylic acid (ACC) as a natural precursor of this
hormone (34), it was found that HRGP biosynthesis in infected Elants
is ethylene-dependent (35). The deposition of protein-bound 1 C-Hyp
in the cell wall closely parallels the biosynthesis of ethylene,both
being simultaneously inhibited or stimulated.

The role of cell surface interaction in promoting, via ethylene,

HRGP biosynthesis is evidenced by the presence of ethylene elicitors
on fungal cell surfaces (Table 111). These elicitors are glyco-
conjugates that we reported for the first time in plant-microorganism
interactions three years ago (32). They elicit the production of
ethylene by healthy tissues 60 to 90 minutes after incubation in
their presence (36) (Fig. 2). Their activity is such that even
microgram amounts induce both the synthesis of ethylene and of l4C-
Hyp rich glycoproteins in otherwise healthy tissues. Ethylene is a
necessary, intermediate step, since inhibitors of the ethylene path-
way also inhibits this elicitation process (37).

This approach provides a means of eliciting in healthy plants

the accumulation of HRGP which normally occurs in diseased plants,
so we can study the host genome activity responsible for this accu-
mulation, without the interfering presence of the pathogen genome
itself. It also suggests that triggering of wall HRGP biosynthesis
HYDROXYPROLlNE-RICH GL YCOPROTEINS 295

Table 111. Produetion of ethylene by healthy petioles ineubated in

the presenee of elieitors extraeted from Colletotriehum
lagenarium. Elieitors eorrespond to a erude extraet
eOfitaining sugars and nitrogen.

Elicitor Activity Crude extract composition

source nl C2 H4 /g petiole/h a mg Glc eq. mg N2

Mycelium 0.27 3.81 1.24

Cell walls 0.14 1.49 0.78
Culture filtrate 0.29
Control 0.06 0 0

apetioles (2g) are incubated in 5 ml of phosphate buffer 10 mM

pR 6.0, with or without elicitors (elicitors from 1 g mycelium).

P
//
/
/
/
/

/
/
/

I
l
I
I
I
I
I
I
I
I
I
/
I
I
I
I
0.1
/
P
/
/
/
/
/
/
/
/
6
/
/
P
I 2 6 8
-0 4 I I

Incubation time (hours)

Fig. 2. Produetion of ethylene by healthy petioles treated with

fungal elieitors. The assay was performed under the eonditions
given in Table 111.
296 M. T. ESQUERRE-TUGAYE ET AL.

in diseased p1ants is media ted by ce11 to ce11 recognition between

host and pathogen.

CONCLUSION

An understanding of the nature of defense reactions occurring

in p1ants is of importance for the practice of crop productivity
and storage. Due to their location as the most outer 1ayer of the
ce11, plant ce11 walls are particu1ar1y exposed to unfavorab1e
environmental conditions. It is the purpose of this paper to
demonstrate that the ce11 wall active1y participates in the defense
of p1ants through the accumu1ation of a Hyp-rich glycoprotein. Be-
cause this glycoprotein is very insoluble, it is, as yet, not c1ear
as to whether it acts as a structura1 protein or whether it fu1fi11s
other functions.

Ethy1ene p1ays a major ro1e in the e1icitation of this defense

response. The fact that this hormone is also invo1ved in aging
processes might exp1ain, in part, why mature p1ants are often more
resistant to disease.

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dans les parois ce1lulaires et le cytoplasme de tiges de
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298 M. T. ESQUERRE-TUGAYE ET AL.

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Toulouse (1977).
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by affinity chromatography on an N, N', N"-triacetylchitotri-
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33. M. Lieberman, A.T. Kunishi, L.D. Owens, Specific inhibitors of
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37. M.T. Esquerre-Tugaye, D. Mazau, A. Toppan and D. Roby, Elicita-
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STRESS METABOLITES

Norman F. Haard

Department of Bioehemistry
Memorial University of Newfoundland
St. John's, Newfoundland, Canada AlB 3X9

INTRODUCTION

A phenomenon, eomparable to indueed immunity in animals, was

reported for the late blight disease of potatoes over 40 years aga
(73). The phytoalexin theory of plant immunity evolved from these
early observations. Aeeording to this theory, a speeifie metabolie
interaetion between a plant host and a ehallenging mieroorganism
may give rise to the formation of phytoalexins (from Greek: to ward
off). The phytoalexins formed in potato aeted to inhibit the growth
of the virulent raee 1 of Phytophthora infestans. Originally, the
theory suggested that phytoalexins are absent in healthy plant
tissues and that the phytoalexin response is highly speeifie with
regard to pathogen and host eultivar. In the example of late
blight disease, potato tubers carrying the gene Rl and an avirulent
raee 0 to I. infestans represented the speeifie metabolie inter-
action. Recent studies have demonstrated that phytoalexins may
oceur in traee quantities in apparently healthy plant tissues and
that a variety of biological agents can trigger the aeeumulation
of phytoalexins in a given plant tissue. In view of these findings,
the phytoalexin concept has been redefined, e.g. "the term phyto-
alexin should serve as an umbrella under whieh chemieal eompounds
eontributing to disease resistanee ean be elassified whether they
are formed in response to injury, physiologieal stimuli, and the
presenee of infeetious agents or are the produets of sueh agents"
(58). It is now elear that these seeondary metabolites may aeeumu-
late in response to a wide array of ehemieal and physieal stresses
in addition to biologieal agents. As a result of these findings
the term "stress metabolite" has been used to deseribe seeondary
plant metabolites whieh aeeumulate in response to varied trauma
to plant tissue. While the terms "stress metabolite" and " phyto-

299
300 N. F. HAARD

alexin" have been used interchangeably, we shall consider phyto-

alexins as a special class of stress metabolites, i.e. substances
which have an implicit role in disease resistance. It should be
noted that additional factors, such as naturally occurring constit-
uents and mechanical barriers also play a role in the resistance
of plants to microbial challenge (114). There are numerous reviews
of work on phytoalexins and their role in plant immunity to disease
(29,55,58,59,67,72,107).

Generally stress metabolites possess some degree of toxicity

to living matter and given that this toxicity can be extended to
humans, one must consider their importance in the context of human
and livestock safety (113). Consideration of stress metabolites
with respect to mammalian toxicity is of continued concern because
of the reality of on-going plant breeding programs and because of
the increased complexity of food processing and handling operations.
The focus of this lecture will therefore be on the occurrence of
stress metabolites in edible plants, the nature of the triggering
mechanism by which these secondary metabolites accumulate in plants,
and our present understanding of their significance with regard to
food safety.

CHEMICAL NATURE AND DISTRIBUTION OF STRESS METABOLITES

Present evidence indicates that stress metabolites are wide-

spread in the plant kingdom. Serious attempts to identify involve-
ment of phytoalexins in host-parasite interactions invariably dem-
onstrate the formation of secondary metabolites possessing anti-
biotic activity. The number of new stress metabo1ites identified
in the past few years is indicative of the considerable activity
in this field of study (3,16,19,20,25,43,45,47,48,50,56,62,67,76,
87,89,98). There appears to be a taxonomie basis to the distri-
bution of phytoalexins in the plant kingdom (49). Members of the
Leguminosae tend to accumulate isof1avanoids and aeeording to a
reeent report (88), there is a dichotomy in phytoalexin distri-
bution amongst the Viceae whieh has taxonomie re1evance. When
the different members of the genera Lathyrus, Lens, Pisum and
Vicia were challenged with Helminthosporium carbonum or Botrytis
cinerea it was observed that Pisum and Lathyrus spp. produced the
isoflavanoid pisatin and Vicia and Lens species produced furano-
acetylenes. Stress metabo1ites have been identified in most plant
families of agronomie importance.

Green beans (Phaseolus vulgaris) produce a number of isof1a-

vanoid stress metabolites including phaseo11in, phaseollidin,
phaseollinisoflavan, 2'-methoxyphaseollinisofalvan, kievitone,
coumesterol, genistein and 2-hydroxygenistein (8). Garden pea
(Pisum sativum) also forms various isoflavanoids in response to
stress ine1uding pisatin, inermin, 3,4-hydroxy-2,3,9~trimethoxy-
STRESS MET ABOLITES 301

pterocarpan, 3-hydroxy-2,9-dimethoxypterocarpan and 2,3,9-tri-

methoxypterocarpan (17). Lima bean roots (Phaseolus lunatus)
produce coumesterol and other coumestans as a result of nematode
infection (86). Broad beans (Vicia faba) produce the isoflavanoid
medicarpin and, in addition, the furanoacetylene compounds wyerone,
wyerol, wyerone acid and wyerone epoxide (38). Lentil seeds (Lens
culinaris) yield a similar family of compounds as broad bean i;:--
cluding wyerone, wyerone epoxide and the isoflavanoid variabilin
(87). Stress compounds identified in soybean (Glycine~)
include glyceollin and its derivatives diadzein, coumesterol and
sojagol (64). Alfalfa (Medicago sativa) produces at least eleven
isoflavanoid derivatives including sativan, sativol, trifoliol,
medicarpin and diadzein (78). Peanuts (Arachis hypogoea) produce
stilbene-like compounds (46) and cowpeas (Vigna sinensis)produce
isoflavonoids such as phaseollin and a 2-arylbenzofuran compound
called vignafuran (82).

The Solanaceae family has also been extensively studied with

respect to stress metabolites. The principle class of compounds
formed in this family are diterpenes although sterols, steroidal
alkaloids and phenylpropanoid compounds are also formed in this
family. White potato (Solanum tuberosum), the tissue examined by
MUller and Börger in their classic study, is now known to form
more than twenty sesquiterpene stress metabolites including rish-
itin, katidinone, dehydrokatadinone, isolubimin, lubimin, oxylub-
imin, phytuberin and rishitinol (93,102). Other examples of ses-
quiterpene stress metabolites in this family include capsidiol in
green pepper (Capsicum brutesceus); 9-oxonerolidol and aubergenone
in eggplant (~. melangena); and rishitin in tomato (~. esculentum)
(31) .

The Solanaceae also contain steroidal alkaloids as natural

constituents (e.g. a-solanine, a-chaconine in potato or tomatine
in tomato) which increase in amounts as a result of stress condi-
tions (19,35). Other alkaloids, such as a- and ß-solamarine are
absent from normal potato tissue but accumulate as a result of
physical stress (94). Phenolic substances (e.g. chlorogenic acid
and scopoletin and sterols (cholesterol, stigmasteral, ß-sitosterol)
are also recognized as stress metabolites in Solanaeceous plants
(79) .

The Umbelliferae family also produce a variety of stress

metabolites. Parsnips may contain the coumarin compound xantho-
toxin as anormal constituent which increases as a result of
stress (53). Sweet potatoes (Ipomea batatas) produce coumarin
derivatives, (e.g. umbelliferone, esculentin), phenylpropanaids
(e.g. chlorogenic acid) and furanoterpenes. Two families of
furanoterpenes are the 15 carbon group, noteably ipomeamarone,
and 9-carbon terpenes such as 4-ipomeanol (32). Carrot roots
produce isocoumarins, noteably 8-hydroxy-3-methyl-6-methoxy-3,
302 N. F. HAARD

4-dihydroisocoumarin and other phenolics which were originally

recognized due to their contribution to the bitterness resulting
from improper vegetable storage (92,101).

Other examples of stress metabolites in edible plants include

betavulgarin and betagarin in sugar beets (Beta vulgaris) (27);
viniferins in grapevines (62); safynol and at least eleven other
polyacetylene compounds in safflower (106); moracins A and B in
mulberry (104); momilactones A and B in rice, wheat and rye,
avenacin in oats and unidentified components in corn (67).

In general, stress metabolites most often occur as homologous

families of structurally related compounds. The ratio of one com-
pound to another may vary with the part of the plant and the nature
of the stress. In addition, different chemical families of stress
metabolites may arise simultaneously or in response to different
stress conditions. A competitive relationship, with respect to
biosynthesis, may occur as is the case for steroidal alkaloids and
terpenes in white potato (11,95). It is important to recognize
the multiplicity of stress metabolites because it undoubtedly
complicates our understanding of their role as phytoalexins and as
potential mammalian toxicants. Furthermore, it is often assumed
(perhaps wrongly) that the most abundant stress metabolite(s) in
a given tissue iso of most importance from the toxicological view-
point.

MECHANISM OF ELICITOR RESPONSE - BIOTIC ELICITORS

Considerable effort has beenmade towards elucidating the

molecular basis for elicitation of stress metabolite(s) formation
in plant tissues. When sweet potato root is challenged by Ceroto-
cystis fimbriata the further invasion of the host (black rot) is
checked by a layer of necrotic tissue and by a layer of furanoter-
penoid phytoalexins in the adjacent healthy tissue. In general,
fungal or insect extracts can also elicit stress metabolite forma-
tion (18,57,60). Most often the race specificity of the pathogen
is lost as a result of cell disruption of the pathogen and much
lower levels of metabolite are often observed. Specific cellular
fractions or pathogens have also been shown to possess the capa-
bility of switching on stress metabolite synthesis in the host.
Numerous studies indicate that cell wall components are active in
eliciting stress metabolites (1,3,14,34,40,60). Chitosan, a poly-
mer of B-(l + 4) glucosamine isolated from Fusarium solani cell
walls is an effective phytoalexin elicitor (33). The elicitation
by chitosan can be potentiated by host chitosanase (75). The cell
walls of Clasdosporium fulvum contain a glycoprotein which elicits
rishitin accumulation in tomato fruit (22). Aß-glucan, isolated
from the cell walls of Phytophthora megasperma and yeast, served
to stimulate isoflavanoid accumulation in kidney Qean and soybean
and terpene accumulation in potato (14).
STRESS METABOLITES 303

Other studies have related fungal metabolites or other intra-

cellular components of the pathogen to the elicitor response (18,
55, 97). There has also been an attempt to relate limited host
cell necrosis to the elicitor response. While most biotic or
abiotic stress conditions generally cause a hypersensitive response,
this is not always the case (11). However, it was recently demon-
strated that phaseollin and phaseollidin were formed in live bean
hypocotyl tissue when incubated in contact with dead bean hypocotyl
tissue (37). The authors provide evidence that a water soluble
(elicitor) component is released during host cell damage.

In most cases the specificity (race or cultivar) associated

with the native systems is lost with molecular elicitors. This
has prompted interest in suppressors of phytoalexin as the basis
of specificity. Suppressors may be molecular components contained
in the pathogen. For example, glucans (ß-(l + 3), ß-(l + 6»
isolated from compatible races of !. infestans are more active in
suppressing the hypersensitive reaction than those from incompat-
ible races (60). Similarly, Mycophaerella pinodes, a pathogen of
pea, contains a low molecular weight peptide which acts to suppress
phytoalexin formation by the host (97). These studies indicate
that virulence of the pathogen may relate more to its ability to
provide suppressors of phytoalexin formation rather than to a lack
of an elicitor. Other factors such as ageing time after cut injury
appear to influence the elicitor response. Potato slices preincu-
bated for 5 hours are no longer responsive to abiotic elicitors (11)
or to !.infestans (95). This may be related to the spontaneous
formation of glycoalkaloids accumulation resulting from cut injury
in the absence of a terpene elicitor. Other factors such as mem-
brane barriers (76), excessive necrosis and soil components can
suppress the elicitor response in certain systems (11,36).

Another factor which may influence the specificity of the

phytoa1exin response is the finding that the pathogen can metabo-
1ize phytoa1exins (26,42,61,63,65,99,110). Generally, phytoa1exins
appear to be metabolized to more polar compounds. This would appear
to be another means of achieving race specificity for the pathogen.
Moreover, it is known that the host may also metabolize phytoa1exins
(9,51,112). Often, the formation of specific metabolites is tran-
sient indicating further metabo1ism (11). Many of these stress
metabo1ites are phytotoxic so their further metabo1ism by the host
may serve an adaptive function.

ABIOTIC ELICITORS

It is now c1ear that a variety of physical and chemical

stresses can evoke the accumulation of phytoalexins in plant tissues.
304 N.F.HAARD
While it was earlier argued that substances which accumulate in
response to abiotic stress are not true phytoalexins, it is now
clear that most, if not all, phytoalexins may accumulate as a re-
sult of such non-specific elicitors.

Physical

Mechanical 1nJury in the form of bruising or slicing is gen-

erally necessary to elicit stress metabolite formation. In most
instances mechanical damage alone is insufficient to trigger stress
metabolite formation although this is the only factor involved with
glycoalkaloid accumulation in potato (94); phaseollin accumulation
in bean pod (24); or coumarin synthesis in carrot (92). Other
physical stresses, in combination with cut injury, can evoke stress
metabolic accumulation. For example, exposure of tissue slices to
ultraviolet light for a brief time triggers furanoterpene accumula-
tion in sweet potato (32), diterpene accumulation in potato slices
(11), isoflavanoid accumulation in lucerne (21), and bean pod (24),
soybean (84) and cowpea (69). Other physical factors known to
evoke stress metabolite accumulation are chilling injury, visible
light, anoxia and reduced atmospheric pressure (32). In all
examples other than chilling injury, preparation,of tissue slices
appear to be aprerequisite for elicitation.

Chemical

There is now abundant evidence that a wide array of chemical

agents can evoke accumulation of stress metabolites in plant
tissues. In many instances the chemical treatment evokes a reac-
tion causing localized tissue necrosis, although this hypersensi-
tive response is not always observed (11). Certain chemical agents
appear to elicit stress metabolite accumulation in all tissue
systems when applied under controlled conditions of time, tempera-
ture and concentration. For example, heavy metal salts of copper
or mercury trigger ipomeamorone accumulation in sweet potato (32),
rishitin accumulation in potato (11), vestitol accumulation in
lucerne (21) and phaseollin in bean (36). Actinomycin D and other
transcriptional and translational inhibitors also appear to be
effective elicitors of stress metabolites when applied und er care-
fully controlled conditions (12,13). Metabolic inhibitors such as
sodium flouride, detergents such as sodium lauryl sulfate, various
sulfyhydryl reagents and salts such as 5 mM sodium acetate are
additional examples of chemical agents known to evoke stress meta-
bolite biosynthesis (13). Moreover, recent studies indicate various
pesticides act to elicit phytoalexin accumulation (74, 108) indicat-
ing that the efficacy of certain fungicides and insecticides may be
partly dependent on switching on a native biological defense
mechanism of the plant.
The findings that relatively innocuous conditions can give
STRESS MET ABOLITES 305

rise to stress metabolite formation is further supported by reports

that produee obtained from wholesale and retail markets may eon-
tain signifieant quantities of toxie stress metabolites (10,15,25,
70). There is a need for more information on the oeeurrenee of
stress resulting from postharvest handling and storage of produce
on metabolite accumulation.

Role of Ethylene

We have discussed three factors which influenee the aceumula-

ti on of stress metabolites in plant tissues - elicitors, suppres-
sors and catabolism of the induced metabolite by the hast or invad-
ing organism. Various lines of evidence point to the involvement
of ethylene gas in the accumulation of stress metabolites by plant
tissues. The subject has recently been reviewed (32). It has
long been recognized that ethylene of endogenous or exogenous
origin is necessary for the accumulation of isocoumarin in earrot
root (101). Ethylene gas can potentiate the accumulation of
terpenes resulting from heavy metal treatment (32) and influence
the balance of different terpenes resulting from the fungal induced
reaction in sweet potato (32) and white potato (2). Phenylpro-
penoid accumulation in carrot slices is also potentiated by exogen-
ous ethylene (92) and the isoflavanoid pisa tin accumulates in
garden pea as a result of ethylene treatment.

It has been suggested that ethylene acts to alter stress

metabolite accumulation via its suggested role (100) in cyanide
resistant respiration (2,32). Whether this apparent relationship
is a direct one or is incidentally related will have to await
further study.

There is relatively little information on the role of other

plant hormones in stress metabolite accumulation. Synthetic
auxins can influence phaseollin accumulation (23) and abscisic
acid acts to prevent rishitin and lubimin accumulation in f.
infestans infected potato but acts to increase the levels of these
terpenes in f. cucumerimun challenged potatoes (41). It has also
been reported that the highly unsaturated eicosapentaenoic and
arachidonic acids elicit antimicrobial stress metabolite accumu-
lation and cause necrosis in potato tuber slices (5) and this
relate to the wound hormone traumatin (115). There would, accord-
ingly, appear to be a need for additional information on the inter-
play of hormones in the elicitation of stress metabolites.

TOXICITY OF STRESS METABOLITES

Most, if not all, stress metabolites possess antibiotic

activity which is more or less specific. For example, the iso-
flavanoid stress metabolites from French bean are selectively toxie
306 N. F. HAARD

to gram positive but not to gram negative bacteria (28). The sensi-
tivity of microorganisms to the isoflavanoids pisatin and phaseollin
is dependent on the growth conditions employed (111).

Members of the various chemical classes of stress metabolites

are also phytotoxic at microgram concentrations. Examples where
phytotoxicity of stress metabolite has been characterized include
pisatin. (96), phaseollin (83), rishitin (66) and wyerone (39). The
phytotoxicity of stress metabolites is probably related to some
lesion formations in postharvest fruits and vegetables.

The toxic characteristics of stress metabolites in mammalian

systems is not very weIl understood although there is ample evidence
of toxicity for certain compounds. The general concern about stress
metabolites with regard to food safety has been reviewed by Wood
(113). Concern was heightened following Renwick's hypothesis that
blighted potatoes containing diterpene stress metabolites such as
rishitin are linked to the severe birth defects spina bifida and
anencephaly (85). While some experimental evidence has supported
this hypothesis (80), later studies did not confirm the involve-
ment of diterpenes in this response (81). Later studies have
implicated glycoalkaloid stress metabolites in teratogenic effects
(54). The current status of the Renwiek hypothesis is complex
and was recently reviewed (52).

The glycoalkaloids a-solanine and a-chaconine have been

responsible for isolated cases of human death and innumerable in-
stances of food poisoning (52). In many instances, minor, unidenti-
field alkaloids, occurring in potatoes have been implicated in food
poisoning (71).

The sweet potato furanoterpenoids have been responsible for

several instances of livestock death and one suspected case of
human death (32). Ipomeamarone, the major stress metabolite in
black rot infected roots is a hepatotoxin and 4-ipomeanol, the
major component in chilI injured and Fusarium infected roots is. a
lung toxin which has been extensively studied as to toxicology in
recent years (6). It has also been suggested that excessive
dietary consumption of sweet potato may be linked to lung cancer.

Other studies on the toxicology of stress metabolites are

preliminary in nature and their significance to mammalian organisms
is not clear. Pisatin and related isoflavanoid compounds like
phaseollin and medicarpin are reported to lyse human and anima 1
erythrocytes (77). Certain steroidal alkaloids, such as a-tomatine
are known to complex with membrane sterols and cause lysis (90).
Phytoalexins formed in rye grass infected with f. rathayi were
found to be acutely toxic to mice at relatively low doses (103).
Accordingly, there is sufficient evidence to justify concern about
the toxicological influence of stress metabolites on livestock
STRESS METABOLITES 307

and people. Virtually nothing is known about possible toxicity of

these substances resulting fram long-term consumption of sub-acute
doses. It may weIl be that the body has adequate detoxification
systems to handle the low levels of these agents which may be
associated with marketable vegetables

CONCLUSlON

Recent evidence indicates that stress metabolites, a family

of noxious compounds, may accumulate in edible plant tissues as a
result of relatively innocuous circumstances of stress and, in the
few vegetable systems studied, may accumulate in blemished areas
of marketable produce. Current toxicological da ta does not justify
alarm but there is presently sufficient information to justify
caution in the use of new storage, handling and processing tech~
niques since they may comprise a sufficient trauma to the tissue
to evoke accumulation of these noxious agents. Similarly, plant
breeders should recognize the benefits of disease resistance
achieved in new cultivars may be accompanied by a greater propen-
sity of the tissue to form stress metabolites or to the formation
of new substances.

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 STRESS MET ABOLITES 313

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MYCOTOXINS AS A DETERIORATING FACTOR IN STORED CROPS

Palle Krogh

Department of Veterinary Microbiology

Purdue University, West Lafayette, IN 47907, USA
and
Department of Microbiology
Royal Dental College, Copenhagen, Denmark

INTRODUCTION

Mycotoxins are secondary fungal metabolities which by ingestion

in man and animals cause intoxication, termed mycotoxicosis. Already
in 1968 more than 100 toxin-producing mold species were known (1),
mainly belonging to the fungal genera Aspergillus, Penicillium and
Fusarium, and this list has since been considerably extended. In-
formation on occurrence of mycotoxins in foodstuff is however limited,
and survey data are only available for a few mycotoxins. Similarly
the number of recognized mycotoxicoses in domestic animals is limited
and amounts to less than ten, and in man even fewer mycotoxicoses
are known. Thus four mycotoxins or groups of mycotoxins were con-
sidered to be causally associated with disease in humans, proven or
hypothetically, according to arecent publication by the World Health
Organization (2). These mycotoxins have been selected for detailed
description in this chapter.

MYCOLOGICAL AND CHEMICAL NATURE OF SELECTED MYCOTOXINS

The aflatoxins are produced by Aspergillus flavus and Aspergil-

lus parasiticus. They are derivatives of furano-coumarin, and al-
though 17 compounds have been identified, aflatoxin BI' B2 , GI and
G2 are the most commonly occurring in natural products. Aflatoxins,
as most of the mycotoxins, are low-molecular weight compounds (2)
(Table 1), and they are heat stable so that they will survive most
cooking conditions. Lactating animals ingesting aflatoxin contaim-
inated feed excrete metabolites in the milk called aflatoxin M.
315
316 P.KROGH

Tab1e 1. Chemica1 Characteristics of Af1atoxins

Mo1ecu1ar Mo1ecu1ar Me1ting

Af1atoxin formu1a weight point

B1 C17H1206 312 268-269

B2 C17H1406 314 286-289
Gl C17H1207 328 244-246
G2 C17H1407 330 237-240
Ml C17 H1207 328 299

Ochratoxins are produced by a number of species within the

genera Aspergillus and Penicillium, with Penicillium viridicatum
being the dominating producer. The ochratoxins constitute a group
of close1y related derivatives of isocoumarin linked to an amino-
acid, L-ß-phenylalanine. Of the ni ne toxins only ochratoxin A is
occurring in natural products. Zearalenone is a phenolic resorcylic
acid lactone, produced by a number of Fusarium species, e.g.
Fusarium graminearum and Fusarium moni1iforme. The trichothecenes
possess the tetracyclic l2,13-epoxy-trichothec-0-ene skeleton, and
more than 30 trichothecenes have been identified, with T-2 toxin,
nivalenol and deoxyniva1eno1 being most commonly found. They are
produced by a number of species belonging to the funga1 genera:
Fusarium, Cephalosporium, Myrothecium, Trichoderma and Stachybotrys.

FORMATION OF MYCOTOXINS

Mycotoxin production can occur in most plant products, but

cereal and oil seeds constitute the crops most likely to be contam-
inated. Apart from the toxin-producing fungal organism mycotoxin
formation is. influenced by a number of factors, the major ones being
temperature and water activity; other factors are : mechanical
damage, nature of substrate and invertebrate vectors (3). The water
activity and temperature conditions for ochratoxin A production is
i11ustrated in Fig. 1 (4). A water activity of 0.83 appears to be
the minimal va1ue also for af1atoxin production, but the minimal
temperature for aflatoxin production is much higher (10°C), compared
to ochratoxin A (5,6), pointing to the fact that whereas aflatoxin
is a contaminant of crops from warmer areas, ochratoxin has been
encountered in products from temperate climatic zones. The water
activity and temperature conditions for production of zearalenone
and trichothecenes are inadequately known.
MYCOTOXINS AS A DETERIORATING FACTOR 317

P. viridicatum
Rate of Growth (mm/day) Ochratoxin A (mg)

5.0 0.4

2.5 0.2

o I

Water Activity Temperature °c

o
Fig. 1. Growth and ochratoxin A production by astrain Penicillium
viridicatum on maize sucrose agar under various conditions of
temperature and water activity.

OCCURRENCE OF SELECTED MYCOTOXINS IN FOODSTUFF

In reporting on contamination of foodstuffs with mycotoxins

two parameters are used, namely, concentration and frequency. Mon-
itoring foodstuffs for mycotoxins is therefore dependent on the
availability of chemical methods of analysis, which can identify
and quantify individual mycotoxins, whereas biological procedures
are unreliable for that purpose. Chemical methods of analysis are
available only for a limited number of mycotoxins. In the following
the occurrence of four mycotoxins is reported, namely, aflatoxins,
ochratoxins, zearalenone and the trichothecenes, because these
toxins have been associated with disease in man or animals. Other
mycotoxins for which causal associations in animal diseases have
been documented, such as sporidesmin causing facial eczema in rumi-
nants (7), are not included because no survey data have been report-
ed.
318 P. KROGH

Aflatoxin

By far the most information on mycotoxin contamination is

available for aflatoxine The occurrence of aflatoxin (aflatoxin
Bl + B2+Gl+G2) in a number of plant products is illustrated in the
following ~ables 2-4. Aflatoxin was first detected in the 1960's
as a contaminant in groundnuts, and this is a commodity likely to be
contaminated, with frequencies up to 49% (Table 4). Also maize is
often contaminated, with frequencies up to 97%, and concentrations
up to 15.6 mg/kg have been reported (Table 2). In addition to the
commodities mentioned here, aflatoxin has been found in treenuts
(often heavily contaminated), copra and spices. lt is evident from
Tables 2-4 that aflatoxin contamination occurs in crops from tropical-
subtropical areas. This reflects the fact that aflatoxin-producing
Aspergillus flavus strains are found mainly in tropical-subtropica1
areas, where also the optimal temperature conditions for af1atoxin
production are present. Contaminated crops can then be shipped to
other areas, and thus practica11y all countries are faced with the
problem of af1atoxin contaminated foodstuffs. As a resu1t, many
countries, in the af1atoxin producing areas and outside, have
established to1erances for af1atoxin in foods and feeds (9). The
use of af1atoxin-contaminated feed for 1actating animals resu1ts
in carry-over into the milk of hydroxy-metabo1ized compounds,
af1atoxin M1 and M2' with almost unchanged properties. As indicated
in Table 5 marke ted milk samp1es have been found contaminated up to
80%, when very sensitive methods ab1e to detect 0.01 ~g af1atoxin/1
have been used. It is also evident from Tab1e 5 that af1atoxin-
contamination of milk occurs in a number of North-European countries
(Belgium, East- and West Germany, the Nether1ands, U.K.) where
aflatoxin is not usua1ly produced, but mere1y imported, in contami-
nated crops used for anima1 feed. In addition, af1atoxin residues
may occur in the carcass and organs of pigs, as indicated by experi-
mental data as we11 as findings in marketed products.

Ochratoxin

As indicated in Tab1e 6 ochratoxin Ais, 1ike af1atoxin, a

contaminant of grain in particu1ar, but has also been found in cof-
fee beans and peanuts. Feed samp1es are more frequent1y contaminated
than foods. The high frequency (above 50%) of ochratoxin contamina-
tion in feeds in Canada and in Denmark is due to biased samp1ing
plans being used in the Canadian study, whereas on1y samp1es associ-
ated with porcine nephropathy were screened in the Danish investi-
gation. It is evident from Tab1e 6 that ochratoxin A is found in
the temperate c1imatic areas of Europe and North America, the 1ead-
ing producer being Penici11um viridicatum. Whether ochratoxin A
occurs in tropica1-subtropica1 crops, produced by the more thermo-
phi1icfunga1 producers in the Aspergillus ochraceus group, cannot
be estab1ished at present, as no survey data are avai1ab1e from
such areas, but the observation that coffee beans have been found
MYCOTOXINS AS A DETERIORATING FACTOR 319

Tab1e 2. Occurrence of Af1atoxin in Maize (8)

No. of % Contam- Range of Con-

Country Sampies inated centration (~g/kg)

USA 1594 2.5 3 - 37

USA 2866 8.2
USA(South- 60 35.0 6 - 348
East)
Thailand 35.0 average: 400
Uganda 40.0 average: 133
Philippines 97.0 average: 213
India 5 250 - 15600
France 461 0.2 52

Table 3. Occurrence of Aflatoxin in other Cereals (2,8)

No. of % Contam- Range of Con-

Country Sampies inated centration (~g/kg

USA 66 (sorghum) 3.0 13 - 50

USA 157 (rice) 0.6 5
Uganda 69 (sorghum) 23.0 average: 152
Viet Nam 139 (rice) 31.0
USSR 169 (wheat) 0.6 100
USSR 138 (wheat) 17.4 10 - 333
USSR(Ka-
----~
zakstan) 50 (wheat) 4.0 5 - 10
.~--------~~~~~------------------~--~~------

Table 4. Occurrence of Af1atoxin in Groundnuts (8)

No. of % Contam- Range of Con-

Country Samp1es inated centration (~g.kg)

USA 361 15.0 5 - 50

Sudan 173 41.0 5 - 1000
Thailand 49.0 average: 1530
Uganda 17.0 average: 363
320 P. KROGH

Table 5. Occurrence of Aflatoxin MI in Milk (2)

No. of % Contam- Range of Con-

Country Sampies inated centration (~g/kg)

Belgium 68 61.8 0.02 - 0.2

DDR 36 11.1 1.7 - 6.5
FRG 260 45.4 0.05 - 0.33
FRG 419 18.9 0.05 - 0.54
India 21 14.3 up to 13.3
Holland 95 77.9 0.09 - 0.5
UK 278 30.6 0.03 - 0.5
South Africa 56 0
USA 320 7.5 O.J, - 0.4
USA 302 63.6 up to 3.9

contaminated with Aspergillus ochraceus-produced ochratoxin A may

support this assumption. Residues of ochratoxin A may occur in food
of animalorigin particularly from pigs and poultry (10).

Zearalenone

Fusarium-elaborated zearalenone is found predominantly in maize

as indicated in Table 7. Only a few surveys of foodstuffs have been
carried out, in a restricted number of countries. However,the
compound is surely occurring in many other areas, and in other
cereals, as indicated by reports on the associated disease in pigs,
the estrogenic syndrome, which have been reported from many coun-
tries.

Trichothecenes

Very few reports on the trichothecenes have been published so

far (Table 8), so no frequency of contamination can be calculated,
and the understanding of the trichothecenes as contaminants of
foodstuffs is very inadequate. However, recent improvements in
methodology for the detection of trichothecenes in foodstuffs may
soon change the present concept that the trichothecenes are only
rarely occurring.

ADVERSE EFFECTS OF MYCOTOXINS

The adverse effect of mycotoxins in man and animals has been

elucidated through observations of field cases (for man exclusively)
and of experiments. Field cases are of major interest in the con-
 s=
Tab1e 6. Natural Occurrence of Ochratoxin A in Foods and Feeds of Plant Origin (10) n-<
o~
o
X
No. of Sampies Percentage Range of Ochratoxin Z
Commodity Country Ana1yzed Contaiminated A Level (].lg/kg) cn
»
cn
»
o
m
Foods ~
maize USA 293 1.0 83 - 166 m
::c
maize (1973) France 463 2.6 15 - 200 6
maize (1974) France 461 1.3 20 200 ::c
wheat
»~
(red winter) USA 291 1.0 5 - 115 z
G)
wheat ."
(red spring) USA 286 2.8 5 - 115 »n
bar1ey (malt) 50 6.0 ~
9 - 189 o
bar1ey USA 127 14.2 10 - 40 ::c
coffee beans USA 267 7.1 20 - 360
maize Yugos1avia 542 8.3 6 140
wheat Yugos1avia 130 8.5 14 - 135
wheat bread Yugos1avia 32 18.8
bar1ey Yugos1avia 64 12.5 14 - 27
bar1ey Czechos1ovakia 48 2.1 3800
bread U.K.* 50 2.0 210
f10ur U.K. 7 28.5 490 - 2900
rice Japan 2 (100) 230 - 430
beans Sweden 71 8.5 10 - 442
peas Sweden 72 2.8 10

*A11 sampies suspicious of containing mycotoxins W

N
 cu
N
N

Tab1e 6 (Continued)

No. of Samp1es Percentage Range of Ochratoxin

COImllodity Country Ana1yzed Contaiminated A Level (flg/kg)

Feeds
bar 1ey, whea t , Po1and 150 5.3 50 - 200
oats, rye, maize
mixed feed Po1and 203 4.9 10 - 50
maize Yugos1avia 191 25.7 45 - 5125
bar1ey, oats Sweden 84 8.3 16 - 409
wheat, hay Canada 95 7.4 30 -- 6000
wheat, oats Canada 32 56.3 30 - 27000
bar1ey, rye
bar1ey, oats Denmark 33 57.6 28 - 27500

:-r
::D
o"
G>
J:
MYCOTOXINS AS A DETERIORATING FACTOR 323

text of this paper, because the reporting of field cases is the

first step in the epidemiologie calculations providing the basis
for estimation of economic loss. The adverse effects of the four
mycotoxins are listed in Table 9 - 11. Outbreaks of liver damage,
consisting of necrosis and subsequent cirrhosis, have been reported
for man and domestic animals, associated with levels of aflatoxin
in foodstuffs in the range 1 - 10 mg/kg (Table 9). Only for primary
liver carcinoma in man are data on incidence rates available.
Aflatoxin Bl particularly is a potent liver carcinogen in experi-
mental animals, and in areas in Africa and Southeast Asia with high
incidence rates of primary liver carcinoma a strong correlation
between aflatoxin in take and liver carcinoma incidence has been
observed. This does not, however, exclude other factors in the
etiology of primary liver carcinoma in these areas (2). At levels
in foodstuffs below 10 mg/kg ochratoxin A is exclusively a nephro-
toxin, causing nephropathy in single-stomach animals, particularly
pigs (Table 10) and most feed lots analyzed contained less than
10 mg/kg (Table 6). Ochratoxin A is not a proven nephrotoxin in
man, but preliminary data seem to indicate that this food contaminant
may play a causal role in endemie nephropathy occurring in rural
populations on the Balkan peninsula (10). Outbreaks of zearalenone-
induced estrogenic syndrome in pigs has been reported from many
countries (Table 11), and a few outbreaks have been observed of
triehotheeene-indueed intoxieations in farm animals, eharaeterized
by necrosis of the alimentary traet. No eases of zearalenone-
assoeiated disease in man have been reported, and the hypothesis
that triehothecenes were causally involved in Alimentary Toxie
Aleukia (ATA), predominant in areas of USSR in the 1930's and 1940's,
eannot be confirmed, because ATA is no longer eneountered.

ASSESSMENT OF TRE LOSS DUE TO MYCOTOXINS

A1though information on concentrations in foodstuffs of myeo-

toxins associated with disease in man and animals is avai1ab1e as
listed in Table 9 - 11, it is not possib1e to predict disease
occurrence and distribution in human populations and economic 10ss
in farm animals on the basis of that kind of information. The main
reason for this is that the existing toxicologic eoncept for myco-
toxicoses cannot explain the effeet of variation in mycotoxin ex-
posure, and it is evident from Tab1e 2 - 8, that there is a pro-
nounced variation in the frequency of mycotoxin contamination.
Assessment of the economic loss due to mycotoxins in farm animals
can however be made, when data on disease occurrenee and distri-
bution are available. This is unfortunately not generally the ease
for mycotoxicoses, but as evident from Table 10 data on prevalence
rates are available for ochratoxin A-induced nephropathy in pigs.
The temporal and spatial distribution of this disease has been
documented for several countries in Northern and Central Europe
(Table 12) using data obtained from the veterinary meat inspection
324 P. KROGH

Table 7. Occurrence of Zearalenone in Foodstuffs (2)

No. of % Contam- Rang~of Concen-

Country SampIes ination traUon (IJg/kg)

USA 576(maize) 1.0 450 - 800

USA 223 (maize) 17.0 100 - 5000
USA 11 (maize- 81.8 12 - 69
meal)
Canada 1 (maize- 100 13
cornflakes)
Zambia maize-beer 10 - 4600
Lesotho l40(beer) 12.0 300 - 2000

Table 8. Occurrence of Trichothecenes in Foodstuffs (2)

Country Commodity Compound Concentration

(IJg/kg)

USA maize T-2 2000

USA barley T-2 25000
USA barley Nivalenol
USA maize Deoxynivale- 100 - 1800
(3 sampIes) nol

at pig slaughterhouses. Although prevalence rates for the disease,

during endemic and epi4emic periods, are available only for Denmark
at present, preliminary da ta indicate a similar distribution pattern
in other countries listed in the table. In addition, ochratoxin
A-induced porcine nephropathy is likely to occur in many other
countries, when the worldwide occurrence is considered (Table 6).
Associated with the renal damage induced in pigs by ochratoxin A
is a growth depression (observed in many other mycotoxicoses as
weIl), the extent of which has been experimentally determined.
Both the decrease in daily gain and increase in feed consumption
contribute to the economic loss, which amounts to approximately
8% if the exposure is 1 mg ochratoxin Aper kg feed, a moderately
low level, which does induce a kidney damage that can be detected
at the meat inspection. An economic loss of 8% based upon the
price level in Denmark 1980, corresponds roughly to US $ 10 per
pig, affected by ochratoxin A-induced nephropathy (Table 13).
Employing the data from Tables 12 & 13 the economic loss due to
ochratoxin A-induced porcine nephropathy in the husbandry of
MYCOTOXINS AS A DETERIORATING FACTOR 325

Table 9. Adverse Effects due to Aflatoxins (2)

Organ/Tissue Goncentration Epidemiologie

Anima 1 Alteration in Food (mg/kg) Data

Turkey Liver neerosis 1 10 outbreaks

" cirrhosis
Gattle " " 1 2 outbreaks
Pig " "
Dog " " 1 2 outbreaks
(hepatitis X)
Man Liver eareinogen-
esis 3.5-222 ng/kg incidence rates
bw/day 2.0-13.0/10(5)
Man Liver fibrosis 0.25 - 15.6 outbreaks
Enfants Reye's syndrome ? outbreaks

Table 10. Adverse Effects due to Oehratoxin A

Organ/Tissue Goneentration Epidemiologie

Animal Alteration in Food (mg/kg) Data

Pig a) nephropathy 0.2 - 10 prevalenee

(and other
single
stomaeh
animals)
b) nephropathy + above 10 no data
extrarenal
damage
Man? Balkan endemie 0.005-0.140 prevalenee
nephropathy rates
326 p, KROGH

Tab1e 11. Effeets of Zeara1enone and Triehotheeenes (2)

Organ/Tissue Coneentration Epidemiologie

Anima1 Alteration in Food (mg/kg) Data

Zeara1enone

Pig estrogenie 0.1 - 6.8 outbreaks

syndrome
Catt1e inferti1ity 14 one outbreak

Triehotheeenes

Catt1e hemorrhages 2(T-2 toxin) one outbreak

Pou1try a1imentary 25 (T-2 toxin) one outbreak
traet neerosis
Man ATA ? epidemies

Tab1e 12. Oeeurrenee of Myeotoxie Poreine Nephropathy (11,12,13)

Preva1enee Rate (Cases per 10 4pigs)

Country Endemie Level Epidemie Level

Denmark 1 - 10 50 - 80
Sweden ? ?
Czeehos10vakia ? ?
Hungary Ca. 1 ?

Tab1e 13. Eeonomie Loss of Experimenta11y OA-Indueed (14)

Poreine Nephropathy

Feed Level of OA Deerease in' Inerease in Eeonomie Loss

(mg/kg) during Daily Gain Feed Con- in in US$
3 months % sumption % % per Pig

0.2 6.4 5.6

1.0 4.2 4.9 8.0 10
4.0 17.2 19.3
MYCOTOXINS AS A DETERIORATiNG FACTOR 327

several areas of the world has been estimated (Table 14). The
estimates are based upon the assumption that the ochratoxin A-
induced nephropathy does occur in these areas, and that the pre-
va1ence rates and agricu1tural economy in these areas are similar
to those in Denmark. Thus the combined annua1 economic lass in
Europe (EEC), U.S.S.R. and U.S.A. is estimated to be US $ 1.4 mill.
This is a conservative estimate, as higher feed levels of ochratoxin
A than 1 mg/kg will increase the economic lass considerab1y (Table
13), and epidemie preva1ence rates wou1d result in an economic
lass 10 - 50 times greater. If similar estimates of economic lass
in anima1 husbandry were made for other mycotoxicoses it is evi-
dent that the combined lass would be considerab1e. The above
estimates inc1ude on1y the lass due to growth depression. If the
disease resu1ts in death of the anima1s, the lass wou1d be much
1arger. Assessment of the occurrence and distribution of mycotoxin-
associated diseases in human populations has so far been inhibited
by the lack of chemica1 procedures for mycotoxin detection app1icab1e
in c1inica1 chemistry. Recent improvements in methodo10gy for
af1atoxin and ochratoxin quantitation for c1inica1 use may provide
the necessary tool for assessment of the role of mycotoxins in
human populations.

Tab1e 14. Estimated Economic Loss of OA-induced Pareine

Nephropathy in Various Countries

Annua1 No. of Pigs(15,16) Predicted No. Estimated Annua1

S1aughtered in 1979 of Pigs with Economic Loss
Country (x1000) Nephropathy Lass (in US $)

Denmark 13 389 6 690 66 900

EEC 119 850 59 925 599 250

USSR 75 000 37 500 375 000

USA 89 000 44 500 445 000

Total 10ss: EEC + USSR + USA 1 419 250

 328 P. KROGH

ACKNOWLEDGEMENTS

The use of Fig. 1 from M.D. Northo1t and coworkers (4) is

gratefu11y acknow1edged. Pub1ished as paper No.8572 of the
Agricu1tura1 and Experiment Station, Purdue University, West
Lafayette, Indiana, USA.

REFERENCES

1. D. E. Wright, Toxin produced by fungi, Ann. Rev. Microbiol.

22: 269 (1968). - -
2. World Health Organization, "Envirorunental Health Criteria
ll:Mycotoxins" wao, Geneva (1969).
3. C. W. Hesse1tine, Conditions leading to mycotoxin contamination
of foods and feeds, in "Mycotoxins and other Fungal Related
Food Problems," J. V-.-Rodricks, ed., American Chemica1
Society, Washington DC (1976).
4. M. D. Northolt, H.P. van Egmond, and W.E. Pau1sch, Ochratoxin
A production by some funga1 species in relation to water
activityand temperature, ~. Food Prot. 42:485 (1979).
5. M. D. Northolt, H.P. van Egmond, and W. E. Paulsch, Differences
between Aspergillus flavus strains in growth and aflatoxin
BI production in relation to water activity and temperature,
~. Food Prot. 40:778 (1977)
6. M. D. Northolt, C.A.H. Verhu1sdonk, P.S.S. Soentoro, and W.E.
Paulsch, Effect of water activity and temperature on
af1atoxin production by Aspergillus parasiticus, ~.
Milk Food Tech. 39:170 (1976).
7. L. G. Atherton, D. Brewer, and A. Tay10r, Pithomyces chartarum,
A fungal parameter in the aetiology of some diseases of
domestic animals, in: Mycotoxins, I.F.H. Purehase, ed.,
Elsevier, Amsterdam-(1974).
8. L. Stoloff, Occurrence of mycotoxins in foods and feeds, in:
Mycotoxins and other Fungal Related Food Problems, J. V.
Rodricks, ed., American Chemical Society, Washington, D.C.
(1976).
9. P. Krogh, Mycotoxin tolerances in foodstuff, Pure~. Chem.
49: 1719 (1977).
10. P. Krogh, Ochratoxins, occurrence, biological effects and causal
ro1e in diseases, in: "Natural Toxins," D. Baker and T.
Wadström, ed., P'ergamon Press, Oxford (1980).
11. P. Krogh, Epidemiology of mycotoxic porcine nephropathy, Nord.
Vet.-Med.28:452 (1976).
12. L. Rutqvist, N.-E, Björklund, K. Hult, and S. Gatenbeck,
Spontaneous occurrence of ochratoxin residues in kidneys of
fattening pigs, Zbl. Veto Med. A. 24:402 (1977).
13. A. Piskac, R. HalQuzka, ~Drabek,-Y. Mala, and C. Smrcek,
Mykotoxicka nefropatie prasat ve velkochovech, Veterinarstvi
29:253 (1979).
14. P. Krogh, N.H. Axelsen, F. Elling, N. Gyrd-Hansen, B. Hald,
MYCOTOXINS AS A DETERIORATING FACTOR 329

J. Hy1dgaard-Jensen, A.E. Larsen, A. Madsen, H.P. Mortensen,

T. M611er, O.K. Petersen, U. Ravnskov, M. Rostgaard, and
O. Aalund: Experimental porcine nephropathy. Changes of renal
function and structure induced by ochratoxin A-contaminated
feed. Acta path. microbio1. scan., Section A, Supp1ementum
No. 246, 21 pp., (1974).
15. The Danish Pig Meat Sector, "Statistics 1979," Copenhagen (1980).
16. Food and Agricu1ture Organization, "Production Yearbook," Rome
(1975).
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS WHICH INFLUENCE

POSTHARVEST QUALITY, MATURITY AND STOREABILITY OF FRUIT

F. Bangerth

Institut für Obst-, Gemüse- and Weinbau

Universität Hohenheim
Stuttgart, Germany

INTRODUCTION

The work of a postharvest physiologist is usually concerned

with the quality and durability of a product at harvest and in
storage. However, this quality, or potential for quality, is often
affected or determined long before that time, and storage may in many
respects be regarded as the preservation of predetermined quality.
In addition, the way a fruit has to be stored greatly depends on its
preharvest life, e.g. the extent of its accumulation of calcium,
sugars, acids, on cell size and cell number etc. Growth regulators
and phytohormones affect most of these processes and the postharvest
physiologist should be aware of the potential as well as the problems
related to their use.

Although only a few of the growth regulators and phytohormones

applied in modern orchard management are used to affect the quality
and post harvest behaviour of fruits directly, many of them do have
side effects which influence the post harvest life of the treated
fruit. Growth regulators are able to affect quality as well as
ripening of fruits when applied as early as the time of blossoming
or shortly thereafter. Because of the considerable period of time
between most of these treatments and the physiological event, it
seems unlikely that the effect of these growth regulators is a direct
one. More likely, they act via an alteration of physiological
processes occurring during or shortly after treatment (e.g. cell
division, Ca 2+ uptake, assimilate partitioning etc.). Quite often
this may be media ted by an interaction between the growth regulator
applied and the synthesis, metabolism, transport etc. of endogenous
hormones. Whilst we have at least some ideas about the participation
of endogenous hormones in ripening and senescence of fruit (Sacher

331
332 F.BANGERTH

1973, McGlasson et al. 1978, Lieberman 1979, Bruinsma, this Vol.)

our knowledge about the interactions of growth regulators and endo~
genous hormones is still rather restricted. It is this field, how-
ever, which has to be explored if we wish to make progress in our
understanding of how growth regulators act. In addition, the use
of growth regulators may be an excellent tool to modify endogenous
hormone levels, metabolism, etc., thus enabling us to investigate
better their role in growth and development.

Because many details of the practical use of growth regulators

in fruit production in general and their effects on the postharvest.
behaviour of fruits in particular can be found in literature surveys
of Faust (1973), Sharples (1973), and Monselise (1979), this review
is restricted largely to more recent and less recognized developments.
Due to the more limited availability of solid data and our even
greater ignorance of the relevant physiological processes, pitfalls
are likely and guesswork is more necessary. It is hoped, however,
that in this way it is possible to draw the attentionof investigators
to those less developed fields and to stimulate activity in these
areas.

INDUCED PARTHENOCARPIC FRUIT SET AND ITS EFFECTS ON POSTHARVEST

BEHAVIOUR

A number of growth regulators and plant hormones are used in

practice to induce or stimulate parthenocarpic fruit set. Gibberellins
are the most prominent group among them but auxins also have an effect
especially on tomatoes, cucurbits and some other multiseeded fruits
(Dennis 1973). Recentlya combination of GA3. an auxin and a cyto-
kinin has been wide1y used in experiments in the UK on a number of
different pome and stone fruits in order to stimulate fruit set
(Schwabe 1978, Webster and Goldwin 1981). Several of these treat-
ments clearly affect postharvest quality physiology and marketability.
With regard to effects on market standards, it is we1l known that
some growth regulators particularly gibberellins can change the
shape of parthenocarpic pear and apple fruits to such an extent that
they are not further acceptab1e for the fresh market and therefore
have to be processed (Stembridge 1973). Puffiness and malformation
of parthenocarpic tomatoes induced by some synthetic auxins are
other examples of this type of problem (Ry1ski 1979, Bangerth,
unpublished). The hormonal and/or physiological reasons for these
deviations from cultivar specific fruit shapes are barely understood.

As far as nutritional and storage characteristics are concerned

some induced parthenocarpic fruits show clear disadvantages which
are moreserious than the alterations in fruit shape. Parthenocarpic
pears e.g. but also some apple, tomato and sour cherry cu1tivars
ripen faster, have a higher respiration rate, less acidity, firmness
and Vitamin C and are more susceptible to some physiological dis-
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 333

orders (Modlibowska 1966, Kotob and Schwabe 1975, Gil et al.. 1972,
Bangerth unpublished). These alterations (see e.g. Table 1.) are
not simply a GA effect because they can also be observed in tomato
fruits induced with some auxins. These effects seem to be related
to parthenocarpic fruit development, and it would be most interest-
ing to know whether natural parthenocarpic apple, pear or tomato
fruits show a similar behavior. Most of the disadvantages mentioned
are probably related to a reduced Ca 2+ content in parthenocarpic
fruits, as shown in Table 1. Other mineral constituents are obvious-
ly not affected. Ca is one of the most crucial factors in determin-
ing postharvest quality and physiology and has therefore been in-
vestigated in some detail for a number of fruits (Shear 1975,
Sharples and Johnson 1977, Bangerth and Link 1972 and many others).
Calcium affects respiration (Fig. 1), softening, acidity, vitamin
C content, rate of ripening and a number of physiological disorders
including chilling injury (Chaplin and Scott 1980) in a number of
fruits. To increase its concentration in the fruit flesh is one of
the most efficient preharvest methods to improve storage life. Due
to these remarkable effects the transport of Ca 2+ into storage organs
has been the subject of many investigations and it seems that one
component of this rather complex transport pathway is hormonally
regulated (see Bangerth 1979 for lit.). A mutual interaction be-
tween auxin and Ca 2+ transport seems to exist as can be concluded
from results with auxin transport inhibitors, like TIBA or morph-
actins (Bangerth and Firuzeh 1971, Stahly and Benson 1976). To
relate this to parthenocarpic fruits we have measured diffusible
auxins from seeded and induced parthenocarpic fruits and found in
most cases a considerably lower auxin concentration in diffusates
from the latter, which may explain their lower Ca 2+ content (Bangerth
and Sjut 1978, Sjut and Bangerth in prep.). Thus although partheno-
carpy is not an important issue in fruit production it demonstrates
a role for growth regulators and endogenous hormones in determining
the concentration of this very important element in fruits, and
suggests a future possibility for the correction for low Ca 2+
concentration and deficiencies by the application of appropriate
growth regulators. In fact one new growth regulator from BASF can
increase the Ca 2+ concentration in fruits even when applied at low
concentration to young fruitlets. Preliminary experiments in our
laboratory seem to indicate a stimulated auxin diffusion out of
tomato fruits treated with this growth regulator. If this can be
verified it will be a good example of how more fundamental research
into growth regulator-hormone interactions can eventually lead not
only to a better understanding of the physiological processes in-
volved but also to an important practical result.

FRUIT THINNING AND ITS EFFECT ON POSTHARVEST QUALITY

Selection and breeding for highly productive cultivars in

combination with intensive orchard management methods have created
334 F. BANGERTH

Tab1e l. Some Characteristics of .Seeded (contro1) and Induced

Parthenocarpic Fruits at Harvest Time

Fruit Titrat-
Calcium firm- Ascorbic ab1e
Treat- Fruit content ness acid acidity
Fruit ment weight mg/lOOg kg/cm mg/100g meq/g

App1e Contro1 165 27 7.8 9.8 0.82

(Golden GA 4 /7 143 20 6.6 7.2 0.91

DeI.)

Pear Contro1 128 49 10.4 7.0

(Alex. GA 3 204 23 7.7 4.4

Lucas)

Tomato Contro1 48 82 35.0 73.0

GA3 18 63 28.0 65.0

trees which after normal fruit set produce more fruits than they
can support without 10ss of size and qua1ity. Fruit- thinning is
therefore a common practice in modern fruit cu1tivation as far as
app1es, peaches, mandarines etc. are concerned. It wou1d be an
even more useful and desirable technique for many fruits if more
effective and reliable growth regulators could be found for this
purpose. Thinning - mostly achieved by growth regulators like NAA,
4 -CPA, carbaryl, ethephon etc. - reduces alternate bearing and
increases fruit size. In addition, however, and this is seldom
recognized, it improves colour development, soluble solids, ti trat-
able acidity and therefore taste and quality in apples (see Table
2), and also in pears and peaches. These are not specific effects
of a particular growth regulator but are obviously caused by any
substance which reduces a heavy fruit load.

On the other hand it is often claimed that thinning, mainly by

increasing fruit size, reduces the Ca 2+ content of fruits and there-
fore impairs storage life and increasespostharvest physiological
disorders. Link (1973), however, has shown that when fruits from
thinned trees are compared with those from unthinned ones over a
period of several years, physiological dis orders like bitter pit in
fact are reduced due to the more regular bearing of these trees.
So in tota~ thinning produces an overall increase in fruit quality.
This may change, however, if C2H4-releasing chemicals are used for
this purpose more frequently. Even applied at early stages ethrel
accelerates ripening of some fruits.
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 335

~4

~2
A
~

48

46

44

42 •
•
40

•
. 38

736
CI
•
.
~
.... 34
0
,. -0.82** •
u32
CI
~o
36

34

32

30

28

26

24 ,0-0.88**
,
01 .
~300
I

~bo .~o .Jo .JO

7~O
PEEL CALCIUM, ppm
1100

Fig. 1. Relationship of average respiration rate at 21°C to peel

Ca 2+ content of 'Baldwin' apples. A.197l-72. B. 1972-73
(Bramlage et al. 1974).

ASSIMILATE PARTITIONING BETWEEN FRUITS AND VEGETATIVE TISSUE

Fruit are almost entirely dependent on long distance transport

of assimilates from the leaves and therefore subject to competition
with vegetative growth. This competition may not only restrict
yield but also quality. Growth regulators, especially the so-called
growth retardants like alar, CCC or ethrel should be useful tools
to achieve that goal. In reality, however,most of these substances
not only check extension growth but also favour lateral thickening
of twigs and branches (Barden 1968, Firuzeh 1971). thus a significant
336 F.BANGERTH
Table 2. Effect of fruit thinning on fruit size and color, soluble
solids and titratable acidity of 'Golden Delicious' apples
(Link 1967)

Treatment Character

% fruits
with a %fruits meq. titr.
diameter with acidity
larger yellow green % soluble per 100 ml
than 70mm* color solids* juice*

Control 20.2 2 19 ll.8 4.8

Hand 32.5 8 13 12.7 5.8

thinned

Chemically 31.0 10 4 12.5 5.3

thinned

*mean values of 4 year results

redistribution of assimilates in favour of fruits is doubtful. This

is also emphasized by the fact that these growth retardants do not
greatly affect carbohydrate content of treated fruits (Drake et ale
1980, Lüdders and Fischer-Bölukbasi 1980). In addition substances
like alar and ethrel, when applied early, inhibit fruit growth and
are therefore not very popular. especially with cultivars where
fruit size is a problem.

Other substances like some synthetic auxins - especially NAA _

when painted on to pruning cuts in late winter or early spring re-
tard or even prevent the outgrowth of lateral buds around these cuts
and therefore reduce considerably vegetative growth (Jackson et a1.
1978 and Fig. 2). In our experiments (Bangerth, unpublished) these
treatments improved colour, soluble solids content, firmness and
storage behaviour of two apple cultivars in two years. Presently
it is not clear to us whether this was the result of a change in
partioning of assimilates and probably Ca 2+, or of the higher light
intensities reaching the fruits because of the reduced leaf area
especially on the top of the trees.

Chemical pinching agents which kill or temporarily check the

growth of shoot apices are ariother class of growth regulators which
may not only enhance fruit set and hence yield but also diminish
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 337

Fig. 2. "Goiden Delicious" apple trees one year after treatment

of pruning cuts with (A) or without (E) NAA

vegetative competition for Ca2+ and therefore improve fruit Ca 2+ and

probably storage life (Jackson et al. 1978).

These results, when confirmed by additional experiments, may

open a new and promising field for further improvement of fruit
quality and storeability without losses on yield. Special attention
should be paid to a better understanding of the basic mechanisms
of competition and nutrient partioning and how hormones and
growth regulators affect them (Pa trick 1976, Wareing 1978).
338 F.BANGERTH

APPLICATIONS OF GA 3 AND THEIR EFFECTS ON FRUIT QUALITY

GAs have been wide1y used in fruit production for quite differ-
ent purposes and because they affect market -. nutritiona1 -, and
storage qua1ity criteria it seems justified to treat them separate1y
and more extensive1y. It has been a1ready mentioned that GAs are
app1ied to induce especia11y parthenocarpic pome fruits. In addi-
tion they are frequent1y used to increase fruit set by reducing
June - drop e.g. in app1es (Wertheim 1971). citrus (Monse1ise 1979).
stone fruits (Webster and Go1dwin 1981) etc. sometimes in combination
with a cytokinin and/or an auxin. These treatments c1ear1y change
market standards with a number of fruits. In some app1e cu1tivars
e.g. GA3 and Benzyladenine app1ications after fruit shape as we11 as
size in a way that may have some practica1 significance for some
count ries 1ike the USA or Canada (Unrath 1974. Looney 1979). Posi-
tive effects on size have also been reported from peaches (Zucconi
and Bukovac 1978). seed1ess grapes (Sachs and Weaver 1968) 'A1exander
Lucas' pears (Buchloh and Kaya1i 1972, and Tab1e 1) and a few other
fruits. More often, however. GA app1ications to fruit at this ear1y
deve10pmenta1 stage reduce the number of seeds and. probab1y as a
resu1t of this. fruit size (Wertheim 1971. Krezdorn 1973, Bangerth
and Götz 1975).

In seeded wine grapes this GA-induced reduction in the number

of seeds per berry (20-50%) advances maturity and stimu1ates ear1y
sugar accumu1ation and acid catabo1ism. which may be of advantage
in countries with short autumn season (Peynaud and Ribereau-Gayon
1971, Bangerth and GBtz 1975 and Tab1e 3). Currants obvious1y be-
have simi1ar1y (Hartmann. pers. communication).

Apart from these GA-effects on grapes and probab1y currants it

has been shown that gibbere11ins can retard ripening of severa1
c1imacteric as we11 as non-c1imacteric fruits. even when app1ied
short1y after fruit set (see e.g. Webster and Go1dwin 1981). A
more pronounced effect can be observed with 1ater app1ications.
which may be re1ated to the general dec1ine of endogenous GAs to
very 10w levels during maturation (e.g. Jackson and Coombe 1966,
Ebert and Bangerth in press). When app1ied during 1ater stages of
ripening GA retards the deve10pment of a great number of different
fruits such as app1es and pears (Sharp1es 1973), prunes(proebsting
and Mi11s 1966), pineapp1e (Norman 1978), citrus (Monse1ise 1979),
apricots (Abde1-Gawad and Romani 1974). kaki fruits (Kitagawa et a1.
1966) etc.

However, it is surprising that with the exception of citrus

no practica1 app1ications have yet been deve10ped from these
esperimenta1 resu1ts. In citrus preharvest GA treatments are
used quite extensive1y to prevent premature pee1 senescence and
physio10gica1 dis orders re1ated to this process (Coggins et a1.
1960, Coggins 1973). Here again a sharp drop in endogenous
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 339

gibberellins in the peel is probably the reason for the good response
of these fruits to gibberellin applications (Goldschmidt and Galili
(1974». It is noticeable that this effect of GAs on clorophyll de-
gradation is counteracted by ethylene and vice-versa (Dostal and
Leopold 1967, Goldschmidt et al. 1977 and Fig. 3).

Another example of GA/C2R4 interaction is the premature ripen-

ing of pears caused by low night temperatures before harvest (Wang
et al. 1971). A rapid outburst of C2R4 is the result of these low
night temperatures followed by an advanced respiratory climacteric.
Similar observations have been made by Streif (1976) with apples.
Wang et al. have been able to prevent this early autocataltic C2H4
production as weIl as the premature ripening by GA 3 (and alar)
applications (Fig. 4)

Dostal and Leopold (1967) found the effect of dipping tomatoes

into GA 3 solutions to be restricted to chlorophyll degradation and
lycopene synthesis with almost no change in respiration pattern.
Similar observations for citrus have been made by Goldschmidt et al.
(1977) who found much more pronounced effects of GA on chloroplast
reactions than on other ripening parameters. On the other hand in

ORANGE
O.!!

S.E
•

!lOG

1 10 100 1 10
GAJ (mgll) BA (mgll)
GREEN
Fig. 3. Effect of combined GA3-ethephon and BA-ethephon treat-
ments on colour change of mature green harvested orange
fruit. Fruits were stored after treatment for 7 days at
2SoC prior to colour evaluation (Goldschmidt et al. 1977).
340 F. BANGERTH

_ . COOLEO
ETHYLENE 0-0 • '" GA " •• '",.n'
10 0---0 .21\41 GA
A-A • ALA"
.---. HEAT'EO

60 RESPIRATION

50

~ 40
I
o
:11:

"
cJ' 30
u

10

o 2 4 , I 10
Da,. a' 70· F
Fig. 4. Effect of preharvest temperature, GA and a1ar on respira-
tion and ethy1ene production of 'Bart1ett' pears after
picking. (Wang et a1. 1971).

pears and apricot GA app1ications affect respiration as effective1y

as chlorophyll degradation. Species - different reactions, and
more 1ike1y, the different app1ication methods used (dipping of
detached tomato and citrus fruits but preharvest spraying of apricots
and pears) may be responsib1e for the observed variations in GA
effects, emphasizing once more the difficu1ties in comparing the
effects of growth regulator/hormone treatments when different app1i-
cation methods have been emp10yed.

Citrus rind disorders can be a serious problem especia11y when

the fruits have to be 1eft for 10nger periods on the tree or in
storage. Under these conditions physio10gica1 as we11 as patho10gi-
ca1 rind dis orders can be very efficient1y reduced by preharvest GA
app1ications (Coggins 1973, Cha1utz pers_ communication). Similarly
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 341

some physiological apple and pear disorders related to senescence

can be diminished by preharvest GA applications (see Sharples 1973
for lit.). Although not a postharvest disease russeting is a
serious problem with a number of important apple varieties and con-
siderably affects postharvest market standards. Early GA4/7 appli-
cations effectively reduces this abnormal development of the fruits
epidermis (Taylor 1975, Eccher and Boffelli 1981). Some disadvantages
of GA applications like delayed degreening of citrus (Monseliese
1979) or inhibition of flower initiation in apples have to be
mentioned.

ALAR AND ITS EFFECTS ON POSTHARVEST QUALITY

Alar is a good example of a growth regulator not primarily used

to influence postharvest physiology and quality which has, however,
prnounced side effects on these processes. A vast amount of litera-
ture exists about the use of al ar in fruit production (Faust 1973,
LUdders 1973, Sharples 1973), although in retrospect it is difficult
to justify the wealth of scientific attention that has been paid to
this chemical. For this reason only the most fundamental effects
of alar on the postharvest behaviour of fruits will be mentioned
here.

From an applied point of view the retardation of ripening of

pome fruits (especially apples) and the acceleration of ripening
of stone fruits (even when climacteric, like peach) is the most
interesting effect of alar. This allows the grower to extend the
harvest per iod by spraying only part of the orchard and more
importantly the ripening per iod can be concentrated, especially with
stone fruits, allowing the number of pickings to be reduced consid-
erably (see Fig. 5).

This may be particularly important for fruits harvested

mechanically, e.g. for processing. Another desirable effect of alar
is its inhibition of preharvest drop of apples which may be related
to the delayed ripening of treated fruits. Retardation of chloro-
phyll loss and the better firmness of treated apple fruits also may
be related to this effect on ripening.

Alar treated ftuits frequently show a higher colouration due

to a higher accumulation of anthocyanins. This is clearly unrelated
to effects of the chemical on ripening because it is observed in
fruits whose ripening is accelerated (cherries, peaches etc.) as
well as in fruits with an inhibited ripening (apples e.g.). It
seems possible that this is in some way linked to an alar induced
shift in carbohydrate metabolism (Faust 1973), an hypothesis
probably supported by the observation that alar aggravates the
occurrence of two physiological disorders in some apple cultivars
342 F. BANGERTH

e... 100

: 90
>
~IO
l:
... 10-
5
f 60
1o.!IO I> Conlrol
o V 1000 ppm
#- 40 - o 2000ppm
I:t. 4000ppm
'"> • eOOOppm
5-' 20
~
u t I , , I , t , I , ,

, 4 11 6 1 • • 10 " 12 g 14
DAYS FROM FIRST HARVEST

Fig. 5. Effect of alar on 'Redskin' peach fruit maturation when

applied during stage I. (Byers et al. 1972)

(internal breakdown and core flush) which are also related to an

alteration in carbohydrate metabolism. A clearly undesirable effect
of early alar applications is their diminuation of fruit size. It
is still not known whether this is due to a reduction in cell divi-
sion or in cell expansion.

More interesting than its practical application seems to be the

use of alar as a tool to explore in more detail the mechanisms
controlling fruit ripening. Comparative studies on pome versus stone
fruits are particularly interesting since they can show opposite
responses to applications of alar. Surprisingly this opportunity
has so far been used only to a limited extent. Looney (1969) and
Rhodes et al. (1969) have shown that alar treated apple fruits have
a delayed and reduced capacity for C2H4 biosynthesis during ripening
and that exogenous ethylene can overcome this effect of alar on
ripening (Fig. 6). In peaches on the other hand, where alar acceler-
ates ripening, the production of ethylene during the final rapid
growth phase is increased (Looney et al. 1974). Nevertheless the
authors cast doubt on the opinion that this increased C2H4 production
is the cause of the earlier ripening and favour instead a role for
ABA in this fruit. Thus it seems unlikely that.alar is acting
directly on ethylene biosynthesis in apples and peaches.

Other hypotheses of how alar may exert its effects on fruit

ripening include that of Williams and Stahly (1970) who suggested
an interference with auxin metabolism, and Martin et al. (1964) who
proposed an effect on gibberellin metabolism. Both lines, however,
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 343

XETHYLENE
PROOUCTION ON
8/12168
("IAgII.. )
~ CONTROL 14.8
........ 6.3XIO-\A 8·9 6.9
20 ___ 3.2 X 10 - 2M 8.9 none c!eleclobl •
....... 6.3XIO- 2M 8·9 ..

..-.....
~ 15
.
8
'i
XETHYLENE
PROOUCTION
ON 8/12/68
10 (,.IAgIh. )
CONTROL 20.3
6.3XIO-~ 8·9 11.5
3.2X 10-2M 8·9 6.6
6.3X 10-2M 8·9 3.2

Ol~~ __~____~__~____~__~ ® f
@
86 7 8 9 10 11 8/6 7 8 9 10 11
Fig. 6. Respiration patterns of and ethylene production by
'Tydeman's Early' apples. (A), treated with B-9;
B-9 plus supplemental ethylene. (Looney 1969).

have not been followed up in recent years, although with the avail-
ability of modern analytical methods it seems worthwhile to look
again into such growth regulator-hormone interactions. Whether the
opposite effects of alar on fruit ripening of pome - and stone fruits
are media ted through an interaction with endogenous phytohormones or
with other physiological processes, the unravelling of such an inter-
action would be an important contribution to our understanding of the
initiation of ripening in climacteric fruits in general.

REGULATION OF ETHYLENE LEVELS IN FRUITS BY PREHARVEST APPLICATIONS

OF GROWTH REGULATORS

From a theoretical as weIl as from an applied point of view

developments in ethylene physiology have been among the most promis-
344 F. BANGERTH

ing ones in the fie1d of hormones and growth regulators. Because of

the dominating ro1e of this hormone in the ripening behaviour of all
c1imacteric fruits (Biale 1968, Burg and Burg 1965, Sacher 1973,
McG1asson et a1. 1978, Lieberman 1979) effects on qua1ity are c1ear1y
expected. The more recent deve10pments in this area started with
the discovery of ethy1ene - re1easing compounds (Abe1es 1973) and
have culminated with the almost complete elucidation of the bio-
synthetic pathway of ethylene (Adams and Yang 1979). More important,
this biosynthesis can be effectively controlled from both sides:
Endogenous ethylene production can be inhibited by applications of
the rhizobitoxin analogue aminoethoxyvinylglycine (AVG) (Lieberman
et al. 1974, Lieberman 1979) or of the aminooxyacetic acid (AOA)
Amrhein and Wenker (1979), whereas the ethylene precursor l-amino-
cyc10propane-1-carboxy1ic acid (ACC) (Adams and Yang 1979, LUrssen
et al. 1979) or derivatives of this compound (LUrssen 1981) stimulate
ethy1ene production considerably.

As far as the ripening of fruits is concerned a whole catalogue

of more or less desirable effects can be achieved by applying these
chemicals. Presently still dominating is the enhancement and con-
centration of ripening of c1imacteric fruits by ethylene-releasing
compounds like ethrel. For example, when ethrel is sprayed onto
indeterminate tomato plants less pickings are required (Fig. 7)
whereas with determinate cultivars more fruits in the ripe stage
can be harvested, especially when "once-over" mechanical harvesting
machines are emp10yed.

Applied together with a "stop-drop" agent such as NM or

2,4,5 TP, ethre1 advances remarkably the harvest date of apples
or peaches, a110wing the grower to take advantage of the higher
prices of ear1y marketed fruits. Furthermore, red co10uration of
treated fruits is often dramatical1y improved and their flavour is
indistinguishab1e from control fruits harvested 1-2 weeks later at
their normal harvest date (Luckwill and Chi1d 1972 and Table 4).

In fact in our experiments (Bangerth, unpub1.) the aroma and

f1avour of ethre1 (CEPHA) treated ear1y ripening app1es was a1ways
superior to contro1 fruits, even when these were pane1-tasted at
their optimal eating time. More experiments involving analytical
methods in addition to tasting panels are necessary to confirm
this observation.

Ethy1ene c1ear1yplays a decisive role in the production of

vo1atile flavour substances in fruits and higher concentrations of
this hormone are probably necessary for this purpose than for many
other ripening parameters. This may indicate the existence of dif-
ferent ethylene threshold and saturating va1ues for different physio-
logica1 processes re1ated to ripening and there are some indications
for this in the literature. Ethy1ene stimu1ated anthocyanin or
1ycopene production e.g. is more sensitive to low C2H4 concentrations
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 345

100

80

~
01
g'60
";:

I
- 40
l
-#-
20

o~--------------~--------~--------~
6 9
Days trom spraying

Fig. 7. The effect of ethephon applications on the ripening of

'Asic Cross' tomate fruits (Ashby 1972).

(Chalmers and Faragher 1977, Streif and Bangerth 1976) than is

chlorophyll degradation (Knee 1976, Streif and Bangerth 1976) or
aroma production (Blanpied ~nd Blak 1976). Alternatively processes
which apparently require higher C2H4 concentrations to be saturated
may be nearer the end of an C2H4 in1tiated chain reaction and thus
need a longer time to be fully operative.

Commercial applications of ethrel plus auxin sprays to acceler-

ate ripening frequently lead to shorter ripening and harvest per iods
which is not solely of advantage. Efficient harvest as weIl as
marketing methods are therefore necessary to get the fruits to the
consumer at the best time. Treatments with ACC or derivatives of it
probably do not have the disadvantage of a too fast softening
(LUrssen 1981) and should therefore be tested more extensively for
this purpose.

Ethrel or other ethylene releasing compounds not only promote

the ripening of climacteric fruits but also of non-climacteric ones
like currants, blueberries, cherries, raspberries, olives, pineapples,
and even nuts and coffee (Pecheur and Ribaillier 1975). Desirable
effects concerning these fruits are often not so much the accelera-
tion of ripening but more a decrease in "fruit removal force" by the
induction of an abscission layer, which facilitates mechanical har-
vest (Cooper and Henry 1973). Some drawbacks of ethrel sprayings
have also to be mentioned. First of all, as with almost all other
growth regulators, the responsiveness of the plant or plant organ
to ethrel is not only species - but very much cultivar - dependent.
346 F. BANGERTH

Tab1e 4. Effect of 3 growth regulators on fruit qua1ity of

"Worcester Pearmain" app1es 10 days after CEPHA
app1ication. (Chi1d 1973)

Character

Weight of
Fruit Firmness Starch Anthocyanin 100
Treatment (kg) Content Content Fruits (kg)

Contro1 8.5 1.4 170 7.3

CEPHA, 7.7 4.4 220 7.2

750 ppm +
2,4,5 TP
15 ppm

CEPHA 8.5 1.9 270 7.2

750 ppm +
A1ar, 2000ppm

In addition environmental effects during and after app1ication are

considerab1e. Together these factors make the se1ection of the
appropriate time and concentration of ethre1 sprays difficu1t. Even
more serious is the ethre1 - induced gummosis and dieback to twigs
and branches in many stone fruits (Cooper and Henry 1973, StBsser
1978). It will be interesting to see whether the use of ACC or its
derivatives can overcome some of these problems.

Procedures reducing the level of ethy1ene in fruits are as

important as those which increase it. Again this is more important
for c1imacteric fruits. This fie1d has been wide1y opened by the
recent discovery that rhizobitoxin and its ana10gue AVG great1y
diminish C2H4 biosynthesis in app1e fruits (Fig. 8). When sprayed
onto app1e trees AVG prevented autocata1ytic C2H4 formation, the
respiratory c1imacteric, softening and other ripening parameters in
at least 4 apple cu1tivars (Fig. 9). Chlorophyll breakdown was not
prevented to the same extent and very surprising1y red co1our forma-
tion was almost unimpaired. The AVG-induced block in ripening cou1d
be comp1etely overcome by subsequent app1ications of C2H4 at low
concentrations (Ha1der-Doll, pers. Communication). Cold storage
experiments with AVG treated app1e fruits have shown that C2H4 pro-
duction and respiration are great1y reduced for at least five months
whereas differences in firmness have been found to be much sma11er.
Stored AVG treated app1e fruits behave, therefore, somewhere in
between co1d- and CA - stored fruits.

Like some other growth regulators and storage methods AVG

HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 347

300
10··M CYClOHUIMIDI

...
:::)

r:
•
.....
c
...
! 200

..
~
~
z

100

--- 10.
,
jA .... loa'tO., ...I.......,.

10·· M.HIZO'''OlINI+1r MCYClOHlllMIOI

• 7 12

HOUIS

Fig. 8. Inhibition of ethylene production in apple tissue slices

by Rhizobitoxin, Cycloheximide. and a combination of both.
(Lieberman et al 1974)

treatment leads to a distintegration of the various ripening charac-

teristics, e.g. as with hypobaric storage or CA, AVG strongly reduces
aroma development whereas other ripening processes are not affected
to the same extent. Another example of this kind is the enhanced
and accelerated colour development after alar application (see above)
without affecting sugar or acid concentration normally associated
with this process. Such disintegrations of the ripening process
by modern technology need further investigation so that losses in
quality can be prevented.
348 F. BANGERTH

500
I!

~2
.....
N

fh
E
4
5 10 15 20

..
I!

........
Q

~1~ N
E
0.1
.....u
CII
... O.L..-~-"---"-T"-~~-
5 10 15 2 4 6 8 101'2'

Days in storagQ (20°C)

Fig. 9. Effect of preharvest AVG treatments on C2H4 production,

respiration, and fruit firmness of 'Golden De1icious'
fruits. 0 0 Contro1; • • AVG treatment. (Bangertb
1978)

In contrast to app1es, some other fruits, 1ike tomatoes,

apparent1y do not res pond to AVG app1ications to the who1e plant
very much because ripening, as assessed by 1ycopene synthesis and
chlorophyll breakdown, is not de1ayed. Bart1ett pears do respond
to AVG but not to the same degree as most apple cultivars (Bufler,
pers. communication). Because app1e fruits seem to obtain most of
the app1ied AVG via the 1eaves (Bangerth 1978) it is possib1e that
this leaf-to-fruit transport takes p1ace not so readi1y in pears
and toma toes .'

CONLU SIONS

Most review artie1es eoneerning the effeets of preharvest hor-

mone and growth regulator app1ieations on postharvest qua1ity and
store~bi1ity of fruits have eoncentrated almost entire1y on a1ar
and ethrel. It seems, however, that by se1ective use of other per-
haps more appropriate growth regulators or phytohormones more
improvements in fruit qua1ity and maturation can be achieve. A1-
though most of these improvement6 may not be as speetacu1ar as a1ar-
indueed intensified eo10uration, or the dramatica11y enhaneed ripen-
ing caused by ethrel, they may in the 10ng run be even more benefi-
eia1. Mueh more funöamenta1 research is necessary in order to
understand better the sometimes bewi1dering comp1exity of the
HORMONAL AND CHEMICAL PREHARVEST TREATMENTS 349

mechanism, and regulation of processes such as cell division/expan~

sion in fruits, Ca transport into fruits, assimilate partioning be~
tween vegetative and reproductive organs, initiation and cQordination
of ripening etc. Such an improved understanding of the basic physio-
logical processes involved and how they are affected by growth
regulators and phytohormones is necessary if we want to overcome
the hitherto almost putely empirical methods used in this field.
The progress made in the field of ethylene biosynthesis and physi-
ology during recent years and the advances in growth regulator re-
search related to these developments are encouraging, and leave us
with the hope that similar progress in research into other such key
processes will improve our ability to control the growth and develop~
ment of fruits both before and after harvest by chemical treatments
in the fie1d.

ACKNOWLEDGMENT

The author expresses his appreciation to Dr. M. V. Palmer for

reading the Eng1ish manuscript.

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triiodobenzoic acid, and other plant growth regulator sprays,
J. Amer. Soc. Hort. Sei., 101: 120.
Stembridge, G. E. 1973. Effect of growth regulators on the size and
shape of fruits, Acta Horticulturae 34:435
Stösser, R., 1978. Untersuchungen über die Entstehung der Lakunen
bei der Gummibildung des Steinobstes, Mitt. Kosterneuburg, 28:
119.
Streif, J., 1976, EinfluB der Temperatur auf verschiedene
Reifemerkmale von Apfe1n, Erwerbsobstbau, 18: 168.
Streif, J., and Bangerth, F., 1976, The effect of different partial
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354 F. BANGERTH

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80-159.
EFFECT OF POST HARVEST TREATMENTS OF GROWTH AND BIOREGULATORS ON

QUALITY AND LONGEVITY OF FRUITS AND VEGETABLES

Elias D. Dekazos

The Agricultural College of Athens, Votanikos

Athens, Greece

Fruits and vegetables after harvest continue to carry on most

of the life processes that were predominate just before harvest.
Harvested fruits and vegetables are living things, using 02 and
stored substrates while giving off C02' water vapor, and heat in
the process of respiration. The more rapid the rate of respiration
after harvest, the more quickly the fresh produce ripens. The rate
of respiration varies with stage of maturity, kind of produce,
temperature, chemical treatment, and composition of surrounding
atmosphere.

Modern technology has greatly increased the yields of fruits

and vegetables. Higher yields are worth little, however, if the
harvested crops are not consumed by people. Approximately 30% of
the harvested crops per year are lost as they move from the farmer
to the consumer. Such losses are much greater in the developing
countries where storage of food is a perennial problem. A sizable
reduction also occurs in the nutritive values and general quality
of much of the perishables such as fruits and vegetables that do
reach the consumer. In addition, such wastage is reflected in
higher consumer prices. It is of prime importance to preserve
what has already been produced. By preventing or minimizing crop
losses, millions more can be fed. It is highly desirable to inhibit
their ripening and senescence until they are to be consumed.
Ideally we would like to be able to inhibit the ripening process
at will to insure maximum quality at a predetermined time. The
storage life of fruits and vegetables can be prolonged by inhibit~
ing the ripening process either with chemical treatments and/or
with controlled atmosphere storage at lower temperatures.

355
356 E.D.DEKAZOS
During ripening and senescence, the fruits and vegetab1es
pass through aseries of overt changes in color, texture and f1avor
indicating the compositiona1 changes are taking p1ace. Many of the
physio10gica1 and bio-chemica1 changes that occur in fruits and
vegetab1es, as they ripen, have been extensive1y studied. The
endogenous factors that contro1 and regu1ate these chemica1 changes
remain 1arge1y obscure.
The ripening and senescence processes of fruits and vegetab1es
invo1ve plant hormones, a group of chemica1 substances produced by
the p1ants themse1ves. Men have synthesized compounds and have
achieved some success in controlling the vital processes of ripen-
ing and senescence of fruits and vegetab1es. These inc1ude all
categories of plant hormones, auxins, gibbere11ins, cytokinins,
ethy1ene and ethy1ene generators, growth retardants, metabo1ic
inhibitors and other chemica1s. The achievement of specific agri-
cu1tura1 objectives may depend upon the proper balance of natural
and app1ied growth regulators. The balance changes throughout the
growing season. Both timing and concentration are critica1 in
achieving specific responses.

Attention will be given to growth substances and other chemi-

ca1 compounds that have been shown to have a pronounced effect on
the storage 1ife of fresh fruits and vegetab1es. At this point,
it can be said that we are actua11y on1y at the beginning of the
commercia1 exploitation of plant growth substances in the produc-
tion, harvesting, and storage of foods.

Auxins

The application of a synthetic auxin such as 2,4-dichlorophen-

oxyacetic acid (2,4-D) to harvested 1emons and limes before storage
was shown to affect markedly the retention of the buttons in a
green condition, thus preventing the undesirab1e b1ackening of
buttons that frequent1y develop during storage (50,67,96). Compar-
ab1e resu1ts were obtained when the fruit on the tree was sprayed
with these chemica1s before harvest. Some oranges and grapefruit
were also found to have better keeping qua1ities after treatment
with 2,4-D (96). In citrus, storage 1ife was pro1onged by app1ica-
tions of 2,4-D and 2,4,5-T (96,97,98), main1y as a resu1t of de1ayed
degreening (59,60). Storage qua1itiies of 2,4,5-Trich10rophenoxya-
cetic acid (2,4,5-T) treated fruits were also improved. The Monsoon
Coorg mandarin orange treated with 2,4,5-T (25 and 50 ppm) and
ch10rophenoxyacetic acid (CLPA) (25 ppm) 8 weeks before harvest had
34, 35 and 28% more weight, respective1y, than the contro1. When
stored for 35 days at 5.5-7.2 0 C and 85-90% RH, the treated fruits
suffered 1ess physio10gica1 10ss in weight, had high marketab1e
percentage, and 1ess vitamin C 10ss as compared with the untreated
ones (88). It was reported that 2,4,5-T was better than 2,4-D in
promoting ear1y ripening of mandarins (25). Later, using Nangpur
EFFECT ON QUALITY AND LONGEVITY 357

mandarin oranges, it was found that 2,4,5-T was in fact less effec-
tive than 2,4-D in prolonging the storage life of the fruit (74b).

The keeping quality of fruits in storage is a primary concern

of the citrus industry. Prior to the late 1940's Alternaria decay
was one of the most serious fungus diseases of stored lemons. In
both preharvest and postharvest treatments, 2,4,5-T has proven
more effective in control of the disease than 2,4-D, and ester
formulations have been more effective than amine salt formulations.
Although 2,4-D is relatively ineffective in inhibiting the growth
of Alternaria in vitro (46), treatment of fruit with this compound
prior to storage reduces the fungus to a minor role in storage de-
cay (45,62,96,98). The primary reason for success appears to be
that treated lemons are more resistant to the entry of fungus.
Once the fungus has gained entrance, it grows as weIl in treated
as in non-treated fruits (35). Alternaria spores are present und er
the button (receptacle and calyx) of most lemons (5). Once the
button dies, invasion can readily occur. Treatment with 2,4-D
delays the development of the abscission layer, the button remains
in a living condition, and entry of fungus is reduced. The usual
practice is to apply 2,4-D as a final step in the washing procedure
for lemons. Where wax is applied prior to storage, 2,4-D is added
with the wax.

A reduction in black buttons, reduced Alternaria decay, and

delayed aging also has resulted from the use of 2,4-D on oranges
and grapefruits (96). Delayed yellowing of Persian limes (59) and
improved storage life of Clementine mandarin (58) has been effected
by postharvest treatments with 2,4.5-T.

Treatments with 2,4-D also delays aging and reduces external

decay which is probably a result of retardation of the aging
process. Delayed aging is characterized by firmer fruits and a
delay in development of the lemon-yellow color. If the dosage is
too high, the delay in coloring can become a problem.

The effectiveness of 2,4,5-T and l-naphthaleneacetic acid (NAA)

in markedly retarding further ripening of pineapple fruit and thus
extending its marketable life as a fresh fruit has been demonstrated.
As little as 1 ppm of 2,4,5-T has noticeable effect. and 100 ppm
appears optimum for senescence delay (52,54,55). For NAA, 500 ppm
is at optimum level for dipping fruit. Abrief wetting of the fruit
is adequate. The crowns remain in better condition when not treated
with growth regulators. Refrigeration can supplement the effect of
the chemical in retarding senescence. This process extended the
shelf-life of pineapples for several days by delaying senescence.

Among leafy vegetables application of 2,4-D, especially if

combined with N6-benzyladenine retarded yellowing of cauliflower(66)
and other green vegetables (115). Thus, during storage, leaf drop
358 E.D.DEKAZOS
in cauliflower and cabbage was reduced by an application of 100 to
500 ppm 2,4-D, 1 to 7 days before harvest (98).

Delaying effect on ripening was obtained with NAA by Lampe (68)

on tomatoes, by Stewart (98) on Smooth Cayenne pineapples, and by
Soni et al. (93) on Basrai Dwarf banana. After 16 days of storage
at 20.5 to 30.2° C, bananas treated with 100 ppm NAA and 4% wax
emulsion had lower reducing, non-reducing, and total sugars
acidity and total soluble solids than the controls (93).

Storage life of onion bulbs was also prolonged when 0.40 ppm
NAA was used as a foliar spray (15). Cauliflower and cabbage wrapped
with shredded paper containing 50 to 100 mg NAA and stored at 32° F
showed a reduction in leaf abscission and weight loss (98).

Gibberellins

The primary aim of growing citrus, for fresh fruit consumption,

is to produce fruit of impeccable appearance. Applied growth
regulators have already been a commercial success in this field.

Rind quality: The Navelorange is prone to softening, puffy

rinds, sticky rinds, and damage. The incidence of these disorders
may be reduced by applying GA (10-20 ppm) when the fruit is begin-
ning to acquire its orange color (20). The events leading to
senescence in navelorange rind appear to include pigment changes,
softening of the tissue, increases in carbohydrates, some changes
in carbohydrate metabolism, and changes in cation concentrations
(the ratio of potassium to calcium plus magnesium increases) (71).
In Australia, where late harvesting is practiced, 2,4-D, which has
been used for some time to prevent the pre-harvest drop of Navel
oranges, is now applied at color break at 30 ppm plus GA at 10 ppm
(40). Sometimes the albedo breaks down giving rise to a serious
condition known as creasing. The cause is still unknown. Since
ethylene has been reported to increase activity of an albedo
macerating factor (89) then natural ethylene may contribute to
this disorder. Applied GA reduces but does not completely prevent
the incidence of creasing (43,69).

GA delays color maturity in citrus fruits (70). This can be

a disadvantage when using GA to prevent rind damage but the navel
in Navel oranges of GA treated fruit, remains greenish and some
people would consider that this enhances the fruit's appearance.

With late Valencia oranges stored on the tree, regreening of

the fruits is an ever present problem. No growth regulator has
been found that will prevent this or overcome it. Where trees
have been previously treated with GA regreening is increased.
EFFECT ON QUALITY AND LONGEVITY 359
It has been demonstrated that GA delays loss of green rind
pigments by Navelorange, Valencia orange, grapefruit, tangerine,
lemon, lime, and Satsuma mandarin (12,16,17,18,19,84,94). Lemons
from treated trees with GA were greener as early as one month and
for as long as seven months after treatment. Fruits treated with
GA also appeared to develop a lemon-yellow color less rapidly in
storage. Delayed yellowing has been induced by GA applied to lemon
trees at any time of the year. The response reflects an over-all
delay in maturity rather than a simple delay in rind maturity as
appears to be the case with Navelorange. The primary benefits of
GA treatment are a more desirable seasonal harvest pattern in re-
lation to market demands, a large percentage of fruit with a long
storage life and a decrease in the number of small yellow lemons.
Postharvest immersion of 'West Indian' limes in solutions of
gibberellic acid increased chlorophyll retention during subsequent
storage at several combinations of temperature and relative humid-
ity (9).

Size: Several growth regulators increase fruit size through

their effects on the growth of the fruit. 2,4-D increased the size
of Bearss limes (44) and enhanced the growth rate of orange fruit-
lets (83). GA is also known to enhance fruit size, and like kinetin,
tends to give longer shaped fruit (83). Recently, a practical
problem arose with lemons exported from Australia for a specialized
market in Europe. In order to delay maturity and to prevent over-
ripening in transit, GA was applied prior to color formation. Since
GA increased fruit size, the fruit became too large for this market.
This problem can be overcome by applying a combination of GA (10 ppm)
and (2-chloroethyl) trimethylammonium chloride (CCC) (1,000 ppm)
which gives the required color and prevents increase in size. Also
winter crop fruit held on the tree after similar treatment until
midsummer still exhibits excellent rind quality (103).

Storage: GA applied as a preharvest spray (5 to 20 ppm) on

Navel oranges reduces the incidence of storage disorders (8).
Conversely, ethylene treatment increases susceptibility to such
disorders (65). Benomyl is now the preferred postharvest fungicide.
It also exhibits cytokinin-like activity (92) and prevents degreening
of citrus fruit (41). Thus, other cytokinins might be worth investi-
gating for their effects on the storage qualities of citrus fruits.

Residues of plant-growth regulators

The possibility of residues of plant growth regulators remain-

ing in fruit at harvest presents a problem that will become increas-
ingly important as commercial applications are developed for these
compounds. Preharvest sprays of the isopropyl ester of 2,4-D are
registered in some countries for oranges, grapefruit, and lemons.
Postharvest sprays of 2,4-D on lemons also are registered. Gibber-
ellic acid is registered for preharvest use on fruiting lemon and
360 E.D.DEKAZOS

Navelorange trees. GA is employed in preharvest treatments for

the purpose of delaying fruit maturity and rind senescence.

Cytokinins

The purpose of most postharvest research is the prolongation

of market acceptability of fresh fruits, vegetables and cut flowers.
Much attention is being devoted to the influence of cytokinins on
plant respiration. The cytokinins which are N6-substituted adenine
derivatives have been used, both natural and synthetic, to affect
plant growth. N6-benzyladenine delays the senescence of many har-
vested crop plants. Treatment results in a decrease in respiration,
retention of chlorophyll, decreased desiccation and the retention
of an increased proportion of the total phosphorus in an organic
form (82). These factors enhance the postharvest life of perish-
able commodities. N6-benzyladenine was effective over an extremely
wide range of concentrations (0.1 ppm for broccoli to 300 ppm for
apples). However, the most effective concentration varies with the
tissue and method of application (7,91,114). Dedolph et al. (27)
observed that senescence inhibition in treated asparagus with BA
and held in the dark at 21° C was concomitant with respiration
inhibition. However, Lipton and Ceponis (74a) observed a retarda-
tion of senescence of head lettuce sprayed with 10 ppm BA and
stimulation of O2 consumption of treated lettuce tissue. MacLean
et al. (81) found that pre- and post-harvest applications of BA at
a concentration of 10 ppm reduced the respiration rates and extend-
ed the storage life of harvested broccoli at 15° C.

For the both the European and North American markets, limes are
required to be green, with minimum yellowing and wilting (108).
Treatment of limes with certain plant hormones has beneficial effects
on the rate of degreening. Thus, postharvest immersion of lime
fruits in either solutions of kinetin (6-furfurylaminopurine) or
seaweed extracts of known cytokinin activity significantly increas-
ed the degreening time (12,86). Also, postharvest immersion of
limes (cvs. 'Persian' and 'West Indian') in solutions of N6-benzyl-
adenine increased chlorophyll retention during subsequent storage at
severa1 combinations of temperature and relative humidity (9).

It was reported (100) that lower concentrations of BA and higher

concentrations of GA3 effectively reduced senescence in fruits of
Trichosanth dioica Roxb which are generally consumed as vegetables.
Accompanied with delay in senescence, levels of chlorophyll, skin
and pulp pro tein were maintained at higher level than control.

It was known that cytokinins delay citrus rind coloration, in

accordance, with their universal senescence-delaying effects (52,
100). Go1dschmidt et a1. (53) found that there is an antagonism
between ethylene and the senescence delaying regulators GA3 and BA
which seems to operate main1y within the chloroplast. Treatment
EFFECT ON QUALITY AND LONOEVITY 361

of mature citrus fruit (Citrus sinensis) with ethylene induced rapid

chlorophyll destruction, a rise in respiration, arelease of free
amino acids and an accumulation of reducing sugars. BA and GA3
opposed the effects of ethylene on chlorophyll, amino acids, and to
a lesser extent, reducing sugar levels.

Dilley (36) suggested that the ripening process is controlled

by the endogenous cytokinins. Fruit ripening, essentially a
senescence phenomenon, has been shown to be retarded by applied
cytokinins (104). Davey et al. (26) reported that ripening fruits
of anormal strain (Rutgers) of tomate (Lycopersicon esculentum
Mill.) were found to contain lower levels of endogenous cytokinins
than fruits of a non-ripening mutant rin. The non-ripening rin
contained also high levels of zeatin glucoside, a storage cytokinin.

Ethylene and ethylene-generators

Degreening: Early varieties of citrus fruit will usually meet

legal maturity standards before the peel attains the characteristic
varietal color. With oranges and mandarins, the practical aim is
for a deep orange-red color. The characteristic orange color does
not fully develop in warmer regions, and may never be attained in
the tropics. At present ethylene gas is used in degreening. A
concentration of 5-10 ppm in the temperature range 21-30° Cover
aperiod of 4 days is optimal (65). Although the best color is
considered to develop if fruit is exposed to ethylene at 15-25° C
for a longer period of time (109). However, ethylene treatment
has the disadvantage that degreening rooms are required and there
is a delay before color is attained.

It is possible that solutions of ethephon could one day replace

ethylene gas although high concentrations (8,000 ppm) are needed (64).
Preharvest applications of ethephon (CEPA) have been investigated (63)
as an aid to degreening, but the results were variable due to inter-
ference from endogenous growth regulators within the tree. Post-
harvest degreening with ethephon appears more promising than pre-
harvest degreening because it eliminates the problem of defoliation
and its effects are not inhi.bited by such packing house operations
as washing and waxing (49,56). Postharvest dipping in ethephon
solutions has been shown to degree citrus fruits comparable to, or
better than, those obtained with commercial degreening using ethylene
gas (10). Recent findings indicate that preharvest degreening of
'Robinson,' 'Nova,' 'Lee' and 'Dancy' tangerines and 'Hamlin' oranges
can be hastened by spraying with 25 to 500 ppm ethephon (23,81,111,
112). Higher rates of 500 to 1000 ppm were required to induce color-
ing of 'Satsuma,' 'Shamouti' and 'Washington Navel' oranges (39,47,
61). Postharvest dipping in ethephon solutions at 500 to 12,000 ppm
for 1 to 10 minutes induced and accelerated color changes of 'Bearss,
'Eureka' and Taiwan lemons (49,63,101); 'March' grapefruit (49,56);
'Valencia,' 'Hamlin' (56,82), 'Valle Washington' (13), 'Shamouti'
362 E:D.DEKAZOS
oranges and 'Clementine' tangerines (49). Gautam et a1.(51) found
that mature green fruit of Kagazi-1ime dipped in solutions of 500 to
2000 ppm ethephon for 1 minute before storage at room temperature
caused degreening of fruit, which deve10p a ye110w color. Degreening
of ethephon-treated citrus was more efficient at 22.8° C than at
17.2 0 C and was inhibited at 6.1 0 C.

App1ication of ethephon to fruits acce1erated the rate of

chlorophyll breakdown, carotenoid accumu1ation, respiratory activ-
ity, ethy1ene production and abscission of buttons (49,112,113).
Since ethephon will not improve the interna1 qua1ity care has to
be taken not to degreen fruits before minimum qua1ity standards.

It wou1d be of considerab1e interest to determine the effects

of commercia1 fungieides (e.g. benomy1, thiabendazo1e, sodium
orthopheny1phenate) prior to degreening fo110wed by anti-transpirant
spray (Vapor Guard, rate 1 to 100) on the degreening, decay and
storage of citrus fruits.

Ethy1ene promoting compounds app1ied at postharvest will induce

degreening. For examp1e, cyc10heximide, cyc1amic acid and ethephon
(all at 4,000 ppm), have affected the co10ring of summer 1emons (42).

Co10ring of citrus fruits

Exogenous growth regulators have a major impact on co10ration.

The major exogenous responses are as fo110ws. Ethy1ene causes the
10ss of chlorophyll and pro duces some minor changes in carotenoids.
Gibbere11ic acid causes a substantia1 de1ay in the 10ss of chloro-
phyll and substantia1 reduction in the rate of accumu1ation of
carotenoids. 2,4-D and benzyladenine cause delay in loss of chloro-
phyll. However, another c1ass of compounds deserve attention.
2-(4-ch1orophenylthio)-triethylamine hydrochloride (CPTA) and some
re1ated compounds cause high amounts of 1ycopene, and certain other
carotenoids, to accumulate in flavedo and albedo tissues of orange,
1emon, 1ime, grapefruit cu1tivars (21,110). Studies conducted on
the mode of action of the high 1ycopene inducers strong1y ~uggest
that these bio-regulators act at the enzyme level by inhibition
of the cyc1ase(s). No growth regulator, to date, has been found
that specifica11y increasea the level of ß citraurin, the main
pigment responsib1e for the orange color (11). A compound that
promotes the accumu1ation of the carotenoid 1ycopene wou1d clear1y
be a bio-regulator, not a hormone. Fruit color is of great economic
importance to the citrus industry because consumers favor oranges
having a deep orange color over those having a ye110w or ye11ow-
orange color. Treatment of the Valencia orange, Eureka 1emon and
Satsuma mandarin with CPTA caused the fruit to deve10p a tomato-red
color due to accumu1ation of lycopene in the pericarp (rind). Pre-
harvest and postharvest treatments (immersion in CPTA solution) were
equa11y effective.
EFFECT ON QUALITY AND LONGEVITY 363

Daminozide-ethephon interaction

Recent pomological studies include the introduction of growth

regulators for modifying fruit quality and ripening behavior (78).
The growth regulator, succinic acid-2,2-dimethylhydrazide (Damino-
zide, SADH) was found to delay ripening of apples (75), improve
flesh color of peaches (6) and sour cherries (102). Ethephon (2-
chloroethyl phosphonic acid)shows considerable promise for commer-
cial application to accelerate color development, advance fruit
maturity and promote abscission of fruits (11,37,57,87,90).

Application of SADH and/or ethephon as preharvest fruit sprays

to rabbiteye blueberries (Vaccinium ashei Reade cv. T-19) at
specific stages of berry development, resulted in advanced and
concentrated fruit ripening, and quality improvement. Successive
applications of SADH and ethephon were more effective in the pro-
motion of highly-colored, firm fruit. Storage quality of blue-
berries was maintained for 40 days--a prolongation of their
marketable life. The waxy bloom, freshly harvested appearance, of
the treated berries was retained throughout storage (28,31). SADH-
ethephon treatment drastically suppressed the rate of softening of
stored berries (Table 1). Furthermore, the Hunter negative b
values which measure blueness were significantly higher for all
treated berries than for the control. In addition the L values
which measure lightness or darkness were considerably lower for the
control meaning that these berries were darker than the treated
(Table 1). The greatest percentage of marketable fruit after 40
days of storage was obtained from the SADH-ethephon treated berries
and the double ethephon treated (Table 1). The double applications
of ethephon and the application of SADH-ethephon have imparted some
important advantages to the rabbiteye blueberries. The improved
fruit texture obtained from the application of SADH-ethephon will
not only be an asset to immediate fresh market acceptability but
will also enhance storage life of the fruit. Anatomical studies
of treated blueberry tissue showed that a relationship exists
betseen cell wall thickness and texture of sprayed berries.

Ripening and quality of detached fruit

The ripening of detached fruit by treatment with growth regula-

tors has received some attention. Postharvest treatments with
ethephon, ethephon followed by CPTA, and/or controlled storage
conditions for ripening of green, full-sized 'Babygold' peaches
(29, 30) accelerated ripening and color development and produced
peaches of highest quality as measured by the various quality para-
meters (Table 2). Of the quality attributes considered, the most
striking trend was an enhancement of fruit color by ethephon as
exhibited by a decrease in hue angle for both skin and flesh color
and an increase in soluble solids as compared to the control fruit.
The ethephon treated peaches followed by CPTA developed an orange
 w
C»
.j:>.
Table 1. Influence of SADH and/or Ethephon on Color, Firmness, Chemical Characteristics and Stor-
age of 'T-19' Rabbiteye Blueberries after 4Q Days at 3°C and 95+% Relative Humidity

Texture Sol- Titrat- Market-

Treatment & (puncture- uble able able
dates of ap- Hunter color values force) fruit
so lids acidity y
application L a b gm-ern (%) (%) (%)

Control 18.70c z +0.18b -2.5Id 1. 60xl0 3 c 14.2c .84b 70.35c

SADH (500 ppm 20.67b +0.25a -3.0Ic 2.20x10 3a 14.lc 1.03a 85.00b
May 20 &
June 3)

SADH (500 ppm

May 20)
followed by 21.63a +0.18b -3.62a 2. 39xl0 3a 16.3a .65d 94.25a
Ethephon (500
ppm June 3)

Ethephon (500
ppm May 20 20.54b +O.20b -3.25b I. 88xl0 3b 14.7b .76c 93.55a
& June 3)
!'f1
0
zMeans within a column followed by different letters are significantly different at the 5% level. 0
m
YAs percent citric acid. A
»
N
0
Cf)
 m
Tab1e 2. Effects of Postharvest Treatments on Ripening of Immature 'Babygold 7' ""m
c1ingstone peaches as measured by various qua1ity parameters (")
-l
0
Hunter color va1ues Z
p
Skin F1esh Firmness c
Hue Saturation Hue Saturation (Magness- Soluble Citric l>
r
Postharvest angle index angle index Tay10r) solids acid =i
J J (kg) % % -<
treatments (tan-1 b/a) (a 2 + b 2) (tan-1 b/ a) (a 2 + b 2 ) l>
z
0
Immature (Green) 97.52° 18.35 81. 99° 30.30 7.12 9.1 0.36 r
0
Tree-ripened 65.77° 23.51 71.13° 32.40 5.44 10.9 0.37
z
G)
m
23.9°C + 90% RH 77.65° 22.95 70.69° 32.75 5.50 10.9 0.35 <
=i
23.9°C + 90% RH 70.49° 24.19 67.44° 32.56 4.88 10.8 0.35 -<
E 250 ppm
26. rC + 90% RH 72.79° 22.95 68.13° 33.18 5.58 11.3 0.40
26.7°C + 90% RH 64.68° 25.21 65.61° 35.96 5.36 11.3 0.31
E 250 ppm
26. rC + 90% RH 55.89° 25.27 64.99° 33.66 5.37 11.8 0.29
E 250 pp + CPTA
2500 ppm
40.5°C + 90% RH 65.82° 25.37 66.11° 34.91 5.57 11.0 0.29
26.7°C + 90% RH
E 250 ppm
40.5°C + 90% RH 67.19° 25.77 65.70° 34.34 5.48 11.1 0.29
26 .. 7°C + 90% RH
CA)
(J)
c.n
366 E. D. DEKAZOS

color, exhibited the greatest decrease in hue angle in both skin

and flesh color.

As the ripening temperatures decreased from 26.9° to 23.9° C,

the hue angle reading increased in the first quadrant going from
red to yellow. With postharvest ripening of immature peaches, an
intensity of saturation was exhibited. Saturation was proportional
to the strength of a given color. Soluble solids were higher in
fruit treated with ethephon and ethephon followed by CPTA treat-
ments. Softening of fruit was increased and the total acid de-
creased by ripening with ethephon. Peaches treated with ethephon
and placed at 26.7° C and 90% RH resulted in the development of
better color. Fruit dipped with ethephon before being dipped in
CPTA and then placed at 26.7° C and 90% RH resulted in an orange
color. Peaches at 40.5° C and 90% RH prior to the ripening tempera-
tures and ethephon dipping were not only more desirable than the
tree-ripened fruit but were also practically free of decay losses.

These quality peaches were more desirable and contained higher

levels of carotenoids than the tree-ripened fruit. Lycopene
accumulation was stimulated and detected for the first time in this
peach tissue treated with CPTA (Table 3). These peaches developed
an orange color. Ethephon appeared to hasten the development of
carotenoids. The value for total carotenoids reached 2.7 g/lOO g
for fresh peaches. Minor amounts of lycopene were found in the
untreated fruit but lycopene in the treated fruit increased 4.5
times. Lycopene accumulated mainly in the skin but also in the
flesh. Carotenoids were compared with the hue angle and saturation
index. There seemed to be a relationship of hue angle to carote-
noids in the flesh of the postharvest ripened fruit. Peaches held
at 40.5° C and 90% RH for 24 hours (in hot-air rooms) before ripen-
ing at 26.7° C and 90% RH after dipping in 250 ppm ethephon for
1 minute produced one of the most efficient methods of fruit ripen-
ing. Ethephon proved most effective in advancing maturity, that
is, when the immature fruit had developed sufficiently to respond.

Aminoethoxyvinylglycine (AVG)

Ripening inhibitors are essential to prlong shelf life and

to control problems such as premature ripening. Daminozide sup-
presses ethylene production and delays ripening (76,77,79). Cyclo-
heximide inhibits protein synthesis and ethylene production (48) •

The discovery that rhizobitoxine (2-amino-4-(2'-amino-3'hydroxy-

propoxy)-trans-3-butenoic acid) 'can inhibit ethylene production from
methionine in fruit and other plant tissue (73,85) has led to the
investigation of related structural analogs. It was found that
L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (74), equally
inhibits ethylene production in fruit and other plant tissues. Now
when the detailed pathway of ethylene biosynthesis was defined an
 m
Tab1e 3. Carotenoids and Color of Postharvest Ripened 'Babygo1d 7' "Tl
"Tl
Non-me1ting, C1ingstone Peaches m
(')
-l
o
Fresh fruit Z
p
Relative intensity c
of carotenoids Tristimulus Color
»r
=i
Crude -<
carotene Lycopene Saturation
»z
Postharvest O.D. O.D. Hue angle index o
r
Treatment (450nm) (502nm) (tan- 1 b/a) V( a 2 + b 2) o
Z
G')
Immature 0.661 0.057 81.99° 30.30 m
<
Tree-ripened 0.810 0.056 71.13° 32.09 =i
-<
23.9 0 C + 90% RH 0.965 0.109 67.44° 32.56
E 250 ppm
26.7 0 c + 90% RH 0.923 0.105 68.13° 33.18
26.7 0 C + 90% RH 1.090 0.130 65.61° 35.96
E 250 ppm
26.7 0 C + 90% RH 1.340 0.262 64.99° 33.66
E 250 ppm +
CPTA 2500 ppm
40.5 0 C + 90% RH 66.11 ° 34.91
26.7°C + 90% RH
E 250 ppm
40.5 0 C + 90% RH 1.200 0.125 65.70 0 34.34
26.7 0 C + 90% RH

CA)
E ethephon cn
-..J
368 E.D.DEKAZOS
important conclusion emerged--that ACC synthase, which converts
S-adenosylmethionine (SAM) to l-aminocyclopropane-l-carboxylic acid
(ACC) is the rate-controlling enzyme in ethylene biosynthesis and is
inhibited by AVG (2).

AVG inhibits ethylene production on both 'Anjou' and 'Bartlett'

pears (107), green tomato slices (3) and other plant tissue and
increases the longevity of various cut flowers (4,72,106,108). In
the case of broccoli (105), the AVG markedly reduced ethylene pro-
duction and respiration, and effectively retarded its yellowing and
senescence at warm temperature (20 0 C). SampIes of broccoli treated
with 5 x 10-~ AVG retained much of the green color and compactness
and were still in salable condition on the third day.

Aseries of studies were conducted and designed to determine

whether the ethylene inhibitor, AVG, applied after harvest would
effectively delay senescence and extend the longevity of fresh
berries (with or without preharvest treatments) (33).

Rabbiteye blueberries (Vaccinium ashei Reade cvs. 'T-19' and

'Tifblue') sprayed before harvest with daminozide [butanedioic
acid mono(2,2-dimethylhydrazide)] and/or ethephon at specific stages
of berry development and p1aced in storage at 3 0 C and 95-100%
relative humidity effectively maintained storage quality for 2~
months--a prolongation of their market life. The waxy b100m, which
is characteristic of fresh1y harvested blueberries, was retained
throughout storage. The Hunter negative b va1ues, which measure
blueness, were significantly higher for the sprayed berries than
for the control (Tables 4, 5). In addition, the L va1ues, which
measure 1ightness or darkness, indicated that the contro1 berries
were considerab1y darker than the sprayed berries (Tab1e 4, 5).
Firmness of berries stored for 75 days was great1y inf1uenced by
preharvest growth regulator treatments. The daminozide/ethephon
treatment was the more effective, drastica11y suppressing the rate
of softening of stored berries (Tab1e 4,5). The percentage of
marketab1e 'T-19' fruit after 75 days of storage was higher for the
daminozide/ethephon-sprayed berries than for the berries sprayed
twice with ethephon (Tab1e 4). Daminozide, a growth retardant,
decreases ethylene production (121); and this effect may have caused
the increase in 10ngevity. If so, the decrease in marketable fruit
between 75 days and 7 months storage (Table 6) means that the
suppression of ethy1ene production by daminozide dec1ined with time.

AVG minimized weight 10ss (Tab1e 7) of the berries treated with

preharvest growth regulators. AVG was especial1y effective in
lightening the color of the sprayed berries. Treating berries with
AVG as a dip improved firmness and color retention during storage.
The va1ues of texture (firmness) for 'T-19' berries treated with
AVG 2000 ppm and for the daminozide/ethephon fol10wed by AVG 2000
ppm were 2.77 x 103 g-cm and 3.33 x 10 3 g-cm, respective1y, whi1e
 m
-n
-n
Tab1e 4. 1nf1uence of Aminoethoxyviny1g1ycine (AVG), Daminozide and/or Ethephon on Color, Firm~ m
()
ness, Chemica1 Characteristics and Storage Life of 'T-19' Rabbiteye Blueberries after -I
75 Days at 3°C and 95+% Relative Humidity. o
Z
p
Texture Market- c
Hunter Color Values Y (puncture- Soluble Titratable able
»r
Cultivar and force in solids acidityX fruit =i
-<
treatment groups L a b g-cm X 10 3 ) (%) (%) (%) »
z
T-19
o
r
A Contro1 17.49b z +0.16a -2.50c 2.47c 13.90b 0.48b 65.60c o
Z
B 2X Ethephon 19.59a +0.16a -3.l5a 2.68b l3.93b 0.64a 86.l9a Cl
m
C Daminozide + 20.24a +0.17a -3.60a 3.07a l5.55a 0.63a 92 .10a <
ethephon =i
-<
F Control + Tween-20 16.66c +0.19a -2.42c 2.40c l3.40b 0.53b 58.l0d
G Control + Tween-20 18.19b +O.22a -2.69b 2.69b l2.63c 0.52b 79.68c
+ 1000 ppm AVG
H Control + Tween-20 18.69b +0.20a -2.82b 2.77b 13.20b 0.46b 85.80b
+ 2000 ppm AVG
1 2 2 X Ethephon + l8.93b +0.20a -2.94a 2.76b 13.70b 0.60a 92.20a
Tween-20 +
2000 ppm AVG
J Daminozide + 19.53a +0.20a -3.03a 3.37a l4.68a 0.62a 95.00a
ethephon + Tween-20
+ 2000 ppm AVG
ZMeans within a column followed by different letters are significantly different (Duncan's multi-
ple range tests, 5% level).
YHunter L, a b terms: L, defines lightness or darkness; a measures redness-greeness; and b,.
describes yellowness-blueness.
xAs percent citric acid. Co)
0)
co
 (0)
~
o
Tab1e 5. Inf1uence of Aminoethoxyviny1g1ycine (AVG), Daminozide & Ethephon on Color, Firmness,
Chemica1 Characteristics and Storage of "Tifb1ue" Rabbiteye B1ueberries after 75 Days
at 30 C and 95+% Relative Humidity.

Texture Market-
(puncture- Soluble Titratab1e ab1e
Cu1tivar and Hunter Color Va1ues Y force in solids acidityX fruit
treatment groups L a b g-cm X 10 3) (%) (%) (%)

Tifb1ue
A' Contro1 17.78b z +0.26a -2.45c 2.21b 15.23b 0.73a 57.76c
C' Daminozide 21. 29a +0.24a -3.38a 3.14a 16.55a 0.70a 84.50a
+ Ethephon

F' Contro1 + Tween-20 17.30c +0.24a -2.41b 2.15b 14.23a 0.67a 57.24c
G' Contro1 + Tween-20 17.89b +0.22a -2.64a 3.17a 13.60b 0.68a 79.60b
+ 1000 ppm AVG
H' Contro1 + Tween-20 18.13a +0.22a -2.69a 3.19a 14.00a 0.65a 87.45a
+ 2000 ppm AVG

~eans within a co1umn fo11owed by different 1etters are significant1y different (Duncan's multiple
range tests, 5% level).
YHunter L, a, b terms: L, defines 1ightness or darkness; a, measures redness-greeness; and b, !'Tl
describes ye11owness-b1ueness. o
o
xAs percent citric acid. m
~
»
N
o
cn
 m
"TI
"TI
Table 6. 1nfluence of Aminoethoxyvinylglycine (AVG) , SADH and/or Ethephon on Storage Life m
()
of 'T-19' and 'Tifblue' Rabbiteye Blueberries after 7 Months at 3 0 C and 95+% -t
0
Relative Humidity. Z
P
C
Deteriorated fruit »r
fruit (%) =i
Cultivar and treatment groups (%) Marketable -<
»
z
T-19 0
r
A Control 81. 27 0 0
B 2X Ethephon 46.86 35.50 z
G)
C Daminozide + ethephon 50.68 37.00 m
<
F Control + Tween-20 81. 80 0 =i
-<
11 2X Ethephon + Tween-20 + 39.30 49.50
1000 ppm AVG
12 2X Ethephon + Tween-20 + 28.64 61.00
2000 ppm AVG
J Daminozide + ethephon + 30.60 60.00
Tween-20 + 2000 ppm AVG

Tifblue
A' Control + Tween-20 74.24 0
C' Daminozide + ethephon 46.00 40.00

F' Control + Tween-20 74.80 0

G' Control + Tween-20 + 35.10 49.50
1000 ppm AVG
H' Control + Tween-20 + 25.70 62.00
2000 ppm AVG
w
-..J
 w
Tab1e 7. 1nf1uence of Arninoethoxyviny1g1ycine (AVG) , SADH and/or Ethephon, CMC, and ......
N
Storage Upon Weight Loss of 'T-19' and 'Tifb1ue' Rabbiteye B1ueberries.

Fruit characteristic and 1ength of storage

weight 10ss after 2~ mos. Weight 10ss after 7 mos.
storage storage
Cu1tivar and treatment (%) (%)

T-19
A Contro1 6.3 19.0
B 2 X Ethephon 6.3 16.7
C Daminozide + ethephon 4.4 12.3
D 2 X Ethephon + CMC 4.4 11.1
E Contro1 + CMC 5.8 17.5

F Contro1 + Tween-20 6.5 18.2

G Contro1 + Tween-20 + 5.0 14.0
1000 ppm AVG
H Contro1 + Tween-20 + 4.0 11.2
2000 ppm AVG
12 2 X Ethephon + Tween-20 + 3.5 9.8
2000 ppm AVG
J Daminozide + ethephon + 3.0 8.4
Tween-20 + 2000 ppm AVG
Tifb1ue
A' Contro1 9.0 25.2 m
G' Contro1 + Tween-20 + 6.5 15.4 Cl
1000 ppm AVG
Cl
H' Contro1 + Tween-20 + 4.5 12.6 m
A
2000 ppm AVG »
N
0
CI)
EFFECT ON QUALITY AND LONGEVITY 373

the Tween-20 was 2.40 x 10 3 g-cm. At 2000 ppm, AVG tended to enhance
the effectiveness of the preharvest sprays in maintaining fruit
firmness. The effects of AVG and the sprays on fruit color, firmness
and weight were positive and should be of significance to producers,
processors and consumers. Treatment with AVG did not exert the same
degree of inhibition on various ripening reactions. The percentage
of marketable fruit after 75 days of storage was higher for AVG
treated berries than for the corresponding berries without AVG, and
was highest for the berries with preharvest spray treatments plus
AVG (Table 4,5). The daminozide/ethephon-treated berries (with or
without AVG) had the highest soluble solids content and the highest
percentage of marketable fruit. Doubling the concentration of AVG
from 1000 to 2000 ppm increased but did not double the percentage
of marketable fruit (Tables 4,5). AVG, which was effective in re-
ducing ethylene production from methionine in other tissue (72),
extended the postharvest longevity of 61% and 62%, respectively of
'T-19' and 'Tifblue' rabbiteye blueberries to 7 months (Table 6).
An informal panel indicated that the AVG-treated berries had superi-
or taste and retained a fresh appearance. At 2000 ppm, AVG increased
the amount of marketable 'T-19' berries treated twice with ethephon
by about 72% and of 'T-19' berries treated with daminozide plus
ethephon by about 62%. Also, the amount of marketable 'Tifblue'
treated only with 2000 ppm AVG was about 55% greater than that of
'Tifblue' treated only with daminozide plus ethephon. This marked
effect of AVG in increasing storage life of berries is probably due
to its potential in retarding ethylene-induced senescence. Both AVG
treatments (1000 and 2000 ppm) , immediately after harvest, with or
without preharvest sprays of daminozide and/or ethephon effectively
retarded senescence and deterioration of blueberries during storage
at 3 0 C. A direct effect of AVG on increasing longevity was dernon-
strated and is of practical significance. This is the first time
that longevity of rabbiteye blueberries has been appreciably in-
creased by chernical treatments. Of the compounds tested, AVG a
suppressant of ethylene, was the most effective inhibitor of senes-
cence of rabbiteye blueberries. It maintained firmness and increased
the longevity of 'T-19' and 'Ti:blue.' The effect of AVG in
lengthening the storage life of rabbiteye blueberries was marked.

Recently, applications of high concentrations of AVG on intact

branches of 'Tifblue' and 'Woodard' rabbiteye blueberries, and of
'Loring' and "Rio Oso Gern' peaches after the completion of the
rest per iod effectively delayed blossoming; thus, reducing the
risk of spring freeze (32, 34). In addition, in both blueberry
cultivars and the 'Rio Oso Gern' peaches firmness was increased in
fruit from branches treated with AVG. Textural properties are
important attributes and contribute to the overall quality of the
fruit. Controlling CZH4 synthesis, as per example by AVG, is the
key to controlling ripening and consequently the storage life of
climacteric fruits. Thus, for the first time it is possible to
control the quality of the fruit from its bud stage.
 374 E.D.DEKAZOS

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45. Erickson, L. C. 1952. Plant growth regulators for 1emons.
Ca1if. Citrograph 37:179, 201.
46. Erickson, L. C., T. A. DeWo1fe, and B. L. Brannaman. 1958.
Growth of some citrus-fruit pathogens as affected by
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47. Fish1er, M. and S. P. Monse1ise. 1971. The use of ethephon
(2-ch10roethy1phosphonic acid) to promote color deve10pment
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48. Fr enkel , C., I. Klein, and D.R. Di11ey. 1968. Protein synthesis
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43:1146.
EFFECT ON QUALITY AND LONGEVITY 377

49. Fuchs, Y. and A. Cohen. 1969. Degreening of citrus fruits with

Ethrel (Amchem 66-329). J. Amer. Soc. Hort Sei. 94:617.
50. Gates, C. M. 1949. The possibility of 2,4-D for the control
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51. Gau tarn , D. R., R. P. Dhar, V. P. Bhutani, and H. S. Dhuria.
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Indian J. Agric. Sei. 47(6):282.
52. Gortner, W. A. 1963. Delaying senescence of pineapple fruit.
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53. Goldschrnidt, E. E., Y. Aharoni, S. K. Eilati, J. W. Riov, and
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54. Gortner, W. A. 1969. Relation of chemica1 structure to plant
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55. Gortner, W. A. and R. Leeper. 1969. Studies on the relation of
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56. Grierson, W., F. H. Ismail, and M. F. Oberbacher. 1972. Ethephon
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57. Griggs, W. H., B. T. Iwakiri, R. B. Fridley, and J. Mehlschau.
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59. Hatton, T. T. 1958. Effects of waxes and 2,4,5-trichloro-
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61. Hirose, K., M. Yamamoto, and H. Daito. 1970. Studies on
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62. Iwasaki, T., M. Nishiura, and T. Chichijo. 1956. Effects of
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TOKAI KINKI Agr. Exp. Sta. 3:17.
63. Jahn, O. L. and R. Young. 1972. Influence of the tree on the
response of citrus fruit to preharvest app1ications of
 378 E.D.DEKAZOS

(2-Chloroethyl phosphonic acid).J. Amer. Soc. Hort. Sci.

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64. Jahn, O. L. 1973. Degreening citrus fruit with post-harvest
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65. Jahn, O. L., W. G. Chase, and R. H. Cubbedge. 1973. Degreen-
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67. Kessler, K. L. and J. R. Allison. 1948. Use of growth regula-
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68. Lampe, C. H. 1971. Response of tomato fruits to certain growth
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70. Lewis, L. N., C. W. Coggins, and M. J. Garber. 1964. Chloro-
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71. Lewis, L. N., C. W. Coggings, Jr., C. K. Labanauskas, and
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EFFECT ON QUALITY AND LONGEVITY 379

77. Looney, N. E. 1972. Interaction of harvest and maturity, cold

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22.
83. Moss, G. I. 1972. Promoting fruit-set and yie1d in sweet orange
using plant growth substances. Aust. J. Exptl. Agric. Anima1
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84. Nishiura, M. and Y. Iba. 1964. Effects of gibberellin spray
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l.
86. Pantastico, E. B., W. Grierson, and G. Soule. 1966. Peel injury
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Soc. Hort. Sci. 94:443.
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89. Rogers, D. J. and C. Hurley. 1971. Ethylene and the appearance
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plant growth regulators, wax emulsions and their combinations
380 E.D.DEKAZOS

on the storage behavior and physio-chemica1 changes during

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Proc. Amer. Hort. Sei. 77:194.
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106. Wang, C. Y., J. E. Baker, R. E. Hardenburg, and M. Lieberman.
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Physio1. 59:546.
108. Wang, C. Y. and J. E. Baker. 1979. Vase cut f10wers treated
with rhizobitoxine ana10gs, sodium benzoate, and iso-
E.D.DEKAZOS 381

penteny1 adenosine. HortSeienee 14(1):59-60.

109. Wheaton, T. A. and I. Stewart. 1973. Optimum temperature and
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337.
110. Yokoyama, H., C. DeBenediet, C. W. Coggins, and G. L. Henning.
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Preharvest sprays with (2-ch1oroethy1 phosphonic acid)to
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237.
113. Young, R. and O. Jahn. 1972. Ethy1ene-indueed earotenoid
aeeumu1ation in eitrus fruit rinds. J. Amer. Soe. Hort.
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MANIPULATION OF THE POSTHARVEST ATMOSPHERE FOR PRESERVATION OF

FOOD CROPS

David R. Dilley

Department of Horticulture
Michigan State University
East Lansing, Michigan USA 48824

INTRODUCTION

Numerous handling, transportation and storage systems have

evolved over the years for postharvest preservation of fresh fruits
and vegetables. Depending upon the commodity and the specific pre-
servation objective, there is a wide selection of techniques and
systems to choose from (1). Table 1 outlines preservation systems
available für fresh perishables. They vary in simplicity from
common storage involving little or no control of the postharvest
environment to highly sophisticated systems such as hypobaric stor-
age (2,3) controlling within very narrow limits the temperature
and humidity, concentration of oxygen, carbon dioxide and other gases
that may affect product preservation. The choice and successful
application of postharvest preservation technology depends largely
on understanding certain fundamental aspects of biology, engineering
and economics that are important in the maintenance and distribution
of perishable commodities. Biological consideration include; the
physical attributes of the commodity, the physiological response to
the postharvest environment and susceptibility to pathogens. Engi-
neering aspects include heat and mass transfer in maintenance of the
ideal environment of temperature and gas atmosphere and product
protection. Economic principles of supply of and demand for the
product in relation to cost, benefit and practicality will ultimately
determine what postharvest technology will be employed. The emphasis
of this paper will be the biological consideration important in
storage of apple and pear fruits.

383
384 D. R. DILLEY

Table 1. Storage Systems and Techniques for Postharvest

Preservation of Fruits and Vegetables

I Common
11 Refrigerated
111 Controlled Atmosphere
1. Ventilated sum: 02 + CO 2 21%
2. Scrubbed sum: 02 + CO 2 < 21%
a. caustic
b. water
c. dry lime
d. activated carbon
e. molecular sieve
3. Inert gas generators
4. Nitrogen purge
5. Diffusion membrane
6. Modified atmosphere
7. Ethylene scrubber
8. Hypobaric

FACTORS ASSOCIATED WITH MAINTENANCE OF QUALITY OF HARVESTED FRUIT

Apples and pears are marketed from storage in some countries

on nearly a year-around basis. Large production volume of suitable
varieties in relation to the market place, and demand requires
controlled atmosphere (CA) storage. Achieving high quality and
marketability of the product from refrigerated and CA storage re-
quires that the fruits be harvested at optimum maturity and stored
under ideal conditions of temperature and gas composition according
to the expected storage duration. Establishing the proper time to
harvest some apple cultivars to ensure high quality after long-term
CA storage requires special consideration. Quality will be poor if
fruits are harvested too early or too late. This is particularly
true for the early-coloring strains of Red Delicious which are fully
red while still very immature. When harvested before they are mature
they are much more apt to develop scald when stored. Chemicals like
diphenylamine and ethoxyquin used as a postharvest treatment to
control scald are often ineffective when applied to immature fruits.
Bitter pit and shrivel are other common problems associated with
storing immature apples. Furthermore, the apples are likely to be
of low dessert quality from the standpoint of texture and flavor.
Importantly, fruit size and tonnage is reduced by harvesting too
early. The adverse consequences of storing apples and pears for
extended periods after ripening has begun include; excessive soften-
ing and internal breakdown and mealiness - physiological disorders
associated with senescence. Ripe fruits are also more prone to
bruising and decay. Harvesting apples and pears at the proper time
MANIPULATION OF THE POSTHARVEST ATMOSPHERE 385

according to when and how they will be utilized is thus a very

important decision.

GROWTR AND DEVELOPMENT OF FRUIT AND TRE USE OF CHEMICALS IN CONTROL-

LING MATURATION AND RIPENING

The relationship between growth and development and metabolism

for fruits of the climacteric type such-as apples and pears is
shown in Figure 1. This figure also relates how the important growth
regulating chemicals, ethephon and daminozide (Alar) and controlled
atmosphere storage affect fruit ripening. During the first 3 to 4
weeks following bloom, 30 to 80 million cells are formed in the
apple and pear fruit. This is followed by aperiod of several
months during which growth is by cell enlargement. The growth rate
during the last few weeks is about 1% per day and this is accompanied
by a significant increase in the intercellular air space between the
cells. This, in large measure, is responsible for the gradual de-
crease observed in flesh firmness before harvest. It is important
to recognize that this decrease in flesh firmness is a result of
growth rather than ripening and imparts to the apple the desired
textural characteristic of crispness as opposed to the tough and
woody texture of immature fruits.

~
100
100

li.
I "
z
LU
(!)
FRUIT GROWTH I 0

z
<l 1O~
I
u ~
~50
~
z
~ 8
-'
J;
~
LU
Q:
>

~
,,
0.1

O~'------·~~'------~6~'o~/,~I--~13b'O~--~1~~5----~1~~O~--~li5
OAYS

Fig. 1. Growth and development of apple fruits in relation to the

effects of ethylene, ethephon, Alar and controlled atmoß-
phere storage on ripening and storage life.
386 D. R. DILLEY

It has been known for more than 50 years that ethylene gas
produced by the fruits causes them to ripen. The relation of ethy-
lene to fruit ripening and storage life is also shown in Figure 1.
Note particularly the precipitous and logarithmi~ increase in ethy-
lene which marks the beginning of the ripening process. During
the last few weeks of the maturation period the ethylene 'level in
the fruit is fairly constant and mostly below 0.1 ppm (4). This
low rate of production then gives way to a more rapid rate and
when the concentration rises too much above 0.1 ppm it stimulates
further ethylene production in an autocatalytic manner responsible
for the log-linear increase. This marks the beginning of the
ripening process which involves qualitative and quantitative changes
in metabolism and this will not occur without ethylene action.
Metabolism is redirected. New enzymes are synthesized (5) that
catalyze ce 11 wall softening (6,7) and development of flavor and
aroma. New biochemical pathways are initiated and the metabolic
rate, exemplified by the respiratory climacteric, increases dra-
matically as shown in Figure 1. When this occurs, the course of
ethylene action cannot be reversed - only slowed.

Figure 1 then describes important features of apple and pear

fruit growth and development that are essential to understand the
processes of maturation and ripening and the role of growth regu-
lators and storage technology. The time scale roughly approximates
the situation for 'Red Delicious' but it is not fixed.

The effects of ethephon and Alar on apples are also best ex-
plained by hastening or delaying, respectively, the onset of acceler-
ated ethylene production. Ethephon is absorbed into the tissues,
releases ethylene and, if the fruits are sufficiently mature, this
initiates ethylene production and subsequent action. One of these
acts is to stimulate the pathway responsible for production of the
anthocyanin pigments. Considerable enhancement of red color can
sometimes be achieved without overly stimulating ethylene production
by the fruits, particularly whenethephon is applied, to trees sprayed
earlier with Alar. In many instances these highly colored fruits
can even be stored successfully in CA. But this requires knowledge
of ethylene levels in the fruits in order to ensure safe storage.
Ethephon advances and Alar delays the " t ime-line" of ripening as
shown in Fig. 1.

Fruits become more responsive to ethylene as they mature. Thus

if ethephon is applied too early, red color enhancement is minimal.
And when fruits are receptive but ethephon is applied to trees with-
out prior Alar treatment, ethylene production can be overly stimu-
lated resulting in excessive drop.

Studies at Michigan State University on 'McIntosh' apples

MANIPULATION OF THE POSTHARVEST ATMOSPHERE 387

established the delaying action of Alar on fruit maturity (8). Alar

was applied about 60 days prior to the estimated harvest date for
long-term storage, and 2,4,5-TP was applied 21 days before the same
target date. These treatments were employed to ascertain if Alar
could attenuate the acceleration of ripening sometimes observed with
the use of 2,4,5-TP for preharvest drop control.

The effects of treatment on maturity and ripening are seen in

Fig. 2 a,b, and 3. Fruits from all treatments on the harvest of
Sept. 15 had identical and low respiration rates, indicating the
fruits had not reached physiological maturity. At harvest, the
fruits from trees sprayed with 20 ppm 2,4,5-TP entered the climac-
teric sooner than the control fruits which were judged to be of
ideal maturity for long term CA storage on Sept. 22. Fruits which
reeeived Alar alone at 500 or 1000 ppm (1/2 to 1 lb. per 100 gal. -
400 gal. basis) showed no tendency to enter the climacteric even
after 1 week at 20°C. However, 20 ppm of 2,4,5-TP treatment over-
came the retarding effect of the 500 ppm Alar concentration.

The same general response was observed for fruits harvested one
week later. Fruits from all treatments containing Alar were less
advanced, and those receiving 20 ppm 2,4,5-TP more advanced than the
controls. 2,4,5-TP stimulates ethylene production, and in this way
overcomes the influence of Alar unless very high dosages are used.

That Alar delays the onset of the ripening process is clear

from the data in Fig. 3 in which the loss in firmness during a
holding period of 7 days is given for each harvest in relation to
Alar concentration. Here it is seen that fruits receiving Alar at
500 ppm (1/2 lb. per 100 gal.) ripen like control fruits do, bu.t
about one week later. Fruits that received 1000 ppm (1 lb. per
100 gal.) Alar ripen like control fruits, but about two weeks later.

To slow down ethylene production and the events it initiates

in ripening requires prompt establishment of controlled atmosphere
conditions. Storage life diminishes rapidly as fruits gain capacity
to produce ethylene. This is depicted as the dotted line in Figure
1. The ability of CA storage to retard ripening diminishes as the
ripening process is initiated. Carbon dioxide in the CA atmosphere
is a natural inhibitor of ethylene action but it becomes ineffective
when ethylene accumulates to above 1 ppm in the fruit. Oxygen is
required for fruits to make ethylene and low temperature slow down
the rate of the chemical reactions of metabolism. These consider-
ations serve as the basis for CA storage. They explain why CA
storage is most successful, in terms of quality retention, when
this technique is applied to fruits before they begin to produce
much ethylene. CA storage can thereby delay the 'time-line' of
ripening as indicated by the arrow in Figure 1.
388 D. R. DILLEY

a.
MclNTOSH
16
HARVEST 2
September 15, 1966
14

o ALU - 1.4,5 - T'

,ALL TIlIA TIlINTSI
6 ~
o--..8_i!lr.!.'""'I!e~99-t'e~C!>_-E9h0r:1'.-0

01" l'
0 3 4 5 , 7
DAYS AFTER HARVEST '20' CI

b.
McINTOSH ALAlI: 1,4,II-T'
16 HARVEST 3 "'111
September 22,1966
~ 14 0: 10
~
.,
","
0: 0
,12
!
~
~IO 0: 10
Il00: I!O
~..
.
ot: 8
it
~
6
0,10
0,10,10

L
OL
0 2 3' 4 5 , 1
8
DAYS AFTER HARVEST nO'CI

Fig. 2 a, and b, The effect of Alar and 2,4,5-TP on the initiation

of the respiratory climacteric and ripening of Mclntosh
apples.
MANIPULATION OF THE POSTHARVEST ATMOSPHERE 389

MclNTOSH

o CONTROL
;:s 4.0
~ 500 PPM ALAR
2 • 1000 PPM ALAR
~
:! 3.0

....
~

~ 2.0
.,c>
il
~
~ '1.0
iL
:t
tl...
"- 0.0

-,J1 r
, 4
'-8-66 '-'5-66 '-22-66 '-29-66
HARVEST

Fig. 3. The effect of Alar on delaying the loss in flesh firmness

of McIntosh apples held at 20 0 e following harvest.

ETHYLENE AS AN INDEX OF FRUIT MATURITY, RIPENESS, AND STOREABILITY

Bartlett pears provide another example of ethylene receptivity.

This is shown in Figure 4 where ethylene was unable to stimulate
flesh softening of fruits harvested on August 7 while it worked one
week later. In this particular season. it was not until August 21
that the fruits had gained sufficient maturity and ethylene produc-
tion capacity of their own to initiate ripening. There is an im-
portant lesson to be learned from these data. Bartlett pears can
gain as much as 30 percent in weight during the final three weeks
before ethylene begins to accumulate sufficiently to stimulate
ripening. Unfortunately many of the Bartlett pears grown are har-
vested while still quite immature. One of the factors contributing
to this problem is placing too much reliance on the use of flesh
firmness in establishing the harvest date. Studies have repeatedly
shown that physiological maturity correlates poor1y with f1esh
firmness. That is, fruits of 15 to 16 1bs. firmness (5/16" tip)
from one orchard can in fact be 1ess mature than fruits of 18 to
19 lbs. from another orchard. A good example of this can be seen
in Figure 5 where ethy1ene concentration in fruit is plotted over
a sequence of four weekly harvests. Flesh firmness at harvest is
390 D. R. DILLEY

given and the value in parenthesis is the firmness after one

week at ripening temperature. Note in particular that fruits from
the last two harvests had initial firmness of 18 lbs. yet only the
fruits from the last harvest were sufficiently mature and contained
enough ethylene to cause ripening. Fruits harvested during the week
beginning September I ripened very weIl after long-term storage in
the air. This clearly deHlOnstrates that internal ethylene level is

m 1 .a,s
'i!; ilitial

et.,I.I.
-...-
.:,25 ~

-= + 1.I,s

-...
~

-...
!!

....
-'

Fig. 4. Flesh firmness of Bartlett pears at harvest and after one

week at ripening temperature in response to ethylene treat-
ment at 500 ppm for 24 hours.

a better measure than flesh firmness as a criterion of harvest

maturity. Moreover, these data show that Bartlett pears need not
and shou1d not be harvested for storage until at least severa1
fruits in a samp1e of ten contain more than 0.1 ppm of ethy1ene.
Be mindfu1 that fruit size and therefore tonnage increases signi-
ficant1y during the last few days without sacrificing storeability.
This is assuming that good storage and handling procedures are
employed. When properly handled and promptly cooled in storage to
MANIPULATION OF THE POSTHARVEST ATMOSPHERE 391

100
:
:
-
- I>

-
10-=
::-
~ --
-
A.
A.

...
....-Z 1-=
; •
I>
l' .11•
(3)

>- :
-- •
.......
::t I>

.- - I>

«
z -
GI:
... 0.1 __ I"" I>

Z
(")

--=:
•
I>

-
-- I> 221M
(22) i
I> 201~.

(19) •
- •
I>

I> I>
.01
8·17 8·24 8·31 9·7
HARVEST DATE

Fig. 5. Internal ethylene levels in Bartlett pears at harvest at

weekly intervals. Each point is an individual fruit.
Firmness is given at harvest and after one week at 20°C.

to 30°F it takes several weeks before the fruits gain capacity to

produce ethylene.

Ethylene is clearly the best measure to assess maturity, ripen-

ing and storability (9). Extensive studies with apple fruit sub-
stantiate this out and in addition, considerable insight has been
gained that helps explain why orchards can vary greatly from grower
to grower and between years. The leaves of apple (4), and presum-
ably pear, trees produce an inhibitor of ripening that is trans-
located to the fruits in the phloem. There are several lines of
evidence for this. Firstly, fruits ripen more rapidly at the same
ternperature loff' as compared to 'on' the tree. This suggests
that the substance is metabolized. Secondly. and more definitively,
fruits borne on spurs that have been defoliated and girdled ripen
much earlier than fruits on normal spurs. These studies help to
interpret observations of ripening behavior and reinforce the
necessity to make an orchard by orchard and block by block assess-
ment of maturity and ripening development. This is because there
392 D. R. DILLEY

are many orchard management practices and environmental stresses

that can hasten or delay fruit ripening. These include: biotic,
insects and diseases; nutrient deficiencies; wound injury; and
environmental stresses of heat, cold, d~ought and flooding.

Ethylene measurements can now be done in the orchard or ware-

house with a new portable instrument called "Snoopy," developed in
1978 in the Postharvest Research Laboratory of the Horticulture
Department at Michigan State University (10). 'Snoopy' is a high-
ly sensitive portable gas chromatograph that detects as little as
0.1 ppm ethylene in a 1 cc gas sampie taken fram the fruit. The
instrument separates ethylene from the other gases and measures
the ethylene concentration. A 1 cc sampie of intercellular atmos-
phere from a fruit or a composite sampie taken fram several fruits
is injected into the instrument and the response is followed on the
meter or a recorder which provides a permanent record of the analysis.
Analysis takes about two minutes. This instrument is being widely
used by individual growers, fieldmen, storage operators, extension
agents and researchers around the world to assess fruit maturity,
ripening and storability.

All fruits on a tree do not begin to ripen at the same time.

The onset of ripening will vary by variety, and from orchard to
orchard and year to year. When immature, all fruits in the sampie
will show uniformly low ethylene levels of 0.1 ppm or lower. Sub-
sequently, some fruits in the sampie will begin to show accumulation
of ethylene while the majority remain low. The king-blossom fruits
and ~hose on spurs adjacent to vigorous terminal growth will gener-
ally begin to produce ethylene before others. It is important then
to monitor the more advanced fruits on a tree to make the maturity
and ripening asses~ment. Eventually, the majority of fruits in a
sampie will begin to accumulate a significant amount of ethylene
indicating that ripening is weIl underway. Table 2. provides guide-
lines for storability of apples according to internal ethylene
levels.

An example of how ethylene data for the Empire apple was used
to make storage duration decisions for a Michigan orchard in 1979
is shown in Figure 6. Sampies taken up until the first of October
had uniformly low ethylene levels below 0.1 ppm. Although the
fruits were fully red and attractive they were still immature. By
October 2, some fruits began to show significant ethylene while the
remainder were low. Fruits harvested during the ensuing week were
judged to be excellent for long-term CA storage. By October 8,
fruits were still low enough in ethylene for mid-term CA storage
and had flesh firmness of 16 lbs. Not until the 16th of October
were fruits from this orchard judged to be suitable for only short-
term storage. Performance of fruits from this orchard harvested
over a three week per iod beginning September 28th and stored in CA
storage up to 9 months bore out the correctness of the maturity and
ripeness assessment.
 MANIPULATION OF THE POSTHARVEST ATMOSPHERE 393

Table Z. Fruit Ethylene Levels As A Guide For Harvest

And Storage Decisions

No. of Fruits Fruit Ethylene Suggested Action

10 out of 10 Less than 0.1 ppm May delay harvest for better
size, color, and quality.

3 out of 10 0.1 to 0.5 ppm Suited for long-term CA. 'CO Z

treatment'maximallyeffective.

3 out of 10 0.5 to 1 ppm Suited for mid-term CA. 'C0 2

treatment' marginally effect-
ive.

3 out of 10 1 to 5 ppm Suited for short-term CA. 'CO Z

treatment' ineffective.
3 out of 10 5 to 10 ppm Suited for up to 4 months of
refrigerated ro CA storage.
3 out of 10 More than 10 ppm Suited for short-term storage
and processing.

It should be clear from the foregoing discussion that apples

destined for long-term CA storage should be harvested and stored
before they have gained much capacity to produce ethylene. Further,
that fruits intended for shorter storage duration can and should be
allowed to remain on the trees longer in order to gain the extra
measure of quality and sales appeal required of fruits in this more
competitive marketing period.

Even fruits of low ethylene content at harvest begin to make

significant amounts of the ripening gas during several months in CA
storage. This is because ethylene accumulates in the sealed storage
atmosphere. This stimulates the less advanced fruits to initiate
ethylene production and eventually levels of ethylene in excess of
100 ppm are commonly found.

Recent research (11, 12, 13) as well as our own has shown that
ripening of apples, providing the fruits haven't begun autocatalytic
ethylene production, can be markedly delayed during CA storage em-
ploying low oxygen levels of 1 to 1.5 percent and judiciously scrub-
bing ethylene from the storage atmosphere. Current devices for
removing ethylene are ineffective or cost prohibitive. Some acti-
394 D. R. DILLEY

100
.i
.
•
S!
- EMPIRE 1979 •
=
:t:

•
..
~

c 0
u c z
10~ u

•"
:t:
•
-
:t:
--= = •
=
~
-- "9
z SI
:t:
I.
• ••
A.
A.
- •
I.
•
IAI
Z 1-;
.... : •
,.•
IAI

>- :
%
"'"
IAI -- • •
....
z
oe - •
111: •
: 0.1-; • •
- -- • • I
Z :
I •

-- I I I •
- "."NUS .1 '1 ., '5"
01
9·24 9·28 10-2
• •
10·8 10·16
HARVEST DATE

Fig. 6. Interna1 ethy1ene levels in Empire app1es in relation to

harvest date and assessment of storability. F1esh firm-
nessva1ues are given above the harvest dates.

vated carbon scrubbers used to remove carbon dioxide from CA rooms

are margina11y effective in reducing ethy1ene level in the storage
atmosphere about 10-fo1d in comparison to the level in CA rooms
scrubbed with dry 1ime (e.g. 50 vs. 500 ppm).

It is important to keep the ethy1ene level be10w 1 ppm in the

CA storage room to effective1y de1ay ripening. This is one of the
primary benefits derived from hypobaric storage at absolute pressures
of 0.1 atmos. or 1ess (14). By 1977 it was generally conc1uded that
other systems for removing ethy1ene from the storage atmosphere wou1d
be ineffective because the ethy1ene in the interna1 atmosphere of
the fruit cou1d not be significant1y reduced. This concept is being
reeva1uated now that u1tra-1ow oxygen levels (e.g. 1 to 1.5% 02)
are being emp10yed for CA storage of app1es and pears. And it is
MANIPULATION OF THE POSTHARVEST ATMOSPHERE 395

being reco~nized that fruits must enter CA storage before they enter
their ethylene climacteric.

These considerations led to the development of a high capacity

ethylene scrubber based upon catalytic oxidation (9). The system
is hermetically sealed and consists in sequence. of a large air
blower (ca. 200 cfm), a heater to raise the storage atmosphere to
about 200°C, a monolithic catalyst similar to that employed for
engine exhaust emission control, and a heat exchanger to cool the
ethylene-free storage atmosphere as it is returned to the CA room.
The ethylene scrubber was tested on a 20 thousand bushel CA storage
of 'Red Delicious' apples in Michigan in 1980. Figure 7 shows that
the scrubber effectively removed ethylene down to less than 1 ppm.
The high initial ethylene level was due to inefficient propane com-
bustion by a CA generator used for oxygen pull-down. Continuous
operation of the scrubber kept the ethylene level in the CA storage
below 1 ppm. Fruit ripening was prevented during the balance of
the CA storage period. Whether or not this type of ethylene scrubber
becomes a connnercial reality will depend on the cos t/benef i t analysis.-

HYPOBARIC STORAGE

Hypobaric storage deserves special consideration as new tech-

nology for postharvest preservation of perishables. Research was
initiated in numerous laboratories around the world following the
disclosure of this storage technology by Burg and Burg (2) in 1966
and was reviewed recently (3). Hypobaric or low pressure storage
(LPS) is currently being commercially developed by the Grumman/
Dormavac Division of Grumman Allied Industries, Inc. The connnodity
is placed in a vacuum-tight and refrigerated container and evacuated
by a vacuum pump to the desired low pressure which, depending upon
the commodity may vary from lOnnn Hg to 76nnn Hg. The oxygen level
varies in direct response to the absolute pressure. When the desired
low pressure is obtained, fresh air is admitted to the chamber
through apressure regulator and then humidified. In the continu-
ously ventilated partial vacuum, carbon dioxide, ethylene, and waste
volatile by-products of metabolism rapidly diffuse out of the com-
modity and are flushed from the storage chamber. The oxygen content
or partial pressure is set and varies within narrow limits by ad-
justing an absolute pressure regulator. Air, water, and electricity
are the consumable items involved in the system. Hypobaric storage
conditions can be achieved within minutes; so the chamber can be
opened to inspect, remove or add connnodity and then closed again
without any deleterious effects.

Hypobaric storage is the most significant breakthrough in post-

harvest preservation technology since controlled atmosphere was
introduced in England in the 1930's. Extensive investigations have
been conducted on hypobaric storage of a wide range of fruits, vege-
tables and flowers. These studies have confirmed and extended Burg's
396 D. R. DILLEY
1~~~~ ______________________- - ,

100

~
0..
0..

Z
Q
~
oe
lIIt
10

...Z
~

v
Z
0
v
...
...Z
..J
~
::t
...
~

----_------.:-----r--_----I
0.1 ~......
15 30 45
HOURS

Fig. 7. Scrubbing ethylene from a 20,000 bu. CA storage of Red

Delicious apples with a catalytic oxidizer at 200°C and
130 CFM. The high initial ethylene level was from incom-
plete oxidation of propane by an Arcat CA generator during
oxygen pull-down.

hypo thesis that hyponormal ethylene levels and low O2 partial pres-
sure in this rarefield environment are the factors responsible for
delaying ripening and extending the storage life of fresh perishable
commodities.

Hypobaric storage markedly extends the postharvest preservation

period of a wide range of fresh fruits and vegetables beyond the
useful period achieved by conventional storage methods. The princi-
pIes of hypobaric storage apply to commodities which benefit solely
by reducing the oxygen partial pressure and those which benefit
additionally by lowering the internal atmosphere concentration of
ethylene and other by-products of metabolism. Hypobaric storage
maintains apples and pears in their pre-climacteric stage and mark-
edly arrests ripening of post-climacteric fruits. Hypobarically-
MANIPULATION OF THE POSTHARVEST ATMOSPHERE 397

stored apples can become insensitive to ethylene action in ripening

until they are acclimated in air at atmospheric pressure while under
refrigeration. Hypobaric storage: retains sweetness in sweet corn,
delays loss of ascorbic acid in asparagus, prevents internal brown-
ing of mushrooms, prevents scald of apples, maintains freshness of
sweet cherries and blueberries, delays leaf abscission of leafy
vegetables and retards bacterial soft-rot development.

REFERENCES

1. Dilley, D. R. Approaches to maintenance of postharvest integri-

ty, J. Food Biochem. 2:235 (1978).
2. Burg, S. P. and E. A. Burg. Fruit storage at subatmospheric
pressure, Science 153:314 (1966).
3. Lougheed, E. C., D. P. Muir and Luce Berad. Low pressure stor-
age for horticultural crops, HortScience 13:21 (1978).
4. Sfakiotakis, E. M. and D. R. Dilley. Internal ethylene in apple
fruits attached to or detached from the tree. J. Am. Soc.
Hort. Sci. 98:501 (1973).
5. Frenkel, C., I. Klein and D. R. Dilley. Protein synthesis in
relation to ripening of pome fruits, Pl. Physiol. 43: 1146
(1968).
6. Knee, M. Changes in structural polysaccharides of apples dur-
ing storage. Coll. Inter. Centre Recherche Sci.: 238 (1975).
7. Sawamura, Masayoshi, E. Knegt and J. Bruinsma. Levelof endo-
genous ethylene, carbon dioxide, and soluble pectin, and
activities of pectin methyl esterase and polygalacturonase
in ripening tomate fruits. Plant and Cell Physiol. 19:
1061 (1978).
8. Dilley, D. R. and W. W. Austin. The effect of Alar (N-dimethyl-
amino succinamic acid) on maturation and storage quality of
apples. 96th Ann. Rpt. Mich. Hort. Soc. p. 102 (1966).
9. Dilley, D. R. Assessing fruit maturity and ripening and
techniques to delay ripening in storage. 110th Ann. Rpt.
Mich. State Hort. Soc. p. 82 (1972).
10. Dilley, D. R., J. Lee and M. E. Saltveit, Jr. Measuring fruit
ethylene concentrations for proper harvest and storage
decisions. 108th Ann. Rpt. Mich. State Hort. Soc. p. 121
(1978).

11. Forsyth, F. R., C. A. Eaves and H. J. Lightfoot. Storage

quality of McIntosh apples as affected by removal of ethylene
from the storage atmosphere. Can. J. Plant Sei. 49:567 (1969).
12. Stoll, K., F. Hansen and D. Datwyler. The ripening of apples
in CA storage as affected by reduction of the ethylene content
of the atmosphere. Coll. Inter. Centre Nat'l. Recherche Sei.
No. 238:81 (1975).
13. Liu, F. Interaction of daminozide; harvesting date, and ethylene
in CA storage on 'McIntosh' apple quality. J. Am. Soc. Hort.
Sei. 104:599 (1979).
METABOLISM, HEAT TRANSFER AND WATER LOSS UNDER HYPOBARIC CONDITIONS

Stanley P. Burg and Robert Kosson

Grumman/Dormavac Division
Grumman-Allied Industries
90 Crossways Park Drive W.
Woodbury, NY 11797

INTRODUCTION

Evaporative cooling is the dominant mode of heat transfer under

hypobaric conditions. This creates a dilemma in water conservation
for plant products. Can they dispel their respiratory heat and re-
main cold without drying excessively, or doeswater loss ultimately
limit storage? A nodal model for heat transfer is used to analyze
this problem. The present report summarizes results obtained with
this model, and in addition discusses several other important dif-
ferences between hypobaric and atmospheric pressure storage.

DESCRIPTION OF THE HYPOBARIC SYSTEM

The hypobaric method requires a vacuum ~ to store the cargo,

a vacuum ~ continuously withdrawing air from the tank, a pressure
regulator leaking air into the tank at a rate sufficient to maintain
the set pressure, a humidifier which injects enough cool steam into
the incoming rarified air to saturate the atmosphere in the vacuum
tank, and a refrigerator to control the cargo temperature (Fig. 1).

The vacuum/water subsystem of Grumman/Dormavac's intermodal

container is illustrated in Figure 2. The dew-point of air passing
through the vacuum tank is controlled by setting the electrical
energy input to the humidifier. Boiling takes place at a low tem-
perature, between 12 to 47°C when the pressure is 10 to 80 mm Hga.
The vacuum pump, a dry, lobed-blower super-charging a water sealed
stage, is continuously cooled to the temperature of the container
walls. During isothermal compression most of the water contained
in air leaving the container is condensed and recovered in the

399
400 S. P. BURG AND R. KOSSON

HUMIVIFIER

REFRIGERATOR
Fig. 1 - Schematic diagram of hypobaric system

t-o---- ~1I':t%,4IR
(CCM1<OI.J.F/
( tAl<)
o All?
o IVArfl?
• "'/TMM

Fig. 2 - Vacuum/water subsystem

HYPOBARIC CONDITIONS 401

water-seal stage. This distilled water is gravity drained into a

storage tank and passed through a deionizer before it is supplied
to the boiler on-demand. The deionizer only needs to remove salt
from the initial supply of stored water. The boiler is further
protected from scale build-up by automatically draining whenever
the container is shut-down. This nearly-sealed system conserves
enough water to operate continuously for 6 weeks at the most de-
manding combination of pressure and temperature, with only 50 gallons
of water initially stored.

The refrigeration subsystem is illustrated in Figure 3. The

inner aluminum surface of the container is used as a cold-plate,
with refrigerated glycol flowing through closely spaced integral
wall tubes, entering and leaving in paired-tubes to eliminate front-
back temperature gradients. Wall gradients are ± 0.6°C compared to
the average wall temperature. This makes it possible to maintain
the RH close to 96%. (In the hypobaric pressure range, the dew-point
and wet-bulb are equivalent, and both can be converted to RH from
a wet-bulb table.) There is no mechanical air-moving system, ex-
cept the vacuum pump, which draws air diagonally through the con-
tainer's cross section at a net velocity of 0.3 ft/min from the
wall-roof juncture at one side to the floor-wall juncture at the
other. The vacuum pump supplies approximately 100 cfm, sufficient
to change the air in an empty container 3.3 times per hour independ-
ent of pressure and temperature.

DIRECT EFFECTS OF PRESSURE ON METABOLISM

Lowering the atmospheric pressure reduces the cellular hydro-

static pressure, thereby decreasing the cellular water potential.
Although water participates in many biochemical reactions, and the
rates of these reactions are controlled in part by the cellular
activity of water, reaction rates are only slightly altered by a
pressure reduction. The relationship between water activity Ca)
and water potential (1jIw) is:

\['w (Rt/V 1) In a (1)

\['w Water potential
R Gas Constant
T Temperature
VI Liquid volume
a Water Activity

From this equation it can be predicted that changing \['W by 20 at-

mospheres only changes (a) by 3 to 4%.

Changes in turgor pressure (1f p ) also have no direct effect on

chemical and metabolic processes. The electrochemical potential
402 S. P. BURG AND R. KOSSON

Fig. 3 - Refrigeration subsystem

of ions is virtually insensit!ve to physiological shifts in pressure

(about 0.2 mV per atmosphere). To markedly effect the rate of
chemical or metabolie reactions involving solid or liquid phases,
more than 100 atmospheres of pressure are required. Therefore shifts
in turgor can only influence metabolism by altering cellular struc-
tures.

Cells typically possess apressure sensing system which responds

by negative feedback to a change in hydrostatic pressure, setting in
motion aseries of events which res tore the original hydrostatic
pressure. The mechanism by which turgor is sensed is thought to be
a thickening or thinning of the cellular membrane (11), in response
to which the rate of passive permeabi1ity and active transport of
potassium ion are altered in directions which res tore the original
turgor (19,21,48,55,56). This pressure sensing-negative feedback
system participates in such processes as osmoregu1ation, opening
and shutting of stomata, and the movement of leaves on sensitive
p1ants. Another process dependent upon turgor is the growth of wa11-
ed ce11s (43). Turgor affects cell expansion in two ways: apressure
in excess of a critica1 va1ue is necessary for biochemica1 modifica-
tion of the cel1 wall properties, and the subsequent physical ex-
tension of the wall also requires turgor. Growth of fungi and
bacteria precedes by processes similar to those in higher plants,
so far as is known, with the exception that the wall materials are
different and growth seems not to be under hormonal control.

Two methods have been used experimentally to modify turgor:

osmotic up- or down-shock, and changes in atmospheric pressure.
Similar results are obtained in both cases. Increasing the atmos-
pheric pressure by 1 atmosphere doubles the rate of potassium efflux
and reduces potassium inf1ux by two-thirds in Valonia cells (19).
Hypotonie solutions or water enhance and hypertonie solutions
HYPOBARIC CONDITIONS 403

inhibit solute leakage in a wide variety of higher plant products

(10,14,22,31,37,44.51) and these treatments have an opposite effect
on potassium influx (14). Animal cells and bacteria osmoregulate
in like manner (23). Extreme turgor shock with 0.5 M glucose under
appropriate conditions causes the release of many of the components
of the active transport system of bacterial cells (23). During the
upshock period the cells become very leaky, and can be loaded from
external solutions with molecules which normally would not permeate.
Cells in the log phase of growth are particularly sensitive to turgor
shock.

The freezing point of mushrooms, roses, lamb and chrysanthemum

cuttings is elevated nearly 0.5°C under hypobaric conditions. There
is no obvious physicochemical basis for this shift, so the explana-
tion must be biological. The first water to freeze usually is
extracellular where the solute concentration is lowest (40). If the
product osmoregulates in response to a decrease in atmospheric
pressure, it will withdraw solute from the extracellular fluid,
thereby increasing the freezing point of that water.

Products stored under hypobaric conditions typically are not

growing, but microbes must grow to cause decay, and therefore they
are subject to inhibition in response to a pressure-induced change
in turgor. Cells of E.coli in the resting state possess no turgor
and do not osmoregulate. Their contents resemble that of the media
in which they are bathed. They begin to transport actively and
osmoregulate during the stationary phase of growth. The osmotic
content of such cells typically is about 0.15 M, mainly potassium
ion. In response to apressure reduction to < 80 mm HgA, these
cells must increase their osmotic content by nearly 0.045 M. Computa-
tion based on an estimate of the respiratoryenergy available at a
low pressure and temperature, presuming one ATP per potassium taken
up, suggest that even psychrophylic bacteria would require many
hours to res tore their original osmotic balance, aperiod roughly
comparable to their generation time. Subsequently, they can develop
the additional turgor needed for growth, and thereafter hypobaric
storage should not affect their rate of multiplication except by
influencing the p02 and pC0 2 available to support growth. If at a
later time, however, the pressure is returned to atmospheric, the
cell now would need to leak out nearly 0.045 M solute. Theoretically,
by adjusting the frequency of apressure cycle it should be possible
to induce bacteria to pump and leak often enough to consume all of
their respiratory energy, with too little left over to support the
synthetic processes needed for growth. In a preliminary test of this
concept during 8 weeks at 13°C, mature green tomatoes on apressure
cycle of 22 hours at 80 mm Hg - 2 hours at 760 mm HgA, developed
90% less decay than fruits continuously at 80 mm HgA. Surprisingly,
the rate of ripening during storage was slightly lower in the
cycled fruits.
404 S. P. BURG AND R. KOSSON

Another direct effect of pressure reduction is to decrease the

boiling point of certain volatile metabolic products. At ODC,
acetaldehyde boils when the pressure is less than 320 mm HgA. Data
for ethanol is given in Table 1.

PRESSURE-INDUCED CHANGES IN GASEOUS COMPOSITION

The p. (atm) in the vacuum tank's air depends on the total

pressure (F~2and water vapor pressure (P ):
v
PC02 = .00035 (P - Pv ) (2)

At atmospheric pressure, even when the product is ventilated ade-

quately with fresh air, its internal C02 is much higher than that
in the ambient air due to restricted outward diffusion of respira-
tory gas. In addition, C02 accumulates in the air of "sealed"
Controlled Atmosphere (CA) and Modified Atmosphere (MA) storages,
where it may be controlled at apressure as high as .05 atm, but
cannot be reduced below .005 atm with the best chemical or mechanical
scrubbers. Under hypobaric conditions the internal CO 2 is decreased
in proportion to the pressure reduction because of increased gaseous
diffusivity.

Elevated CO 2 has the advantage of suppressing ethylene action

by competitive inhibition, and in addition directly inhibits res-
piration. The first advantage is not beneficial under hypobaric

Table 1. Vapor Pressure of Ethanol at Various Temperatures

Temperature Vapor Pressure

(OC) (mm HgA)

-1 11
o 12
2 13
10 20
13 25
At each indicated temperature, if the total pressure 1-s
equal to or lower than the corresponding vapor pressure
value in the table, ethanol boils from the product. For
example, at ODC, 10 mm Hg, neither ethanol nor aldehyde
can remain in the tissue, and this conceivably could have
a beneficial effect on freezing damage or low 02
(fermentation) injury.
HYPOBARIC CONDITIONS 405

conditions because the outward diffusivity of ethy1ene from the

tissue is so enhanced that the interna1 concentration is kept be10w
a bio10gica11y active level (7,8). With many products, anyaccumu-
1ation of C02 is harmfu1, producing disorders such as sca1d, and in
general a PC02 greater than .05 mm atm is not desirab1e.

Very high concentrations of C02. ranging from 30 to 70% at

atmospheric pressure, have been used to control bacterial decay,
especial1y in meat and fish storage. But before this practice
became common it was known that many bacteria require CO 2 to grow,
and that their mu1tiplication is great1y enhanced by CO 2 in the
range of .0001 to .001 atm (12,38). In fact, C02 incubators are
common1y used to promote bacterial growth! Conceivably, the low
C02 produced under hypobaric conditions may be beneficial for
controlling bacterial decay.

The P0 2 in the vacuum tank's air also is related to P and Pv :

(3)

Regulation of P + 1 mm Hg by an altitude compensated vacuum

breaker (Figs. 1 and 2), and Pv by a precise, controlled boiler
wattage, is accurate enough and the flow of make-up fresh air fast
enough to control the B02 of the vacuum tank's air ±.0003 atm,
irrespective of the product's 02 consumption. This contrasts favor-
ably to the best control under CA conditions, Pe ± .05 atm. More-
over, due to the leak-tightness of the vacuum taÄk and associated
systems, 02 can be kept close to complete anaerobiosis, whereas CA
and MA cannot bring the P0 2 lower than .05 atm. This feature of the
hypobaric system makes it suitable for use with meat and fish,
which require nearly complete anaerobiosis to prevent pigment oxida-
tion and to retard aerobic and microaerophylic bacterial growth.

Low 02 damage occurs under CA conditions when the P0 2. is less

than .01 to .02 atm, but under hypobaric conditions products often
store best at pressures in the 10 to 20 mm HgA range where the P0 2
is .001 to .003 atm. This difference in behavior probably is
caused by an effect of pressure on gaseous diffusivity. At atmos-
pheric pressure the inward diffusivity of 02 is slow enough compared
to the tissue's 02-consumption to create an 02 gradient of .005 to
.02 atm between the product surface and center. Apparently low 02
damage occurs when the center becomes almost anaerobic. Under hypo-
baric conditions the gradient is lowered in relation to the pressure
reduction, so that the surface and center have a closely similar
P0 2 • Even at 10 mm HgA this P0 2 is not low enough to markedly
inhibit cyanide sensitive respiration mediated by cytochrome oxidase,
and therefore does not usually induce substantial fermentation,
but it profoundly inhibits the cyanide insensitive alternative
pathway of respiration, reducing the production of peroxide without
depleting the energy supply or inducing fermentation (47). A
406 S. P. BURG AND R. KOSSON

further advantage gained is that the entire product, surface and

center, is brought to an optimal P0 2 for storage, and in addition
the surface P0 2 is 10wered enough to inhibit the growth of aerobic
mo1ds and bacteria without damaging the product's center.

HEAT LOADS UNDER HYPOBARIC CONDITIONS

The on1y significant heat which has to be transferred at

steady-state from the product to the cold p1ate or changing air
is the respiratory load. This is much sma11er in a partial vacuum
than it is at atmospheric pressure because 1ess O2 is avai1ab1e to
support respiration. The amount of heat produced can be computed
by comparing the 02 or C02 content of the air entering the container
with that 1eaving at the vacuum pump exhaust, assuming 673 Kg-Ca1 of
heat produced per 6 moles of 02 consumed or C02 evo1ved. Data ob-
tained with carnations, corn, app1es and mushrooms show a,- c10se1y
simi1ar relationship between C02 production, 02 consumption and
pressure, and an R.Q. c10se to unity even at very 10w pressures
(Figure 4). Using these data as a nomograph, va1ues at atmospheric
pressure (50) have been adjusted to estimate hypobaric heat loads
at optimal pressure and temperature (Tab1e 2).

Heat infiltration and contro11ed (and uncontro11ed) in-1eakage

contribute 1itt1e to the heat load. The pounds of air which have to
be passed through a container under hypobaric conditions to effect
an air change are much 1ess thanthose needed at atmospheric pressure,
in proportion to the pressure reduction. Therefore the amount of
sensible heat admitted per air change is corresponding1y 10wer. In
addition, the incoming air decreases in RH when it expands because
now the same amount of water vapor is present in a much 1arger
volume. This air does not reach its dew-point even when it is
coo1ed to O°C, and no latent heat must be removed. Assuming ambient
conditions of 80% RH at 38°C, on1y 79 Btu/Hr must be removed to
change the air 3.3. times each hour at an interior temperature of
10°C and apressure of 20 mm HgA. At atmospheric pressure 20,000
Btu/Hr wou1d have to be rejected to accomp1ish the same rate of
air change at 10°C. Uncontro11ed in-1eakage is so sma11 relative
to the contro11ed 1eakage even at 10 mm HgA that it can be neg1ected.
Heat infi1trating from outside is captured at the hypobaric contain-
er's cold p1ate before it enters the cargo area, and therefore does
not norma11y reach the product. At atmospheric pressure in a con-
ventiona1 forced air container this heat infiltrates into the cargo
area and warms the product.

NODAL MODEL OF HEAT TRANSFER

A schematic model for the transfer of heat from a papaya fruit

(P) contained in a box (B) to the container air (C) and container
HYPOBARIC CONDITIONS 407

Table 2. Respiratory Heat Loads at Optimal Storage

Pressure and Temperature
Pressure TelIlP
Product (JIlm HgA) (0f) BTU/Ton/Day

Apple 50 32 252
Asparagus 10 32 2200
Avocado 20 50 2800
Banana 40 56 1656
Carnation (b1oom) 15 36 1950
Chrysanthemum (b1oom) 10 32 462
Corn 50 32 2685
Cucumber 80 55 1050
Lettuce 10 32 550
Lime 150 50 780
Mango 80 55 1500
Me10n (Honeydew) 20 50 1400
Mushroom 10 32 1760
Papaya 20 50 625
Pepper (sweet) 80 50 1848
Pineapp1e 20 50 385
Rose (bloom) 10 32 2376
Strawberry 10 32 726

The heat load at atmospheric pressure and the indicated tempera-

ture (50) has been corrected to an optimal pressure according
to Figure 2.

wall (W) is illustrated in Figure 5. The model assumes uniform tem-

peratures for the papaya (T p )' air in the box (TA), dew~point in the
box (T DP A), box wall (TB)' container air (Tc), container dew-point
(TDP,C) and container wal~ (TW). Other symbols used are for papaya
respiratory heat output (QG), convective heat transfer from the pa-
.
paya to the box air (Qc ,PA). convective heat transfer from the box
.
air to the box wall (Qc ,AB), convective heat transfer from the box
.
wall to the container air (Qc , BC), radiation from the papaya to the
box wall (QR PB)' radiation from the box wall to the container wall
.
(QR , BW)' evaporative heat flux (Qv , PA) from the papaya to the box air,
and evaporative heat flux (QV AC) from the box air to the container
air. All convective couplings are presumed to be natural convection.
408 S. P. BURG AND R. KOSSON

1.2

Z 1.0

°
w
"" 0.6
w
>
~
corn
C02
-0-
°2
..... 0 .•
-.-
~ apples -6- -&-

w carnalions -0- -e-

"" mushrooms

100 200 300

PRESSURE (mm Hg)

Fig. 4 - Relation between respiration rate and ambient pressure.

CONTAINER
AIR (C)
BOX (B)

BOX AIR
(A)

CONTAINER
WALL
(w)

Fig. 5 - Schematic model for heat transfer

HYPOBARIC CONDITIONS 409

The radiative coupling from the box to the all is set o for
interior boxes.

The corresponding nodal model, shown in Figure 6, involves the

determination of 4 nodal temperatures (Tp, TA' TDP A, TB), given a
specified respiratory heat sour ce applied to node P, and three fixed
boundary nodal temperatures (TC, TDP C,TW)' There are seven tempera-
~ure de~endent heat transfer c~upling.-- three convective (QC,PA,
QC ,AB , Qc., BC), two radiative (QR , PB , QR , BW) and two evaporative
(QV,PA, QV,AC). Equations for each of the heat transfer couplings
are presented in the discussion which follows.

CONVECTIVE COUPLINGS

Convection from the papaya to the air is given by:

(4)
with conductance
Kl = hpAA p
when h pA is the convective film coefficient and Ap is the surface
area of the papaya. Va lues for hpA are based on da ta correlations
for natural convection from short vertical plates to air (33) in
the form:
hL
-= (5)
K

where

Constants a, b depend on the value of X as shown below:

X a b
1-10 1.44 0.120
10-10 2 1. 37 0.141
lO L 10 3 1.20 '0.170
10 3-10 4 1.04 0.190
10 4-10 9 0.59 0.250
109_10 12 0.13 0.333

The bouyancy force for natural convection arises from the

changes in density, represented by the (p ß~T) product in the numer-
ator, and, for a perfect gas in the absence of mass transfer, ß =
l/T. With evaporation, there is an additional bouyancy term due to
the lower molecu1ar weight of water vapor compared with air. While
410 S. P. BURG AND R. KOSSON

~ CALCULATED NODES I 3 BOUNDARY NODES

Fig. 6. Where K1,K2,K3,K4,KS,R1 and R2 are

the rate constants for convection and
radiation respective1y, as defined in
Equations 4,17,18,21,23,2S,27

sma11 at atmospheric pressure, this can be significant at hypobaric

conditions where the water vapor represents a much 1arger fraction
of the container air.

The expression for ß is then:

1dp 11dM (6)
ß = -j) dT = T - M dT
where, for a mixture of air and water vapor:
P
M = Ma -(Ma-My) ~ (7)

This expression for ß can be written

1
ß = T (1 + al) (8)

where the bouyance term, a1' is given by

CpKge (dPV)(Tp - TDP,A) (9)
a1=(Ma - Mv) h dt Tp - TA

In this expression, the ratio (Tp-TDP A)/(Tp-TA) accounts for the

fact that evaporation depends on dew point differences, whi1e the ß
term is mu1tip1ied by the dry bulb temperature difference in the
expression for Grashof number. Kga is the effective mass transfer
coefficient, defined by the relatIon:
HYPOBARIC CONDITIONS 411

For papaya, like all other fruits, the skin resistance, r p '
and the natural convective boundary layer resistance act in series,
so that:
(11)

where Kg is the mass transfer coefficient for the convection boundary

layer. The mass transfer coefficient is related to the heat transfer
coefficient through the expression (28):
h PM
= ( _ ) ("'V
Cp 1J pD
) [ ( _ ) (--2)] .
°
67
(12)
Cpp R1JTP" a k 1J
where Pa' the mean air partial pressure in the film, is close to
the air partial pressure in the box. Dv ' the binary diffusion co-
efficient, is inverse with mixture total pressure and proportional
to the square root of temperature:
-D -PR{-f-~ (13)
Dv - vr P T
r

where the subscript R denotes the reference conditions for Dv :

Equation (12) can be rewritten in the form:

K hM
=~ [C(~) (PD
R vR) PfJO.67
Tr
(14)
g CpMP a K R T
from which it can be seen that Kg is roughly inverse with Pa'

Similarly the skin resistance, r p ' which is a function not only

of the geometry of the skin, but also of the resistance to diffusive
flow of vapor through the pores of the skin, can be expressed as:

G~=:::~ J
rp ,R
In
In
fP
[! =P~ :~
PJ (15)

where rp R is the skin resistance measured at a reference pressure

PR for particular vapor pressure values Pv,i and Pv,o inside and
outside the skin, respectively. Most skin resistance measurements
are made at 1 atmosphere. Using equation (15), skin resistance
va lues under hypobaric conditions are computed to be one or two
orders of magnitude lower.

Equations (9) - (15) permit evaluation of ß using equation (8).

Under hypobaric conditions, cases can arise in which the papaya is
being heated by the air (T p < TA) and at the same time losing heat by
DP
evaporation (T p > T A)' In such cases, the sensible and evapo-
rative bouyancy effects are in opposite directions, and it is
necessary to set:
ß =/ß/ (16)
to prevent computational difficulties in evaluating equation (5)
412 S. P. BURG AND R. KOSSON

Convection from the air in the box to the box is given by:

where ABX is the portion of the surface area of a box within the
stack which is exposed to a ventilating air passage (chimney), and
ABE is the additional box surface area for a box at the outer sur-
face of the stack exposed to the container walls. Note that portions
of the box surface which contact adjacent boxes in the stack are
considered adiabatic and are excluded from the heat transfer analysis.
The film coefficient hAB for the inner surfaces of the box is
simply taken equal to that of the external surface of the fruit, hpA,
in part because of the strong coupling between the two natural con-
vective flows, but also because the internal packing and wrapping
geometry is rather complicated for analysis.

Convection from external surfaces of the box to the container

air is given by:

(18)

where h BC is the natural convective film coefficient for exterior

surfaces of the box. Values of hBC are determined using the same
equations as for hpA' except that the characteristic length is taken
as the height of the stack and the bouyancy augmentation term is
given by:

a2=(M a -Mv ) Cp (dPv) (TDP,A-TDP,C)

(19)
R~rBOXh dt (TB - TC)
where r BOX represents the resistance to vapor flow of the box, ex-
pressed as a "skin" resistance.

EVAPORATIVE COUPLINGS

The heat transferred by evaporation from the papaya to the air

in the box is given by:
QV,PA=illvhfg=K4 (Tp-TDP,A) (20)

where the conductance:

dPv (21)
K4 = Ap Kge (dt ) h fg

and Kge is the same mass transfer coefficient defined in equation(ll).

The heat transferred by vapor flow through the box walls is

given by:

KS (TDP,A - TDP,C) (22)

HYPOBARIC CONDITIONS 413

where the conductance:

KS
A ~v
B RflTrBOX
(:i v ) (23)

In this case, AB is taken as the total box surface area (including

surfaces in contact with adjacent boxes). Box resistance, rBOX is
the same value as used in equation (19). Values of rBOX were
obtained directly from box tests under hypobaric conditions using
simultaneous dew point measurements inside and outside the box.
Because of the way the measurements were made, rBOX combines both
box wall resistance and the resistance of the convection films on
the box surfaces, resulting in the slightly simpler form for KS
compared with K4 .

RADIATIVE COUPLINGS
The heat transferred by radiation from the papaya to the box
wall is given by:
• 4 4
QR,PB = R,cr (Tp - TB) (24)

where cr is the Stefan-Boltzman constant (.1714 Btu/(ft 2-hr- OR4»

and the radiation coupling:

(2S)

FpB is the grey body shape factor for radiant exchange between
papaya and box. FpB is set = 0.1 to account for the restrictive
radiative path due to intervening layers of plastic and paper.

Radiant heat transfer from an exterior box surface to the

container wall is given by:
·44
QR,BW= R2 cr (TB - Tw) (26)

where
(27)
and FBW is set = 1

SKIN RESISTANCE MEASUREMENTS

Gas exchange in plants has been studied extensively in connec-

tion with transpiration (water vapor movement), respiration and
photosynthesis (C0 2 and 02 movement), and ethylene evolution. The
respective gases move through the plant product's skin in an air
phase of restricted area, the stomates and lenticles, and then
414 S. P. BURG AND R. KOSSON

through a boundary air layer so the behavior of one gas might be

inferred from that of others by comparing their diffusion coefficients
in air. Water vapor diffuses twice as readily as ethylene and 1.7
times more rapidly than C02 in air.

In leaves, the resistance of the boundary air layer to water

vapor (ra ,v) ranges from 0.4 to 3.5 sec- l cm depending upon air
velocity and the size and shape of the leaf (24,42,46); the minimal
stomatal resistance (r s v) is 0.2 to 17.8 sec- l cm at full opening
(24,42,46); and the cutlcular resistance to water (r c v) is 4 to
170 sec- l cm (24). During hypobaric storage the stomata must be
closed because the product has been harvested and is kept in the
dark. Under these conditions air velocity has little or no in-
fluence because r a v now constitutes a minor part of the total re-
sistence (rv ), whi~h depends mainly on r c ,v (42).

The cuticular resistance to C02 is much higher than to water

r c v (24), and that to ethylene must also be high since the over-
all resistance to ethylene in various types of leaves, coty1edons,
stems and buds under conditions where the stomata must be closed
is large, ranging from 6000 to 18,000 sec- l cm (1,2,5,9,15,18,26,
34,35,54). This is not due to a mesophyll resistance, as defined
in ref. 24 for that term is intended to account for the additional
resistance afforded by liquid water when gases penetrate through
the cellular fluid prior to being metabolized. The ethylene va1ues
do not include this step in permeation since they are based on the
rate of ethylene loss from the leaf surface and the ethylene grad-
ient between the intercellular air spaces and atmosphere. Ethylene
and C02 data clearly are not useful for estimating r c ,v in 1eaves,
so only va1ues for water vapor have been used in the heat transfer
computations.

The surface to mass ratio of leafy tissue is so large that even

the highest reported value of r c v (170 sec- l cm) constitutes an
insignificant constraint on water evaporation when accdunt is taken
of the influence of a low pressure on water mass movement. However,
the surface to mass ratio of fruits is several orders of magnitude
lower, and skin resistance might in this case significantly influence
evaporative cooling and the temperature gradients which arise. Only
a few measurements of overall resistance,rv,have been made with
fruits, but there are many measurements of the resistance to ethy-
1ene and C02 (3,4,6,7,12,27,29,30,32,39,41,49,52). Can these be
used to compute r v ?

The skin resistance of apples to water vapor varies from 116

to 398 sec- l cm depending upon variety and RH (16,25), and that of
oranges trom 56 to 77 sec- l cm (36). These values are far 10wer
than resistances to ethylene and C02, e.g. about 8000 sec- l cm for
apples (4,6,7,9) and 2000 sec- l cm in oranges (3,7). Two dozen
fruits tested all have resistances to ethylene in the range 1600 to
HYPOBARIC CONDITIONS 415

50,000 sec l - cm and in ten cases where the resistances to CO Z and

ethylene have been compared, they are nearly identical.

Why is the resistance to water vapor far lower than that to

other gases in fruits? The resistance of the isolated apple peel
towater vapor (Z5) and to COZ and ethylene (7) is equal to and
accounts for the entire resistance of the intact apple to these
vapors. Isolated pulp slices have very little resistance to COZ
(7), and when the peel is removed from the fruit the normal CO Z
and ethylene gradients in the pulp are dissipated (7,49). Therefore,
an explanation must be sought in the properties of the peel.

Ethylene and COZ pass through the apple peel in an air phase
of limited area. This is proven by the fact that the rate of pass-
age of these gases out of an apple is inversely proportional to
the atmospheric pressure, and also dependent upon the density of
the constituents of the ambient atmosphere (7). If passäge were
limited by a liquid phase, the resistances to CO Z and ethylene
would be inversely proportional to their solubility in that phase;
e.g. much higher to ethylene if the phase were water, much higher
to CO Z if it were lipid, and in no event would the resistance be
similar to both gases, as is the case. Differential permeability
is manifest even when water vapor and the other gases are applied
at one side of an isolated apple peel and measured at the other
side, so it is independent of "pore length." The only plausible
explanation is that the lenticular and sublenticular chambers are
partially occluded with water. This would decrease the pore area
available for mass transfer of all gases except water vapor. Small
amounts of water occluding intercellular spaces markedly increase
the skin resistance to various gases (4,7,13,53); and when fruits
ripen the intercellular spaces progressively occlude with water
and the resistance to gas exchange increases (49). However, the
resistance to water vapor movement in bananas does not increase,
but rather the transpiration at all tirnes is proportional to the
respiratory heat generated, even when the internal Oz falls to
very low values, and the COZ accurnulates to high levels during
ripening (45).

In summary, the 1arge amount of data available on the skin re-

sistance of various fruits to ethylene and COZ cannot be used to
compute a skin resistance to water vapor. For analytic computations
values ranging from 400 to 1Z00 sec- 1 cm at atmospheric pressure
have been used in the papaya computation.

HEAT TRANSFER AND WATER LOSS: ANALYTIC RESULTS

The nodal model described previously was run for papaya fruit
for conditions and properties as given be10w:
416 S. P. BURG AND R. KOSSON

Container conditions:

Pressure Pc = 20 nun HgA

Air Temperature TC 52°F
Dew Point Temperature TDP,C 46 - 52°F
Wall Temperature TW 46 - 52°F

Papaya Properties:

Respiration at 20 nun HgA,~p= 625 Btu/ton/day

Density p= 69.3 pcf 2 3
Surface to Volume Ratio, Ap/V p = 20.5 ft 1ft
Skin Resistance at atmospheric pressure, -1
r p = 400 - 1200 sec cm
Characteristic Length for natural convection,
x p = 0.5 ft

The papaya are assumed to be packed 10 lbs. per box. To de-

termine surface areas, the boxes are assumed to have internal dimen-
sions of 14" x 10" x 6", providing a total inside surface area of
2.94 ft 2 . With the chimney stack arrangement, 10 high, 6 wide,
and 32 long, there would be 1920 boxes, of which half would be in-
terior boxes, surrounded by other boxes or interior gaps. The others,
called exterior boxes, would see one or more walls.

The interior boxes would have the equivalent of 19" of side

length and a 2" x 10" strip on top and bottom surfaces (total area =
0.86 ft 2 ) exposed to the container air. The remaining surfaces
(2.08 ft 2) would be in contact with other boxes. The exterior boxes,
on average, have an additional 0.78 ft 2 of surface area facing a
wall.

The effective resistance to vapor transport of the box surfaces

was determined from cool down tests with simultaneous interna1 and
external dew point measurements. A value for rBOX = 10.9 sec-: cm
was determined at 20 nun HgA, equiva1ent to a value of 660 sec 1 cm
at 760 nun HgA.

Some representative results for an assumed skin resistance at

one atmosphere of 400 sec- 1 cm are shown in Table 3. The results
for 100% relative humidity (TDP,C = 52°F) show that the papaya
temperature is very close to air temperature, and the weight 10ss is
on1y 0.51 to 0.61% for exterior and interior boxes, respective1y,
in 3 weeks. The results for 86% relative humidity (TDP C= 48°F)
show a markedly higher weight loss, with the exterior box rising
to 4.0%, weIl above the va1ue of 1.2% calculated for the interior
box in 3 weeks.

The high weight loss at 86% relative humidity is due to the

10w resistance to vapor loss of both the skin and box under hypobaric
HYPOBARIC CONDITIONS 417

conditions (11 and 7 sec- l cm respectively). The papaya tends to

cool by evaporation to a temperature close to dew point temperature
and weIl below the container temperature. In this condition the
papaya receives heat by convection from the air in the box and by
radiation from the box itself. This heat, in addition to the
respiratory load, leads to a high evaporation rate. The exterior
box receives more heat from its surroundings than does the interior
box, leading to a higher weight loss.

Figure 7 (left and middle) shows the heat loss by mode for
interior and exterior boxes as a function of dew point temperature.
At 100% relative humidity (T DP C = 52°F), the papaya is marginally
warmer than its surroundings, with all three modes of heat transfer
in the same direction, and evaporation the dominant mode of heat
loss. At lower dew point temperatures, radiation and convection
provide heat gains, and evaporation increases substantially. The
results shown in figure 7 (middle) are for the wall temperature
equal to the air temperature of 52°F. The low relative humidity
in this case would be due to air being supplied to the container
in a relatively dry state. The results in figure 7 (middle) indicate
the importance of proper humidification to keep evaporation rates
low.

If saturated air was supplied to the container hotter than the

temperature of the walls, the container dew point would be kept very
close to wall temperature by condensation on the walls. This case is
illustrated in figure 7 (right). Exterior boxes, in this case, can
radiate some heat to the container walls and hence are colder than
interior boxes. The colder box provides less heat to the papaya,
reducing the evaporation heat loss. The evaporation rate for in-
terior boxes is shown in figure 7 (right) for comparison.

The effect of 3-fold increase in skin resistance is indicated

r
by the dashed curves in figur 7 (left and middle). The effect is
modest, because the 1200 sec- cm value at atmospheric pressure
drops to only 21 sec- l cm at 20 mm HgA. A somewhat stronger effect
should be exhibited at higher storage pressures.

While not directly shown, the papaya weight loss during storage
is directly proportional to the evaporative heat loss. Expressed
as a percentage, for a 21 day storage period, the weight loss is
simply the evaporative heat loss in Btu/hr multiplied by 4.73.

The weight loss problem with leafy products such as cut flowers
is even more severe than that with fruits because the surface/mass
ratio of the flowers is so much higher. To prevent excessive weight
loss it is essential that the flowers not be allowed to decrease in
temperature below that of the container air and wall. This can be
accomplished by adding additional resistances such as a plastic wrap
in series with the papaya skin and box resistances. The effect is
418 S. P. BURG AND R. KOSSON

Table 3. Analytic Results, Papaya at 20 mm HgA

TDP,C= 52°F TDP,C= 48°F

Computed Values Interior Exterior Interior Exterior
Box Box Box Box
Papaya Temp., T 52.13 52.10 48.31 48.99
p
Box Air Temp., TA 52.13 52.08 48.40 49.73
Box Temp., TB 52.12 52.03 48.71 51.08
Box Dew Point, TDP,A 52.03 52.02 48.06 48.16
21 Day Wgt. Loss, % 0.61 0.51 1.19 4.00

Container Air (TC) = 52°F (11.11 C); Walls (Tw) = 52°F (11.11 C);
Container Dew P01nt (T DP C) = 52°F (102% RH) or 48°F (8.89 C, 86%
RH); Papaya Skin Resistal\ce = 400 sec- cm @ 1 Atmosphere. 6.9
sec- 1 cm @ 20 mm HgA.

simi1ar to that shown in figure 7 (left and midd1e) for the two
va lues of skin resistance. By se1ecting an appropriate total re-
sistance of the box plus p1astic wraps it is a1ways possib1e to
prevent weight 10ss in excess of that required to dispel respiratory
heat without causing the product to overheat.

The respiratory process generates on1y about 9% of the water

needed to remove respiratory heat by evaporative cooling. The re-
mainder must be drawn from the cellular reserve, but there is a
limit to how much can be lost without adverse consequences. Weight
loss in excess of about 5% not on1y causes wilting, but also hor-
monal changes such as enhanced ethy1ene production and increased
ABA levels, with concomitant loss of storage and she1f-life. Due
to the marked reduction in heat generation under hypobaric condi-
tions, water loss need not limit storage life provided it does not
exceed that needed to dispel the product respiratory heat load.

If the only air movement in the container was the f10w induced
by the vacuum pump from the roof to f100r, the dew-point of the air
passing through a stack of papaya boxes wou1d progressively increase
in that direction. One might expect that the upper layers of the
stack would be cooler and experience noticeab1y more weight 10ss
than the lower layers. Observations made during a 2l-day papaya
test indiate that this does not occur. Since the net f10w must be
vertica11y downward, it wou1d appear that there is significant re-
circulation within the stack, with downward flow in the narrow gap
regions and near the box surfaces, and upward flow in the center of
the chimneys, due to the bouyancy effect.
HYPOBARIC CONDITIONS 419

0.4 1.6 OA

12
co:
....
u..
V>
z
«

t~Ol ,;:;i'/
V>
co: V>

: ~ 0.1
«

~ -;;;..----~
01"""....;;;;.-;;;;.-;;..-

• -0.1

"-0 2 08 -02

46 48 50 52 46 48 50 52 46 46

Figure 7. (LEFT) Papaya heat transfer by mode (Interior box).

10 1bs/box; rbox = 10.9 sec- 1 cm at 20 mm HgA; TC= TW=
52°F; 20 mm HgA pressure; rskin at 1 atmosphere = 400
sec- 1 cm ( - - ) or 1200 sec- 1 cm (-----).
(MIDDLE) Papaya heat transfer by mode (Exterior box).
Same conditions as in Fig. 7 (left).

(RIGHT) Papaya heat transfer by mode. 10 1bs/box; rBOX;

rBgX = 10.9 sec- 1 cm at 20 mm HgA; Twall = TDPC ; TC
52 F. Exterior box (---); Interior box (-----).
420 S. P. BURG AND R. KOSSON

COOLDOWN

The mechanism discussed for steady-state condition also apply

with minor reservations to the cooldown process. TheQretical1y,
during evaporative coo1ing, the load loses about 1% of its weight
per 5.6 '1 C decrease in temperature to remove sensible heat. During
a 6.7 '1 C cooldown at 15 mm Hg, mushrooms lost the expected 1.0 ~
1.2% of their weight in a container-load test.

In a cooldown of carnation f10wers from 15.6'1 to 1.7 '1 C with the

pressure regulator set to open at 10 mm HgA, product water f1ashed
at 12 mm HgA. During the next five hours the water vapor pressure
in the container (computed from dew-point measurements), total con~
tainer pressure, and vapor pressure of water at the product tempera-
ture all were equa1 and slow1y dec1ined as the product coo1ed.
During this period the cooldown rate was 1imited on1y by refrigera~
tion capacity. When the productls temperature decreased be10w
11.7 '1 C, all pressure reached 10 mm HgA and the vacuum breaker opened.
Thereafter, in the absence of product "boi1ing", the cooldown was
much slower, 1imited by the rate of heat transfer from the product
to the cold p1ate and air.

Vacuum coo1ing is used routine1y and successfu11y with many

products, but we do not recommend cooldowns in Dormavac which cause
the product's water to "flash" and "boi1.'" The duration of time
that a product withstands anaerobic conditions depends in part on
the temperature. Carnations are not damaged by 0.4 to 0.5% 02 at
atmospheric pressure during 6 to 9 weeks at O.6 '1 C, or 3 weeks at
7.2 to 10°C, but they are damaged in a matter of days above 10 '1 C
(20). Experimenta11y we have determined that under hypobaric condi-
tions carnations withstand 2 to 3 hours at their boi1ing point when
the temperature is 11.1 to 15.6°C, but in 5 hours they are damaged.
With fu11 loads it may take 10nger than 5 hours to bring the tempera-
ture below 11.1 °c. We have also observed damage with chrysanthemum
cuttings during hypobaric cooldown, but roses are more tolerant.
To avoid this problem either the product should be precooled, or
else it must be coo1ed in Dormavac at an initial set-pressure sub-
stantially higher than the vapor pressure of water at the initial
product temperature, fo110wed by a reduction in pressure after the
product has coo1ed. The 1atter procedure provides a re1ative1y
slow cooldown, however, and, in our experience, precoo1ing is c1early
preferab1e.

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HYPOBARIC CONDITIONS 421

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422 S. P. BURG AND R. KOSSON

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HYPOBARIC CONDITIONS 423

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424 S. P. BURG AND R. KOSSON

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MAINTAINING NUTRITIONAL AND PROCESSING QUALITY IN GRAIN CROPS

DU RING HANDLING, STORAGE, AND TRANSPORTATION

Philip C. Williarns

Canadian Grain Commission

Winnipeg, Canada

INTRODUCTION

Production of grain in excess quantities sufficient for export

and distribution to other areas where it is in demand for human
and animal food is only practiced in a few areas of the World at
present. North America, including Canada, Australia, and Argentina
account for over 95% of the World's grain exports. The development
of other areas with the potential of growing grain surpluses and
the distribution of this grain to food-poor regions of high popu-
lation density is essential to lasting World peace. Significant
steps are presently in progress to achieve this aim. The initiation
and continuity of the "Green Revolution" has been largely respon-
sible for drarnatic improvements in the grain production of coun-
tries such as Mexico and Turkey, and in the establishment of
International Centers for Agricultural Research, suchlas C.I.M.M.Y.T.,
I.C.R.I.S.A.T., I.R.R.I., I.I.T.A., and I.C.A.R.D.A., which are
aimed at the improvement of agricultural production in the regions
covered by their respective mandates. The successful completion
of such projects depends not only upon the production of continu-
ally increasing quantities of grains, pulses, root crops, and
other food raw materials, but also (a) on their proliferation at

lC.I.M.M.y.T. - Centro Internacionale por Majorifiacion de Mais

y Trigo.
I.C.R.I.S.A.T. - The International Centre for Research in the Semi-
arid Tropics.
I.R.R.I.- The International Rice Research Institute.
I.I.T.A.- The International Institute for Tropical Agriculture.
I.C.A.R.D.A. - The International Centre for Agricultural Research
in the Dry Areas.
425
426 P. C. WILLIAMS

quality levels suitable for processing and acceptability in socio-

economic patterns, (b) on the provision of incentives to growers
of the increased quantities of food by developing an efficient and
equitable system for marketing, distribution, and processing of
the food materials which is both economically and politically
acceptable, and (c) on the development and maintenance of practi-
cable methods for transportation of the food materials from their
source of origin to the points of distribution.

GRAIN TRANSPORTATION SYSTEMS

An efficient system for transportation and distribution must

consist of several components, which are summarized in Table 1.
This paper will limit its content to matters concerning the
quality and handling of grains and seeds of all types. The most
formidable task facing the overall improvement of agricultural
production and distribution of food materials unquestionably features
post-harvest technology, rather than the relatively more simple
aspect of growing two grains where one grew before. The biggest
reasons for this lie (a) with the complexity of the concept, out-
lined briefly in the first paragraph, and also (b) with the cost of
improving post-harvest technology. We are not asking for ploughs,
drills, tractors, combine harvesters, or even the extra staff to
operate them. These are relatively inexpensive. Instead post-
harvest technology calls for building grain silos of many different
sizes and degrees of complexity, also building roads and railroads,

Table 1. Components of a grain transportation system

1. Collection of grains from farms into consolidated

deposits.
2. Facilities for storage--short- and long-term.
3. Loading, unloading, and conveying systems.
4. Methods of packaging or bulk handling.
5. Roads, railways, and waterways.
6. Systems for grading the commodities into categories
of different visual quality and establishing equit-
able price scales.
7. Systems for servicing and maintaining equipment
and facilities for all aspects of the system.
8. Systems for recruiting and training personnel for
operation and administration.
9. Systems for education and extension of information
to farmers, grain merchants, and other personnel
involved wi~h the overall handling operation.
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 427

improving wharf facilities, and establishing marketing and pricing

systems and concepts which do not conflict with the individual view-
points and aspirations of the people called upon to formulate them.
In other words, post-harvest technology is expensive and complicated,
a very tough job. At present, losses of grain annually amount to
about 10-20% of World production. This proportion will certainly
increase if post-harvest technology does not keep pace with increased
production. In order to derive full benefits from the wealth of
expertise and equipment presently devoted to increased crop pro-
duction, post-harvest technology must improve at a much greater rate
than crop production itself.

GRAIN QUALITY

Quality in grains is a term which may be subdivided into at

least three categories; namely, nutritional quality, processing
quality, and marketing quality. These in turn are also divisible
into different aspects of each quality category, and the overall
picture is summarized in Table 2. These aspects of quality are
not listed in order of importance since all are of equal importance.
Nutritional quality includes actual eating quality and is affected
by chemical composition, texture, color, flavor, and general
appearance of the grain. It can also be seriously affected as a
result of infestation with fungi, insects, or rodents, which them-
selves may produce toxic alkaloids and other substances. Process-
ing quality means the usefulness of the grains for industrial or
domestic processing into other substances which may be the end
products themselves, such as edible oils, or intermediate products
such as flour, semolina, or malt. Finally, the marketing of grains
can probably be more seriously affected by storage and transporta-
tion than either nutritional or processing quality, since market-
ing involves vendor-customer relationships which may depend on
rather subjective evaluation of the potential quality of the
commodity before negotiations are completed.

Nutritional Quality

Changes in nutritional quality of a grain as a result of

storage and transportation are easier to define than those in
processing or marketing quality. The chemical composition of the
grain has usually been established before purchase or assignment
of the grain to a particular purpose, so after storage and delivery
and prior to use, the grain must be inspected to verify that it
meets the specifications defined for its particular end use. If
it does not meet the specifications, either it can be used only
by modifications of processing technique or diet formulation, or
it cannot be used for the purpose originally specified, and must
be assigned to other uses. The moisture content of grains is a
chemical constituent, and often changes during storage and trans~
428 P. C. WILLIAMS

Table 2. Aspects of grain quality

A. Nutritional B. Processing C. Marketing

l. Chemical l. Physical l. Appearance

composition condition
2. Flavor 2. Chemical 2. Physical
composition condition
3. Appearance 3. Physico- 3. Chemical
chemie al composition
properties
4. Toxicities 4. Inclusions 4. Infestation
(foreign
material)
5. Infestation 5. Infestation 5. Inclusions
(foreign
material)
6. Financial
considerations

portation. Changes in moisture proportionally introduce changes

in all other constituents so that changes may become necessary
in the formulation of diets. Also, the amounts of minor but
important constituents such as vitamins can change significantly
as a result of adverse conditions between harvest and utilization.
These changes also call for modifications in diet or feed formula-
tion. OVerall appearance and infestations can be readily detected,
and if deterioration has occurred, either in appearance or due to
infestation, further testing for possible toxic or otherwise harm-
ful contaminants must be carried out. Of recent years, consider-
able emphasis has been laid on the detection and determination of
aflatoxin and other mycotoxins. The flavor of a grain, or the
products derived therefrom can be affected by the development of
taints caused by packaging, mold development, bacterial develop-
ment, or by the application of protection chemicals such as bag
preservatives, pesticides, fungicides, and fumigants. Nutritional
quality is affected by insect infestation in two ways: first,
the repugnant effect the sight of crawling insects or larvae exerts
on the consumer and second, the actual composition of the grain
may be changed due, for example, to preferential consumption of
the germ areas of grains by many types of insects and by contamina-
tion by excreta, secretions, and insect parts. The accidental
incorporation of insects into foods during processing also affects
the nutritive value of the food, since insect parts are often
heavily chitinized, which makes them difficult to digest by mono-
gastrics.
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 429

Processing

Processing quality is affected to a marked degree by changes

induced during storage and transportation. Breakage and abrasion
of grains affect such procedures as tempering, which is a pre-
requisite of flour and semolina milling, and also forms sites for
preferential attack by fungi, bacteria, and insects. This last
aspect is of particular importance in the case of crops which are
harvested and stored at moisture contents in excess of 14-15%, since
molds can establish themselves very rapidly in such material. Ch~
ical composition can change in the same way as described above.
Changes in moisture content affect such procedures as tempering of
wheat prior to milling and steeping of barley prior to malting.
Milling of other cereals such as sorghums is also affected by the
moisture content of the grain. Heating of grains during storage
usually causes discoloration and usually affects the state of
pro teins and fats causing varying degrees of denaturation and
polymerization. This in turn affects the physicochemical behaviour
of the end products, and flours that have been subjected to these
conditions often will not perform satisfactorily in the baking of
breads or sweet goods. Foreign material such as straw, chaff, weed
seeds, sticks, stones, soil, and bits of metal affect the physical
makeup of end products, for example, by changing particle size
distribution and maybe also causing serious damage to equipment
such as mill rolls. Pieces of metal or stones mayaIso genera te
sparks during processing, which can cause fires or explosions in
dust-charged atmospheres. The removal of foreign material from
grains is inherent to most processing plants that handle grain as
a raw material. Some of the foreign materials, such as straw,
chaff, and weed seeds, can be recycled into other products, but
inorganic inclusions have to be disposed of as detritus, and their
presence is deleterious to processing quality. Finally, infesta-
tions by insects or rodents seriously impair processing quality
in most cases. Both spoil rather than consume the commodity, but
the processing of infested grain calls for removal of the causes
of infestation by such processes as fumigation and extra cleaning,
and the extra costs and losses involved represents a direct depre-
ciation in processing quality.

Marketing

Marketing of grains can probably be affected by storage and

transportation more seriously than either processing or nutritional
quality. Grains in many countries are priced according to their
physical condition in terms of size, soundness, and general appear-
ance. Grain size is not likely to be affected by either storage
or transportation, but both soundness (in terms of density) and
physical appearance can undergo drastic changes. Storage for even
short per iods under conditions of excessive moisture and tempera-
tures above lODe can result in heating and mold development.
430 P. C. WILLIAMS

Heating may cause disco10ration which may extend through the grain,
and und er extreme cases, the grain can ignite. The physica1 con-
dition of grains can deteriorate due to breakage, abrasion, and
the introduction of foreign material during both storage and trans-
portation. The introduction of 1% foreign material such as dust
or soi1 and stones means that the customer has purchased 1% of
rubbish for the same price as the grain, and furthermore, has to
pay more to remove the extra 1%. A1though 1% may not appear to be
a significant increase, 1% of a 10,000 ton shipment is 100 tons of
rubbish which has to be removed both from the grain and from the
c1eaning area. Some grain exporting countries impose upon them-
se1ves rigid specifications for foreign material; for examp1e, the
Government of Canada stipu1ates maximum total content of foreign
material of 0.75% inc1uding cerea1 grains other than wheat, in their
top mi11ing grade of hard red spring wheat. A concise and we11-
monitored system for separating different grains into grades based
on visua1 appearance and soundness ~s fundamental in marketing of
grains.

Moisture Content

Changes in moisture content affect the physica1 amount of

grain present in a parce1. Moisture may change by up to ±5% or
more during storage and transportation. Most grain marketing
agencies recognize this and negotiate compensatory c1auses based
on moisture tests at the time of purchase and de1ivery. The
deve10pment of taints and odors as a resu1t of mo1ds increases in
free fatty acids, and rancidities impair marketing qua1ity and
usua1y deve10p in storage rather than during transportation.
Infestation by insects or rodents both reduces the marketabie
quantities of grains and reduces the market value. Though fumiga-
tion may destroy all insects, 1arvae, and even eggs, it will not
disguise holes bored in grains by weevi1s and boring-type beatles.
Fina1ly, financial considerations can affect marketing quality
indirect1y. Inequitable pricing and generally inefficient systems
for buying from producers or merchants and distribution to customers
and processors can prolong the necessity for storage, and increase
the 1ikelihood of deterioration by any of the causal agencies to
be out1ined be10w. This is particularly true of grain marketing
in hot c1imates.

The remainder of this paper deals with the principa1 causes of

damage to grains during storage and transportation, and the steps
to be taken to e1iminate or minimize these factors. The agencies
are not discussed in order of importance since under individual
circumstances practica11y any of the agencies may become the most
important. Space and time limit the scope of the paper to make
no more than abrief 'introduction to the many aspects which have
to be considered in order to maintain quality. Several excel1ent
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 431

monographs exist, notably references 1-12, which between them list

a comprehensive reference library.

CAUSAL AGENCIES OF DAMAGE

Storage

Tab1e 3 summarizes the principa1 sources of damage to grains

during both storage and transportation. Bacteria1 infection is
generally not a serious hazard, and is not discussed further in
this paper. In a1phabetica1 order, the first factor to be discussed
is comp1acency and ignorance. Other than genuine charitab1e
organizations, most grain-handling invo1ves some sort of agreement
for payment in money or concessions, which becomes the foca1 point
of the transaction. The assumptions are usua11y made that grain
can and will be co11ected from p1ace A and de1ivered to p1ace B
in accordance with the terms of the agreement. Peop1e who make
the agreements are often remote from the grain itse1f, and the
possibility of damage or spoilage of the grain frequently falls
under the "it can't happen to us," or "it's going to
------
(country) and they'll never notice" category. The ignorance
component usua11y means simp1y a lack of know1edge either of the
hazards of grain storage, or of the state of either the grain or
the storage container prior to storage. A 1963 estimate put grain
storage los ses in the U.S. at between 13 and 23 million tons (13)
which occurred in spite of the wea1th of know1edge and sophisti-
cated equipment availab1e. Complacency also inc1udes the attitude
preva1ent among some organizations to increase productivity of

Table 3. Sources of damage to grain during storage

and transportation

Storage Transportation

Complacency and ignorance Complacency and ignorance

Containers Fungi
Conveying Handling (loading and
Dockage unloading)
Fungi Insects
Insects Moisture
Material itself Rodents
Moisture Temperature
Rodents Transport (rai1raods,
roads, ships)
432 P. C. WILLIAMS

grains without consideration of what will happen to the grain after

it has been grown. Actually, if all of the resources presently
being devoted to increased productivity in the next 5 years could
be directed instead to improvements in storage and damage prevention,
the increase in grain available for food would probably be far great-
er, and the on-going advantages of the improved conditions and tech-
nology would be beneficial to further researches on improved pro-
ductivity.

Containers include storage receptacles from sacks to bulk

storage bins. Bins, pits, and cribs can vary in size from a few
quintals at the farm level to hugh flat storage bins which are
really covered grain piles, the biggest of which accommodates up
to 100,000 tons. The shape, age, and the construction material
are the most important features of a container affecting the
possibility of damage to grain during storage. Covered pits and
mud walled stores are satisfactory for short-term storage,
especially where moisture levels are low. Several types of such
storage are described in (4). Bags made of hessian or other" materi-
al allow free passage of air to and from the contents and are highly
susceptible to attack by insects and rodents. On the other hand,
plastic bags restrict moisture-movements, and although they offer
more protection, they can also con~titute a hazard when temperatures
are liable to fluctuate, due to condensation inside the bags.
Insect eggs and larvae and mold spores persist well in the seams
and interstices of bags, and the bags themselves can constitute
sources of infestation. Grain bins made of wood are usually square
and by virtue of their construction possess a multitude of cracks,
crevices, and angles which are havens for insect eggs and larvae.
Furthermore, certain types of insects bore into the wooden fabric
itself and emerge fram the actual walls to infest new charges of
grain. Concrete bins are usually round, star-shaped, or hexagonal.
Star-shaped bins possess crevices which can harbor grain residues
constituting a source of infestation. Furthermore, concrete
itself possesses certain sorptive powers and chemical reactivity,
and unless coated with an impervious material such as paint, the
walls of concrete bins can interfere with normal fumigation
procedures. Nevertheless concrete bins, particularly the more
modern hexagonal type, are probably the safest form of long-term
storage for grain. Metal bins are usually round and again may be
very large. They carry few crevices and do not react significantly
with protective chemicals and are also a very safe method of
storage. Airtight storage has been in use for storage of high
moisture grain in temperate climates. Success depends on reduction
of oxygen and inhibition of respiration which restricts all infes-
tations. This type of storage has not been widely used in hot
climates to date. Neither concrete nor metal allows interchanges
between stored grain and the atmosphere, and moisture movement
due to temperature fluctuations, convection, and condensation can
result in extensive deterioration and even internal combustion of
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 433

grains. Due to the superior conductive properties of steel, this

is particularly true of metal bins.

Conveying of grain into and out of storage areas may be

achieved by hand, bucket elevators, augers, belts, box conveyors,
gravity, or pneumatics. Of these systems, hand carrying, bucket
elevators, and belts are least likely to cause physical damage to
the grain, since the grain itself is static when in transit. The
other four methods involve movement of the grain, and this can cause
abrasion and breakage of kerneis. There are about 30 million kerneis
in a ton of hard red spring wheat, so a conveyor, such as an auger
or box conveyor which moves grain at about 100 metres aminute, can
generate considerable kernel to kernel abrasion. Bucket conveyors
and augers possess several areas where grain residue may accumulate
and cause a source of infestation. Belts pneumatics, air slides
(gravity flow), and box conveyors are more or less self-clearning

Dockage is foreign material other than the grain itself. It

consists of grains of other cereals, weed seeds, bits of straw,
chaff, wood, and metal, stones and soil, string, and many other
minor items. Weed seeds and fibrous dockage increase the severity
of infestations by insects (14,15) and fungi. Fine particles of
dockage, such as grain dust, blocks the interstices between grains,
inhibits movement of air, and changes convection patterns. Fibrous
dockage such as chaff and straw are often contaminated with fungal
and bacterial spores. Stones and bits of metal increase the degree
of abrasion of grains during movement.

The fungi which affect grains can be subdivided into field

fungi and storage fungi. Field fungi attack crops in the field
before harvest and in the swathe or stook. They exist in spore
form on the grains either in the crease or in the interstices of
the outer layers of the seed coats, and cause discoloration of the
seed coat (and eventually of flours and other derived products),
affect germination capacity, and may produce mycotoxins which affect
the subsequent nutritive quality of the grain. Principal field
fungi include the general Alternaria, Fusarium, Cladosporium, and
Helminthosporium. Storage fungi include several species of
Aspergillus and Penicillium. Storage fungi vary widely in their
conditions for optimum growth, and Table 4 summarizes the lower
limit of grain moisture levels for growth for several species, to-
gether with principal damage caused. The data are taken from
reference 1

Insects

Insects have been estimated to cause annual losses in grain

of over half a billion dollars (16). Insect pests cause proportion-
434 P. C. WILLIAMS

Table 4. Principa1 species of storage fungi responsib1e for damage

to wheat

Minimum grain
moisture content
for funga1 Principa1 types
Genus and species deve10pment of damage
Aspergillus restrictus 13.5-14.5% "Mustiness," discolors
and kills germs
A. glaucus 14.0-14.5% As for A. restrictus.
Also creates favorable
conditions for other
species
A. candidus 15.0-15.5% As above, rapid action,
causes heating and
decay
A. ochraceus 15.0-15.5% Discolors & ki11s germs
A. f1avus 18.0-18.5% Discolors & ki11s germs,
causes heating
Penicillum sp. 16.5-19.0% Ki11s & discolors germs,
causes mustiness, may
cause some heating

a11y greater losses in tropica1 and subtropica1 c1imates. The most

prominent insect pests are tabulated in Tab1e 5. Of these, five
species develop inside the kerne1s. Weavi1s actua11y deposit
their eggs inside the grains; the grain borer and Angoumois moth
lay eggs on the surface of the grains and their larvae bore into
the kernels. The larvae of all these organisms feed on the endo-
sperm of the grains so grain which appears unharmed may, in fact,
be heavily infested with organisms hidden beneath the surface.
The beet1e species, such as the rusty and f1at grain beet1es, and
the red and confused flour beet1es deve10p outside the kerne1s
so infestations are more readily apparent. All insects contami-
nate grain with excretion, cast skins, and body parts. In addition,
they may produce odoriferous secretions which can taint the entire
parcel of grain. Most of the insect pests attack the germ prefer-
ential1y since this is both the softest part of the grain and con-
ta ins the richest concentrations of nutrients.

The material itself; i.e., the type of grain, affects the

type and extent of damage which can occur to it. The size and
shape of grains affects the area of interstitial space between the
grains. There are about 30 million grains in 1 ton of wheat, but
over 120 million in a ton of mustard seed, with consequent increase
in surface area. This affects the movement of air and moisture in
the grain masse In general, softer grains such as soft wheat and
corn, sorghums, and the softer 1ines of chickpeas and lentils are
more readi1y attacked by insects. Oi1seeds such as flax, rapeseed,
MAINTAINING NUTRITIONALAND PROCESSING QUALITY 435

and sunflowers are rather less suseeptible to inseet damage. Sinee

these erops are hydrophobie, due to their high oil eontent, their
moisture eontent at time of storage ean be very important sinee
moisture is lost rapidly and ean eause serious aeeumulation of
moisture at different areas of bins due to eonveetion and eonden-
sation. As a result, heating ean develop quite rapidly in bins
eontaining oilseeds, partieularly if the doekage eontent is high.

Moisture

Moisture is probably the most important single faetor affeeting

damage of all types to grain during storage. Grains eonsist of
stareh and protein, with smaller proportions of oil and eellulose-
type substanees. Stareh and pro tein ean absorb, respeetively, 75%
and 200% of their own weight of water, and the pentosan/eellulose-
type materials whieh make up mueh of the external layers of the
grain ean absorb relatively even greater quantities. Consequently,
the seeds of most eereals and pulses are fairly hygroseopie. Oil-
seeds are generally hydrophobie and do not absorb water as readily.
The interehange of water between grain and the atmosphere is zero
when the partial pressures of the water in the grain and the air
immediately surrounding it are equal. This state of equilibrium is
not statie and will depend on temperature ehanges within the grain
mass, relative moisture eontents of different areas of grain within
the mass, movements of the grain, and seeondary influenees set up
by infestations with inseets or fungi. Generally, the equilibrium

Table 5. Prineipal inseet pests of stored grain

Speeies Common name

Aearus siro (L) Grain mite
Crypolestes ferrugineus (Stephens) Rusty grain beetle
C. pusillus (SehBnherr) Flat grain beetle
Oryzaephilus surinamensis (L) Saw-toothed grain
beetle
Plodia interpunetella (HUbner) Indian meal moth
Rhizopertha dominiea (F) Lesser grain borer
Sitopnilus granarius (L) Granary weevil
S. oryzae (L) Riee weevil
S. zeamais (Motsehulsky) Maize weevil
Sitotroga eerealella (Olivier) Angoumois moth
Tenebroides mauritanieus (L) Cadelle
Tribolium eastaneum (Herbst) Red flour beetle
T eonfusum (Duval) Confused flour beetle
Trogodema granarium (Everts) Khapra beetle
436 P. C. WILLIAMS

within a large bulk storage bin will change fairly extensively dur-
ing the initial period of storage, then will settle down and und er-
go little further change provided no such infestations develop.

The matter of whether the environment within a storage area

is safe for storage or likely to lead to heating, mold developments,
and spoilage depends mainly on the initial moisture content of the
grain at the time of storage, together with the relative humidity
of the atmosphere within the storage area (the environmental rel-
ative humidity or ERH), with a rather lesser but still important
input from the temperature of both the grain and the storage area.
Due to their individual composition and structures, different
grains have different critical moisture levels above which pro-
longed storage is unsafe. Prolonged storage in this paper means
storage for more than 2 weeks, but und er conditions of high
moisture content and high temperature, the period after which
spoilage can occur may be only a few hours. The "safe" moisture
levels are generally recognized to be the moisture levels of the
grains which are in equilibrium with an ERH of 70% for temperate
climates and 60% for tropical climates. Table 6 gives the approxi-
mate levels of moisture at which several commodities are in
equilibrium with ERH of 60 and 70% at 20° and 30°C. Two features
are apparent: first, the significant difference between oilseeds,
and cereals and pulses; second, the relatively small influence of
temperature. These figures have been derived as a result of pains-
taking determinations of absorption isotherms for the various
cereals. Practical use is made of these during grain inspections
in North America for establishing legal levels of moisture above
which grain cannot be accepted for storage unless it is predried,
the expense of which has to be borne by the producer or the vendor
of the grain.

Temperature

Temperature affects the movement of air in grain stored in

bins and other storage areas. Water tends to move from warmer"
areas to cooler areas of the grain mass. The movement of air
affects the movement of water and eventually the accumulation of
areas of higher moisture in the grain mass. The temperature of a
grain mass is affected by the ambient temperature of the storage
area at the time of storage, by the temperature of the grain
itself, by the size of the storage container, and by the material
of which it is made. Sheet metal, for example, conducts heat
weIl, so that the effects of wide fluctuations in air temperature
outside the storage container are likely to be transmitted into
the grain inside, in contact with the walls, with consequent
secondary effects on the movement of air currents and water witnin
the grain mass. Concrete, mud, and wood are less effective con-
ductors. Grain itself. partly due to the air-filled interstices
within the grain mass, is an effective insulator and is not a good
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 437

Table 6. "Safe" moisture levels for storage of grains

at 25°C

70% ERH 60% ERH

Grain 20°C 30°C 20°C 30°C

Wheat l4.5 a 13.5 13.0 12.5

Barley 15.0 14.5 14.0 13.5
Oats 14.5 14.0 13.0 12.5
Corn(maize) 14.5 13.5 13.0 12.5
Sorghum 14.5 13.5 13.0 12.5
Millet 16.5 16.0 15.5 15.0
Riee 13.5 13.0 12.5 12.0
Soybean 12.0 11.5 10.0 9.0
Beans 15.5 15.0 14.5 14.0
Cowpeas 15.5 15.0 14.5 14.0
Flax 9.0 8.5 8.0 7.5
Sunf10wer 8.0 7.5 7.0 6.5
Mustard 8.5 8.0 7.5 7.0

aTo nearest 0.5% moisture

eonduetor of heat. As a resu1t, grain entering a storage bin

immediately after harvest at a temperature of possibly 30° or 40°C
will likely retain its heat for a eonsiderable per iod after entering
storage. Due to differential temperatures near the top and sides
of grain in storage, whether in bins or piles, moisture movements
by eonveetion tend to result in inereased moisture eontents in
these areas. When eonditions reaeh a eritieal stage, molds develop.
This may oeeur at other areas randomly distributed within the grain
mass, and the inereased temperature eaused by respiration and other
metabolie aetivities of the deve10ping fungi lead to the development
of "hot spots" in the grain. Hot spots, or loealized areas of
higher temperature within the grain mass, may develop in two ways.
The first oeeurs at relative1y low moisture levels, for examp1e
12-14%, and is usually eaused by inseet metabolism. Temperatures
of about 40°C may be reaehed. This is sometimes referred to as
"dry grain" heating. At higher relative humidity levels, fungal
infestations deve1op. These produee higher temperatures of up to
and over 60°C, and the eondition is known as "damp grain" heating.
In assoeiation with this, and due to the favorable eonditions of
temperature and moisture, germination of grain may take plaee, and
the inereased metabo1ism of the grain may eause even greater heating.

Rodents

Rodents are the final eausa1 faetor of damage to grain in

438 P. C. WILLIAMS

storage and transportation. Rats and mice of the genera Rattus and
Mus are the rodents which most frequently infest grains. Damage
is caused by direct consumption of grain; by fouling the grain
with faeces, urine, hairs, and dead bodies; by transmission of
diseases in body excreta; and by destruction of containers and bags.
Indirect damage may be brought about by fires caused by rats gnawing
through electrical cables. Losses in individual stores can amount
to 70% of the total grain, due to direct spoilage and contamination.
Mouse faeces are particularly difficult to remove from grain because
they are approximately the same size as grains of wheat, rye, and
barley. In 1953, it was estimated that rats alone caused an annual
loss of approximately 1 billion dollars in terms of consumption and
spoilage of grain and other foods and damage to property. The most
predominant rodent pests are the Norwegian rat, !. norvegicus; the
black rat,..!: rattus rattus; the grey-bellied rat, !. rattus
alexandricus; the Polynesian rat, R. exulans; and the house mouse,
Mus musculus. All of these rodents are capable of climbing and can
easily enter storage sheds, flat storage bins, and warehouses, and
infestation of terminal elevators is possible through basements. The
interstices of galleries, elevator boots, and other areas can house
colonies of rodents, and contamination of grain moving along the
belts or conveyors can become continuous.

Transportation

Turning to transportation, damage to grains ar~s~ng as a result

of transportation follows basically the same pattern as for storage.
Of the agencies associated with storage, most of them are equally
applicable to transportation conditions. This applied to complacencj~
fungi, insects, moisture, rodents, and temperature. Conveying i n i
the transportation of grains usually involves loading and unloading,~
of ships, trains, and trucks, and lateral movement of grains from'"
collection points into and out of elevators. Physical damage can
occur to the grains at any stage of conveying. Also, sources of
infestation can be introduced. Ships and railcars can serve as
sources of infestation due to small amounts of residual grain in
holds and railcars. Also, certain types of insect larvae bore into
the woodwork of wooden railcars. On long trips or when cars are at
rest in sidings awaiting unload, the larvae can infest the new grain
in the car.

PREVENTION OF DAMAGE

Storage of grain can be short-term (up to 2-4 weeks) or long-

term (up to several years). In general, long-term storage is of
more concern in temperate climates and in countries producing large
surplus es of grain for export. In developing countries, storage
periods are generally shorter, parce1s of grain are smaller, and
rather more use is made of pit- and mud-wa11ed silos and bags for
MAI NT AINING NUTRITIONAL AND PROCESSING QUALITY 439

storage and transportation. In many cases, grain marketing in

developing countries is carried out on a large scale but with small
scale facilities--horse and oxen-drawn carts and smaller trucks--
with bag storage as an interim between producer and consumer or
processor. Longer term storage occurs in private homes and village
communes where grains may be stored up to 1 year between crops.
Another reason for fairly long-term storage in developing countries
concerns those countries which are obliged to import significant
quantities of grain for subsequent distribution within the country.
Damage to grains over short-term storage is usually limited to phys-
ical damage, or rapid fungal degeneration of the material due to
excessive moisture content. Insect generations usually take several
weeks to develop and are sensitive to physical activity and changes
in environment. As a result, movement of the grain out of temporary
storage may decrease damage due to reduction of the insect population
at the larval and pupal stage. Temporary storage facilities if al-
ready infested or contaminated may cause new infestation to any grain
passing through the facilities. The most effective measures for
prevention of damage to grains are listed in Table 7.

Aeration

Aeration is an effective method of reduction both of moisture

content and temperature. It is particularly applicable to sma11er
storage faci1ities and finds its forte in the"open corn-crib type
of storage. Turning of grain in bins is practicedin farm storage
and in elevator bins. In some areas, grain elevators have bui1t-in
aeration faci1ities. Grain stored in piles packs itse1f fair1y
c1ose1y and movement of air inside the pile is restricted. Turning
such piles can effective1y reduce moisture content by 1-4%, depend-
ing upon the initial moisture level and may reduce insect and rodent
infestation.

Design of storage facilities can assist in prevention of

damage. Avoidance of sharp ang1es and unnecessary recesses and
crevices, choice of bui1ding materials, use of conveying equipment
such as pneumatics, which causes 1ess damage to grain and provides
1ess scope for infestation all he1ps in prevention of damage. This
extends from the 1argest, most sophisticated storage faci1ities
down to the farm storage bin, grain pit, or bagged grain warehouse,
where we11-p1anned stacking is important. New designs of rai1cars
of the hopper-type have reduced time for loading and un10ading and
made the hoppers se1f-c1eaning, so that the risk of infestation
is minimized. The use of the tanker-type ship for moving grain
introduced the concept of many sma11 ho1ds (tanks) which does not
serious1y impair 10ading and un10ading and reduces infestation risks
since one or two sma11 infested tanks are easier to res tore than a
hold with a capacity of 2,000 tons. The extra work invo1ved in
c1ean-up and fumigation of the many sma11er tanks is offset by the
increased effectiveness and f1exibi1ity.
440 P. C. WILLIAMS

Table 7. Measures for prevention of damage to grains

during storage and transportation

Measure Area of Application

Aeration Storage bins, short-term and long-

term
Moisture control Storage bins and containers
Temperature con- Storage bins and warehouses
trol
Fumigation Storage bins, warehouses, ships'
holds, railcars, trucks, sacks
Fungicides Storage bins, warehouses
Insecticides Storage bins, warehouses, ships'
holds, railcars, trucks, sacks
Design Storage areas
Hygiene All areas
Legislation All areas

Fumigation

Fumigat10n and treatment with protective chemicals are widely

used and are effective methods for prevention of infestation.
Fumigants act in the gaseous or vapor states. They can be applied
by several methods, and storage areas such as collections of bins
can be fitted permanently with equipment for periodical fumigation
at the same time as aeration. Fumigants penetrate to all parts
of the storage area, including tiny crevices and ideally are
effective against all stages of insect life including eggs, larvae,
pupae, and adults. The most widely used fumigants include methyl
bromide, which is effective against eggs, larvae, and adult insects
of many species (but rather less effective at the pupal stage).
Phosphine in the form of aluminum phosphide is another excellent
fumigant which can be applied in tab let form. Hydrogen cyanide
and dichlorvos, an organic phosphate, are two other commonly used
fumigants. Fumigants can be applied to piles of grain stored under
tarpaulins or in bags by covering the pile with a gasproof sheet
such as polyethylene or polyvinyl chloride. The fumigant is applied
under the sheet, and the ends sealed down with tubes of fabric filled
with sand or any suitable weight. Strict precautions must be ob-
served to protect workers from exposure to the fumigant. Gas masks
and other protective clothing must be worn by workers responsible
for removing tarpaulins, etc. after fumigation.

The efficiency of fumigation is affected by temperature,

moisture, the presence of dockage, the type of container, the
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 441

dosage rate, the types of insects, and the characteristics of the

grain itself. Temperature affects efficiency at both ends of the
scale. At high temperatur es , the fumigant tends to evaporate too
quickly without realizing maximum effect. At low temperatures,
permeation is affected and the dosage reaching the insects may
be sublethai. However, for practical purposes these drawbacks
are of relatively little moment, since most insects cannot survive
at temperatures much above 40°C, and at temperatures below 10°C
(50°F) grains do not require fumigation. High moisture reduces
the effectiveness of fumigants by increasing the sorptive capacity
of the grain. The presence of excessive dockage reduces aeration
space between kerneis and therefore the ease of permeability by
fumigants, and also absorbs significant amounts of the fumigant.
The efficiency of fumigants and the overall ease of sanitation
is facilitated by minimizing crevices and rough surfaces of
containers.

Fungicides are not generally recommended as being amoung the

most satisfactory methods for controlling fungal infestation of
stored grains, mainly due to residues toxic to animals, difficulty
of application and most of all, limited efficiency in combating
the fungi. Insecticides on the other hand are widely used to
control a variety of insects. The most effective of these are
malathion, pyrethrins, lindane (although use of this is decreasing
due to legislation against residues) and dichlorvos. Malathion
may be applied as sprays, aerosols, or dusts directly to the grain,
or during the filling of bins, etc. Dosages depend upon the amount
and type of grain, storage area, type of insect, and environmental
conditions. Pyrethrins are one of the oldest forms of insecticide
and have been in use for more than 100 years. Recent research has
increased their effectiveness by the use of synergists, such as
piperonyl butoxide, which significantly increases toxicity.
Pyrethrins are often applied as space treatment; i.e., to empty
bins, ships, or railcars in advance of the grain, as weil as direct-
ly to the grain itself. Dichlorvos is an organophosphate which has
great potential as a conventional insecticide, particularly for use
in areas where the insect populations are exhibiting resistance to
malathion. It is also effective when applied directly to bagged
grain. Resistance to malathion and some other pesticides is in-
creasing and necessitates continuous research into the development
of new insecticides. Legislation against infestations of all types
provides a negative incentive to grain-handling organizations and
companies to install, improve, and maintain protective measures.
Inspections, heavy fines, and condemnation of grains has the effect
of forcing intensified measures to combat infestation by insects
or fungi. Hygiene means simply keeping all areas and equipment
free of infested and otherwise contaiminated grain and other resi-
dues. This is achieved by sweeping, vacuum cleaning, aspiration,
and washing. Grain-handling areas are very dusty, and dust settles
over all equipment and floor, wall, and ceiling space providing
442 P. C. WILLIAMS

ideal locations for insect multiplication. Exhausting of dusts,

frequent sweeping of floors, etc. can go a long way to reduce the
incidence of infestation. Hygiene can also be interpreted to
include removal of dockage from grain, which is beneficial to the
grain itself by improvement of the value and purity of the com-
modity and by improvement in pest control. In addition, the clean-
ings (screenings) may be sold or processed as animal feed.

Temperature

Grain stored at low temperatures (up to 10°C) and low moisture

content (up to 12-13° for cereals and pulses and 9% for oilseeds)
will maintain its quality for very long periods. Hard red spring
wheat tested for baking quality after storage for up to 13 years
in Western Canadian wooden grain elevators showed no signs of
deterioration in quality, in terms of loaf volume or such parameters
as fat acidity (18). By far the most effective methods of prevention
of infestation or spoilage of grains involve the control of moisture
and temperature, and of these, moisture is the most important single
variable. Temperature control of large masses of grain is difficult
without excessive and expensive bin-turning (moving grain from
one bin to another) due to the insulating properties of grain. When
grain enters bulk storage, even on a relatively small scale, the
temperature on the inside of the bulk remains fairly stable, and
in the absence of infestations, appreciable changes in temperature
may take several months. If grain harvested at 40°C is immediately
placed in storage, the temperature of the bulk grain or bags will
remain close to 40°C for a considerable time. Similarly, if grain
enters storage, for example, from railcars or ships at low tempera-
tures, these temperatures will prevail with only very slow change.
For grain to be stored safely at elevated temperatures (30°C and
higher), the moisture content must be reduced to at least 1% below
the values given for the equilibrium moisture content of the grains
at given ERH. High ambient temperatures are advantageous to
moisture reduction, and under field conditions in warm climates,
moisture reduction to quite low levels can be attained simply by
allowing the grain to lie in the sun for a day or two before or
after harvest.

Moisture

Moisture control is overall the most effective method of pre-

vention of many types of damage and infestation to grain. High
moisture grain is more readily damaged physically during elevation
and conveying then lower moisture grain (provided that the moisture
content is not sufficiently low as to make the grain brittle).
Neither insects nor fungi can survive sufficiently well at low
moisture levels (below 12-13% for cereals and 9% for oilseeds) to
cause a serious infestation. Moisture content can be reduced by
direct exposure to radiant heat, such as the sun, by aeration, and
MAINTAINING NUTRITIONAL AND PROCESSING QUALITY 443

by forced draught hot air, as in grain driers. It can also be re-

duced to a certain extent by blending high moisture with low mois-
ture grain, provided that the equilibrium moisture content is likely
to be sufficiently low (4). Grain drying by means of various types
of crib or open storage container is weIl suited to areas where
sunshine and high temperatures are abundant, and to crops which are
harvested in cobs or in sheaves when the heads can be exposed to
the action of hot sun and wind. The drying of bulk or bagged grain
cannot be achieved in this manner. Bagged grain can be dried by
aeration, either naturally (e.g. by stacking in narrow rows in open-
sided covered sheds) or in batch driers. Batch hot-air driers are
efficient, but the bags must be moved periodically to avoid over-
heating and heat damage to bags that are closest to the heat source.
The most efficient method of drying bulk grain is the grain drier,
of which many versions exist (1,17). These are available for use
at farm or commune level, as weIl as for use in large grain ter-
minals, and all employ some form of hot-air forced-draught aeration.

It is important to obtain an accurate test result for the

moisture content of the grain before it is placed in storage and
during its period in storage. Themethods for testing for moisture
are legion, but most accurate, modern moisture testers utilize
some form of electrical conductivity, with capacitance measurement
foremost among the inexpensive instrumental methods. Sampling
for moisture testing is rather more important than the actual test
method employed, since the error of sampling large bulks of grain
is far greater than the error of the moisture test itself. Sampling
of large bins and grain piles can be carried out by vacuum probes,
which can withdraw sampies from any part of a bin or pile. It is
also important to record the actual points and depths from which
the sampies have been taken, in order to map the moisture/temperature
relationships of the grain mass as a whole. Grain which has been
dried should not be tested for moisture by electric meters too soon
after drying, since moisture may migrate outwards from the center
of the kernei, resulting in significant increases in moisture until
about 16 hours after drying (19). During the period in storage
for the greatest protection, grains should be tested periodically
for moisture to verify that significant buildup of moisture in the
upper levels of bins and piles is not taking place. Monitoring of
temperature throughout the grain bulk using remote sensing is
another effective preventive precaution, since the development of
"hot spots" can then be detected and the corrective precautions
taken.

In summary, this paper has presented some of the hazards in-

herent in storage and transportation of grain, the causal agencies,
and methods for remedy. To reach for the ultimate objectives of no
spoilage and losses is practically and economically unrealistic,
but the establishment of and strict adherence to sound preventive
maintenance and hygiene practices will achieve highly significant
444 P. C. WILLIAMS

annua1 savings in grain and food production and uti1ization.

REFERENCES

1. C. M. Christensen, ed., Storage of Cerea1 Grains and Their

Products, The American Assoc. of Cerea1 Chemists, St. Pau1,
Minn. (1974).
2. C. M. Christensen and H. H. Kauffman, Grain Storage, Univ. of
Minnesota Press, Minneapolis, Minn. (1969).
3. S. S. Easter, ed., Preservation of Grains in Storage, F.A.O.
Agricu1tura1 Series No. 2, Washington, D.C. (1948).
4. D. W. Hall, Handling and Storage of Food Grains in Tropica1
and Subtropica1 Areas, F.A.O. Agricu1tura1 Deve10pment
Paper No. 90, Rome, Ita1y (1970).
5. L. A. Trisviatsky, Storage of Grain, Nat. Lending Library for
Science and TechnQ10gy, Boston Spa, England (1969).
6. T. A. Ox1ey, The Scientific Princip1es of Grain Storage, The
Northern Pub1ishing Co. Ltd., Liverpoo1, England (1948).
7. A. Wex1er, ed., Humidity and Moisture Measurement and Contro1
in Science and lndustry, Vo1. 4, Van Nostrand, New York,
N.Y. (1965).
8. C. M. Christensen and H. H. Kauffman, Grain Storage - The Ro1e
of Fungi in Qua1ity Loss, Univ. of Minn. Press, Minneapo1is,
Minn. (1969).
9. G. Wogan, ed., Mycotoxins in Foodstuffs, Mass. lnst. of Techno1.
Press, Cambridge, Mass. (1965),
10. R. T. Colton, Pests of Stored Grain and Grain Products, Burgess
Pub. Co., Minneapo1is, Minn. (1963).
11. H. E. Hinton and A. S. Corbet, Common Insect Pests of Stored
Products - A guide to Their Identification, Brit. Museum
Economics Sero S., London, England (1955).
12. E. A. Parkin, Insects and Stored Food: Wor1d Losses and Contro1
Measures Surveyed, Food Manuf. 34:164 (1959).
13. w. H. Paw1ey, Possibi1ities of Increasing Wor1d Food Production,
F.A.O. Basic Study No. 10, Rome (1963).
14. R. T. Colton, H. H. Wa1kden, G. D. White, and D. A. Wi1bur,
Causes of Outbreasks of Stored Grain Insects, Kansas Agr.
Exp. Stn. BuH. No. 416 (1960).
15. H.E. MacGregor, Preference of Tribo1ium Castaneum for Wheat
Containing Various Percentages of Dockage, J. Econ. Entomo1.
57:
16. USDA ARS Handbook No. 291, Losses in Agricu1ture (1965).
17. A. P. Gerzhoi and V. F. Samochetor, Grain Drying and Grain
Driers, trans 1a ted by B. Shapiro, Office of Tech. Serv~es,
U.S. Dept. of Commerce, Washington, D.C. (1960).
18. K. H. Tipp1es, Offsite Storage Study, Grain Research Lab.
Report. unpub1ished (1972).
19. P. C. Wi11iams and J. T. Sigurdson, Grain-drying Study, Grain
Research Lab. Report, unpublished (1974).
NEW POST-HARVEST TREATMENTS OF HORTICULTURAL PRODUCE AND DEVELOP-
MENTS TO MAINTAIN QUALITY AND TO PREVENT DAMAGE IN WESTERN EUROPE
WITH SPECIAL REFERENCE TO THE NETHERLANDS

W. S. Duvekot

Sprenger Instituut
Wageningen, The Netherlands

INTRODUCTION

The purpose of this paper is to describe in short the present

situation of post-harvest techniques in Western Europe, as to stor-
age, pre-cooling, sorting, grading, packing and transport.

Competition on the European markets compels producers and

the whole trade to offer high quality produce, attractively and
safely packed and in such a stage of maturity, that the proper
taste and flavor will develop fully.

Existing practices are going to be extended and new ones are

introduced and/or developed. Restrietions made by public health
and envirenmental autherities force in seme cases to discontinue
established practices and to develop new ones in order to maintain
the required high quality of the produce.

STORAGE

In the field of storage of perishables attention is paid more

and more to a better control of the storage climate, so the de-
crease of quality can be reduced and produce of high quality, with
a reasonable shelf life can be offered to the consumer. Attention
has been and still is focused at relative humidity, temperature,
C.A.-conditions, and the rate of air-circulation with respect to
quality, waterloss and energy consumption.

Traditionally the range of relative humidities recommended for

fruit, vegetables and cut flowers is 90-95%. In the last seven

445
446 W.S.DUVEKOT
years however research workers and commercia1 experiences have
shown, that very high relative humidities of 98-100%, so near
saturation, may reduce weight 10ss and the rate of senescence of
some vegetab1es. Even in severa1 cases rot and decay were 1ess,
compared to produce that was stored under 10wer relative humidities.
For cut flowers it is found, that storage and transport at relative
humidities, near saturation, pro10ng the flowering per iod at the
vase and increase the ornamental value and pleasure. However, for
some varieties of apples relative humidities, near saturation seem
to stimulate the occurrence of core flush. Due to these resu1ts a
diversification in the installation of refrigeration plants can be
observed. For maintaining very high relative humidities, the so-
called wet-cooler systems, sometimes based on the ice-bank system,
are being introduced. Particularly for long-term storage of some
winter vegetables as cabbage, beets, carrots, turnips, celeriac
and chicory roots, wet-cooler systems are going to be installed
more and more. The same is true for storage of cut flowers, same
flower bulbs, and ornamental trees and shrubs. The wet-cooler
systems are preferred above the jacketed store, because of the
uniform climate which is produced and can be maintained.

For cut flowers the wet-cooler system also came into use as a
pre-cooling system. Practical experience has made clear however,
that in that case the flowers that have been pre-cooled previously,
should be kept in a separate cold room. The changes in temperature
occurring during loading qf the pre-cooler do harm the quality of
already cold flowers, mostly due to condensation.

C.A.-storage is still practically reserved for the long-term

storage of fruit, apples and pears. Commercia1ly vegetables are
nearly not stored under CA conditions. In the last years it has
become evident, that for certain varieties of apples the oxygen
concentration in the CA atmosphere could be decreased below 3% to
2% and even 1.5%. Commercial practice in the United Kingdom, the
USA, and in the Netherlands, has proved that the storage time of Cox's
Orange Pippin can be prolonged for several months in an atmosphere
of 2% oxygen and 0.1% carbon dioxide. Oxygen levels lower than
1.8% do effect taste and flavor in an unacceptable way and increase
the danger of the forming of a1cohol and low oxygen injury. The
variety Boskoop appears to keep under the same conditions also weIl.
The occurrence of core-flush is then retarded considerably.

In case this method is going to be introduced on a large

scale this will make high demands upon measuring instruments and
regulations of the atmosphere. In the United Kingdom a large
cold store with many CA rooms is the first enterprise in Europe
that has been equipped with computer registration and computer
regulation of temperature, carbon dioxide and oxygen. The who1e
complex is campletely automatically controlled by a computer. The
NEW POST-HARVEST TREATMENTS 447

very high investment necessary will be a handicap for a rapid growth

of this type of operation. A further impediment of introducing this
way of working will be the gas tightness of CA rooms and the capac-
ity of existing scrubbers. Though the gas tightness of CA rooms
has been improved considerably, it can be stated, that about 40% is
not operating as it should be.

The scrubbers of the activated carbon type, are mostly calcula-

ted for atmospheres containing 3 - 4% carbon dioxide, so they have
too small a capacity if a level of 0-1% carbon dioxide has to be
maintained. In commercial practice the insufficient capacity of the
activated carbon scrubbers is compensated by placing dry lime sacks
in the rooms. In some cases the scrubbing is done exclusively by
means of dry lime.

As to the construction of refrigerated- and CA rooms the

development goes into the direction of ready insulated panels cover-
ed with metal or plastic. In the case of CA rooms here the problem
of leaking seams exists, which demands utmost attention. In cases
where rural or city regulations do forbid the application of panels
and require brick buildings, gas tightness is performed by spraying
inner walls and ceilings with a special elastic p.v.c. compound,
resulting in a seamless cover, which lasts for years without
problems. As to the capacity of new cold- and CA rooms it must be
mentioned, that there is a tendency towards rooms of 80 to 150 tons,
the average being around 100 tons. Reasons for this practice are
that special pre-cooling rooms for fruit nearly do not exist.
Filling the rooms in 5 to 7 days, thus bringing in a warm lot every
day, has consequences for the already cold fruit and the same is
true for the unloading period, particularly after long-term storage.
For cooling down the produce as soon as possible, a rather high
refrigeration capacity is necessary, but when the fruit is at the
required storage temperature a small part of the compressor capacity
is needed. Therefore central cooling plants are installed with
capacity regulations instead of installations for each room separat-
ely. So during the storage per iod only one or two large compressors
are operating. This saves a considerable amount of energy. In the
last few years owners of cold stores have experimented with the
running time of fans in order to save on energy. Part of the fans
were stopped when no cold was asked for, so maintaining a slow air
circulation. According to the last results of some experiments
it must be possible to maintain an air circulation of 15 times/
hour the capacity of the room during the whole storage period.
This could result in a saving of energy of about 40%, and at the
same time give a decrease of weight loss because of slighter dif-
ference in temperature and relative humidity between the micro-
and macro climate. This practice of a decrease in air circulation
will undoubtedly be continued. A further point in maintaining a
uniform storage climate is that a development is going on towards
continuous operating installations, instead of the traditional
448 W.S.DUVEKOT
on-off installations. Though continuous operating refrigeration
plants are installed already on modern refrigerated containers for
trans-atlantic transport of flower bulbs, the same investments in
land plants are going very slowly. Research of the last year has
shown, that in fruit stores ethylene is produced at low temperatures,
even at lOC. In The Netherlands it has been measured, that in CA
stores filled with Golden Delicious apples and scrubbed with dry
lime and in ventilated CA stores the ethylene concentration was
between 1800 and 2700 ppm. In CA stores scrubbed with activated
carbon concentrations have been found varying from 350 to 500 ppm.
So from a view point of quality, it could be worthwhile to consider
scrubbing of ethylene in the future. In The Netherlands an experi-
mental scrubber, oxidizing ethylene by means of ozone has been
developed, but the apparatus has not been applied due to its very
high energy consumption. New storage techniques, such as the Low
Pressure Storage System and the French PRAC system (pre-cooling
under vacuum and CA storage) and scrubbing of ethylene are in Europe
still not applied commercially and it is not to be expected that the
introduction will proceed quickly. Thelow pressure storage system
requires high investments and as long as good results are obtained
with scrubbed CA storage and storage with a very high relative
humidity it is doubtful whether this system will come into existence
in Europe.

Regarding the introduction of the PRAC system it is not clear

whether the required working method gives a better result than
existing systems and whether it is to be realized commercially. The
produce has to be in a vacuum cooler at the latest six hours after
harvest. The produce has to be packed on pallets. The pallets are
enveloped with a large plastic bag which has an opening which after
vacuum cooling is closed with a special filter of silicone treated
polythene (according to the Marcellin patent). This filter, which
lets pass in a selective way oxygen and carbon dioxide has a surface
which has to be in proper relation to the product concerned (rapid
and slow respiration). It will keep the atmosphere in the bag at
a maximum of 5% carbon dioxide. The bag itself takes care of a very
high relative humidity inside it. As said the system works on the
condition that the produce is brought to a vacuum cooler maximum
six hours, but preferable two to four hours,after harvest. It
should be cooled down in the vacuum cooler to the desired storage-
or transport temperature. The feature of the system is that the
intercellular spaces are sucked empty and that after the vacuum is
abolished and air of normal composition has entered, very soon a
CA condition comes into being with a maximum of 5% carbon dioxide,
a low partial pressure of oxygen and a very high relative humidity.
The results according to French research are similar to those of
the Low Pressure system. Whether this PRAC system, which has been
applied up till now on a very small commercial scale will grow in
importance, will depend completely on the practical possibility
of getting the produce into the vacuum cooler in a very short time.
NEW POST -HARVEST TREATMENTS 449

PRE-COOLING

In Europe pre-eooling of hortieultural produee is applied to

a limited extent. For lettuee and other leafy vegetables vaeuum
eooling is applied, but mainly in exporting countries. Tomatoes,
eueumbers and other vegetables are pre-eooled by means of foreed
air eireulation and cut flowers to a very limited extent by means
of pressure eooling. This again is praetieed only in exporting
countries and often for produee that has to be transported over
long distanees only. Sinee, however, the eustomer is asking for
produee whieh is as fresh as possible, the interest in, pre-eooling
is growing rapidly and it may be expeeted, that pre-eooling in the
not too distant future will be an established practice for all pro-
duce that has to be transported over middle and long distances.
In some countries pre-cooling is also applied on a limited scale
for uncooked food, cut vegetables in consumer units, and for this
purpose too this activity will be growing. For the pre-cooling of
large hauls cooling by evaportation is now under experimentation.

SORTING AND GRADING

Sorting and grading on quality and size are progressively

demanding special attention. The trade and consumers do ask for
uniform produce of high quality, weIl graded and sized and without
bruises. In many cases the existing equipment does not meet these
needs, particularly not in small growers enterprises. An electronic
ball, measuring impacts in 3 dimensions has been of great help in
finding the hazardous spots on the machinery. For larger packing
houses great efforts have already been made in improving the accuracy
of sizing and avoiding of bruising. Protection against bruising has
been obtained by applying better cushioning materials and by replac-
ing mechanical dumpers and conveyor belts by water dumpers and water
gutters and by using weight sizers releasing the fruit in water.
The accuracy of the sizing and grading is improved tremendously by
combining electronic weight sorting and colour sorting. Fruit is
categorized both by its size and colour and the sorting can be pro-
grammed to release a given category of fruit at any cross conveyor
or gutter. Besides the programming feature facilitates the layout
and positioning of down-line packaging or bulk-filling machinery.
This type of equipment is also used for pre-grading prior to storage.
Despite these improvements the sizing is still not as satisfying as
it should be. At present sizing requirements are such that fruit
has to be measured by its largest diameter. The correlation between
weight and largest diameter is not so precise, that packing in trays
and cell pack can be done right away from the machine without partly
reslzlng. In this respect the development of an electronic shadow
sizer, measuring the largest diameter, the smallest diameter and the
height of a fruit may be mentioned as the possible proper solution.
Experiments with this electronic shadow sizer have given very hopeful
450 W.S.DUVEKOT
results. As to economics it has to be stated, that the large elec-
tronic, computer-steered graders and sizers require a high invest-
ment. Consequently these machines can be opera ted economically by
large central packing-houses only. For individual growers,this type
of equipment is as yet not within reach. In our opinion this does
not have to be a disadvantage. There is a tendency towards improve-
ment of quality of all horticultural produce. The consumer is de-
manding a constant high and reliable quality and uniform products as
to size and color and these demands require at its turn efficient
operating packing houses and an alert trade apparatus. In such a
concept, small enterprises of individual growers, with their own
interpretation of uniformity and quality, do not fit very weIl.
They will undoubtedly have to join cooperatives or private opera ted
large enterprises or find their own market, a market for lower quality
products.

TREATMENTS AFTER HARVEST

In using water dumpers and graders, it is very weIl possible

to add chemicals to the water to control rot and possible storage
diseases, as for instance scald on apples. This system is practiced
at several places in the world. In the European Economic Community
(Common Market) however there is a strong drive to exclude all
chemicals for treatment of fruits and vegetables after harvest. In
several European Countries application of chemicals after harvest
for controlling rot and storage diseases are forbidden for reasons
of public health or pollution of the environment. This may have
far reaching consequences for storage life, shelf-life and final
quality of produce. As an example it may be mentioned, that in the
Common Market the residue tolerance for the scald controlling chemi-
cal Ethoxyquin has been dropped per January 1, 1981 from 3 to 0 ppm.
Only when the manufacturers of these types of chemicals are able
to prove, that there is nothing to fear at all for human health nor
for pollution of the environment and that after the treatment the
remaining chemical can be collected safely from the water and des-
troyed, the authorities concerned may consider admission. Since
this type of research is very expensive and time consuming, and the
quantities of the chemicals concerned that can be sold relatively
small, most of the manufacturers have little or no interest to
undertake such research. A similar attitude of public health and
environmental authorities can be observed as to the application of
artificial waxes.

For cut flowers a post-harvest treatment with bactericides

and nutritional substances is going to be common practice. A
treatment against the injurious influence of ethylene with silver-
thiosulfate is up till now only applied for carnations.

Also here environmental authorities are ready to issue

NEW POST-HARVESTTREATMENTS 451

restrictions. It is necessary and advisable that research is

carried out to find substances that are friendly to the environ-
ment. Maybe growth regulators could provide an acceptable solution.

PACKING

The developments in the distribution channels, the wishes of

the consumers to be able to buy good looking, good tasting un-
damaged products, the demands of the produce as to packages and
the necessity to reach an optimal degree of loading of transport
means and warehouses, forces the packaging industry to appear with
new designs, better materials and better constructions. This is
particularly true for cardboard packages. The manufacturers of
paper and cardboard do have already reasonable wet-strength
material, but it needs improvement. The manufacturers have re-
search underway to increase the stacking strength of corrugated
cardboard by using thicker flutings, by applying different material
for the fluting, as semi-chemical and by laminated flutings. It
is not to be expected, that the height of the flutes, a very impor-
tant factor as to the strength, will deviate very much from the
current ones. The packaging industry has already developed better
liners, wet-strength liners, with resin impregnated ones, liners
coated with plastics and laminated liners. Experiments with poly-
thene laminated liners have shown, that a high stacking-strength can
be achieved. Combinations of different packing materials such as
cardboard, wood and plastic have been tried, but did not prove sat-
isfactory as a whole. It may be expected, that cardboard, solid and
corrugated will be used in the near future to a greater extent than
nowadays. Cardboard is preferred to wood and plastic by the majority
of the trade, especially the retail trade. Wooden and plastic pack-
ages provide at the points of sale problems as to storage and trans-
port of the empties. Cardboard can be pressed in bulk and recycled.
Many supermarkets and chain-store enterprises do have paper-presses
at their points of sale. The old-paper trade collects regularly,
sometimes daily the pressed paperbulk, so the retailer gets easily
rid of all packaging material. In several branches of industry the
manufacturers of cardboard packages and their clients have stimulated
the system-approach, by which cardboard and machines for setting up
the packages will be offered as adeal. The packages are delivered
flat and set up at the point of dispatch according to need. This
system saves on costs of transport, warehousing and administration.
In horticulture this system-approach is hardly known. It is applied
on a limited scale for packing of flower-bed plants, but it is
expected to gain much interest, specially when products as tomatoes
are going to be packed in cardboard packages. The majority of this
product still moves in the international trade in wooden trays.

In recent years a certain standardization of bot tom dimensions

of packages has been accomplished. The Economic Commission for
452 W. S. DUVEKOT

Europe of the United States at Geneva has, after long years of

discussions and commercial trials, recommended to standardize the
bottom dimensions of fruit and vegetable packages in the dimensions
60 x 40 cm, 50 x 30 cm and 40 x 30 cm. In many countries these
recommendations have been followed and the majority of the packages
moving in the international trade do have more or less the recommend-
ed bottom dimensions. These packages fit the international standard
of I.S.O. fixed pallet dimensions of 100 x 120 cm and 80 x 120 cm.
However problems arise with refrigerated loads over long distances.
Even when the loads are properly pre-cooled, there must be air
space available between the packages in order to remove the heat
genera ted by the produce. These air spaces cannot be created when
bottom dimensions of the packages, loaded on standard pallets, are
up to the recommended ones. Therefore in practice bottom dimensions
are chosen mostly close to the recommended dimensions, and weIl in
this respect, that they are slightly, one or two cm smaller. Only
wooden packages with extending corner posts do as a rule conform to
the recommendation, since the extended corner posts allow for suf-
ficient air movement over and through the produce.

TRANSPORT

The majority of fresh horticultural produce is transported by

road. At present only a minor part move in open or not completely,
closed vehicles. Depending on the distances that have to be covered
and the demand of the receivers, closed, insulated or refrigerated
vehicles are used. For the sake of efficiency the use of pallet
loads is increasing rapidly. To achieve a stable load, pallet loads
are usually shrink wrapped in large polythene bags wh ich are put
over the pallet load. This method has the disadvantage, that venti-
lation and air circulation in the shrunk bag are very much limited.
In many cases condensation will occur, which can harm the produce.
This is avoided by pinching several holes in the shrunk bag,
particularly in the top. Since recently a low-priced machine be-
came available, pallet loads are wrapped also in stretch nets, so
no problems of air circulation and condensation exist any more.
In general there is a tendency towards insulated and refrigerated
trucks and trailers, especially thin-walled ones, equipped with 7 cm
high floor racks, so sufficient air circulation under the load is
guaranteed. As to standardization of pallets it has to be mentioned,
that at present there is no unanimity as to which pallet dimensions
are to be preferred. For many reasons the 100 x 200 cm pallet, which
is used world wide, is to be preferred, compared to the 80 x 120 cm
pallet. On the long-run it is cheaper as to handling and energy
consumption. Loading or unloading a certain quantity of goods on
a 100 x 120 cm pallet demands less moving and handling of fork-lift
trucks than when the goods are stacked on 80 x 120 cm pallets.
Besides the 100 x 120 cm pallet has more possibilities for cross
stacking and consequently, that pallet loads will be more stable
NEW POST-HARVEST TREATMENTS 453

and safe. Though both pallet sizes are fixed by ISO standards the
European Union of Railway Companies has established a palletpool
with the 80 x 120 cm pallet. And following the railway policy for
not very clear reasons, the largest importing country on the con-
tinent has also chosen for the 80 by 120 cm.pallet. This decision
has given ground for heavy discussions between producers, trade
circles, receivers, transporters and even international organizations.
Undoubtedly these discussions will continue for years before a
reasonable, and for all parties concerned, acceptable solution will
be achieved. Due to increasing costs, the movement of piece-goods
is increasing in the roll on - roll off system by railroad. Whether
horticultural produce is going to be transported this way, will de-
pend on the development of costs of energy and the speed the service
and the guarantee the railway companies can give. In Europe an
international drive exists to come to standard procedures for trans-
port conditions. Proposals are worked out to come to an obligation
for registration of the transit temperature, which could be of
importance for the receiver in connection with further handling
and distribution. At present the use of generators on road vehicles
for maintaining certain conditions in the load is not a common
practice. They are used to a limited extent on containers for over-
seas transport. A rather new development is the application of
ethylene absorbing devices, like impregnated blankets and small
towers with potassium permanganate. Particularly for mixed loads
of vegetables, fruit and cut flowers purafill and exten-O-life
blankets have proved to have some favorable effects. According to
our latest research results they are most effective at about 15°C.

QUALITY AND CONSUMER

The applications of quality and grading regulations shall,

through a better and above all an uniform inspection in all
countries, guarantee that trade and consumer can buy a reliable
product. For this purpose a central training course is established
at Saragossa, Spain. The demand for a top quality product shall
induce the further development of test methods for the internal
quality. Disappointments with regard to internal quality are grow-
ing, particularly of products that have been stored too long or of
imported produce from many countries that have been harvested far
too early. The consumer is getting more and more quality conscious
and is also asking more information of the produce. As a result of
this the Common Market has made the decision that after 1982 all
foodstuffs sold in shops have to be provided with eight basic data,
namely: name, list of ingredients, net weight, names of manufacturer
or producer, packer or importer, origin and directions for use. For
a number of products exceptions are made as to the mentioning of
certain da ta on the label or the package. For fresh fruit and
vegetables it is not obliged to mention an outmost date of sale and
a list of ingredients. Consumer organizations however have announc-
454 W. S. DUVEKOT

ed already, that they have the strong wish to know at least in the
not too distant future what the most important ingredients are of
fresh fruit and vegetables with regard to the nutritional value.
Whether this will materialize in the future has to be waited for.

SUMMARY

-In the field of storage of perishables, attention is focused on

improvement of the climate in refrigerated stores and CA rooms.
Special attention is paid to the relative humidity and CA storage
under low partial pressures of oxygen und er absence of carbon
dioxide.
-It is not to be expected that new storage techniques as storage
under low pressure and the French PRAC system will be introduced
rapidlyon a large commercial scale.
-Pre-cooling will grow in importance, particular for middle- and
long-distance transport and for long-term storage of fruit.
-In sorting and grading plants the accuracy has been improved
by the introduction of computer controlled electronic machinery.
-It is rather likely that chemical treatments after harvest will
have to be replaced by different techniques, due to requirements
of public health and environmental authorities.
-Packing materials, in particular cardboard will be improved as
to quality and stacking strength.
-Standardization of packages and pallets still form a subject
of discussion as to dimensions.
-In transport the development go es towards closed, insulated and
refrigerated thin-walled vechicles.
-Labeling of packages with the aim to inform the customers will
put a heavier responsibility on producers and trade.
POSTHARVEST QUALITY MAINTENANCE OF FRUITS AND VEGETABLES IN

DEVELOPING COUNTRIES

Adel A. Kader

Department of Pomology
University of California
Davis, CA 95616

QUALITY COMPONENTS

Quality of fruits and vegetables is a combination of attributes

or properties that give them value in terms of human food. Compon-
ents of quality include appearance, texture, flavor, and nutritive
value (Table 1). Growers and shippers are concerned that their
commodities have good appearance and few visual defects. But to
them a useful cultivar of a given commodity must score high on yield,
disease resistance, ease of harvest, and shipping quality. Plant
breeders have given these characteristics higher priority over flavor
and nutritional quality. To receivers and market distributors,
quality of appearance is most important; they are also keenly inter-
ested in firmness and long storage life. Traditionally, postharvest
biology and technology research has concentrated on using appearance
and texture as parameters for quality evaluation (28). Consumers
see quality fruits as ones that look good, are firm, and offer good
flavor and nutritive value. Although they buy on the basis of
appearance and feel, their satisfaction is dependent upon good eating
quality.

Fresh fruits, nuts, and vegetables play a very significant role

in human nutrition, especially as sources of vitamins, minerals, and
dietary fiber (4, 12, 25, 30). Postharvest losses in vitamin
content, particularly vitamin C, can be substantial. These losses
are enhanced by extended storage, higher temperature, low relative
humidity, physical damage, and chilling injury (8). A large volume
of data on composition and compositional changes of fruits and
vegetables is available (14, 15, 29). But many gaps exist and
further research is needed. As more becomes known about human

455
456 A. A. KADER

Table 1. Quality Components of Fresh Fruits and Vegetables.

Main Factors Components

A. Appearance 1. Size: dimensions, weight, volume

(visual) 2 .. Shape & form: diameter/depth ratio,
smoothness, compactness
3. Color: uniformity, intensity
4. Gloss: wax
5. Defects: external, internal

a. Morphological
b. Physical & mechanical
c. Physiological
d. Pathological
e. Entomo1ogical

B. Texture 1. Firmness, hardness, softness

(feei) 2. Crispness
3. Succu1ence, juiciness
4. Mea1iness, grittiness
5. Toughness, fibrousness

C. Flavor 1. Sweetness
(taste and 2. Sourness (acidity)
smell) 3. Astringency
4. Bitterness
5. Aroma (vo1ati1e compounds)
6. Off-f1avors and off-odors

D. Nutritive 1. Carbohydrates (inc1uding dietary

value fiber)
2. Proteins
3. Lipids
4. Vitamins
5. Minerals

E. Safety 1. Natura11y-occurring toxicants

2. Contaminants (chemica1 residues,
heavy metals, etc.)
3. Mycotoxins
4. Microbia1 contamination
QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 457

nutrition, additional compositional data will be needed. For example,

it is not adequate to know how much total sugars are contained in a
certain fruit. Information about individual sugars is important from
the human nutrition as weIl as the sweetness standpoints.

Flavor is a complex sensation that involves perception of the

tastes and aromas of many compounds. It is difficult to deal
effectively with flavor in research programs because meaningful
widespread taste testing is virtually impossible. An effective
approach is to define the crucial components of flavor and then look
at how these are affected by genotypes, cultural practices, and post-
harvest handling procedures (18, 26). Objective analytical evaluation
of critical components coupled with subjective evaluations by a taste
panel can result in meaningful and useful information about flavor.

Numerous methods are available for evaluation of color, texture,

and other quality attributes (1, 3, 6, 9, 10, 11, 20). However, there
is a need for developing new objective and non-destructive methods
for quality evaluation. It is also important to better define the
interrelationships among various components of quality (appearance
including color, texture, flavor, nutritive value) in various fruits
and vegetables. In each case, an attempt should be made to correlate
subjective and objective methods of quality evaluation. Such infor-
mation is essential for selection of new cultivars by plant breeders,
choice of optimum production practices by production physiologists,
and redefinition of optimum postharvest handling procedures by post-
harvest biologists. This total effort will no doubt result in the
best quality fruits and vegetables possible for the consumers.

MATURITY AND QUALITY INDICES

Maturity indices are important for deciding when a given com-

modity should be harvested to provide some marketing flexibility
and to insure the attainment of acceptable eating quality to the
consumer. These two goals are not always compatible. The frequent
need for shipping fruits and vegetables long distances has neces-
sitated harvesting them at less than ideal maturity. This, in turn,
has resulted in less than optimum quality to the consumer. Indices
used for determining the legal maturity of fruits in most cases
coincide with their minimum palatability.

Maturity Indices Currently Used and Their Limitations

For decades substantial effort has been directed by horti-

culturists towards the evaluation of maturity indices. Extensive
data are available on morphological, physiological, and biochemical
changes in fruits and vegetables during development, maturation,
and ripening (14, 15). However, onlya small portion of these da ta
has been used in the establishment of maturity standards. In the
458 A.A.KADER

U.S. standards for grades, maturity is considered as one parameter

of quality for many fruits and vegetables. It is defined as "that
stage which will ensure proper completion of the ripening process."

Table 2 includes a listing of maturity indices used for

selected fruits and vegetables. It is necessary for some commodities
to define maturity indices for specific cultivars, production areas,
and seasons. Although numerous objective indices for maturity are
available, very few are actually used in practice because they are
in most cases destructive and difficult to do in the field or
orchard. Emphasis is on appearance factors, i.e., harvesting stage
is determined by experience and judged largely by the visual appear-
ance of the commodity.

Maturity vs. Quality

Most maturity indices are also factors of quality, but there

are many important quality indices which are not used in determining
optimum harvesting stage. The eating quality of fruits and veget-
ables cannot be accurately determined by appearance factors alone.

Timing of harvest (based on maturity indices) is complicated

by the great differences which occur in the rate of development
and maturation of individual plants, or organs on the same plant,
bush or tree. This variability in maturation and ripening is
especially important when once-over mechanical harvesting is used.
Variability is related to preharvest cultural practices and environ-
mental factors.

With a few exceptions (e.g., pears, avocados bananas), all

fruits reach peak eating quality when fully ripened on the plant.
Because of the constraints of the postharvest distribution system,
fully-ripe fruits cannot be successfully delivered to the consumer
except for roadside or pick-your-own type marketing situations.
So compromises between optimum maturity and optimum quality have
to be made.

For many vegetables, the optimum eating quality is reached

before full maturity, e.g., leafy vegetables, immature fruits
(cucumbers, sweet corn, green beans, peas, etc.). In this case,
the problem frequently is delayed harvest which results in lower
quality.

FACTORS INFLUENCING QUALITY AND ITS MAINTENANCE AFTER HARVEST

Many pre- and postharvest factors influence the composition

and quality of fresh fruits and vegetables; these are:

1. Genetic factors: selection of cultivars, rootstocks.

QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 459

Table 2. Maturity Indices for Selected Fruits and Vegetables.

Index Examples
Elapsed days from full bloom
to harvest Apples, pears

Mean heat units during fruit

development Peas

Development of abscission layer Cantaloupe

Surface morphology and structure Cuticle formation on grapes,

tomatoes
Netting of cantaloupes
Gloss of same fruits (develop-
ment of wax)

Size All fruits and many vegetables

Specific gravity Cherries, watermeions, potatoes

Shape Angularity of banana fingers

Full cheeks of mangoes
Compactness of broccoli and
cauliflower

Solidity Lettuce, cabbage, brussels sprouts

Textural properties
Firmness Apples, pears, stone fruits
Tenderness Peas
Toughness Asparagus

Color, external All fruits and most vegetables

Internal color and structure Formation of jelly-like material

in tomato fruits
Flesh color of some fruits

I
Compositional factors
Starch content Apples, pears
Sugar content Apples, pears, stone fruits,
Acid content grapes, pomegranates, citrus,
sugar/acid ratio papaya, melons
Juice content Citrus fruits
Oil content Avocados
Astringency (tannin content) Persimmons, dates

Internal ethylene concentration Apples

460 A.A.KADER
2. Preharvest environmental factors
a. Cltmatic: temperature, light, wind, rainfall,
pollutants, etc.
b. Cultural conditions: soil type, nutrient and water
supply, mulching, pruning, thinning, control of pests
and diseases, time and method of harvest, etc.

3. Harvesting stage: maturity, ripeness, physiological age.

4. Postharvest treatments: environmental factors, handling

methods, duration between harvesting and consumption, etc.

5. Interactions among various factors.

Both quantitative and qualitative losses take place in horti-

cultural crops between harvest and consumption. Dur aim is to
minimize these losses and to do so we must: 1) understand the
biological and environmental factors involved in deterioration,
and 2) use those postharvest technology procedures which will slow
down senescence and maintain the best possible quality (13, 24, 27).
Qualitative losses include loss in edibility, in nutritional quality,
in caloric value, and in consumer acceptability of the products.
Qualitative losses are much more difficult to assess than quantita-
tive losses, especially since standards for quality and consumers'
purehase power in developing countries are different than those in
developed countries. For example, elimination of defects for a
given commodity before marketing is much less rigorous in developing
than in developed countries. This, however, is not necessarily bad,
since appearance quality is somewhat over-emphasized in developed
countries. A fruit or vegetable which is misshaped or has same
blemishes may be as tasty and nutritious as one that is perfect in
appearance. Any produce that is not spoiled (rotten) or totally
unusable will have a market, if the price is right, in developing
countries (16). Miles (21) proposed that an investigation should
be conducted to determine the degree to which current trends in the
consumption of fresh fruits and vegetables in the U.S.A. are
indirectly the result of retailer demand for cosmetic qualities that
are unrelated to flavor and nutritional quality; the degree to which
there would be consumer acceptance of less blemish-free fruits and
vegetables; the effect that lowered quality standards would have on
the prices of fresh fruits and vegetables; and the effect that lower
prices would have on consumption.

Biological and Environmental Factors Involved in Deterioration

Fresh horticultural crops are diverse in morphological structure

(roots, stems, leaves, flowers, fruits, etc.), in composition, and
in general physiology. Thus, commodity requirements and recommend-
QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 461

ations for maximum postharvest life vary among various groups of

commodities. However, they are all subject to the following
biological (internal) causes of quality deterioration:

1. Metabolic changes associated with development, maturation,

and senescence (respiration, compositional changes);

2. Mechanical injuries (cuts, bruises, abrasions, etc.);

3. Transpiration or water loss (shrivelling, wilting,

desiccation);

4. Growth and development (sprouting and rooting in some

commodities, elongation, etc.);

5. Incidence of physiological disorders (sunburn, freezing

injury, chilling injury, etc.); and

6. Pathological breakdown (decay caused by bacteria and fungi).

The rate of biological deterioration depends on various environmental

(external) factors such as temperature, relative humidity, atmos-
pheric composition, pressure, etc. (7, 13, 24, 27). The relative
importance of the deterioration factors depends upon the commodity
(Table 3). Losses in quality and quantity in fresh fruits and
vegetables can occur throughout the harvesting and postharvest
handling systems and are cumulative (Table 4). Losses closer to
the consumer level are more costly because they include not only
direct losses of the commodity but also losses of energy and natural
resources used in postharvest handling (16).

There is a need for an accurate and specific identification of

the causes and extent of los ses in quantity and quality for each
commodity at each stage between harvest and consumption. Such
detailed information is essential to pinpoint problem areas in the
handling system and to set priorities for loss prevention efforts in
every developing country. The principal objectives of postharvest
technology are: 1) to maintain quality of the eommodity between
harvest and eonsumption, and 2) to reduee losses during harvesting,
preparation for market, transport, storage, and marketing operations.
In general, the level of teehnology eurrently used in postharvest
handling of fruits and vegetables in developing countries is not
adequate for realizing the above-stated objectives (2, 5, 7, 16, 22,
23). Adoption of new technological proeedures is badly needed pro-
vided they fit loeal eonditions. Transfer of "advaneed" technology
such as that used in developed countries without some adaptation and
modification to suit specifie loeal eonditions can be counter-
productive.
462 A.A.KADER

Table 3. Principal Causes of Postharvest Losses and Poor Quality

for Various Groups of Fruits and Vegetables.

Group Examples Principal Causes of Postharvest Losses

and Poor Quality
(in order of importance)

Root Carrots l-Mechanical injuries

vegetables Beets 2-Improper curing
Onions 3-Sprouting and rooting
Garlic 4-Water loss (shrivelling)
Potato 5-Decay
Sweet Potato 6-Chilling injury (subtropical and
tropical root crops)

Leafy Lettuce l-Water loss (wilting)

vegetables Chard 2-Loss of green color
Spinach 3-Mechanical injuries
Cabbage 4-Relatively high respiration rates
Green Onions 5-Decay

Flower Artichokes l-Mechanical injuries

vegetables Broccoli 2-Yellowing and other discolorations
Cauliflower 3-Abscission of florets
4-Decay

Immature- Cucumbers l-Overmaturity at harvest

fruit Squash 2-Water loss (shrivelling)
vegetables Eggplant 3-Bruising and other mechanical injuries
Peppers
Okra 4-Chilling injury
Snap beans 5-Decay

Mature- Tomato l-Bruising

fruit Melons 2-0ver-ripeness and excessive softening
vegetables Citrus at harvest
Bananas 3-Water loss
Mangoes 4-Chilling injury (chilling sensitive
Apples fruits)
Grapes 5-Compositional changes
Stone fruits 6-Decay
QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 463

Table 4. Most Common Causes of Postharvest Losses in Fresh Fruits

and Vegetables in Some Developing Countries.

Postharvest
Handling
Operation Causes of Losses

Harvesting I-Immaturity or overmaturity of the commodity

2-Inadequate field containers
3-Mechanical damage due to improper harvesting
methods
4-Failure to protect the commodity from the sun
5-Delays before delivery to packinghouse or
transporting to market

Preparation for I-Failure to sort-out produce with serious defects

market (in the and decay; inadequate cleaning
field or at the 2-Inappropriate packaging resulting in mechanical
packinghouse) damage, inadeuate ventilation and cooling, and
increased decay
3-Failure to remove field heat (lack of cooling
prior to shipment)
4-Lack of sanitation

Transport I-Rough handling causing increased mechanical

injuries
2-Lack of proper management of temperature,
relative humidity, and ventilation during
transit
3-Mixing of non-compatible commodities in the
transport vehicle (different types of con-
tainers which are not easily stackable
together, different temperature requirements,
ethylene-producing and non-producing com-
modities)
4-Delays during transport

Handling at I-Rough handling during loading and unloading

destination 2-Exposure to undesirable enviornmental con-
ditions
3-Delays in getting the commodity to the consumer
4-Improper ripening and storage practices
5-Lack of sanitation

Handling at I-Delays before consumption

horne 2-Improper storage (lack of horne refrigerators
or other means of storage)
464 A.A. KADER

Non-bio1ogica1 (Socio-economic) Factors Invo1ved in Deterioration

Severa1 indirect and non-bio1ogica1, but very important, factors

contribute to postharvest qualitative and quantitative losses of
fresh fruits and vegetab1es (16). These inc1ude:

1. Inadequate marketing systems. Growers can produce 1arge

quantities of good-qua1ity fruits and vegetab1es, but if they do
not have a dependab1e, fast, and equitab1e means of getting such
commodities to the consumer, losses will be extensive. This problem
exists in many locations within deve10ping countries. It is
accentuated by lack of communication between producers and receivers,
and lack of market information.

Marketing cooperatives shou1d be encouraged among producers of

major commodities in important production areas. Such organizations
are especia11y needed in deve10ping countries because of the
re1ative1y sma11 farm size. Advantages of marketing cooperatives
inc1ude: 1) providing centra1 accumu1ation points for the harvested
commodity, 2) purchasing harvesting and packing supp1ies and
materials in quantity, 3) providing faci1ities for proper prepar-
ation for market and storage when needed, 4) faci1itating trans-
portation to the markets, and 5) acting as a common se11ing unit
for the members, coordinating the marketing program and distri-
buting profits equitab1y.

Alternative distribution systems such as direct se11ing to the

consumer (roadside stands, produce markets in cities, loca1 farmers'
markets in the countryside, etc.) shou1d be encouraged. Production
shou1d be maintained as c10se to the major population centers as
possib1e to minimize transportation costs.

Who1esa1e markets in most of the deve10ping countries are in

desperate need of improvement in terms of faci1ities and sanitation.
These are overcrowded, unsanitary, and lack adequate faci1ities for
loading, un1oading, ripening, consumer packaging, and temporary
storage. In severa1 countries, there are plans to bui1d better
who1esa1e marketing faci1ities, but their imp1ementation has been
de1ayed more because of socia1 and po1itica1 than because of
financia1 considerations.

2. Inadequate transportation faci1ities. In most deve10ping

countries, roads are not adequate for proper transport of horti-
cu1tura1 crops. Also, transport vehic1es and other modes,
especia11y those suited for fruits and vegetab1es, are in short
supp1y. This is true whether for loca1 marketing or export to
other countries. The majority of producers have sma11 holdings
and cannot afford owning their own transport vehic1es. In a few
cases, marketing organizations and cooperatives have been ab1e to
QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 465

acquire transport vehicles, but they cannot do much about poor road
conditions.

3. Governmental regulations and legislations. The degree of

governmental controls especially on wholesale and retail prices of
fresh fruits and vegetables varies from one country to another. In
many cases, price controls are counter-productive. Although intended
for consumer protection, such regulations encourage fraud and provide
no incentive for producing high quality produce or for postharvest
quality maintenance. On the other hand, regulations covering proper
handling procedures and public health aspects during marketing are,
if enforced properly, very important to the consumer.

4. Unavailability of needed tools and equipment. Even if the

growers and handlers of fresh fruits and vegetables were convinced
of the merits of using some special tools and/or equipment in
harvesting and postharvest handling, they most likely will not be
able to find them on the domestic market. This is true of harvesting
aids, containers, equipment for cleaning. waxing and packing, and
cooling facilities. Most of these tools are neither manufactured
locally nor imported in sufficient quantity to meet demand. Various
governmental regulations in some countries do not permit direct
importation by producers of their needs. It is imperative that the
tools that will enable handlers to use recommended technology for a
given situation be available for them to use. In many cases such
tools can be manufactured locally at much lower cost than the
imported ones.

5. Lack of information. The human element in postharvest

handling of fruits and vegetables is extremely important. Most
handlers directly involved in harvesting, packaging, transporting,
and marketing in developing countries have limited or no appreciation
for the need for or how to maintain quality. An effective and far-
reaching educational (extension) program on these aspects is criti-
cally needed now and will continue to be essential in the future.

6. Poor maintenance. In many developing countries some good

facilities which were built a few years ago are currently "out-of-
order" or not functioning properly because of lack of maintenance
and unavailability of spare parts. This problem is especially true
of public sec tor facilities. Any new project should include in its
plan adequate funds for maintenance to ensure its success and
extended usefulness.

Kriesberg and Steele (19) stated that at each stage of the

marketing system in developing countries there are forces exogenous
to it which influence its development. Among political and economic
factors are public policies, the general stage of technology, and
466 A.A.KADER

income levels and their distribution. Among social and cultural

factors are urbanization, education and population growth and its
characteristics. Other factors working more directly on the ma~et­
ing systems include the kinds and quantities of commodities avail-
able for market, and consumer demand and commodity preferences.

All non-biological factors influencing quality deterioration

are much more difficult to change than the environmental factors
which control biological deterioration. However, to be successful,
any improvement program roust take all factors into consideration.

QUALITY STANDARDIZATION AND INSPECTION

Grade standards are developed to identify the degrees of quality

in the various commodities which aid in establishing their useability
and value. They are important tools in the marketing of fresh fruits
and vegetables because of the following factors: a) they provide a
common language for trading between growers, handlers, processors,
and receivers at terminal markets; b) they assist producers and
handlers in doing a better job of preparing fresh horticultural com-
modities for market and appropriate labeling; c) they provide a basis
for making incentive payments for better quality; d) they serve as
the basis for marketing reporting; and e) they help settle damage
claims and disputes between buyers and seIlers.

The first U.S. Grade Standards were developed for potatoes in

1917. Currently there are more than 150 standards covering 80 dif-
ferent commodities. In addition, several states also have mandatory
minimum quality standards. The International Standards for Fruits
and Vegetab1es, which have been defined by the Economic Commission
forEurope (since 1954) and the Organization for Economic Cooperation
and Development, have provided the basis for EEC standards current1y
in effect for 38 commodities. Developing countries must use these
quality standards for their commodities which are exported to EEC
countries. With very few exceptions, no quality standards are used
for fresh fruits and vegetables destined for local marketing. The
establishment of simple grade standards and their use for local
distribution in developing countries can be major steps which would
help reduce fraud and deception in packaging and encourage high and
uniform quality. Enforcement of these standards will require a well-
trained group of inspectors in each country and an effective exten-
sion program to inform all producers and handlers about the stan-
dards. They need to be convinced that proper standardization and
inspection would promote trust and encourage commerce.

POSTHARVEST TECHNOLOGY PROCEDURES IN RELATION TO QUALITY

Commodity requirements and recommended conditions for optimum

quality maintenance and postharvest life are the same regardless
QUALITY MAINTENANCE IN DEVELOPING COUNTRIES 467

of the distribution system (direct marketing, local marketing,

export, etc.). However, the type of appropriate technology needed
to provide such conditions will depend upon the distance and time
between production and consumption areas as weIl as intended use
(fresh vs. processing). In selecting the proper postharvest tech-
nology procedures, one should remember the following:

A. The technology used elsewhere is not necessarily the best

for use under conditions of a given developing country.
Many of the recent modifications in postharvest technology
in developed countries have been in response to the need to
economize in labor, materials, and energy use, and to
protect the environment. It is useful to study the cur-
rently used practices in other countries and to select
those which are appropriate for local conditions.

B. Expensive equipment and facilities without proper manage-

ment are useless. People who operate such facilities are
more important than their level of sophistication.

C. Commodity requirements can be provided using simple and

inexpensive methods in many cases. For example, proper
temperature management procedures include:

1. Protection from exposure to the sun,

2. Harvesting during cooler parts of the day or even at
night,
3. Adequate ventilation in containers and non-refrigerated
transport vehicles,
4. Possibly simple and inexpensive cooling procedures such
as evaporative cooling and use of cool-night ambient
air, and
5. Expedited handling.

D. Mechanical injuries are major causes of losses in quality

and quantity of fresh horticultural commodities in all
handling systems. Their incidence and severity can be
greatly reduced by simple modifications in harvesting and
handling procedures and by informing all personnel involved
about the need for careful handling.

Solving the postharvest technology problems in a given country

will require cooperation and effective communication among all the
research and extension personnel involved. Postharvest horticultur-
ists need to coordinate their efforts and to cooperate with pro-
duction horticulturists, agricultural marketing economists, engine-
ers, food technologists, and others who may be involved in various
aspects of the marketing systems. In most cases, solutions to
existing problems in the postharvest handling system require use
468 A.A.KADER
of available information (17) rather than any new research. Follow-
ing is a proposed program for improving the postharvest handling
system in a developing country:

1. Survey the magnitude and causes of losses in quality and

quantity during harvesting and postharvest handling of
major commodities.

2. Survey available tools and facilities for harvesting,

packing, transport, storage, etc., for each commodity in
its important production seasons and areas.

3. Evaluate the impact of simple modifications in the handling

system (picking stage and method, type of containers,
quality sorting, etc.) on quality maintenance and losses.

4. Extend information about recommended harvesting and handling

procedures to all those who can use it. All appropriate
extension methods for the intended audiences should be used.

5. Identify problems which need further research, carry out

research and extend any new information when completed.

FUTURE RESEARCH NEEDS

Additional research in both developed and developing countries

is needed to improve our understanding of quality and its postharvest
maintenance in fresh fruits and vegetables. The objectives of this
research should be to:

A. Better define components of quality (appearance, texture,

flavor, nutritive value) and their interrelationships for
various fruits and vegetables destined for the fresh
market or for processing.

B. Develop objective and non-destructive methods for determin-

ation of appearance and textural quality and optimum
maturity which are related to their flavor and nutritional
quality.

c. Evaluate the effects of preharvest factors (genetic,

environmental, and cultural) on flavor and nutritional
quality of fruits and vegetables.

D. Develop multiple maturity indices related to distance to

market and intended use of the commodity (fresh market,
processing, etc.).

E. Relate maturity indices at harvest to the final organoleptic

aUALITY MAINTENANCE IN DEVELOPING COUNTRIES 469

acceptabi1ity by the consumer and to nutritiona1 quality.

F. Study the effects of any proposed changes in harvesting and

postharvest handling practices on qua1ity and safety attri-
butes.
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48.
27. Tinda11, H. D. and F. J. Proctor. 1980. Loss prevention of
horticu1tUl:al crops in the tropics. Prog. Food Nutr.
Sei. 4(3-4):25.
28. Watada, A. E. 1980. Qua1ity evaluation of horticu1tura1 crops:
the problem. HortScience 15:47.
29. Watt, B. K. and A. L. Merri11. 1963. Composition of foods,
raw, processed. USDA, Agr. Hb. No. 8, 190 p.
30. White, P. L. and N. Se1vey (eds.). 1974. Nutritiona1 qua1ities
of fresh fruits and vegetab1es. Futura Pub1. Co., Mount
Kisco, NY. 186 p.
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY OF AGRICULTURAL

CROPS

Karl H. Norris

Instrumentation Research Laboratory

BARC, ARS, USDA
Beltsville, MD 20705

INTRODUCTION

Many instrumental techniques have been developed for measuring

the quality of agricultural products. However, instrumentation
specialists often feel frustrated in this effort because quality
as related to agricultural products is difficult to define in terms
of physical parameters which can be readily quantified. In general,
it is the chemical composition of the product which really deter-
mines the quality, but most often, we define the quality by our
senses of sight, touch, or smell, rather than by the composition.
It has been our experience that it is much easier to develop
techniques to measure composition than it is to measure quality.
Therefore, this paper will concentrate on the measurement of com-
position as a means of indicating quality. Spectrophotometry by
transmission, reflectance, and fluorescence offers the best tech-
nology for instrumental measurement of composition, so we will
examine the visible and near-infrared region of the spectrum for
this purpose.

NEAR-INFRARED REFLECTANCE (NIR)

Diffuse reflectance spectrophotometry is now widely used for

rapid prediction of composition of grains and oilseeds. This
technology is based on the fact that each of the major constituents
of grains and oilseeds has definitive absorption characteristics
in the 1000 to 2600 nm region, and these absorption characteris-
tics can be sensed by diffuse reflectance. The spectra for the
major constituents are shown in Fig. 1. The data are plotted as
log (l/R) where R is the diffuse reflectance. The spectra plotted

471
472 K. H. NORRIS

r,
STAR~H j\
,., I(~t "",\ \
[\

WATER:i\; ~J
1
. \. I / \~ \./'\, .\. /
. \ I I ,

r'\/~ l/', x'

~ I~/ ! \

ROTEIN
/ \
.1\"
"U'" ./
/(l',j "~I . . . . . . . . . . . .
I f'V \. !

I 1\ i

•1 I I / '. i
\. ,jJ '. :
''-.../1; ..... ....... ...... l
--.' li

~~~ttatt""~~LLLLLLll+uLllllllll~WWWWWWwWWwwwwwwwuuh
------

12Ba. 14BB. 16Ba. 1BBa. 2BBa. 22Ba. 24Ba. 26021.

WAVELENGTH nm
Fig. 1. Spectra of major components of grains and oilseeds.

as log (l/R) are very similar to absorption spectra, and it has

been found that log (l/R) varies linearly with concentration of a
specific absorber in a mixture with other materials.

These spectra were recorded with an inhouse-designed com-

puterized spectrophotometer. This instrument is built around a
Cary Model 14 prism-grating monochromator with optics optimized
for the infrared. The monochromator is coupled to a digital
computer for collection and analysis of data. The instrument is
opera ted in a single-beam mode with a reference spectrum stored in
the computer. The sample, packed into a cell and covered with a
quartz window, is illuminated through the window and diffusely
reflected radiation is collected with four lead sulfide cells
surrounding the sample. Several different materials have been
used as reflectance reference standards, but we now use a ceramic
material because its reflectance is the same for all wavelengths.
Solid teflon, powdered telfon, calcium carbonate, magnesium carbo-
nate, and barium sulfate, previously used as standards, all exhibit
absorption bands within the 1000 to 2600 nm region. The signal
 INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 473

C,!)
o
H

1200. 1400. 1600. 1800. 2000. 2200. 2400. 2608.

WAVELENGTH nm
Fig. 2. Spectra of a sampIe of wheat ground with three different
grinders to produce mean partic1e sizes of 330, 200, and
160 ~m respective1y.

from the detectors is amplified, digitized, and fed to the computer.

The computer corrects for the system response, inc1uding the
ref1ectance of the ceramic standard, and processes the da ta to
log (l/R) for storage on disk or magnetic tape. The scanning speed
is 10 nm per second with data col1ected at 0.2 nm interva1s. The
data processing includes smoothing by averaging of 10 adjacent
points on either side of each data point and shrinking of points
to a maximum of 1024. The resulting spectrum at 1.6 nm between
points has sufficient resolution to resolve the sharpest absorp-
tion bands which have been observed in grains and oilseeds.

The near-infrared diffuse ref1ectance spectra of grains and

oilseeds are affected by many factors, including: particle size,
particle shape, packing density, sampie temperature, and the com-
position of the sampie. The effect of particle size is shown in
Fig. 2 for a wheat samp1e ground with three different grinders to
produce three samp1es vlith the same composi tion having mean
 474 K. H. NORRIS

.81

-. 01 1111I1111I1111111I11II1111111111111111111111111111111111111111111111111111111111

o
o
H

128B. 14fJB. 16fJB. 188B. 288B. 228B. 2488. 2600.

WAVELENGTH nm
Fig. 3. Spectra of a wheat sampIe at 15°C - - and 25 D C
Upper curve is the difference curve (25°-15°).

partic1e sizes of 160, 200, and 330 ~m,respective1y. As the

partic1e size increases, the apparent absorption increases causing
an increase in log (l/R). The effect of partic1e size is greater
for strong absorbers than for weak absorbers, so the effect is
greater at the longer wave1engths. A simi1ar effect is observed
for packing density with more tight1y packed sampIes having 10wer
log (l/R) va1ues.

SampIe temperature affects the near-infrared spectra because

the water absorption band is temperature sensitive. This effect
is difficu1t to observe in Fig. 3 for a wheat sampIe at two differ-
ent temperatures. However, a plot of the difference curve,
subtracting one curve from the other, c1ear1y shows the temperature
effect. The greatest effect is in the region of the water absorp-
tion bands at 1450 rum and 1940 nm. The dip in the curve at 2100 nm
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 475

o
o
H

128B. 148B. 168B. 188B. 288B. 228B. 248B. 2688.

WAVELENGTH nm
Fig. 4. Spectra of two wheat samp1es . ..... 20.0% protein,
----- 11.8% protein. Upper curve is the difference
curve (20.0%-11.8%).

suggests an interaction between carbohydrates and water with respect

to temperature, but this has not been exp1ored.

Re1ative1y 1arge differences in protein cause on1y a sma11

change in the log (l/R) curve for wheat. Fig. 4 shows the curves
for two samp1es with 20.0 and 11.8% protein, respective1y. The
difference curve, subtracting the curve for 11.8% pro tein from the
curve for 20.0% protein, more c1ear1y shows the effect of the pro-
tein difference with major effect in the carbohydrate band at
2100 nm and the pro tein bands at 1980, 2060, and 2180 nm. It
shou1d be noted that an increase in protein in wheat is accompanied
by a decrease in carbohydrates, and the difference curve shows this
relationship.
476 K. H. NORRIS

1200. 1400. 1600. 1800. 2000. 2200. 2400. 2600.

WAVELENGTH nm
Fig. 5. Wheat spectra at four moisture levels.

Differences in moisture content are readi1y apparent as shown

in Fig. 5 for wheat at different moisture levels. The greatest
effect is in the regions where water absorbs at 1940 and 1450 nm.
Another factor which affects NIR spectra is the form of the con-
stituents. The spectra of two breakfast cerea1s (Fig. 6) are quite
different, a1though they are very simi1ar in composition. The
difference between the two spectra resu1ts from the fact that the
sugar is in the amorphous form for one samp1e and in the crysta11ine
form in the other. The spectrum of crysta11ine sucrose shows all
the sharp spectra1 features which distinguish the spectrum of one
cerea1 from the other. The sharp spectra1 features of crysta11ine
sucrose have not been observed with natural products, even those
with very high sucrose content, such as dates. However, this type
of effect cou1d occur with processed products.

The problem of ana1yzing the diffuse ref1ectance curve of a

samp1e to predict its composition is obvious1y not a simple problem
because of the many factors which affect the curve. Severa1 methods
of data treatment have been used to ana1yze NIR spectra. The
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 477

1200. 1400. 1600. 1800. 2000. 2200. 2400. 2608.

WAVELENGTH nm
Fig. 6. Spectra of two breakfast cereals with the same sucrose
content (36%), and the spectrum of sucrose.

simplest is a measurement of log (l!R) at each of the wavelengths

for the maximum absorption of the constituents. These data are
then combined in a multiple regression program to produce a cali-
bration for each constituent. For example, the prediction of oil,
moisture, and pro tein is made with measurements at six wavelengths.
These are: 2308 nm for oil, 2180 nm for protein, 1940 nm for water,
2100 nm for stareh, and then two reference wavelengths of 1680 nm
and 2230 nm at which the absorption is aminimum. Varying com-
binations from three to six of these wavelengths are used for each
of the constituents of oil, moisture, and pro tein in grain and
oilseeds.

A second data treatment uses the difference signal at two

wavelengths for each constituent, where one wavelength is at a
peak absorption and the other is at a minimum absorption. Measure-
ments at 2308 nm and 2230 nm are used for oil, 2180 nm and 2230 nm
for protein, and 1940 and 1800 nm for water. These three sets of
differences are then combined in a multiple regression program to
produce a calibration for oil, protein, and moisture.
478 K. H. NORRIS

19.

17.
PROTEIN IN WHEAT
S.E.P. = 0.14%
N = 50

11.

9.~~~~~~~~~~~~. . . . . . . .~~~~~~~~~
9. 11. 13. 15. 17. 19.
% PROTEIN BY KJELDAHL
Fig. 7. Scatter plot of protein predicted from NIR spectra of
ground wheat.

Amore complex data treatment uses the first or second deriva-

tive of the log (l/R) data, with two to four first or second
derivative terms combined in a multiple regression equation. The
second derivative at 2308 nrn is most sensitive to oil, 2180 nm is
most sensitive to protein, and 1940 nm is most sensitive to water.
More than four derivative terms can be used for ca1ibration, but
overfitting of the data may occur if the number of sampies is
inadequate. It is recommended that the nurnber of sampies shou1d
be greater than fifteen times the number of regression terms.

A single-term regression using a ratio of first derivatives

at two wavelengths has been very successful for predicting the
pro tein content of wheat sampies with wide variation in particle
size, temperature, and moisture content. The wave1engths used
are 2160 nm for the numerator and 2270 nm for the denominator.
This data treatment gave a standard error in predicting the protein
content of Hard Red Spring wheat of 0.14% protein for sampies
varying from 10.1% to 18.7% pro tein (Fig. 7).
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 479

WATEr V"', I \
STARGH....

/\\ \ I \~ROTEIN
I I ~ \
I !\ \

I I I\1\'\ \ \
I' \/'
I

1 / \\\
II ! I "

J
l.

) i"\ I I/I \'\

i 7 \\ !I
1I \
\ \
\
I \ \ I' \

/ rJ\/ZJV
.i I I .........
\)/ \\
~ I \
~/
-
.......................-:;:. \,. I
~
/ ......./~ \

L8~~~~LLLL~~~LLLl~~LLLL~~~,~
88L 118L
WAVELENGTH nm
Fig. 8. Short-wavelength absorption speetra of oil, water,
stareh, and protein.

Other data treatments include multi term first or second deriva-

tive ratios, curve fitting, and factor analysis. The optimum data
treatment appears to be dependent on a number of sample-related
parameters, and on the available computer capability.

Three manufacturers are now marketing NIR instruments. They

are: Neotec Instruments Division of Pacific Scientific,
Silver Spring, MD; Dickey-john Gorp., Auburn, IL.; and Industrial
Systems Division of Technicon Gorp., Tarrytown, NY. These three
firms market filter instruments designed for analyses of grains
and oilseeds but their designs are applicable to other products
as well. In addition, Neotec and Technicon market computerized
spectrophotometers for research applications.
480 K. H. NORRIS

NEAR-INFRARED TRANSMITTANCE

The initial work in rapid infrared analysis used transmittance

of a thin slurry of sampie and carbon tetrachloride to measure
moisture content (Norris and Hart, 1965; Ben Gera and Norris,
1968). The magnitude of the absorption coefficients over the 1400
to 2700 nm region limits the sampie thickness to less than 1 mm
for dry powders and the problems of working with a hazardous
chemical has limited the application of the transmittance technique.
However, the major constituents of grains and oilseeds also have
definitive absorptions in the 800 to 1100 ~m region. In this
region, the absorption coefficients are weak enough to permit trans-
mittance measurements on relatively thick sampies. The relative
absorption characteristics of stareh, protein, oil, and water are
compared in Fig. 8.

Large area, high sensitivity, silicon detectors make it

possible to measure the transmittance of 10 cm of clear water or
oil, 2 cm of potato tissue, 2 cm of whole grain, 1.5 cm of homoge-
nized meat, and 0.3 cm of finely ground grain or other dry powder
in the 800 to 1100 ~m region. Therefore, this region offers the
possibility for a rapid analysis on a wide range of agricultura1
products. This region has been explored for the measurement of
moisture and protein of samples of intact wheat. Spectral trans-
mittance measurements were made on 2.1 cm thick samples of whole-
grain wheat of 8 different c1asses. A second derivative ratio
ca1ibration for each c1ass of wheat was deve10ped using log (l/T)
data where T is the transmittance relative to air. These ca1i-
brations were used to predict the protein content of another set
of sampies with the resu1ts as shown in Table 1. The mean error
of 0.29% pro tein is more than adequate for many applications. An
instrument based on this deve10pment is now marke ted by Trebor
Industries, Gaithersburg, MD.

Table 1. Pro tein Errors for Eight C1asses of Whole-Grain Wheat.

C1ass HRS HRW SRW DURUM Semi- Western White Soft

Dwarf White Club White
S.D. a .93 1.11 .62 1.09 1.89 1.59 1. 79 1.31
S.E.P. b .207 .291 .232 .305 .216 .288 .316 .446
BIAS -.022 -.065 .053 -.04 -.02 .003 -.010 .026

aS. D. is the standard deviation for the pro tein content within
each c1ass.
bS•E. P • is the standard error in predicting the protein compared
to the Kje1dah1 results.
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 481

Transmittance measurements have been used on individual

seeds to measure moisture content of peanuts (Norris and Hart,
1965) and of corn kernels (Finney and Norris, 1978). This type
of measurement is now being explored in the author's laboratory
to measure the oil, protein, and moisture content of individual
seeds.

Reflectance measurements can be useful in this region

although the reflectance signals are relatively weak. The fat
content of ground beef has been measured by reflectance in this
region using a gallium arsenide infrared-emitting diode as the
radiation source (Massie, 1976). In this work, the temperature
of the diode was changed to scan a limited spectral region so
that no filters or other type of spectral-discriminating elements
were needed. Infrared-emitting diodes, being developed for this
region, may greatly enhance the possibilities for low-cost instru-
ments to measure the composition of agricultural commodities.

VISIBLE TRANSMITTANCE AND REFLECTANCE

Reflectance measurements are widely used to evaluate color

and to detect defects. Many products are sorted automatically
based on reflectance measurements in the visible region. Because
of the wide availability of easy-to-use color-measuring instruments,
they are often used to predict composition of agricultural products.
However, it should be noted that such instruments were not designed
to measure composition, and for this reason, they do not represent
the optimum design for composition analysis. The computerized
spectrophotometric techniques developed for NIR can be applied
in the visible to analyze for anthocyanins, chlorophyll, carote-
noids, and other pigments.

Much of the effort for transmittance requirements in the

visible has concentrated on intact commodities. Multiplier-type
phototubes provide the sensitivity needed to measure the light
transmitted through an object such as an apple, peach, or tomato.
A computerized spectophotometer has been developed for such
measurements (}1assie and Norris, 1975) and a two-filter instrument
has been described for specific measurements (Birth and Norris,
1965). A four-filter instrument, using a silicon detector in
place of the phototube, has been marketed by Neotec. The silicon
detector limits the range of the instrument both in wave-
length and density of sample which can be measured. The Neotec
instrument is most useful in the 600 to 1000 ~m region, and for
samples with an optical density less than 6.0.

The visible absorption spectra of intact tomatoes (Fig. 9)

show strong chlorophyll absorption in the 600 to 690 nm region
for the mature green stage. The chlorophyll absorption which
482 K. H. NORRIS

7.

6.

5.

2.

1.

a~~~~~~~~~~~LL~~~~~~~"~~~.
48a 68a
WAVELENGTH nm
rig. 9. Absorption spectra of a ripening miniature tomato.
The numbers are the hours of storage at 22°C after
harvesting the fruit in the mature green stage.

peaks at 675 nm, gradually declines as the tomato ripens. The

increase in absorption in the 500 to 600 nm region is mainly from
the lycopene which shows its peak absorption at 570 nrn. Another
pigment, probably a carotenoid, having a maximum absorption at
526 nm also increased with time as the tomato ripened. These
spectra of ripening tomatoes illustrates a problem which is cornrnon
to all spectrophotometric measurements of biological tissue. The
spectra observed on biological tissues do not match the spectra
anticipated for the pigments known to be present. This results
from the fact that our knowledge of absorption spectra for pig-
ments are based on spectra of the pigments in a solvent and
solvent interactions change the absorption spectra. Thus, we
find lycopene in tomatoes with maximum absorption at 570 nm rather
than at 510 nm as we observe in a solvent. The pigment absorbing
at 526 nm may be S-Carotene although the solvent spectra for
S-Carotene is at a much shorter wavelength.
INSTRUMENTAL TECHNIQUES FOR MEASURING QUALITY 483

These spectra were measured with a computerized spectro-

photometer bui1t around a Cary Model 14 monochromator optimized
for the visible region. This spectrophotometer uses a 1arge area
multiplier-type phototube to measure the light transmitted by the
samp1e. Dense 1ight-scattering samp1es such as tomatoes are
measured using the princip1es described by Butler and Norris,
1960; and Norris, 1965. Data handling for this visible spectro-
photometer is the same as for the NIR instrument discussed ear1ier.

Light-transmittance techniques have been used to measure

the chlorophyll and carotenoid content of intact tomatoes (Watada,
et a1., 1976); the chlorophyll content of intact peaches (Sidwe11,
et a1., 1961); and chlorophyll content of app1es (Yeatman and
Norris, 1965). These techniques have also been used to detect
defects such as water core in app1es (Birth and 01sen, 1964);
ho11ow heart in potatoes (Birth, 1960); and sca1d damage in cherries
(Yeatman, et a1., 1961). In general, any interna1 defect which
causes a general change in the interna1 f1esh color can be detected
by visible transmittance measurement.

SUMMARY

Computerized spectrophotometric measurements provide a

techno1ogy for rapid prediction of the major constituents of agri-
cu1tura1 commodities. Protein, oi1, starch, and moisture content
have been measured on grains and oi1seeds using NIR techniques
on ground samp1es. Pro tein and moisture content have been measured
on wheat samp1es without any samp1e preparation using near-infrared
transmittance. Chlorophyll, anthocyanin, and catotenoid content
have been measured on intact fruits and vegetab1es using visible
transmittance. These deve10pments make it possib1e to measure the
composition of a product and this compositiona1 information can
then be used to indicate qua1ity.

REFERENCES

Ben Gera, I. and Norris, K. H., 1968, Determination of moisture

content in soybeans by direct spectrophotometry, Israel
J. Agric. Res. 18:125.
Birth, G. S., 1960, A nondestructive technique for detecting
interna1 disco1orations in potatoes, Amer. Potato J.
37:53.
Birth, G. S. and 01sen, K. L., 1964, Nondestructive detection of
water core in De1icious app1es, Proc. Amer. Soc. Hort.
Sci. 85:74.
Birth ~S. and Norris, K. H., 1965, The difference meter for
measuring interior qua1ity of foods and pigments in
bio1ogica1 tissues, USDA Tech. Bu11. No. 1341.
484 K. H. NORRIS

Butler, W. L. and Norris, K. H., 1960, The spectrophotometry of

dense light-scattering material, Arch. Biochem. and Biophys.
87:3l.
Finney, E. E. and Norris, K. H., 1978, Determination of moisture
in corn kernels by near-infrared-transmittance measurements,
Trans. ASAE 21:581.
Massie, D. R., 1976, Fat measurement of ground beef with a gallium
arsenide infrared emitter, in: "Quality Detection in Foods,"
ASAE Publication.
Massie, D. R. and Norris, K. H., 1975, A high intensity spectro-
photometer interfaced with a computer for food quality
measurements, Trans. ASAE 18:173.
Norris, K. H., 1965, Measuring and using light transmittance
properties of plant materials, in: "Electromagnetic Radiation
in Agriculture," Illuminating Engineering Soc. and ASAE,
New York.
Norris, K. H. and Hart, J. R., 1965, Direct spectrophotometric
determination of moisture content of grain and seeds,
in: "Humidity and Moisture," Vol. 4, "Principles and
Methods of Measuring Moisture in Liquids and Solids,"
Reinhold Publishing Corp., New York.
Sidwell, A. P , Birth, G. S., Ernest, J. V., and Golumbic, C.,
1961, The use of light transmittance techniques to estimate
the chlorophyll content and stage of maturation of Elberta
peaches, Food Tech. 15:75.
Watada, A. E., Norris, K. H., Worthington, J. T., and Massie, D. R.,
1976, Estimation of chlorophyll and carotenoid content of
whole tomate by absorbance techniques, J. of Food Sei.
41:329.
Yeatman, J. N., Birth, G. S., Ernest, J. V., Bender, R. W., and
Sidwell, A. P., 1961, Spectrophotometric evaluation of
anthocyanin pigment development and scald damage in intact
red tart cherries, Food Tech. 15:521.
Yeatman, J. N. and Norris, K. H., 1965, Evaluating internal quality
of apples with new automatie fruit sorter, Food Tech.
19:123.
POST HARVEST LOSSES IN PERISHABLE FOODS OF TRE DEVELOPING WORLD

D. G. Coursey

Tropical Products Institute

56/62 Gray's Inn Road
London WClX 8LU England

INTRODUCTION

The majority of the papers presented to this Advanced Study

Institute have been related primarily to situations relevant to the
technologically advanced countries of Europe, North America and
Australasia. This paper will do something to redress the balance,
for it must be remembered that about a third of the world's popula-
tion lives within the Less Developed Countries (L.D.C. 's); that it
is generally within the L.D.C. 's that living standards and nutri-
tional standards are the lowest; while rates of population increase
tend to be the greatest. Thus, it is within these countries, mostly
within the tropics, that there is the most urgent need to increase
food availability. This is not solely an ethical issue; whatever
an individual's, or a government's, political or moral stance may
be it must be accepted that a situation where such a large propor-
tion of the world's population are living under severely sub-optimal
conditions is highly unsatisfactory, and is a destabilising force
within the established world economic order.

The world's population is expected to increase by around 50%

between 1980 and the end of the century with a disproportionately
large increase in the tropical world: a similar increase in food
availability is necessary merely to maintain even the current
position, which is itself seriously inadequate. It is a matter of
speculation as to how far this increased food availability will be
achieved, but it is generally accepted that only in a few limited
areas is there much scope for bringing new lands into cultivation.
Increased food production will need to be derived to a greater
extent from the intensification of agricultural production, both
extension and intensification of agriculture may have severe

485
486 D. G. COURSEY

environmental impacts. A large part of tropical food production

is derived from small scale farmers operating at or near the sub-
sistence level, with only modest excess production to supply the
rapidly growing urban centres. These farmers usually have no, or
severely limited, access to such inputs as credit, mechanical
power or fertilizers. These constraints will tend to increase
with increasing energy costs, rendering intensification diffi-
cult (71).

A partial alternative to increased agricultural production as

a means of increasing food availability is provided by improved
storage and conservation, leading to reduced post harvest loss.
Food loss reduction is normally cheaper than equivalent increases
in food production, whether the cost is expressed in terms of
economics, energetics, or adverse environmental impact. Neverthe-
less, only recently has serious scientific attention been given to
post harvest problems of the tropical world, compared with those
of agricultural production. Good conservation of fresh produce is
especially important in the tropics. where the proportion of food
which is processed is much lower than in technologically advanced
countries. Post harvest studies initially developed from the need
to protect the large bulk grain reserves held in Europe during the
2nd World War, and subsequent work has concentrated largely on the
problems of "durable" or "dry" produce, such as grains, grain
legumes, and oilseeds, which have essentially similar storage
problems, mainly attack by external factors such as insects,
rodents and spoilage microorganisms (28,29).

Attention to post harvest food loss reduction as a significant

means to increase food availability was first given prominence at
the international level at the World Food Conference in Rome in 1974,
following which the 7th Special Session of the U.N. General Assembly
in 1975 passed aResolution calling for a 50% reduction of post
harvest losses by 1985. This recognition of the potential va1ue
of post harvest 10ss reduction has found practica1 expression in
the continuing debate among a number of international and national
institutions, and severa1 practica1 initiatives have been taken,
aimed at making an effort to reduce losses at all the post harvest
stages of the food production process. In the Food and Agricu1ture
Organization (F.A.O.), food loss prevention became a priority area
in 1978, but initially concentrated on grain storage. Perishables
received some attention at aseries of meetings held in 1977 by the
U.S. National Academy of Sciences (41) but the first formal, inter-
national recognition to the importance of post harvest los ses in
perishable foods came only when an Expert Consultation on the
Reduction of Food Losses in Perishab1es of Plant Origin was held
joint1y by F.A.O. and the United Nations Environmental Programme
(U.N.E.P.) in 1980 (26). Severa1 individual studies on the subject
had neverthe1ess been made ear1ier, notab1y at the Tropica1
Products Institute on a 1imited sca1e, before this, (11,14,16,17)
POST HARVEST LOSSES 487

but it attracted only limited interest among a relatively small

number of workers.

THE MAGNITUDE AND SCOPE OF THE PROBLEM

After grains and similar durables, perishable plant foods are

the next in importance, especially staple foods such as cassava
and cooking bananas followed by fruits and vegetables. According
to recent F.A.O. statistics production of perishable plant foods
in L.D.C. 's represents 85% of cereal production. The comparable
global figure is only 70%, indicating the high er relative impor-
tance of perishables in the tropics. These figures, expressed as
simple tonnages, somewhat overstate the case for perishables, as
they have higher water contents than the grains. If this is allowed
for, their contribution to L.D.C. diets in calorific terms is of
the order of 29% of that of the grains. Total production in
L.D.C. 's of the principal types of perishable plant foods are in-
dicated in Table 1, the grand total being 372,300,000 tonnes (24),
and, allowing for the substantial production of unrecorded minor
"backyard" vegetable crops, the actual production is probably sub-
stantially higher. A classification based on utilization may be
convenient, although a commodity may sometimes fall into more than
one group.

Staple foods. In many tropical regions, perishable staples

take the place of grains in providing the calorific base of
the diet. Cassava is the most important in spite of the
problems associated with its toxicity (42), followed by
cooking bananas, yams, sweet potatoes, potatoes, aroid root
crops, breadfruit and some minor staples. Root crops alone
were estimated a decade ago to provide staple food for around
400 million people (16): the figure today would probably
be more than 500 million. Under humid tropical ecosystems,
many of these crops are the most efficient producers of
carbohydrate (72), being far more productive than grains.

Secondary foods. This includes the culinary category of

'vegetables': e.g. green or salad vegetables, on ions ,
tomatoes, brassicas, legumes and a great variety of others,
many unfamiliar to European diets; and also fresh dessert
fruit. Although consumed in smaller quantities than staples,
they are of importance in balancing the diet by providing
minerals and vitamins; in adding flavour and variety to make
it more attractive; and making minor contributions to
calorific and pro tein nutrition.

Export crop. Substantial quantities of fruit, e.g. bananas,

pineapples, citrus, mangoes, avocados and out-of-season
vegetables are exported from the tropics to the developed
 ~
TABLE 1 00
00

Production of Vegetab1e and Fruit Crops in Less Deve10ped Countries

(after F.A.O., 1979)

Production Production
Commodity Commodity
('000 metric tons) ('000 metric tons)

Cassava 110,617 Tomatoes 15,161

Potato 32,053 Me10ns 13,257
Sweet potato 16,324 Onion (dry) 7,612
Yams and miscf11aneous Cabbage 3,980
29,931
root crops Pumpkins, squashes
and gourds 2,685
Root and tubers total 188,925
Green chi11ies and
2,691
Grapes 13,078 peppers
Oranges 22,139 Eggp1ant 1,601
Other citrus 5,602 Cucumbers and gherkins 1,735
Mango 13,771 Cau1if1ower 1,046
Pineapp1es 5,884 Carrot 865
Bananas 37,383 Green peas 566
Plantains 20,584 Green beans 598
App1es 5,038 Gar1ic 1,355
Dates 2,525 Artichokes 178
Peaches and nectarines 1,319 Vegetab1es and me10ns
Avocado 1,196 53,330
total
Papaya 1,526
c
Fruits (exc1uding G)
130,045
me1ons) total o
o
C
1 Yams (Dioscorea spp.) constitute about 19 million tons, and taros, tannias, etc. most of the ::D
remainder. cn
m
-<
POST HARVEST LOSSES 489

countries. Although these contribute to the nutrition of

the developed world, they are valuable earners of foreign
exchange for the exporter countries, many of which are net
food importers.

Within the general category of "perishables," the post harvest

characteristics of the different comrnodities vary enormously. At
one extreme, some leafy vegetables and soft fruit keep only a day
or two under tropical ambient conditions, while at the other extreme,
yams and potatoes will store under the right conditions for many
months.

DEFINITIONS OF LOSS

The definition of loss has been a subject of considerable

debate, and the situation is especially complex with perishable
food products, owing to factors as the high water content, which
permits substantial transpiratory weight loss with only minor loss
of actual food; senescence, or sprouting, which can reduce the
quality of the food product without necessarily rendering it a
total loss, and the varying levels of quality deterioration at
which a food product becomes unacceptable in different societies,
or even at different social levels within a society. Some studies
on the subject (20) concentrated on the economic aspects of loss,
which is difficult to relate to actual food loss as the product
acquires added value at every stage of the complex chain from
producer through purchaser, transporter, wholesaler, retailer(s) to
final consumer at a greater rate than it suffers deterioration or
loss. The economic loss is thus inflated in relation to the loss
of food.

At the recent F.A.O./U.N.E.P. Expert Consultation (26), con-

siderable effort was devoted to establishing a manageable definition
of "post harvest food loss," and the form ultimately determined
follows:-

'Post Harvest' begins at the moment of separation of the

edible comrnodity from the plant that produced it by a de-
liberate human act with the intention of starting it on its
way to the table. The post harvest per iod ends when the
food comes into the possession of the final consumer.

'Food' means weight of wholesome edible material that would

normally be consumed by humans, measured on a moisture free
basis. Inedible portions such as skins, stalks, leaves and
seeds are not food. Potential foods (e.g. leaf protein) are
not foods; they do not become food until they are accepted
and consumed by large populations. Feed (intended for con-
sumption by animals) is not food.
490 D. G. COURSEY

The method of measuring the quantity of food in the post

harvest chain should be on the basis of weight expressed
on a moisture free basis. There will be times when in-
formation on losses in nutritional units and economic
losses will also be needed but these should not be the
prime means of measuring post harvest food losses.

'Loss' means any change in the availability, edibility,

wholesomeness or quality of the food that prevents it
from being consumed by people.

Some comment is needed: although it is difficult to estimate

loss once the product is in the hands of the final consumer, losses
occurring at this stage are of importance, even in market economies.
Within L.D.C.'s, substantial proportions of the populations live
within the subsistence agriculture sector, where most food produced
on the farm is consumed within the extended family, and most food
eaten is home-grown, so that there is virtually no distinction
between producer and consumer. That food loss must be calculated
on a moisture free basis is of the greatest importance, especially
with the perishables, which are usually of high moisture content:
nevertheless, gross changes in water content, although not
necessarily destroying the nutritional value of the produce, could
render it unacceptable, and so a loss.

A briefer definition (5,30) seems to be adequate for practical

purposes, although perhaps a trifle simplistic.

',that weight of wholesome edible product (exclusive of

moisture content) that is normally consumed by humans
and that has been separated from the medium and the
site of its immediate growth or production by deliberate
human action with the intention of using it for human
feeding but which, for any reason, fails to be consumed
by humans"

CAUSATIVE FACTORS IN POST HARVEST LOSS: TECHNICAL

Perishable produce differs in physical and biochemical char-

acteristics from durable produce, as summarized in Table 2, and
the mechanisms and causative factors of loss are thus quite dif-
ferent within the two groups. Loss in durable produce is very
largely brought about by external factors, mainly moulds, insects
and rodents: endogenous factors are of minimal importance, and if
the produce is of adequate quality and is protected from external
biodeteriogens it may be kept for very long periods: certainly
longer than from one harvest to the next, which is all that is
usually necessary, and under optimal conditions for many years.
Perishable produce is, however, still metabolically active, and
POST HARVEST LOSSES 491

Tab1e 2. Characteristics of Durable and Perishab1e Crop Products

(adapted from F.A.O./U.N.E.P., 1981)

Durables Perishab1es

Low moisture content, usua11y High moisture content,

10% to 15% or 1ess. typica11y between 50% to 90%.

Sma11 unit size, typica11y Large unit size, typica11y 5

1ess than 1 gram. g to 5 kg., occasiona11y
even 1arger.
Very 10w respiration rate,
with very sma11 generation of High to very high respiration
heat. rate, heat production is
therefore high.
Hard texture, not easi1y
damaged. Soft texture, easi1y damaged.

Stab1e, natural she1f 1ife of Perishab1e, natural shelf

severa1 years 1ife a few days to at best
severa1 months, according to
Losses main1y caused by type of produce.
externa1 agents, such as
mou1ds, insects and rodents. Losses caused partly by
external agents, e.g. rotting
by bacteria and fungi and
part1y by endogenous factors,
respiration, senescence and
sprouting.

the very metabolic processes necessary for its survival are them-
selves a cause of loss, a1though externa1 factors can also be of
importance. The very term "perishable" implies that such produce
cannot be kept for any very great length of time, hard1y ever as
long as from one harvest to the next, and in many cases on1y for a
few days or weeks.

This leads to the concept of inherent storage life, which is

of fundamental importance in the understanding of the post harvest
behaviour of perishables. Although much can be done to reduce
post harvest losses in these commodities, and extend their storage
1ife, there is a limit beyond which they cannot effectively be
kept, when the produce is either shrivelled or rotted to destruc-
tion, or has been so changed by its own metabo1ism as to have be-
come unacceptable as food. This inherent 1ife is related to the
natural biological function of the plant organ, and may be only a
few days, or up to several months, but in every case the life of
492 D.G.COURSEY
the produce as an acceptab1e food item will eventua11y be terminated.

Thus, to minimize losses in perishab1es, the first essential

is to maintain the physica1 and physio10gica1 integrity of the
detached but still 1iving plant organ, as losses arise from
assaults on its integrity - a principle first clearly enunciated
by the writer and his colleagues (13,17). Secondly, the natural
1ife may be prolonged within certain limits, by the provision of
optimal environments, or by manipulation of the physiological state
of the material. Thirdly, there should be selection of material
for storage that is entirely sound, and also at the ideal state of
maturity. Thus, los ses are minimized by choosing healthy material
and keeping it healthy, but at the same time it must always be
remembered that all living material will eventually die, and some
detached organs inherently live longer than others. The factors
that affect the health and 1ength of life of perishable produce,
and so post harvest loss, can be considered under three main
headings:

Physical Considerations

The most obvious, but frequently ignored, factors in deteriora-

tion are purely physical. Mechanica1 injury takes many forms and
arises at all stages in the his tory of the produce from pre-harvest
operations, through harvesting, handling, grading, packaging, trans-
porting and storage to exposure in the market and finally at the
hands of the consumer. Losses due to mechanical injury are frequent-
1y overlooked and although difficult to estimate, are no doubt seri-
ous. Mechanically injured produce will normally deteriorate much
more rapid1y than sound produce and shou1d never be considered for
long-term storage. An example of the possible magnitude of 10ss
through direct mechanica1 injury was revealed in recent surveys in
Britain (54) which showed that up to the stage of grading on the
farm, 33% of the potato crop is seriously damaged.

Pre-harvest attack by predators must be mentioned, but as al-

ready indicated, damaged produce should normally be diverted to
immediate use. The harvesting process is liable to inflict wounds
on the produce, except in those relatively rare cases where abscis-
sion leads to a situation where the organ can be removed from the
parent plant without forming a wound. Usually, removal of the
edible organ at harvest is a traumatic process, necessitating the
formation of a wound where it is detached from the plant. After
harvest, perishable produce is often as thoughtlessly and roughly
hand1ed as are bags of grain and extensive bruising or breakage of
the individual organs frequently takes place: the damage caused
greatly enhances further deterioration from both physio10gical and
phytopathological causes. When unsuitable containers are used to
remove the produce from the field, severe cuts may be inflicted on
hitherto sound produce. During transportation, especially to a
POST HARVEST LaSSES 493

remote market, package design and construction needs to be optimized,

if further damage by bruising or cutting is not to be incurred (40,
43). Apart from damage caused by the harvest trauma, transportation
is probably a major cause of mechanical damage. As an example, the
effect of transportation on mechanical damage to bananas in Jamaica
is shown in Table 3. This example is typical of the kind of funda-
mental information on loss which is often needed, but seldom avail-
able. Different types of mechanical damage differ in the serious-
ness of their effects: with tubers and starchy storage organs,
bruising or surface abrasions may initially be less obvious than
breakage or cutting, but can be far more damaging during long-term
storage. Decay will often spread from a bruise through the entire
tissue, but a clean cut may be healed (cured) und er favourable
conditions (2,51).

Impairment of the produce's life processes from extremes of

heat and cold must be considered. Produce exposed to the tropical
sun can rise far above the ambient, to as high as 40° or even 50°C,
which can have major deleterious effects (58,59). Damagingly low
temperatures seldom occur in the tropics, but the phenomenon of
chilling injury provides a major constraint on the use of refrig-
eration for the storage of tropical produce (21,22). Extremes of
relative humidity can also adversely affect produce. Excessively
low humidity can bring about rapid wilting of leafy vegetables, and
shrivelling of the softer fruit, while at the other extreme, al-
though high relative humidity may help to maintain turgor and reduce
water loss, it can also favour the growth of pathogens.

Physiological Considerations

A distinction must be made between the unavoidable losses

during storage of perishable produce that arise from the endogen-

Table 3. Damage to Bananas Caused During Transportation (after

Thompson, 1972)

% Damage
Type of transportation
Scarring of fruit Damage to 'neck'

Head-loading 1.1 0.4

9-mile 10rry journey 7.1 15.1

45-mile lorry journey 25.1 12.2

494 D.G.COURSEY
ous metabolie processes of the detached plant organs and avoidable
losses which are brought about by exposure to adverse environ-
mental factors. All perishable produce consists of organs, which,
although detached from the parent plant, are living, and whose
metabolie processes must continue. The essential metabolie pro-
cesses are energy-consuming, and depend on the energy supplied by
respiration. This being a process which requires the availability
of oxygen, adequate ventilation of stored perishable produce is
obviously a fundamental requirement. Tropical ambient temperatures
generally lie between 25° and 35°e, compared with the 10° to 20 0 e
of temperate summer temperatures, and near or below zero of the
temperate winters through which much long term storage of temperate
perishable crop products takes place. The endogenous metabolie
processes of tropical produce therefore proceed at substantially
higher rates than those of temperate produce stored under its own
native ecological conditions. The requirment for good ventilation
is thus high er and the losses of weight also higher. than in
temperate crops. This is illustrated by studies on losses in
yams (9, 52) where respiratory losses, representing actual destruc-
tion of carbohydrate, were between 0.01 and 0.1% per day, repre-
senting from around 7% to as much as 35% of the total weight loss,
according to the temperature and state of dormancy (Table 4): these
total weight losses are about an order of magnitude higher than
those reported for potatoes, stored under temperate winter condi-
tions (8). Associated with destruction of carbohydrate matter by
respiration, is water loss by transpiration: in the same example
of the yams, this constitutes the other main factor in weight loss.
However, water is of no nutritional value, and so, using the defini-
tion of loss already given, this factor should not be considered,
except in the situation where the water 10ss is sufficient1y great
to affect the acceptability of the produce as food. Losses of
weight associated with transpiratory loss of water may, of course,
be·of substantial economic importance in a trading context, where
they are often referred to as shrinkage. Thus, perishable produce,
even when kept under ideal conditions, will always suffer some loss
of weight during storage from the combined effects of respiration
and transpiration: this is in marked contrast to durable produce,
where losses are miniscule under optimum conditions.

Reference has been made to inherent storage life, which is

normally determined by physiological factors. All fruits mature,
then ripen and finally senesce, and when the process of senescence
is sufficiently far advanced, the flavour and textural character-
istics will have so changed that the produce is inedible, and so
must be regarded as a total loss. The onset of ripening, and se-
nescence can often be delayed, for example in bananas by techniques
for the removal of ethylene (62) and more recently by the use of
gibberellin treatment (35) but even though senescence may be de-
layed it will eventually occur. In organs of dormancy, such as
bulbs and tubers, the endogenous period of dormancy is terminated
POSTHARVESTLOSSES 495

Table 4. Relative Contribution of Respiration to Total Weight Loss

of Yam Tubers During Storage at 25°C and 35 Q C
(after Passam et al, 1978)

Daily weight
Total daily weight loss due to
loss (%) respiration
(%)

Condition of tubers 25°C 35°C 25°C 35°C

Immediately after harvest 0.22+0.02 0.36+0.02 0.058 0.108

Dormant 0.15+0.03 0.28+0.06 0.011 0.028
Sprouting 0.21+0.02 0.34+0.07 0.074 0.068

by regrowth and sprouting: sprouting leads to rapid transfer of

both dry matter and water from the edible organ into the sprouts,
and so to massive loss. A number of sprout suppressants are ef-
fective in delaying the appearance of sprouts in potatoes (8) but
in the yam have little or no effect (49) probably because the bud
primordia are formed beneath the skin only shortly before the end
of dormancy. The dormancy of onion bulbs can be extended to some
extent by the careful selection of temperature (66) but sprouting
will eventually occur. Fruit which has started to senesce, or
bulbs or tubers that have passed the natural breakage of dormancy,
increase greatly in their susceptibility to phytopathological
attack; become softer and so more liable to mechanical damage; and
develop undesirable flavour characteristics, which make them un-
acceptable as food. Thus sorne loss will always occur in perishable
produce as a result of its natural physiological activity, and its
storage life ultimately terrninated for physiological reasons.

These physiological processes are normal, and inevitable.

Additionally, consideration rnust be given to abnormal physiolog-
ical losses, which arise frorn the stresses of adverse environ-
mental factors already outlined. Mechanical damage usually has
only fairly moderate physiological effects, wounding or especially
bruising leads to increased respiration rate, and enhanced loss of
dry matter: evaporation of water through the damaged surface will
also be increased. Wounds can be cured, but considerable physio-
logical losses will occur before the curing process is complete.
This effect has been weIl demonstrated in the yarn (51). An impor-
tant exception is that of cassava. When the roots are damaged, when
they are cut from the plant, or o therwise , the primary phase of
deterioration is purely physiological. Known as vascular discol-
496 D.G.COURSEY
ouration, it consists of darkening of the vascular bundles, and
the formation of occlusions within them, involving migration of
material from the xylem parenchyma. Only after this physiological
response has occurred is there any involvement of microorganisms,
which eventually lead to total decay (3,45,60).

More generally however, abnormal physiological deterioration

is caused by extremes of heat and cold or of relative humidity.
Unlike temperate perishable produce, which can be stored at temp-
eratures only marginally above zero, and is usually damaged only
by actual freezing of the tissue, most tropical produce is liable
to "chilling injury," at temperatures weIl above zero. The temp-
erature below which chilling injury occurs is often around 10°C,
but in some crops it is lower, but still weIl above zero, and
occasionally it is as high as 14° or 15°C. There is much variation
in chilling sensitivity between cultivars, with the state of ripe-
ness, maturity or dormancy, within a species or cultivar. Visible
symptoms of chilling injury vary greatly from one type of produce
to another. In bananas, the first signs are discolouration of the
under-peel vascular bundles, followed by a failure to ripen normally,
and a grey-green peel colour when ripe (39); avocados also show
vascular discolouration; in yams, there is greyish discolouration
of the tissue followed by water-logging arising from breakdown of
the Na+ and K+ pump mechanisms, leading to massive phytopathological
invasion (10): rather similar symptoms occur in sweet potatoes and
electrolyte leakage is also involved in chilling in tomatoes. The
ultimate cause is believed to arise from phase changes in the lipid
structure of the cell and mitochondrial membranes, wh ich although
reversible, can lead to irreversible changes in enzyme activity,
and thus to energy supply within the ce11, and relative metabolite
levels (21,22,36,37).

At the other end of the temperature scale, attention has

already been drawn to problems of high temperatures developed in
produce when it is exposed to insolation (58, 59). The effects are
greatest in dark coloured produce, which absorbs radiant heat more
readily than light coloured: in some of the darker items, such as
aubergines, potatoes and yams, internal temperatures during the
afternoon can c1ose1y approach the "black bulb" temperature. Little
information is availab1e as to the mechanism of the deteriorative
processes involved, but, especially in the larger organs it is
likely that oxygen deficiency in the tissues may result in a change
to anaerobic respiration.

Phytopathological Consideration

These probably constitute the largest single factor in bring-

ing about avoidable post harvest loss in perishable produce, a1-
though endogenous physiologica1 processes will always be the cause
of some unavoidab1e losses. It is particularly important to dis-
POST HARVEST LaSSES 497

tinguish between quantitative and qualitative lasses, as these will

be of greatly differing relative importance in different situations.

Quantitative phytopathological lasses result in rapid, exten-

sive and eventually complete breakdown of hast tissues by micro-
organisms. The pattern of attack is typically an initial infection
by one or a few specific pathogens, which establish the initial
lesions in the previously healthy tissue: there follows a massive
infection by a broad spectrum of non-specific saprophytes which are
only weakly, if at all, pathogenic but are capable of surviving on
the dead or moribund tissue rernaining from the primary infection
and enhancing the damage done (14,69). These secondary invaders
thus playamajor role in post harvest pathology by multiplying and
extending the darnage initiated by the primary pathogens. Qualita-
tive pathogenic lasses result from blemish or surface disease which
render the produce less attractive and so less marketable, though
little actual destruction of food matter has occurred. Such dis-
eases are particularly important in fruit and vegetable export
industries where increasing emphasis is being placed on visual
quality. In the developed countries, much research in post harvest
phytopathology has been devoted to these purely "cosmetic" factars
and similar efforts in L.D.C. 's are needed if exports to sophisti-
cated markets are to be successful.

Bacteria are often the most important casual factor in the

spoilage of vegetables, the bacterial soft rot group (Erwinia spp.)
being the cornrnonest, but in the case of fruit and root crop spoil-
age, fungi pathogens are more frequently involved. The taxonomy
can be very complicated: over 20 different fungi have been asso-
ciated with post harvest decay in tomatoes or in crown rot of ba-
nanas. Virus diseases, while seldom of post harvest importance,
may render produce less marketable, for example, "internal brown
spot" of yams (14). Nernatodes have been shown to affect the quality
and storage life of yarns (65). Pathogens differ in their response
to the host, to environment, to various control measures, and in
their time and mode of infection. Successful disease control thus
depends on a thorough knowledge of these factors in each individual
situation.

Post harvest diseases may also be classified, according to

whether initial infection occurs before harvest; or during or
after harvest. The latter are often caused by wound pathogens,
associated with mechanical darnage or physiological injuries. Con-
trol of the former may be effected by the pre-harvest use of pro-
tectant or eradicant sprays in addition to post harvest treatments
after infection has occurred. Control measures against the latter
are restricted to post harvest treatments, either physical or
chernical. Physical treatments include the use of high and low
temperatures to reduce the growth of the pathogens, and curing.
Low ternperature storage, widely used for many cornrnodities, slows
498 D. G. COURSEY

down the metabolism of both host and pathogens and so frequently

arrests rotting temporarily, but when the produce is returned to
ambient temperature, rotting may recommence rapidly. Heat treat-
ment suffers from the drawback that certain pathogens are heat
tolerant and the high temperatures required to achieve control can
damage the produce. The pathogens and disease symptoms of the
major tropical perishables, and chemical control measures have
been summarized (14).

Insects, rodents and birds are usually of minor concern in

the storage of perishable produce, compared with their important
role in the storage of durables (14).

CAUSATIVE FACTORS IN POST HARVEST LOSS: NON-TECHNICAL

The whole problem of post harvest loss in perishables is

greatly affected by factors of an entirely non-technical nature,
which are of even greater importance in L.D.C.'s than in developed
countries. These problems arise ultimately from the shortage of
managerial and administrative skills that still exist in many
L.D.C.'s; inadequate extension services; lack of appropriate
educational facilities; and, in many cases, shortages of capital
or of foreign exchange facilities. These are sometimes compounded
by the advice or technical/managerial inputs supplied by "experts"
from the technologically advanced countries, which are not always
relevant, properly motivated or based on appropriate experience.
Arecent F.A.O. publication (25) has drawn attention to the need
for improved co-operative facilities, transport systems and espe-
cially training in post harvest loss reduction, as weIl as to
various technical considerations: the importance of training, and
of publications for the dissemination of information, specifically
in connection with losses in perishables was re-emphasized in
another (26). Shortage of administrative and managerial expertise
tends to lead to situations in which staff are often over-graded
and through lack of experience and even of basic knowledge mistakes
and errors of judgement occur more frequently than they should: this
can apply from national administrators to packhouse operatives,
and every level in between. Farmers are often unaware of the basic
requirements for produce for sophisticated storage systems, or
high-quality markets, although their traditional post harvest
technology may be weIl adapted to the conditions of subsistence
economies (12). The produce presented to buying organizations is
often merely the excess production of the subsistence agriculture
sector: supplies thus tend to be irregular, and of variable quality.

The operation of these factors can be seen clearly in many

cold storage operations set up in L.D.C. 'so Firstly, at national
planning level, the installation of cold stores is often seen as
a universal panacea for all situations, even when it is not - an
attitude often encouraged by the less scrupulous representatives
POST HARVEST LOSSES 499

of manufacturing organizations in the developed world. Secondly,

it is seldom realized that a cold store, even where it is appropri-
ate, is often useful only as a link in the "cold chain" - including
cold stores in the production areas, refrigerated transport to urban
centres and different cold stores in those centres. Thirdly, at
store manager level, there may be lack of knowledge of the various
technical requirements for produce storage. Even the concept of
chilling damage may be unknown, and highly chill-sensitive produce
packed into stores maintained near or even below zero. Incompatible
produce can be mixed together in the same store. Fourthly, even the
best machinery eventually breaks down. Local mechanics are un-
familiar with it: the operating manual (if any) may be written in a
language which no one locally understands; the firm that sold the
entire plant in the first instance has no agent in the country who
stocks spare parts; and no allocation of foreign currency can be
made available to obtain the parts from abroad, even though they
may cost only $100 or so. Even when the plant is working, farmers
may receive no advice of the type, quantity or quality of produce
required or to supply them or advise them on suitable seed, plant-
ing regimes or harvesting techniques, and often there will be little
financial incentive for them to produce what is required. When all
these factors are taken into account, it is not surprising that the
tropical world is littered with cold stores that are either shut
down; broken down; grossly under-utilized or maintained at a loss
by official subsidies. The worst possible situation is described
here in order to bring out the essentials of the case most effec-
tively. Many highly efficient cold storage and cold chain systems
do exist and function in many developing countries, but a combina-
tion of any or all of the factors mentioned above do, only too
frequently, apply.

Reference has been made to training and information supply

needs. Although most of the L.D.C. 's now have excellent univer-
sities and institutions of higher learning, the courses available
there are usually academically oriented. There is a serious need
for relatively short term, academically rigorous, but practically
oriented and relevant training schemes in the essentials of the
post harvest technology of perishable produce, geared to various
levels of seniority and responsibility, and conducted by those
who have genuine and appropriate experience of L.D.C. conditions.
An important aspect of such training needs to be guidance on
national and international sources of up-to-date information, and
on its interpretation to suit local conditions (26).

TRE MAGNITUDE OF LOSSES

Although this topic has been the subject of very considerable

debate over the last decade, remarkably little reliable information
is yet available on the magnitude of post harvest los ses of perish-
500 D. G. COURSEY

ab1e produce. It has been conservative1y estimated from the very

1imited data scattered in the literature that the global 10ss is of
the order of 25% (11,17). However, these ear1y papers are based
very 1arge1y on what might be described as anecdota1 information,
that being all that was avai1ab1e at the time. An independent esti-
mate (48) for Africa and India is 30%.

Even in the wor1d's most high1y deve10ped and non-tropica1

country, the United States of America, perishab1e produce has been
described (6) as "the victim of phenomena11y high waste because of
incredib1y poor handling practices," and "the 10ss rate as a resu1t
of multiple handling ••••• is frightful." The annua1 economic 10ss
in that country arising from deterioration during shipment, storage
and marketing was estimated 15 years aga at $200 million (20).
Estimates of material 10ss in fruit and vegetab1es as percent of
total production were made by the U.S. Department of Agricu1ture
as 11% for the period 1942-1951 and more recent1y for 1957-1960 at
8% which may indicate the benefits that resu1ted from a decade
of research and app1ication (17). However, another analysis of the
situation in the U.S.A. over the past 30 years (53) conc1uded that
a1though there had been some reduction in post harvest 10ss with
some commodities, with others losses had actua11y become more
serious. Arecent review (30) quotes a survey of produce in New
York markets, se1ected data from which are given in Tab1e 5. Total
losses range from as 1itt1e as 1.7% for app1es to 22.9% for straw-
berries: most surprising1y the commodity which exhibited the second
highest 10ss was sweet potatoes (15.1%), which wou1d be considered
by most to be re1ative1y rugged.

In the tropica1 wor1d, the situation is doubtless worse than

in the developed world, but even less hard information is avail-
able. A number of specific cases have been reviewed (17). The
meeting concerning perishables in the series on post harvest losses
held by the U.S. National Academy of Sciences (41) opera ted in the
absence of any hard factual information, on the De1phi Princip1e of
summarizing the estimates (or guesses) of a number of professionals
who have some standing or authority in the fie1d. Some of the
figures produced by this process are given in Tab1e 6, but few, if
any, of these are based on rea11y re1iab1e original data. Perhaps
the most usefu1 outcome was to estab1ish that losses in perishab1es
are virtua11y impossible to quantify except with reference to a
particular commodity and 10ca1 situation. This concept was more
fully developed at the F.A.O/U.N.E.P. Expert Consu1tation (26)
where it became evident that under different conditions and 1ength
of storage and with different commodities, almost any 10ss figure
between 0% and 100% may genuinely be found. Overall, therefore,
losses of perishab1e plant foods are extreme1y serious, and losses
in the deve10ping wor1d wou1d appear most frequently to be between
10% and 30%, according to commodity and 10cation-specific storage
conditions. Each 10ca1 situation needs investigation and analysis
POST HARVEST LOSSES 501

Table 5. Post Harvest Losses in Selected Fruits and Vegetables

Sampled at Wholesale, Retail and Consumer Markets in
the New York Market Area (selected from Harvey,1978)

Loss in indicated markets

Wholesale Retail Consumer Total loss
Commodity (%) (%) (%) (%)

Apples, Red Delicious 0.2 0.2 1.5 1.7

Cucumbers 5.0 2.9 7.9
Lettuce, Iceberg 4.1 4.6 7.1 11. 7
Oranges, Navel 1.9 1.9 2.3 4.2
Peaches 2.3 4.5 8.1 12.6
Pears, d'Anjou 2.5 1.6 4.1
Peppers, Bell 7.1 9.2 1.4 10.6
Potatoes, Katahdin 1.3 3.6 4.9
Strawberries 5.9 4.9 18.0 22.9
Sweet potatoes 5.7 9.4 15.1
Tomatoes, packaged 6.3 7.9 14.2

on a particular individual basis, and broad global figures of loss

such as have been quoted are of value mainly in indicating the
magnitude of the problem that exists, which in turn implies the
need for urgent action.

REDUCTION OF POST HARVEST LOSS

Traditional subsistence agriculture systems account for a very

large proportion of agricultural production in L.D.C. 's (23) and
within these systems exist many simple techniques for the avoid-
anee of post harvest loss in traditional erops, which are often
highly effective within the situations in whieh they were devel-
oped (12), but are nevertheless subject to severe limitations.
Where multinational companies are involved in the plantation scale
production of perishable crop products for export, relatively few
problems arise. Production of bananas in Central America is,
perhaps, the best example. The multinationals have, through their
own research departments, access to the latest scientific informa-
tion. The most serious problems arise at the interface between
subsistence and market economy sectors. The crops being grown
are often unfamiliar to the farmers, as may be the concept of
growing crops, especially perishable crops, for sale, rather than
subsistence. The tropical cash crops that have been developed over
the last century, primarily yield durable products, directly or
after simple farm-level processing. The concept of exacting qual-
ity requirements may well also be absent. At this interface, and
502 D. G. COURSEY

Table 6. Non-Grain Staples, Vegetables and Fruits. Losses Reported

by Commodity (adapted from N.A.S., 1978)

Estimated
Commodity Loss
(Percent)

ROOTS/TUBERS

Potatoes 5 - 40
Sweet Potatoes 35 - 95
Yams 10 - 60
Cassava 10

VEGETABLES

Onion 16 - 35
Tomatoes 20 - 50
Plantain 35 -100
Cabbage 37
Cauliflower 49
Lettuce 62

FRUITS

Banana 20 - 80
Papaya 40 -100
Avocado 43
Peaches, apricots and nectarines 28
Citrus 23 - 33
Raisins 20 - 95
Apples 14

within the marketing economies that are developing within the

L.D.C.'s, numerous technical problems arise in connection with
perishable food crops (68). These problems, and some solutions
that do not involve highly sophisticated technologies, will now be
discussed.

Pre-Harvest Consideration

Cultivars of individual crops have differing, genetically

controlled post harvest behaviour due to differing inherent phys-
ical, biochemical and physiological characteristics. Plant breed-
ers have seldom exploited these potential differences, although
work has recently been carried out on the genetic factors control-
POST HARVEST LOSSES 503

ling the storage life of tomatoes (67). Evaluation of post harvest

characteristics is a factor which should be, but seldom is, consid-
ered in the early stages of any breeding programme. A masssive
banana breeding programme carried out to develop tetraploid bananas
in order to reduce dependence on triploid clones did not take such
factors into consideration until a late stage, when the new clones
were found unsuitable for trans-Atlantic transportation (44).

There is also scope within screening trials for the evaluation

of difference cultivars of a crop for significant differences in
their storage potential. Red onions, for example, usually store
better than white (61, 66). However, a major problem in many trop-
ical areas is the availability of regular supplies of viable seed
of cultivar which are adapted to local conditions in any respect,
including good post harvest characteristics. Few tropical countries
have adequate facilities for breeding, selection and multiplication
and commercial sources of seed are limited and often of unsuitable
cultivars. Many growers rely on their own resources for seed pro-
duction, which is often responsible for the low yields obtained and
the variability in market and storage life of produce from variable
seed.

Appropriate timing of sowing is important for annual vegetable

crops, in order to optimize quality; crops which are sown late may
not mature adequately before the required harvest period. Many
annual vegetable crops are grown during the dry season and heavy
rainfall occurring before the crops have fully matured can promote
post harvest fungal infection. Although little is known of the
effects of irrigation on post harvest quality, water stress during
growth is generally deleterious to produce quality, as weIl as to
yield. Rates of fertilizer application can also affect post harvest
qualfty and especially crops subjected to high levels of nitrogen
are likely to yield produce of diminished life.

Crops which have become infected with diseases in the field

may subsequently deteriorate rapidly due to the development of
these initially pre-harvest infections during storage. Field hy-
giene, the removal and destruction of diseased plant material and
dead haulms, and the adequate spacing of crops to promote air cir-
culation all have positive effects in reducing disease levels in the
field. Preventative crop protection techniques are therefore a
major requirement for the production of crops of good quality with
good storage potential.

Harvesting and Field Handling

Produce of optimum quality is obtained only when harvested at

the optimal stage of maturity. Fruits, when harvested immature,
will be of poor quality and ripen erratically: a delay in harvest-
ing may increase susceptibility to disease. Where produce is to be
stored for protracted periods it is often preferable to harvest
504 D.G.COURSEY

mature, but unripe. Maturity indices may be indicated by visual or

physical means, chemical analysis, computation of time from flower-
ing and physiological factors (47) although at the subsistence level,
only visual or physical methods of fruit selection are practicable.
Bananas may be harvested on the subjective basis of the shape of
the fruit fingers, which are angular when immature but fill out
while they are still green to a rounded or full shape, this latter
stage is frequently too mature for shipping. The condition of the
stem can be a basis for determining maturity in melons, while a
direct relationship between skin colour and soluble solids content
is an index of maturity in papaya (1). Maturity in root crops, such
as yam, sweet potato and potato is usually indicated by senescence
of the annual leafy growth (vines or haulms). These crops are often
harvested immature for immediate use, but immature tubers are not
suitable for storage. Cassava is often left in the ground until
required, as it has an extremely short storage life, although the
crop then occupies land which could be used for alternative crop-
ping (31). Alternative storage systems are being investigated.
Mechanized harvesting techniques for tropical root crops have not
been developed far, and the manual techniques still widely used
generally inflict less damage to the produce. Yams, culturally a
highly esteemed crop, are traditionally harvested and handled with
the greatest care, but other root crops are often as roughly handled
in the field as are durables, negating the benefits of manual har-
vesting.

It is customary and desirable for harvesting operations to be

carried out early in the day, especially for leafy green vegetables
fruits and fruit-type vegetables, which would otherwise acquire
excessive "field heat" leading to rapid deterioration. The treat-
ment which produce receives immediately after harvesting is a major
factor contributing to its deterioration in quality. Produce
should be collected from the field in suitable containers and not
allowed to be exposed to sun, rain or wind. Exceptions are onions,
which may be sun-dried or "cured" on the ground after harvesting,
and root crops, especially yams, which benefit from being left in
the sun for a few days (but not longer) to harden the skin, and to
dry adhering soil so that it may be brushed off. A collecting
point convenient to the field for the reception of produce is
desirable. This should be shaded; some leafy vegetable crops also
need to be sprinkled with water to maintain leaf turgidity, and
produce may be washed at this stage, to remove adhering soil. Ru-
dimentary grading, selection and trimming of produce may be under-
taken here, to avoid unmarketable material being transported
further.
Curing
With starchy root crops and onions, a "curing" process imme-
diately after harvesting can substantially reduce post harvest loss
POST HARVEST LOSSES 505

and prolong storage life. The term is rather loosely used: in the
case of root crops, it implies a wound healing process brought about
by a few days' exposure to high relative humidites and tempera-
ture (2), while with bulb crops it involves simply a drying out of
the skin and outer layers. The curing of Irish and sweet potatoes
and the drying of onions are weIl established practices in developed
countries but they are often not effectively undertaken in L.D.C. 'so
Curing involves the suberisation of the skin, followed by the
development of a wo und periderm which effectively retards water loss
and acts as a barrier against infection. The drying of onions, al-
though a different process, has a somewhat similar effect (66).
Methods of curing potatoes under tropical conditions have been re-
viewed (4) while it has been shown that curing yam tubers can signi-
ficantly reduce decay (51). Curing is essential to avoid the initial
physiological phase of the deterioration of cassava. The process
can often be carried out under tropical conditions at little cost
by stacking and covering the produce so that the temperature and
relative humidity rise naturally.

Transportation and Packaging

Many types of containers, quite unsuitable for the transport

of perishable produce, are nevertheless commonly used, and few
attempts are made to standardize the design or capacity of those
which are satisfactory. The use of containers of unsuitable di-
mensions or design or which are in poor condition leads to exces-
sive mechanical damage to produce in transit. Alternative designs
for wooden containers for produce, which can be manufactured locally,
have been published, but these designs and modifications on them
have not been effectively evaluated in developing countries (46.70).
Even where only a limited range of local materials is available,
modifications to design can produce a saving in product quality.
Very few studies appear to have been conducted on this subject.
Investigations in India have evaluated existing and improved designs
of container for the transport of both mango (34) and grapes (38).
In Ghana (57), when tomatoes were packed and shipped to urban cen-
tres in conventional "inverted cone" baskets, transportation losses
were around 15%. When "upright cone" baskets, i. e. flat-bottomed
with sides converging towards the top were used, and the tomatoes
packed on a thin layer of dried grass, losses in the same handling
chain were reduced to about 3%. Chili i peppers, which have very
high rates of respiration, are packed for marketing in South East
Asia in baskets provided with a central open chimney thus allowing
effective removal of the heat of respiration.

The quality of transport facilities in L.D.C. 's varies greatly

and it is difficult to identify the factors that contribute to dam-
age during transport. Containers are over-filled as transport
charges are often related to the total number of packages of a
consignment. The poor condition of road surfaces, and that lorries
506 D.G.COURSEY
are often driven much too fast, are two factors which result in
much mechanical damage being caused to produce consigned by road.
Road conditions in the tropical world are slowly improving, but
little can be done to educate drivers and middle-men in better
practices, as immediate monetary gain is involved. More appro-
priate containers, and their propermaintenance and handling, could
doubtless lead to substantial reduction in losses. Proper packag-
ing is, of course, especially important in connection with air-
freight exports, where the farm-gate price of the produce repre-
sents only a small fraction (~lO%) of the final selling price (55).
Investment in appropriately light, but strong packaging is there-
fore worthwhile (43). Many items of produce, particularly those of
large individual size, low unit cost and high bulk density such as
water melon, plantains and pineapple, are often transported without
containers. Systematic stacking within the lorries reduces the
transit damage, as also does lining a lorry with protective materi-
als such as grass or leaves, and covering the produce during transit
to protect it from the weather.

Storage Structures

However simple the materials from which a structure is designed

to be made, it must achieve certain requirements. It must afford
protection from sun and rain, allow adequate ventilation, permit
inspection of the produce to detect early any incidence of disease,
or infestation by pests, and achieve the lowest practical ambient
temperature. Many traditional storage structures have been develop-
ed for medium and long-term holding of commodities such as yam,
Irish potato, onions and pumpkin, under tropical conditions (56).
Huts and underground pits and clamps, adapted particularly to potato
storage in tropical highland conditions are also used for short
term holding of root crops in the lowland tropics. In upland pro-
duction areas, low night temperatures offer the opportunity of
further development of ventilated stores, using either natural con-
vective ventilation or by controlling intakes of the cool night air
under a forced air ventilation system. Overall, the use of appro-
priate designs for storage structures for perishables can make a
major contribution towards loss reduction.

Chemical Treatments

These fall into two main categories: biocides that control

or limit the attack of bacterial or fungal pathogens, and those
which affect the physiology of the produce, with the aim of extend-
ing storage life. The use of crop protection chemicals prior to
harvest has been referred to as a means of reducing the incidence
of pre-harvest infections that lead to post harvest deterioration;
chemical and physical treatments applied to the post harvest phase
to prevent losses due to pests and diseases are also important:
they may be applied as dips, dusts, sprays and fumigants. They
POST HARVEST LOSSES 507

must only, however, be used in conformity with recognized legal

standards. Post harvest chemical applications need not be exces-
sively costly. A simple fungicide applicator (7), originally de-
signed for the control of crown rot on green bananas, could be
readily adapted for other commodities for the application of system-
ic fungicides such as benomyl and thiabendazole, or other water-
dispersible biocides. Similarly, work in Colombia on simple appli-
cation systems for the volatile fungicide 2-aminobutane, has been
developed as an effective low cost disease control system for
citrus (27). Waxes and plastic coatings are useful for extending
shelf life and retarding respiratory activity in fruits and vege-
tables (33), although these techniques are generally only suitable
for application on a large commercial scale. The application of
wax to citrus is usually carried out in combination with a post
harvest fungicidal treatment (18). Their use could probably be
extended to small scale production, if the value of the crop
justified the additional expenditure on materials and the labour
involved.

The second group of chemicals whose use may improve post har-
vest storage of perishables are the growth-regulatory substances.
Sprout suppressants are in common use in the temperate world to
control sprouting of Irish potatoes, and sprouting and root develop-
ment of onions, but their possible application under tropical condi-
tions has yet to be fully explored. The breakage of dormancy in
many tropical root crops is a major constraint on storage life,
and compounds effective on potatoes are not generally effective
with tropical root crops (49). Post harvest applications of
gibberellins A4 to A7 to bananas in order to delay ripening, im-
prove the control of crown rot and thus improve post harvest life,
have been successful (35), and they have also been used to extend
the green life of limes (50) and the dormancy of yams. Their use
in the preservation of tropical perishable produce seems to merit
further attention, especially as they are relatively cheap. Ex-
tension of shelf life of pineapples by the immersion of fruits in
growth regulatory chemieals, mainly alpha-naphthalene acetic, have
recently been reported in Australia (63), and it may be that this
technique could be extended to other tropical fruits.

REDUCTION OF LOSS BY ENVIRONMENTAL CONTROL

In developed countries, a major factor in loss reduction in

perishable produce has been the use of refrigerated storage, and
more recently of even more sophisticated techniques, such as con-
trolled atmosphere and hypobaric storage, in which an optimal or
near-optimal environment is provided for the produce have been
used. Reduction of temperature reduces the level of metabolic
activity of the produce, and so extends inherent storage life. The
activity of any pathogens is diminished, although once the produce
508 D.G.COURSEY
is returned to ambient re-growth will usually commence. The effec-
tive use of cold storage usually implies the existence of a com-
plete "cold chain" of linked storage and transportation facilities,
and unless such a "cold chain" system can be evolved, the use of
isolated, individual cold stores may weIl be contraindicated, as
the return of cold produce to ambient may often lead to condensa-
tion problems; a greater degree of damage than if it had never been
cooled, and enhanced liability to pathogenic attack. Within the
L.D.C.'s, although the same basic considerations apply, there are
major limitations on the use of cold storage, or its more sophis-
ticated variants. The administrative and infrastructural problems
or organizing and managing cold stores under L.D.C. conditions
have already been discussed and the importance of these problems
cannot be over-emphasized. The maintenance of a high relative
humidity is also important for the preservation of produce quality;
many fruits and leafy vegetables are particularly sensitive to low
humidity conditions that can arise in cold stores. However, high
relative humidities may pre-dispose produce to infection by micro-
organisms, so a compromise has to be reached between adequate air
movement by ventilation and a moderately high relative humidity.

The application of crushed ice, by the 'top-icing' process,

is used for maintaining the quality of many leafy vegetable and
other crops during transportation, particularly in South East
Asia. Produce which would be damaged by direct contact with ic.e
can be packed in containers so that the ice is adjacent to, but
not in contact with, the produce and low temperatures still main-
tained.

There are some co-operatives and progressive market retailers

in the tropics who do provide cool storage facilities for horti-
cultural produce, but it is only on a relatively l~rge commercial
scale that the unit value of fruits and vegetables justifies the
investment and for the relatively low-cost perishable staple foods
it is rarely, if ever, justified. Most traditional market systems
are adapted to a rapid throughput of perishable produce; in partic-
ular, many markets in the tropics are active during the very early,
relatively cool hours of the morning with produce being rapidly
sold. Cool storage facilities for produce intended for local dis-
tribution tend therefore to be limited to the larger markets. How-
ever, for export crops the justification for cool storage facilities
becomes more realistic in view of the high prices anticipated, and
the correspondingly high quality standards that need to be main-
tained (40, 55, 68).

Additionally, a fundamental constraint of the use of cold

storage facilities for tropical perishable produce arises from the
fact that many types of tropical produce are liable to chilling
damage at temperatures, usually around 10°C. This not only means
that maximum benefit in reduction of metabolie activity to low
POST HARVEST LOSSES 509

levels cannot be achieved, but also that de1icate contro1 o{ temp-

erature is needed in order to secure the maximum benefit possib1e
without risk of chi11ing damage.

Refrigeration neverthe1ess has an important ro1e to p1ay in

the reduction of post harvest losses of some tropica1 perishab1es,
so 10ng as sound management is avai1ab1e, and proper attention is
given to the temperature requirements of different types of pro-
duce. Optimum holding temperatures for various tropica1 crops pro-
duce have been given. Simi1ar general considerations app1y to the
more sophisticated systems, such as contro11ed atmosphere and hypo-
baric storage. The va1ue of increased levels of carbon dioxide or
nitrogen, reduced levels of oxygen and of 10w pressure for extend-
ing the storage 1ife of temperate fruits and vegetab1es has been
extensive1y investigated and a number of systems are in commercia1
use in the more techno10gica11y advanced countries. It is fe1t,
however, that the app1ication of such techniques to deve10ping
countries is 1ike1y to be slight for some considerab1e time, with
the possib1e exception of sea freight export industries where some
of these systems may be app1icab1e. Recent1y, much attention has
been directed to the use of the high degree of insolation typica1
of the tropica1 wor1d for solar-powered refrigeration systems: a1-
though still at the experimental stage, the fact that most tropica1
perishab1es require holding temperatures we11 above zero may we11
faci1itate this deve10pment (21,22,32,36).

Some simpler app1ications of contro11ed atmosphere princip1es

may be of va1ue, especia11y in connection with ethy1ene. Its re-
mova1 from a system can therefore extend the pre-c1imacteric 1ife
of the fruit. The removal of ethy1ene from bananas during sea
transportation, through the use of high levels of ventilation, is
an important factor in ensuring that the produce reaches its des-
tination in the required green pre-c1imacteric condition. Investi-
gations (62) on the transportation of bananas at ambient tempera-
tures by packing them in sea1ed po1ythene bags containing an ethy-
1ene absorbant has shown that transportation can be extended by up
to 18 days under tropica1 conditions. The 1imiting factor then is
disease and the best resu1ts were obtained by combining ethy1ene
absorption with fungicide to contro1 crown rot.

CONCLU SION

Perishab1e plant foods - fruits, vegetab1es, root crops - are

of great importance in the nutrition of the L.D.C.I S of the tropi-
ca1 wor1d, to a rather 1arger extent than in temperate countries.
Incteased avai1abi1ity is essential to the improvement of diets,
or because of population growth, to maintaining existing standards.
Reduction of post harvest 10ss provides an attractive alternative
or supplement to increased food production. These are current1y
very severe: in individual situations almost any 10ss between 0%
510 D.G.COURSEY
and 100% may take place. losses commonly lie between 10% and 30%.
The concensus of opinion suggests that around a quarter of perish-
able produce grown in the tropics is lost before it can be consum-
ed. These losses arise from a number of inter-related technical
factors. most of which can be classified as physical; physiologi-
cal; or phytopathological. The degree of loss from any or all of
these technical factors can be greatly compounded by the lack of
adequate administrative infrastructures, technical expertise and
educational facilities that often prevail in L.D.C. 'so

Various precautions, many of a simple nature, can be taken

which can very substantially reduce post harvest losses. A largely
unexplored field, is the inclusion of desirable post harvest qual-
ities in breeding or selection programmes. This is a long-term
approach; in the short-term, good pre harvest field hygiene; sound
and appropriate harvesting practices; the use of curing techniques
and/or of appropriate chemical treatments; the avoidance of me chan-
ical damage and thermal stress; the use of suitable containers and
storage structures can all greatly reduce loss: these measures aim
at minimizing any attack on the physical or physiological integrity
of the detached plant organs that constitute the produce. Refrig-
eration, especially when used throughout an entire cold chain, and
even more sophisticated technologies such as controlled atmosphere
and hypobaric storage, have a role to play, especially with high
quality produce destined for high-cost markets, but their role
may be only a somewhat limited one, for both technical and infra-
structural reasons. In many areas, further research could improve
understanding of the processes involved in loss, but more immediate-
ly there is probably a greater need for training and extension
activities, so that the information and expertise that a1ready
exists can be put more widely into practical application.

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development and total soluble solids in papaya. Horticultural
Sci., 6:567 (1971).
2. R. H. Booth. Post harvest deterioration of tropical root crops:
losses and their control. Trop. Sci., 16:49 (1974).
3. R. H. Booth and D. G. Coursey. Storage of cassava roots and
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Storage, I.D.R.C. Monograph, I.D.R.C. -031e, Int. Develop.
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4. R. H. Booth and F. J. Proctor. Considerations relevant to the
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5. M. C. Bourne. Proposed definition of post harvest 10ss. Proc.
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6. A. L. Brody and S. Sacharow. Flexible packaging of foods.
Crit. Rev. Fd. Techno1., 1:71 (1970).
POST HARVEST LOSSES 511

7. o. J. Burden and P. J. Griffee. A simple machine for the app1i-

cation of fungicide to harvested green bananas. PANS, 20:
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8. W. G. Burton. The Potato. Veenman and Zonen, Wageningen (1966).
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POST HARVEST LOSSES 513

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SOLVING THIRD WORLD FOOD PROBLEMS: THE ROLE OF POST-HARVEST PLANNING

Martin Greeley

Research Fellow
Institute of Development Studies
University of Sussex, Brighton, Sussex

INTRODUCTION

There is a substantial body of literature which argues that

developing countries' food shortages can be reduced, even removed,
by preventing post-harvest food losses. It has spawned major
research and development and extension initiatives, often funded
through aid, that seek to improve farm-level post-harvest systems.
Attention has focused on the major cereal staples which are the
subject of this paper. Section I examines why the role of post-
harvest planning has been characterized in this way. Section 11
challenges the conventional assertion of high farm-level food
losses using evidence from recent field studies. Section 111
provides a reassessment of the opportunities for and consequences
of farm-level post-harvest technical change.

SECTION I

The literature on post-harvest losses dates back at least as

far as Pliny (1) but until very recently it has enjoyed only a
narrow readership. With a few exceptions (2) the post-1945 effort
to increase food availability in developing countries concentrated
on increased production and it was not until after the Kissinger
initiatives of 1974 and 1975 (3) that post-harvest loss prevention
became a widely acknowledged alternative. Kissinger's speeches were
essentially opportunist, and, whilst they were undoubtedly influ-
ential in encouraging politicians' support, there were at least
four more substantive causes of the heightened interest in loss
prevention.

515
516 M. GREELEY

First, the repeated assertions (4) of high food losses in tradi-

tional post-harvest systems suggested an easy option for solving the
growing problems of hunger. There were few systematic studies of the
size and causes of losses at farm level, and in their absence, gen-
uine but untypical 'horror' stories of high losses were influential
in channelling funds to solve post-harvest problems of food losses.

The science of loss-assessment was primitive and reports that

did provide "estimates" of losses were often methodologically weak,
omitting definitions of loss and details of measurement and even
failing to specify which operations were included.

These problems led to some odd results - losses of 105 per-

cent! (5) - and even the most widely quoted set of figures had a bias
in presentation which gave a total loss figure seventeen percent
greater than the study actually found (6). These deficiencies, even
if recognized, were rarely acknowledged - and official reports that
did so, were not always published (7) - for the problems of hunger
were real, and growing, and post-harvest research offered a new
dynamic in the search for higher food output.

In many countries, neither Ministries of Agriculture concerned

primarily with increased food production nor Ministries of Food
concerned with better food distribution, had been responsible for
farm-level post-harvest problems; knowledge of this neglect further
strengthened the case for intervention.

Secondly, the introduction of HYVs(high yielding varieties),

notably with rice, but also maize and wheat, had sometimes resulted
in higher food losses, for both biological and physical reasons.
Frequently, the new varieties were softer or thinner husked than the
traditional ones and were more susceptible to insect attack (8).
Dormancy, a primitive genetic characteristic, was often reduced in
the breeding process and the risk of premature germination increased,
particularly when, as often was the case, a second rice crop was
grown under irrigated conditions during the dry season and harvested
at the beginning of the monsoons. These biological changes increased
the risks of deterioration during post-production operations and they
were accompanied by an increased risk of physical los ses as output
per hectare increased. These problems were feIt at farm-level where
space, labour and bullock constraints caused delays in harvesting
and threshing but they were most acute in market centers, for the
proportionate increase in marke ted surplus was very much larger than
the increase in output per hectare. The marketing and purchasing
operations in the North Indian wheat markets were a particularly
extreme case, (9) and the documentation of these losses, suffered due
to inadequate market facilities and storage space, further strength-
ened the case for a higher priority to post-harvest research and
development.
THE ROlE OF POST-HARVEST PlANNING 517

Thirdly, despite the regional problems of surplus continuing

national food shortages kept food aid, notably of course PL 480, at
high levels and the deterioration of these stocks prior to distribu-
tion was becoming a cause for grave concern. The US Senate Foreign
Relations Sub-Committee on Foreign Assistance was receiving mission
reports (10) of substantial losses in storage and was questioning the
value of continued food assistance. This highly sensitive issue -
and the only influence in the debate that encouraged LDCs (less de-
veloped countires) to show low food los ses - was exacerbated by re-
peated quality problems with grain before dis charge at ports that
resulted in expensive litigation.

Fourthly, multinational companies manufacturing post-harvest

equipment and anxious to promote sales in LDCs were using the loss-
prevention argument as a sales strategy. Both chemieals (11) and
machinery (12) were marke ted in this way. The products were often
unsuited for the job - early Japanese rice milling equipment in
countries growing indica rice varieties being a notorious and widely
quoted example - but sales pressure, tied aid and the sense of urgen-
cy genera ted through food deficits and suggestions of high losses
frequently resulted in their purchase. These commercial interests
were instrumental in propagating the view of high food losses in
traditional post-harvest systems and were the strongest proponents
of mechanization of these systems.

The response in some LCDs was the development of local manu-

facturing capabilities for post-harvest equipment (13) and more
generally there was a concern with 'appropriate' technology (14)
but the high loss figures were accepted and it was an attempt at
alternative solutions not reformulation of the problem.

Thus, through a variety of mutual1y reinforcing developments

in the 1970s, post-harvest planning became a widely debated issue.
The overwhelming view was that the allocation of funds by donors
and LDCs national programmes could result in post-harvest innovation
that would reduce food 10ss and even remove the food deficit. The
FAO had taken an early initiative in the 1ate 1960s with its War
on Waste programme and during the 1970s expanded its efforts through
the Prevention of Food Loss programme; the World Bank has developed
its own programme, a major part of the United Nations University
World Hunger Programme is concerned with post-harvest losses, several
large bi-lateral agencies - inc1uding the American, British, Canadian
and German - have increased funding to post-harvest R & D and many
LDCs (15) have developed their own programmes.

The few earlier post-harvest programmes had been concerned

1arge1y with the handling of imports and the marke ted surplus; im-
proved urban food distribution rather than loss prevention was the
objective. These new programmes concentrated on farm-level post-
518 M. GREELEY

harvest systems and within that upon storage. Between sixty and
and ninety percent of cerea1s output was retained at farm-level and
if substantia1 increases in food avai1abi1ity were to be achieved by
10ss-prevention, then they wou1d necessari1y be through innovation
at farm level. Traditiona1 farm-level methods were regarded as in-
efficient not because of any vety specific evidence of high losses
but because they were characterized by "ancient" often rudimentary
practices, uninfluenced by modern food science and technology. Sev-
era1 agencies directed funds to deve10pment of more systematic and
re1iable 10ss assessment methods (16), but in the meantime the need
for modernizing technica1 change had become a conventiona1 wisdom.

This 'wisdom' for some was rooted in the supposed neg1ect or

carelessness of peasant farmers; for others it was their lack of
basic know1edge of food science; for others it was their lack of
access to improved methods and for others it was their shortage of
resources to invest in improved methods. Imp1icit in all was the
view that a latent demand for improved techno10gy to prevent losses
existed and that by meeting it and thereby improving food avail-
ability there wou1d be fewer hungry people. A further attraction
of farm-level post-harvest innovations was the apparent income dis-
tribution advantages, through the concentration of benefits on sma11
producer-consumers who formed the majority of the wor1d's poorest.
Severa1 reports (17) argued that sma11 farmers suffered higher losses
than 1arge ones and would therefore benefit more. Furthermore, farm-
level storage, and more generally post-harvest improvement, wou1d
reduce risk - removing uncertainty over 1ean season stocks and 1im-
iting the need for expensive 1ean season food purchases - and for
tenants, was more equitab1e than production innovation since the
benefits accrued to them rather than the landlord.

SECTION II

The opportunity to increase food avai1abi1ity was the core of

the deve10ping interest in LDC farm-level post-harvest techno10gy.
This genuine concern - notwithstanding 1ess a1truistic commercia1,
professional and bureaucratic inf1uences - was based on four
assumptions:

1. That traditiona1 farm-level post-harvest techno10gy was

the cause of substantia1 food losses •••• And therefore
2. That techniques were avai1ab1e or cou1d be developed that
wou1d prevent these losses;
3. That it was profitable for farmers to adopt these new
techniques;
4. That food consumption of the hungry wou1d increase once
new techniques were adopted.
Recent evidence suggests that these assumptions are more often
THE ROLE OF POST-HARVEST PLANNING 519

false than true, and that policy-oriented R & D would be better

servedby an alternative set of assumptions:

1. That the most serious food losses occur off-farm in the

public sector in times of large surplus;
2. That farmer motivation to change post-harvest techniques is
cost-reduction which may not involve loss-reduction; techniques con-
cerned with loss-prevention would have to be simple, low-cost improve-
ments to existing practices;
3. That with improvement in land productivity the development
of market relations and the improvement of the rural infrastructure
will determine the rate of adoption and the distribution of costs
and benefits of adopting new techniques;
(And for South and South East Asia particularly)
4. That technical change is invariably labour displacing and
hence often worsens income (and food) distribution; the labour
displaced is sometimes from the poorest rural households and
threatens the livelihood and survival of these families.

These assumptions do not denigrate the role of post-harvest

systems in influencing rural development. They do suggest though
that their village-level impact on food consumption will be
influenced by broader questions of employment and income-distribution
rather than food loss prevention. These we discuss in Section 111
and the remainder of this section discusses recent research which has
established beyond reasonable doubt the low average farm-level food
losses in post-harvest operations.

These studies provide results on all operations for two coun-

tries, on all operations except drying and milling for one country,
on storage only for eleven countries and for harvesting operations
only for three countries. They have been selected from the numerous
figures available because they provide measured (except Malaysia,
based on a farmers' questionnaire) estimates of physical loss based
on field surveys of operations under normal farmer practice, and
provide details of the methods of assessment used. The results are
summarized in Table 1 - and references in the text to country stud-
ies are from the sources given there.

Losses during farm-level storage have received the most atten-

tion in the literature; a loss of ten percent is commonly quoted as
a conservative estimate and some 'experts' consider they run as high
as 40 percent (18). Why then do the resu1ts from careful measurement
in thirteen countries show that losses are below five percent and
often very much lower (see Table l)? The chief explanation is the
acceptance of convenient but unfounded guesses, but another important
set of reasons is that the methodological difficulties in accurate
10ss assessment tend to lead to upward rather than downward biases
in estimation.
Tab1e 1. Measured Physica1 Food Losses in Traditiona1 Post-Harvest Practices at Farm and Vi11age U1
Level o'"

country Commodity Operation Per Cent Loss Note

(rounded to one
decima1 p1ace)
l.Africa Maize Storage 1.6 Insect 10ss on1y; an average of
8 countries
2. Bang1adesh Rice Cutting to 6.9 Storage losses 2.6%
consumption
Wheat Cutting and
threshing 1.7
3.India Rice Storage 4.3 Preventab1e 10ss 3.2%
Wheat Storage 1.5 - 4.3
4. Indonesia Rice Standing crop 3.8 A further 4.5% remains uncut
to threshing much of which is recovered during
gleaning
5.Korea Rice Cutting to 11 Preventab1e 10ss 7%, storage
consumption 10ss 2-3% (2% preventab1e)
6.Ma1aysia Rice Standing crop 7.3 All operations except drying and
to consumption mi11ing. Storage 10ss 1.2% -
data based on farmer question-
naire.
7.Nepal Rice Harvesting 1.2 Figures reported as incomp1ete
Threshing 1.9
~
8.Nigeria Sorghum Storage 4
Cl
& Mi11et :Il
m
m
9.Philippines Rice Shattering before be10w 1 r
m
and during cutting -<
10. Zambia Maize Storage 2 - 5
 Sources: -i
:c
1. M.P. Miracle, Maize in Tropical Africa, University of Wisconsin Press 1966, p. 243. m
::0
2. M. Greeley, Rural Technology, RuralInstitutions and the Rural Poorest: The case of rice or
processing in Bangladesh. Paper presented at the Post-Harvest Technology Workshop, IARI, m
Delhi, Jan. 1981, p. 10. o
3. R. A. Boxall et al. The Prevention of Farm-Level Food Grain Storage Losses in India: ."
"
A Social Cost-Benefit Analysis, IDS Research Reports, 1978, p. 58, and B. P. Khare, o
Cf)
Insect Pests of Stored Grain and Their Control in Uttar Pradesh, G. B. Pant University -i
of Agriculture and Technology Pantnagar, Research Bulletin No. 5, 1972, p. 1 ±
>
::0
4. D. Gaiser, A Brief Summary of Paddy Loss in Indonesian Rice Post-Harvest Systems. Paper
presented at the Regional Grains Post-Harvest Workshop, Philippines Jan. 1981, p. 6.
<
m
Cf)
5. C. J. Chung, Post-Production Rice Systems in Korea, Final Report of Phase 11, College of -i
."
Agricultuxe, Suweon, 1980, pp. 4-8. r
6. N. H. Mohamed, Rice Grain Losses from Kada Area (Malaysia): An Overview vis-a-vis Traditional >
Z
Storage System. Paper presented at the Regional Grains Post-harvest Workshop Philippines Z
Jan 1981, p. 11. Z
G')
7. S. K. Bhalla and T. B. Basnyat, Post-Harvest Technology and Its Impact on the Rural Poor
in Nepal. Paper presented at seminar on Rural Technology, RuralInstitutions and Rural
Poorest, Comilla Feb. 1981, p. 7.
8. P. H. Giles, The storage of cereals by Farmers in Northern Nigeria, Trop. Agric. (Trin)
41(3):197-212, 1964.
9. R. B. Calpatura, Variety-Maturity and Length of Straw Cutting Interaction in the Grain
Losses of Rice During Harvesting. Paper Presented at the Regional Grains Post-Harvest
Workshop, Philippines Jan. 1981, pp 1-2.
10. J. M. Adams and G. W. Harman. The Evaluation of Losses in Maize Stored in a Selection
of small farms in Zambia with particu1ar reference to the Development of Methodology,
Tropical Products Institute, 1977, p. 4.

U1
N
522 M.GREELEY
First, all the figures quoted are averages based on a number of
experiments, whereas high losses, if they are based on any sort of
measurement, are often selected examples of extreme cases that should
be used to illustrate the worst, but often get quoted as the usual.
[Ideally estimates should specify the standard deviation and sampIe
size to allow calculation of a confidence interval, which would in-
dicate the reliability of the estimate. This is only available for.
LDCs (India (1.33) and Bangladesh (0.63» of the cases in the tables,
but if these are typical then in ninety-five percent of cases further
estimates of losses will be very close to the obtained mean.]

Secondly, when grain is periodically removed there is often

confusion over the summation of different sampIe estimates made over
the period of storage (19). When no allowance is made for periodic
removal and the final sampIe result is taken as the total loss figure
there is an upward bias in the estimate. Grain stored at farm level
for consumption is usually removed gradually over the storage period;
an even removal and a smooth incremental loss rate would give an
average true loss of only half the final sampIe loss. Where losses
increase exponentially the bias will be even greater.

Thirdly, insect damaged grains are sometimes regarded as lost

grains when the damage is only partial (20). For most cereals esti-
mates of damage are a poor guide to actual weight loss.

Fourthly, the poor supervision or organization of fieldwork will

frequently lead to overestimates. Farmers are unlikely to add grain
to a store long after harvest but may very weIl have to remove some
in the absence of field staff; unless staff are weIl trained and
weIl motivated. these removals will not be accounted for and esti-
mates of loss will be larger by the quantity removed. Similarly if
sampIes, once collected from a farmer's store are not handled care-
fully, they can suffer further deterioration between the time of
sampling and time of analysis. There are several problems like these
which make accuracy difficult and expensive to achieve. Avoiding
them often results in other biases, notably the selection of sampIe
stores primarily from large farmers who can cooperate with less in-
convenience but consequently may remove grains less frequently, store
for a longer per iod and take less care than the average farmer,. there-
fore suffering untypically high losses.

These methodological problems are only examples and there are

several more areas - selection of stores, grain sampling techniques,
simulated trails, observer bias etc. - where errors in design tend
to bias results upwards.

When studies are carefully designed, like those quoted in Table

1. they involve extended fieldwork and the one characteristic most
commonly distinguishing high from low estimates is the time spent in
the field. Field experience suggests that even though farmers'
THE ROLE OF POST-HARVEST PLANNING 523

stores are often crude in construction the critical considerations

of store hygiene, temperature and humidity control are competently,
often ingeniously, catered for. Anthropologists (21) have been a
useful source of knowledge here though their papers are rarely
accessible to - or their approach acceptable to - the more techno-
cratic post-harvest sciences.

The cases reported here confirm studies (22) on other aspects

of peasant economy in showing that poor farmers are efficient. The
levels of storage investment that would be justified on the basis
of los ses alone are small and in many cases current practices are
optimal in that the costs of loss prevention are greater than the
value of losses.

However, they do not always preclude carefully selected low cost

innovation, and two of the studies quoted (India and Zambia) recomm-
ended specific extension efforts to encourage small modifications of
existing practices; (none of them suggested priority to the intro-
duction of new types of store such as metal bins). The prohibiting
costs in these programmes of modification are in the training and
motivation of field staff and village artisans to extend the concepts
(of modified cribs, rodent-proof platforms etc.) rather than the
actual cost of construction/manufacture. In practice as with much
'appropriate' technology (23) the marginality of benefits and the
organizational strains imposed on government bureaucracies as well
as peasant economy result in little progression beyond pilot pro-
grammes.

The adoption of metal bins where total capacity is being in-

creased or where large and untypical farmers are the purchasers, of-
ten for a variety of non-economic reasons, in parts of India and in
some African countries does not contradict these conclusions; apart
from the subsidies they often receive, there are cost considerations-
labour, materials, loading/unloading time, durability, and space/size
criteria - which are independent of 10ss levels; there are also
conditions of storage - long per iods for soft grains such as wheat
in the indian Punjab - where losses may be on average higher than
the results in Tab1e 1 and high enough to make moderate investments
profitable. (Although the changes in farmer practices required may
lead to unanticipated results as happened in Andhra Pradesh, India;
farmers using meta1 bins did not always dry their paddy down to the
required 12 percent as opposed to their usua1 14 percent moisture
content and the lack of aeration led to premature germination of
seed paddy.) These programmes are exceptions however and the evi-
dence overwhelmingly refutes the stereotyped characterization of
high food losses in farm-level cereal storage.

Of all the post-harvest operations, storage has received the

most attention in the literature and in R & D programmes - and this
is why we have singled it out - but it is threshing, and milling
524 M.GREELEY

(grinding) where technical change is being most rapidly promoted

and therefore where the assumptions regarding its effects are most
critical. [Crop drying has received much attention from engineers
but there is little evidence on drying losses which is a topic we
take up in the next section.]

Evidence from empirical research suggests that in these cases

also the high losses hypothesis is inappropriate. To illustrate
Threshing: The evidence on threshing losses (Bangladesh 1.79%,
Indonesia 2.38%, Malaysia 2.15% and Nepal 1.87% - the Korean data
is not sufficiently disaggregated) in traditional beating and/or
bullock trampling systems is restricted to rice, excepting the
Bangladesh study which gives a figure of 1.13% for wheat. Variation
around these averages is due to differences in straw length, variety,
maturity, condition of the threshing floor/tub and care of the farmer
which affect the amount lost in the mud and cracks of the floor and
the amount remaining unthreshed on the straw. Table 2 (part A)
shows the variability in loss in Bangladesh and also that the intro-
duction of an 'improved' technique, the pedal thresher, actually
increases food losses. The reduction in labour from 110 to 20 man-
hours per ton (and the speeding up of the operation when supervisory
time and space are scarce) outweighs the costs of these additional
los ses though and the thresher is spreading into areas where short
stiff-strawed HYVs are grown. Clearly, the level of losses was not
the determining factor in the adoption decision. The chief cause of
loss is grain remaining unthreshed, because typically the care taken
in drying out, covering, compacting and sweeping the threshing floor
minimize, to immeasurable proportions, the levels of scattering loss.
These. unthreshed grains are in principle easily recoverable simply
by taking more time in the threshing operation or by hand-stripping
the straw - which poor farmers sometimes do when they purchase straw
for livestock feed. The constraint is the time involved not the
absence of a technique; the loss is preventable but it is not econom-
ic to do so.

Milling For non-rice crops where the whole grain is ground and
where - in village-level systems - no separations are made, physical
losses are negligible, but the estimation of rice milling losses is
complicated by the difficulty in establishing appropriate criteria
for measuring outturn. The percent total rice recovered and the
percent unbroken rice are the two criteria commonly used; some
authors (24) have combined them to estimate a composite index of
milling efficiency. These are arbitrary judgements though for the
degree of polish and the level of brokens have price effects which
reflect consumer preferences and which are unrelated to nutritional
(food-value) losses. The Korean study for example shows a total
loss in milling of 4-6% but of this 1.5% is due to poor pre-milling
quality control which should properly be included elsewhere, 2% is
due to consumer preference for overpolished rice and only 1.5% of
the loss is due to the poor performance of the milling equipment.
THE ROlE OF POST-HARVEST PlANNING 525

Table 2. Technica1 Change and Food Losses in Bang1adesh

Labor
Per cent Samp1e hrs Standard
10ss size per ton error
A. Threshing
Bu110ck treading Long 2.54 40 0.29
(Broadcas t) straw

Hand beating fo110wed Short 0.60 38 110 0.08

by bu110ck treading straw
(Transplanted)
Long 1.45 20 0.28
straw

Pedal Thresher 1 Short 1.82 10 20 0.39

(Transplanted) straw

Long 3.49 9 0.27

straw
Labor
Per cent Per cent hrs Samp1e
milling ~ie1d broken grains Eer ton size
B. Husking and 2
Polishing

Dheki 72.02 28.28 240 20

(0.46) (4.50)

Eng1eberg-type 69.94 30.54 2.8 20

hu11er rice mi11 (0.33) (4.52)
Laboratory model 71.87 8.89 20
modern milling (0.28) (1. 91)

1
There was only one case of a broadcast crop being pedal threshed
and the measured loss was 7.3 per cent.
2
Figures in brackets are standard errors.

Source: p. 26, M. Gree1ey, Rural Techno10gy, RuralInstitutions and

the Rural Poorest: The case of rice processing in Bang1a-
desh, 1981, plus previous1y unpub1ished data on labor use
from the Institute of Development Studies Post-Harvest
Project.
526 M.GREELEY
The most satisfactory criterion, reflecting economic loss to
the producer, would be the effect that poor milling has on price
except that in the majority of cases subsistence production prevails
and the rice is consumed on the farm. There are no satisfactory
criteria for measuring absolute loss in this situation but comparisons
between methods can still be usefully made. In Bangladesh (see
Table 2, part B) the change to Engelberg hullers from manual methods
increases losses by 2% measured by the reduction in milling yield
and has a negligible effect on brokens - increasing them from 28 to
30 percent. As with threshing, the reduction in labour costs more
than compensates for the reduction in yield. The traditional system
also compares favourably to modern milling methods (rubber roll
huller and truncated stone cone polisher) in total eutturn because
of consumer preference for undermilled rice. The high level of
brokens has no economic implications where rice is consumed on
the farm - and indeed in the market there is little premium for whole
rice because of these consumer habits. At present most of Bangla-
desh's off-farm milling capacity is in Engelberg-type hullers which
continue to increase in number and the transition to rubber-roll
hullers with separators and polishers is in an early stage compared
to most Asian countries. The rate of transition is determined by
the commercial opportunities to invest in larger scale processing
units which are limited by the size and regularity of paddy supplies.
There is no evidence that the change from manual methods can increase
food availability or that the direction and the rate of technical
change are influenced by differences in the level of food-losses
which are a marginal influence on the economic viability of different
milling systems.

SECTION 111

Most of the reports cited in Table 1 are of very recent

origin; preliminary reports from continuing research programmes
support their findings and a new concensus is developing; the myth
of high average food losses in farm-level post-harvest systems is
being slowly but firmly dispelled. A retrenchment because of this
would be wrong for at least two reasons. First, however, inaccurate,
it was the almost universal belief of high los ses that attracted
the funding for research and which has in turn helped in assessing
more carefully what needs to be done; prior to this exposure the
progress on improving loss-assessment methods had been slow and
systems improvement was based on educated guesswork. The research
results are still providing essential information and if anything
their significance is enhanced because they are so different to the
preliminary hypothesis. Secondly, the evidence does not show that
farm-level post-harvest technical change is unimportant only that
food loss as a motivating force iso Post-harvest operations are
still the major source of rural employment outside of direct crop
production and collectively the ancillary activities associated
THE ROLE OF POST-HARVEST PLANNING 527

with them are commonly the largest rural industry. With the
quantity and quality of field data now becoming available it is
possible to identify what changes in the system are most important
for development and to reassess future priorities. Three issues are
of obvious relevance to this reassessment: the opportunities for low-
cost innovation, the effects of cost-reducing technical change and
the implications of increased food output.

Low Cost Innovation

Even if food losses are low, the gains from prevention of small
losses will be large if they are widespread. This has anyway been
the position adopted by many (25) who have been unwilling to rely
upon old statistics of dubious value and have taken a 'conservative'
view of losses; the argument has been (26) that if losses are ten
percent and we can save only half of them there would be an extra
40-45 million tons of food available annually worth over $7 billion ••.
and therefore investment of $1 billion at least to achieve this is
a priority.

The evidence of field studies shows that even a five percent

reduction is optimistic; when low losses are spread across many
operations, was tage in any one operation is small and the effective
margin for improvement is often negligible. Moreover, the statistic
that matters is not total loss but preventable loss. Even with a
very sophisticated post-harvest system it is virtually impossible
to restrict losses to zero.

Assuming a technique is available, the decision to adopt will

depend upon the costs of and benefits to the farmer; with small
grain savings only a low cost improvement will yield a positive
return. In storage this will normally me an modification to
existing structures or use of insecticides; the difficulties posed
for extension and training by this type of programme have been
discussed briefly above in the context of the Indian and Zambian re-
sults and similar difficulties have been identified in Bangladesh,
Ghana, Kenya, Mexico, Senegal and Tanzania (27). The Tanzanian case
study was unique amongst these in trying to promote a self-help
programme based on local farmers' understanding of storage problems
developed through group discussions. This was very much a pilot
programme - and has remained so since the response was limited
though similar programmes may be developed elsewhere - but a further
binding constraint was the staff training requirements to operate
Frierian or similar methods on a bigger scale. This staffing and
management problem is a recurrent theme in self-help programmes and
in practice prohibits the approach for national strategies of exten-
sion. With well-trained and motivated extension staff the Indians
have demonstrated through their Save Grain Campaign (28) that more
traditional extension methods can have some effect but their exper-
iences also show that it is easier to sell finished products, such as
528 M. GREELEY

metal bins, to less poor and prestige-conscious farmers than it is

to motivate small farmers with scarce resources - including their
own labour time - to invest in marginal improvements. Cash con-
straints - and economic rationality - prevent these farmers from
costly innovation but with minimallosses any form of innovation in
storage is low on their prlorities. Questionnaires on farmers'
attitudes sometimes give different results to the actual outcomes
when extension staff begin work but there is now sufficient experi-
ence with storage extension to know that it suffers from vapidity
more than rapidity.

The discussion of these storage programmes has assumed, reason-

ably, that low cost improvements are available but in other opera-
tions, such as crop drying, this is not always the case. Led by
international research institutes, there can be few departments of
agricultural engineering that have not tried to develop crop dryers
for use at farm-level. Wet grain - rice in particular - is commonly
cited in their papers as a major cause of deterioration and food
loss in the tropics yet 'farm-level' driers are extremely uncommon
off campus. The engineers have been successful technically - the
principles of safe crop drying are weIl established - the failure
has been to identify economic applications.

The frequency with which the same farmer suffers sufficient

post-harvest damage due to rain to warrant investment in a new
technique performing a function now provided free by the sun is the
critical and very complex question that engineers need to answer.
Experience from Bangladesh (29) in measuring wet season los ses has
shown that this condition is very infrequent and even co-operative
ownership of driers has proved uneconomic there (30). For many
farmers the quantity produced is smal1 a110wing discretion over
harvesting dates, use of rooms for spreading the threshed crop
and use of short periods of sunshine. The economic risk is further
reduced because most production is retained for self-consumption
where qualitative deterioration has no cash imp1ications and, on
small farms, grain for sale is usually marketed at the ear1iest
opportunity, (i.e. when it is still wet) to meet cash requirements.
These practices all reduce the potential benefits from owning or
using driers and most traditional designs, requiring blowers,
heaters and purchased fue1, are hopelessly unprofitable at farm-
level. With the high 10ss-high cost solution discredited, engineers
have been examining more carefully the opportunities for very low
cost improvements, through solar drying (31) or natural draught
drying (32) using crop residues for fuel; these designs have created
a new generation of problems re1ating to technica1 efficiency,capac-
ity, space requirements and operating skills and even in situations
where losses would justify a low-cost innovation these technical
difficu1ties have prevented any widespread app1ication.

The experience with innovation in storage and drying leaves

THE ROlE OF POST-HARVEST PlANNING 529

little room for optimism about low-cost solutions for low-loss

situations. There is still a requirement for some carefully located
research to explore areas where these general conditions may not
apply but the efficiency of labour-intensive subsistence production
greatly limits these opportunities.

Cost Reduction

Unlike crop production, where innovation increases total output,

innovation in post-harvest operations cannot increase output except
by reducing losses; with low food losses the profitability of techni-
cal change depends on cost reduction. This has two implications of
general significance in planning technical change. First, since
traditional post-harvest practices are extremely labour-intensive,
cost-reducing change will necessarily be labour replacing. Apart
from storage structures and hand pounding equipment there are no
major items of equipment and even these two often only require labour
time rather than capital investment. Obtaining increased labour use
would depend upon innovations such as the use of locally-produced
crop driers which, with low food losses, are not economic.

In situations of labour shortage or where family labour is

being released for other productive tasks displacement will have
no costs. Frequently however, post-harvest labour is a substantial
source of earnings for the landless - especially in South Asia; the
Bangladesh study identified reduced earning opportunities for land-
less women as the chief cost of the change from traditional to
mechanical milling. We have seen (Table 2, part B) that the change
increases food losses but that it also reduces the labour hours per
ton from 240 to 2.8. With the constraints of purdah only a small
number (about 8%) of rural women work for wages but in their house-
hold they contribute over twenty-five per cent of family income, half
of which was traditionally earned through rice processing (33). The
larger farms that employed these women are the first to start using
rice mills - which are increasing in number as the rural electrifi-
cation programme develops - and the lost jobs threaten the survival
of these anyway very poor families. Alternative work places are
difficult to develop because there are social restrietions on female
mobility and lack of assets prohibits productive employment off the
farm or homestead.

Contrary to the usual assertions, the implications of technical

change are a reduction in food availability overall and a reduction
in the earnings and therefore food-purchasing power of the poorest.
This is a particularly extreme example in a very poor country but
labour-displacing technical change is a general feature of post-
harvest innovation. Changes in storage structure from units locally
produced by village artisans to factory-made units, typical of the
thrust of current extension activity, also reduce labour earnings
for a minority and poor group.
530 M.GREELEY
Whilst some technical change, such as pedal threshing in
Bangladesh, can solve genuine seasonal labour scarcities even in
surplus labour countries the implications for labour use need
careful evaluation in each case. The problem of hunger in
developing countries is shortage of purchasing power as well as
production and labour-displacing post-harvest innovation will reduce
purchasing power.

Secondly, with a few exceptions such as the hand-held maize

sheller, cost-reducing change will also change the ownership pattern
of post-harvest equipment. The introduction of capital equipment -
threshers, mills, perhaps driers eventually - allows and sometimes
requires them to be opera ted on a custom hire basis; in other
words, the potential throughput, or with lumpier investments such
as mills, the minimum profitable throughput, is larger than the
output of most individual farms. The significance of the change
in ownership is the opening up of opportunities it provides for
entrepreneurial activities that did not exist so long as post-harvest
operations were without larger units of capital equipment.

The rate of adoption will depend upon the cost reductions

obtained; in addition to labour saving this will be influenced by
the effects on farm management and transportation. The easing of
farm management by reducing the time per operation is an additional
benefit when space (for threshing particularly) and supervisory time
are binding constraints; but the critical adoption issue is trans-
portation. Costs per unit of output in threshers and mills includ-
~ the additional transport costs (for the machine or the grain)
have to be lower than traditional practices to allow adoption.
Therefore the development of the rural infrastructure particularly
roads (and canals/railways) and seasonal and size variation in
transport costs will jointly determille the rate of adoption. Where
these developments have oecurred post-harvest meehanization has been
a major - and in some eases the major - source of rural agro-
industrial growth. This has been particularly true for the poorest
LDC economies where the shortages of purehasing power have preeluded
entrepreneurial initiatives in manufacturing; crop processing does
not require the development of a market for onee value added in the
new techniques is greater than in traditional practices, a captive
market exists. Moreover, value added in post-harvest operations
varies between twenty-five and forty-five percent of total value
added from seed to consumption; therefore, decisions on support
(subsidies/eredit/fiscal benefits) and control (manufacturing
and operating licenees/zoning/import restrictions) of post-harvest
technology ean have a sweeping influenee on the pace and structure
of rural growth.

The separation of proeessing fram production also allows the

development of programmes for the landless by supporting their,
individual and collective, ownership of equipment like threshers
THE AOlE OF POST-HAAVEST PlANNING 531

and mills. In land-scarce economies, recent analyses (34) argue that

these sorts of initiative are necessary constituents of rural growth
because of their demand linkages. This type of involvement can turn
the cost-reducing (labour-displacing) nature of technical change to
the advantage of labour and actually increase their share of income
from post-harvest activities. Programmes of this sort are now opera-
ting (35) and their further development may weIl be the most important
dynamic of post-harvest planning.

Increased Food Output

The efficiency of traditional post-harvest systems depends to

a large extent upon the ordinary (careful) management of small
quantities of output by farmers with adequate labour resources and
few fixed assets. As land productivity increases it becomes increas-
ingly difficult to maintain these levels of efficiency. The fixed
assets, - particularly farm-yard space, supervisory time and draught
power for transport and threshing - are insufficient to maintain
throughput; at the same time labour bottlenecks appear bidding wages
up and making it more expensive to get the same job done. These
high er marginal costs lead to larger marginal food losses - such as
inadequate threshing, because it is now too expensive to avoid them,
and thereby increase the return from cost-reducing innovation. High-
er costs and delays also encourage producers to seIl grain early -
before drying and at low harvest time prices - and in so doing they
pass the risk of deterioration to traders and the public sec tors
procurement agencies.

The severity of these effects depends upon the magnitude of

production increases and relative scarcity of the fixed assets and
labour. Evidence from the Indian Punjab (36) and from the MUDA scheme
in Malaysia (37) shows that they can result in high losses; threshing
losses measured for rice in the MUDA scheme were over nine percent.
Harvesting more susceptib1e HYVs in the rainy season with labour
shortages is characteristic of a substantial part of the increased
land productivity in Asia and in some African countries, and the
experience of MUDA is being repeated now even in parts of Bang1adesh
where sma11 farm size and surplus labour reduce the risk of such
105ses. In these situations the adoption of new techniques such as
mechanical threshers or even combine harvesters to reduce 1abour
costs can, but may not a1ways, also reduce losses. However, farmers
can and do re1ieve both their 1abour and 10ss problems by se11ing the
crop. The marke ted surplus will anyway normally increase by more
than the increase in production and as early experience has shown
the most severe risks of deterioration occur with this surplus,
particularly when it is wet.

Commercia1 traders respond to price incentives and their

investment in better drying and mi11ing comp1exes will depend on
the price premiums for good qua1ity grain/f10ur. This is not the
532 M. GREELEY

case with the public sec tor food corporations who are often operating
support prices and whose commercial incentives are remote from daily
transaction. Faced with sudden sharp rises in marke ted surplus, they
become involved in market operations lacking the storage space, the
equipment, technical staff and management capacities to maintain good
quality stocks. The public sector is usually a residual element in
the system and provides the backs top in periods of glut. At these
times, the level of losses in stocks surplus to their carrying ca-
pacity can be very high because of the difficulties in obtaining
reasonable temporary storage. The evidence on los ses is very thin
because these agencies are sensitive about their performance and
frequently they only quote selected accounting records of loss - i.e.
losses not within the corporation's allowable losses. However, these
problems are weIl known and al ready the subject of intensive develop-
ment assistance, especially in the construction of public sec tor
storage capacity.

The low levels of farm-level storage loss provide an alternative

solution to the problems of surplus. By the use of seasonally step-
ped incentive prices that provide a margin for storage costs, risk
and a small profit farmers could be persuaded to hold stocks until
after the immediate post-harvest period. Alternatively the grain
could be purchased by the government but held as a lien (a prior
claim) by the farmer (38) until storage space was available. Such
schemes would take advantage of the low levels of farm-level food
losses and save the investment costs in large-scale capital intensive
central storage facilities. These are suggestions that would only
operate satisfactorily in times of surplus but that is when they are
needed. Decentralization offers more effective food security at
lower cost and with the continuing need for large public sector food
stocks offers a viable long-term po1icy strategy for future post-
harvest p1anning.

REFERENCES

1. K.D. White, "Roman Farming, v Thames Hudson (1970), pp. 196-97.

2. "Reducing Post-Harvest Food Losses in Developing Countries,"
FAO (1975), pp. 2-4 and Appendix 11. The Tropical Products
Institute, London, is one of the few other organizations
that has been regularly active in loss prevention programmes.
3. M.C. Bourne "Post-Harvest Food Losses - The Neg1ected Dimension
in Increasing the World Food Supp1y," Cornell International
Agriculture Mimeograph (1977), pp. 2-3.
4. Examples are numerous since they preface most papers on the
subject and a short list of examples from government, volun-
tary agencies, international donors and private companies is
given on pp. 4-5, "Rural Technology, Rura1Institutions and
The Rural Poorest: The case of rice processing in Bangladesh,"
(Martin Greeley 1981).
THE ROlE OF POST-HARVEST PlANNING 533

5. James Boulware, USDA attache in New Delhi drew attention to

this anomaly of Indian reports, cited in Lester Brown,
"Seeds of Change," Pal1 Mal1 London (1970).
6. These are figures originally published internationally in
"Rice Post-Harvest Problems in Southeast Asia," International
Food Technologists Meeting, Philadelphia, June 1977, and else-
where they are cited as IRRI and FAO figures. They give total
maximum losses of 31.43%, but several papers, e.g. H.A. Parpia,
More than Food Would be Saved, Ceres, December 1977 report
37% losses because they fail to take account of reductions in
the quantity available due to loss at earlier stages.
7. For example, Final Report of the Expert Committee on Storage
Losses of Foodgrains During Post-Harvest Handling, Government
of India (1971), unpublished.
8. See M. Sriramalu, Studies on the Varietal Resistance of Paddy
to Lesser Grain Borer, Rhizopertha Dominica (Fab.), MSc.
Thesis, Dept. of Entomology, Agricultural College, Bapatla,
1973 (unpub.); S. Srinivasan, Studies on Degree of Infest-
ation by Rice Weevil Sitophilus Oryzae L. in Different
Varieties of Paddy and Rice, MSc Thesis, Dept. of Entomology
Agricultural College, Bapatla, 1972 (unpub). Both demonstrate
the need for plant breeders to take account of resistance to
store insect pests by showing how some modern thin-husked
rice varieties are more susceptible to the rice weevil
(Sitophilus Oryzae L.) and the lesser grain borer (Rhizopertha
Dominica (Fab.». This is supported by field observations of
the effects of this factor on the timing of sales of, e.g.
RP4-14, by paddy cultivators in West Godavari, A.P. Though
see also, "Resistance to Storage Insects in Wheat Grain,"
IARI Research Bulletin No. 28 (1980).
9. K. S. Gill, "Wheat market behaviour: emerging problems of wheat
marketing in Punjab and Haryana post-harvest period," Punjab
Agricultural University, Ludhiana (undated).
10. For example, "Comrnodity Storage Conditions in Bangladesh," a
staff report to the Subcomrnittee on Foreign Assistance of
the Committee on Foreign Relations Uni ted States Senate,
(1976).
11. For example, "Protec ting the World' s Crops ," Shell Brief ing
Service (1977).
12. For example, Robert Satake, "Status of the Rice Milling Sector,"
AMA IX, No. 2, Spring 1979.
13. For example The Comilla Co-operative Karkhana Ltd., Ranirbazar,
Comilla, Bangladesh manufacturing pedal and mechanical
threshers.
14. 'Appropriate' is really shorthand here for low-cost, simple to
use, local skill and raw material intensive techniques.
A good example of the approach is "Small Farm Grain Storage,"
Appropriate Technologies for Development, Volunteers in
Technical Assistance, Vita (1976).
534 M. GREELEY

15. India has been very active both in research with the Indian
Grain Storage Institute and the All India Co-ordinated
Post-Harvest Technology Scheme and in extension with the
Save Grain Campaign. In Africa, Zambia, Kenya and Swazi1and
all developed extension programmes quite early on and in
Nigeria the West African Stored Products Research Unit has
made important contributions to research from an early date.
16. Of special importance was the preparation of K.L. Harris and
C.J. Lindblad, "Postharvest Grain Loss Assessment Methods,"
USAID. (1978).
17. For example, Cost-Benefit Analysis of Crop Storage Improvements:
A South Indian Pilot Study, IDS Discussion Paper No. 56,
University of Sussex (1974), by M. Lipton.
18. S.K. Majumdar and H.A.P. Parpia, Possible Losses of Foodgrains
in India, 1966 reprint from Vijnan Karmee - The Journal of the
Association of Scientific Workers in India, Vol. 18, No. 4.
19. Bourne, op. cit. pp. 17-18.
20. A.M.A. Karim and H. Rashid, Extent of Loss of Boro Paddy during
Post-harvest Operation: A Study Conducted in Bahadurpur Village
of Mymensingh District, Agri-Varsity Extension Project,
Bangladesh Agricultural University, Publication No. 10. (1979).
21. H. Guggenheim, Who is the Loser in Post-Harvest Losses? The
Wunderman Foundation, (undated) --
22. T.W. Schultz, "Transforming Traditional Agriculture," New Haven,
Yale UP (1964).
23. Appropriate Rural Technology: Recent Indian Experience with
farm-level Foodgrain Storage Research, Martin Greeley, Food
Policy, (February 1978). --
24. The ~echnical and Economic Characteristics of Rice Post-produc-
tion systems in the Bicol River Basin, Bicol River Basin
Deve10pment Program (1978).
25. "Postharvest Food Losses in Developing Countries," National
Academy of Sciences, Washington (1978).
26. Most recently presented in South, No. 3 Dec. 1980, p. 27.
27. A. K. Fazlul Huq, Rice in Bangladesh: Estimation of Food Losses
in Farm-level Storage at a Workshop sponsored by the Food
Science and Technology Division, Bangladesh Council of
Scientific and Industrial Research, Dacca December 1980. p.12-
13; Post-harvest Food Losses in Developing Countries, Develop-
ment Digest Vol VIII No. 4 Oct 1980, p. 107; W.D. Rolston, .
The Post-Harvest Food Grain System in East Africa (A Kenyan
Case Study), International Developme~t Research Centre,
Edmonton (1975), p. 10; W.D. Rolston, The Post-Harvest Food
Grains System in West Africa (A Senegalese Cast Study), IDRC,
Edmonton (1975), pp 11-14; Mexico, the Post-Harvest Maize
System in Two Pider Micro-Regions, The World Bank Rural
Development Division, (1978), p. 14 and Appendix 3; Appro-'
priate Technology for Grain Storage, Report of a Pilot Pro-
ject, Community Development Trust Fund of Tanzania (1977),
Section 2.
THE ROLE OF POST-HARVEST PLANNING 535

28. K. Krishnamurthy, Save Grain Campaign - Objectives and Plan of

Work, Post-Harvest Technology Workshop, New Delhi, Jan 1981.
29. M. Greeley and S. Rahman, Wet Season Post-Harvest Food Losses,
Paper Presented at the Post-Production Workshop on Food
Grains, December 1980.
30. Danida Drying Project in Comilla, Project Report 1974.
31. R.H.B. Exell , Basic Design Theory for a Simple Solar Rice Dryer,
Renewable Energy Review Journal, Voll. No. 2, Jan. 1980.
32. Rice Post Harvest Technology Project, 1978 and 1979, Report,
TPI/BRRI, Dacca Bangladesh (1980).
33. S. Begum and M. Gree1ey, Women, Employment and Agriculture:
Notes from a Bang1adesh Case Study, IDS, University of
Sussex (1980).
34. G.D. Wood, How the Interests of the Rural Poor Can be Included
in the Second Five-Year Plan, Dacca (1980).
35. Through the Bangladesh Academy for Rural Development, Comilla.
36. K.S. Gill, Post-Harvest Market Technology for Cereals (Paddy
and Wheat) - Needed Improvements (The Punjab Case) , paper
presented at Rural Technology, RuralInstitutions, and the
Rural Poorest, Comilla, Bangladesh, Feb. 1981.
37. D.J.B. Calverley, P.R. Street, T.J. Cree, D.A.V. Dendy, Post-
Harvest Losses of Rice in Malaysia, Conference on Food and
Agriculture, Malaysia 2000 (undated).
38. M. Lipton, Post-Harvest Techno10gy in the Context of the Reduc-
tion of Hunger, Workshop on Farm-Level Post-Harvest Technol-
ogy for Prevention of Food Losses, New Delhi, Jan. 1981.
UTILIZATION OF AGRICULTURAL WASTES - SOME GLOBAL CONSIDERATIONS

A. J. Vlitos

Group Scientific & Agricultural Director

Tate & Lyle, Limited
Reading, Berkshire, England

INTRODUCTION

We are living in a World where the future comes sooner than it

used to, and in a World where change is as inevitable as death and
taxation. My lecture this evening therefore will not be devoted
to post-harvest physiology or for that matter to the specific
techniques for converting agricultural wastes to useful products.
Instead I would like to make some guesses about the place of
agriculture, worldwide, say in 20 years' time - by the year 2001.
No one really knows what sort of place the World will be in 20
years time. All we can do is guess. And the guesses have to be
based on what is known today and on trends - and on the likely in-
novations which will occur as the result of the research wh ich is
underway today. So, what sort of world will it be in 20 years'
time?

It will be more crowded, more polluted, shorter of food, a

more expensive place to live in than it is today. Every long-term
global study has come up with the same conclusion. Only the degree
of overpopulation, the amount of pollution and the exact figures of
inflation are at issue between one study and another. The "Global
2000 Strategie Study" which was prepared for President Carter can
be summarized as in Tables 1 to 10.

GENERAL

If the economic gap between the Third World and the developed
world is to become even greater than it is today, one can predict
that certain carbohydrate crops, such as sugar cane, will assume

537
538 A. J. VLiTOS

Tab1e 1. G1obal-2000

1. More peop1e (i.e. 6.35 billion)

2. Poorer
3. More pollution
4. More vulnerable to disruption
5. Outlook for food and other necessities
of 1ife no better than now

(Reference: Global 2000 Report to President

of U.S.A.)

Tab1e 2. Population Growth

Added each year

1975 4 Bi11ion------ 75 million
2000 6.35 Bi11ion------100 million
2030 10 Bi11ion------120 million

(90% of growth in population in the

poorest countries).

Tab1e 3. Wor1d Food Production

% Increase in
Production
1. 1870 to 2000 90% (i.e. 15% global
per capita).
2. Most of this increase goes to countries
which have high per capita food consump-
tion (i.e. 'richer' ones).
3. Per capita consumption in LDC's scarce1y
improves or actua11y dec1ines.

Table 4. Wor1d Food Production

1. Arab1e land increases by 4% by 2000

2. Most increases in food has to come
from increased yie1ds on existing
acreages.
3. Almost all prices to consumer will be
higher.
UTILIZATION OF AGRICULTURAL WASTES 539

Table 5. Fisheries
1. 1970 to 1975--.~ 70 million metric
tons per annum.
(i.e. 60 million tons marine:
10 million tons freshwater).
2. Required to keep pace with projected
increases in population - 115 million
tons per~.
3. Assuming catch of marine and freshwater
fish rises to unlikely level of 100
million tons and that yields from aqua-
culture double (to 12 million tons) -
the 112 million tons is not enough to
provide per capita pro tein from fish
provided today.

Table 6. Estimates of World Forest Resources, 1978

and 2000.
2
Closed Forest Growing Stock
(millions of (bi11ions cu m
hectares) overbark)

1978 2000 1978 2000

U.S.S.R. 785 775 79 77

Europe 140 150 15 13
North America 470 464 58 55
Japan, Australia,
New Zealand 69 68 4 4
Sub total 1464 1457 156 149
Latin America 550 329 94 54
Africa 188 150 39 31
Asia and Pacific
LDCs 361 181 38 19
Subtotal (LDCs) 1099 660 171 104

Total (world) 2563 2117 327 253

Growing Stock
per Capita
(cu m biomass)
Industrial countries 142 114
LDCs 57 21
Global 76 40
aClosed forests are relatively dense and productive forests.
They are defined variously in different parts of the world.
see Global 2000 Technical.Report, footnote, p. 117.
540 A. J. VUTOS

Table 7. Energy

1. By 1990 - energy demand to increase by 58%.

2. Nuclear and hydrosources increase most rapidly
226%.
3. Oi! by 58%.
4. Natural gas by 43%.
5. Goal by 13%.

Oil remains leading energy source through 1990's (46-47%).

Real price of oil increases 65% over 1975 - 1990.

Table 8. Environmental Gonsequences

Impacts On Agriculture : Acceleration of -

1. Soil Erosion
2. Loss of nutrients
3. Gompaction of soils
4.* Increasing salinisation of both irrigated
land and water used for irrigation
5. Loss of high-quality cropland to urban
development
6. Grop damage due to increasing air and
water pollution
7. Extinction of local and wild crop strains
needed by plant breeders
8. More frequent and more severe regional water
shortages - especially where energy and
industrial developments compete for water.

* 6 million hectares per annum (area size of Maine)

lost through desertification. This rate likely
to increase by 2000.
UTILIZATION OF AGRICUlTURAl WASTES 541

Table 9. Impacts of Forest Losses

1. If present trends continue forest of 80uth

Asia, Amazon basin and Central Africa will
be reduced by one-half by year 2000.
2. About one billion people live today in
heavily farmed alluvial basins and valleys
depending on forested mountain watersheds
for their water.
3. Diversity of species lost after extensive
cutting.

Table 10. Impact on Atmosphere & Climate

1. Further pollution virtually certain: (80 2

particulates, N0 2 , CO, etc.).

2. pH value of rainfall dropping from 5.7 to

4.5 due to 8 and Nitrogen oxides combining
with water vapours.

3. Combustion of fossil fuels increasing CO

concentration ("greenhouse" effect). Alters
temperatures and precipitation patterns?

4. Impacts of nuclear waste disposal?

even a more important place in tropical agriculture than they enjoy

today. There are two reasons for this prediction. First1y, the
major increases in population are 1ike1y to occur in the poorer,
tropical nations. The per capita consumption of sugar in these
nations is presently amongst the lowest in the World. The major
increases in consumption are 1ike1y therefore to occur here. There
will be more people eating more sugar in the Third Wor1d and more
of that sugar will be grown there. 8econd1y, most of the tropica1,
Third Wor1d nations are deficient in fossil fue1s. There will be
compe1ling reasons to increase the acreages devoted to sugar cane
for the production of power a1coho1. And by the year 2001 a 1arge
proportion of the a1cohol produced in Third Wor1d countries by the
fermentation of cane juice will go into ethylene producti.on emp10y-
ing sma11-sca1e converters. The ethy1ene thus produced will serve
for the establishment of re1atively sma11-sca1e, almost "cottage-
type" 'downstream' chemical industries - turning out some of the
specia1ity chemica1s (high added-va1ue products) such as plastics,
detergents, 1ubricants, etc. present1y imported into Third World
Countries from Europe and North America. By the year 2001 the cost
of energy and of the 'feedstocks' used in industrial chemical plants
542 Ä. J. VLiTOS

of Europe and North America will be so high that a shift to the

tropics will be attractive. A simple diagram illustrating the key
position of ethanol and ethylene by 2001 in this chain of events is
shown in Table 11.

There are of course several pre-requisites for this to happen.

The first of these is financial investment. Over the past eight
years there has been aglobai shift of wealth from the nations which
manufacture finished products to those nations which supply the
energy needed to manufacture such products. By the year 2001 it is
hoped that the OPEC nations, wh ich by then, shall have most of the
World's money invested, will have placed a major portion of the in-
vestment in Third World agriculture. But the major funding agencies
today encourage most of the investments in Third World agriculture
to go into local food crop schemes rather than into major sugar
projects. Although one can understand why a few years ago it did
not consume the amounts being produced, this situation is unlikely
to be repeated between now and the year 2001 - in view of the
attraction and potential for sugarcane as a raw material for fer-
mentation. I believe a very strong case could be made to the World
Bank, and to other funding agencies, for the encouragement of
"carbohydrate agriculture" in nations which lack fossil fuels and
which also lack any hope of establishing small-scale agricultural
industries unless an energy source is readily available. Sugar cane
and alcohol and ethylene from alcohol offers hope of lessening the
reliance on imported fuels. Encouragement to utilize fermentable
fruit and vegetable residues is another area offering considerable
scope.

A second pre-requisite is, of course, a commitment by the Third

Wor1d nations themselves which will encourage carbohydrate crop
cultivation for domestic consumption as much as for export. Today,
of course, there is a heavy dependence by sugar cane producers on

Table 11. Third World Scenario for Sugar cane

by year 2001

Food
/ (and)
Sugar ca ne -Fermentation- Power Alcohol

Ethanol -------.. Ethylene-------.. Plastics + Lubri-

cants + Other
Chemica1s
(Liquid Fuel)
UTILIZATION OFAGRICULTURAL WASTES 543

exports and on guaranteed prices for exported cane sugar. Indeed

without export markets it is unlikely that the cane industry in
most ACP (African/Caribbean/Pacific) Nations could survive. But is
that likely to be the case in 20 years' time? It might weIl be that
the traditional markets for raw can sugar in North America, in
Europe and in Japan may not be as attractive then as they are now
for a variety of reasons which I will be discussing momentarily.
The likely result of these changes in external markets is that in
20 years' time more cane sugar will be refined in the countries of
origin and most of the cane will be consumed either as food or
used industrially in those countries in which it is produced. The
change is likely to be gradual rather than precipitous - but it will
depend a great deal on the position in 20 years' time of sugar beet
in Europe and of maize in N. America and Europe, and of new, non-
nutritive sweeteners in the industralized nations.

Although the predictions for the growth in population by the

year 2001 in Europe and in North America are not as frightening as
those for the Third World, there will nevertheless be increases in
population and with the accompanying problems, high energy costs
and consequent inflation and increasing pollution. There are good
reasons for expecting sugar beet to remain a major crop in Europe,
but here again there are some pre-requisites to be met. Firstly,
the cost of energy in processing beet sugar is already quite high
and in 20 years' time it could be the most serious obstacle to the
crop's survival - unless of course-a-major research thrust begins
soon which can offer significant savings in the costs of processing.
Another unknown is whether new European varieties of maize, in 20
years' time, can begin to become attractive alternatives to beet -
and whether high fructose syrups will make inroads into European
beet. Consumer pressures could be more significant in Europe in
20 years' time and we should not underestimate them, especially
if new varieties of European maize offer cheap stareh, and if
European farmers find growing maize more attractive than growing
beet, or if a non-nutritive sweetener becomes attractive. So the
variables in the future of beet are more difficult to assess than
those for cane. These are the qualitative scenarios and thus far
I have avoided trying to guess the quantitative impact on total
world production of sugar by 2001. The FAD figures of cane and
beet production for 1979 are shown in Table 12.

By 2001 the chances are that total world production for cane
cou1d exceed the 1979 figure by a minimum of 25% which would me an
737 + 184 = 921 thousand tons but with yie1ds per hectare increased
by at least 15% giving 56 x .15 + 64.5 t/ha. which would still be
weIl below the Australian figure of 75 tons per hectare. Although
improvements in average beet yields especially in Europe are possible
- say from 38 tons/hectare to 45 or 50 tons per hectare - the total
acreage devoted to beet by 2001 could be about the same as now or
slightly decreased.
544 A. J. VLITOS

Tab1e 12. Total produetion (10 6 t) and yie1ds (t.ha- 1 ) of the

major sugar and stareh erops.

These figures are on a erop basis exe1uding the eonsiderab1e amounts

of erop wastes, straw ete. The eerea1 figures exe1ude such erops
grQwn for forage silage.

a) Sugar
Cane Beet
Total Yie1d Total Yie1d

World 737 56 290 32

Deve10ped count ries 70 79 263 32
Deve10ping countries 667 54 26 30
U.S.A. 25 82 23 46
Canada 1 39
Europe 0.3 63 143 38
U.S.S.R. 93 25
Asia 305 52 25 31
S. Ameriea 187 57
Afriea 60 64 16 32
Oeeania 26 75

Another aspeet of the energy erop eoneept is as fo11ows: By

the year 2001 we may see a eompetition for land to be used either
to grow food or to grow erops for energy. In the past when erops
were grown ehief1y for food we measured effieieney by the number of
peop1e emp10yed on the land to feed so many peop1e in the eities.
Thus, as we see in Tab1e 13 a very small pereentage of the popula-
tion in the U.S.A. and Austra1ia feed a mueh larger pereEmtage of
peop1e in eities. This is possible beeause a very high input of
energy is put into agrieultural produetion - fertilizers, maehines,
ete. But when erops are to be grown for energy we won't be able
to put more energy into the erop than we ean retrieve onee the erop
is harvested. Thus, I have deseribed in Table 14 four levels of
agrieu1tura1 praetiee. You will note that the natural eeosystem
requires the lowest inputs of energy. Energy erops of greatest
potential interest will be those requiring the lowest inputs of
energy. That's something else whieh we sha11 have to deal with by
the year 2001.

SUMMARY

Now, the attraetions of uti1izing fruit and vegetab1e wastes

as fermentab1e substrates are many. First of all, the energy inputs
in produeing the erop have a1ready been paid for in most eases.
However, there remains the problem of eolleeting the wastes and
UTILIZATION OF AGRICUL TURAL WASTES 545

Table 13. People Employed in Agriculture on a Global Basis

1965 1980
Percent of Percent of
POEulation POEulation

Worldwide 54 45

Africa 70 67

S. America 45 38

U,S,A, 4.0 2.5

Australia 3.5 2.0

Table 14. Levels of Agricultural Practice and Energy Inputs

~ Characteristics

Natural ecosystem - High yields;

no human influence. forest.

Labour intensive; Subsistence farming -

no inputs from (Third World Countries
external sources. absence of cash inputs,
low yields, shifting
cultivation, destruction
of forests.

Present-day 'average' Yields increased by

agriculture, some increasing inputs of
mechanization reason- energy via more fertili-
able soil and water zers, more mechanization,
management. etc. Yields high in
relation to people
employed.

Optimal production based

on theoretical use of similar to above
incident solar energy.
546 Ä. J. VLiTOS

transporting them to the fermenter. In Brazil an energy-efficient

farming system is being devised in the Mato Grosso which utilizes
trucks and tractors running on 100% ethanol produced from sugar
cane - the distillation being fueled by the cane by-product, bagasse.
One can envisage a collection system for other agricultural wastes
using vehicles running on alcohol. Secondly, most agricultural
wastes are of negative value. The only major cost is in collecting
and transporting them. Thirdly, in most tropical countries the
wastes are rich in fermentable sugars. In the temperate zones many
of the wastes are cellulosic, but the technologies for converting
cellulose to fermentable sugars are being developed rapidly and in
20 years' time will be economical. In Table 15 I have listed some
of the common substrates which are being experimented today. To
these may be added the numerous fruit residues, ranging from pine-
apple to figs, which in future will be sought as sources of alcohol.

Finally, a word about the conversion of carbohydrate wastes to

microbial proteins. As you know the major producers of microbial
pro teins use methanol as the primary substrate. The chances are
that in future methanol will prove too valuable as a liquid fuel to
convert to pro teins and it may prove difficult to compete with
vegetable pro teins such as soya.

Therefore to leave you with the final prediction - negative

value agricultural wastes - especially those right in carbohydrates
will become increasingly attractive as substrates for fermentation
to produce microbial proteins as non-ruminant animal feeds. Oscar
Wilde once remarked that "it is dangerous to prophesy - especially
about the future" - and with that sentiment I humbly agree!

Table 15. Biomass of Interest

Sugar Starch Crop residues

Molasses Cellulose Rice husk

Coffee husk

Bagasse Straw Spoiled grains

Shell-fish Vegetable wastes
wastes
 PARTICIPANTS

Adams, D. O. Postharvest Physiology Laboratory, U.S.

Department of Agriculture, BARC-W, I
Beltsville, Maryland, 20705, U.S.A.
Apeland, J. Department of Vegetable Crops, Agricultural
University, Norway, P.O. Box 22, 1432 AAS-
NLH, Norway
Atwa, A. A. 21 Murad Street, Giza, Cairo, Egypt
Baker, J. E. Postharvest Physiology Laboratory, U.S.
Department of Agriculture, BARC-W,
Beltsville, Maryland, 20705, U.S.A.
Bangerth, F. Institute für Obstbau, University of Hohenheim
Stuttgart 70, Federal Republic of Germany
Barreiro, G. Departmento de Fisiologia Vegetal, Estacao
Agronomica Nacional, Quinta Do Marques,
2780 Oeiras, Portugal
Ben Arie, R. Division of Fruit & Vegetable Storage,
Ministry of Agriculture, P.O.B. 6, Bet Dagan
20-500, Israel
Bohling, H. Federal Research Center for Nutrition
Engesserstr 20, Karlsruhe D 7500, Federal
Republic of Germany
Bruinsma, J. Agricultural University, Arboretumlaan 4,
Wageningen, The Netherlands
Bufler, G. Institut Für Obstbau, University of Hohenheim
7000 Stuttgart 70, Federal Republic of
Germany
Burg, S. P. 3770 Kent Court, Miami, Florida,- 33133, U.S.A.
Cabral, M. L. Departmento de Fisiologia Vegetal, Estacao
AgronomicaNacional, Quinto Do Marques,
2780 Oeiras, Portugal
Carmona, M. Quinto Do Marques, 2780 Oeiras, Portugal
Chalutz, E. Institute for Technology & Storage, P.O.B. 6
Bet Dagan (20-500), Israel
came, D. Laboratoire de Physiologie des Organes
Vegetaux Apres Recolte, 4ter Route des
Gardes, 92190 Meudon, France
Coursey, D. G. Plant Food Commodities Department, Overseas
Development Administration, Tropical
Products Institute, 56-62 Gray's Inn Road,
London, WC2X8LV, England
Cowan, A. M. National Program Staff, PHST, U.S. Department
of Agriculture~ BARC-W, Beltsville, Md.
20705, U.S.A.
Dekazos, E. Department of Pomology, College of Agricultural
Sciences, Votanikos, Athens 301, Greece
547
548 PARTICIPANTS

De Leo, P. Centro Richerche Bonomo, Via Sparano, 141,

70126 Bari, Ita1y
Dilley, D. R. Department of Horticu1ture, Michigan State
University, East Lansing, Mich. 48824,
U.S.A.
Duvekot, W. S. Sprenger Institute, P.O. Box 17, 6700 AA
Wageningen, The Nether1ands
Eckert, J. W. Department of Plant Patho1ogy, College of
Agricu1ture Sciences, University of
Ca1ifornia, Riverside, CA 92521, U.S.A.
Efthimiades, P. Agronomy Department, Agricu1tura1 College
of Athens, Votanikos 75, Athens 301,
Greece
Esquerre-Tugaye, M-T Universite Pau1 Sabatier, Centre de Physiologie
Vegeta1e, Laboratoire Associe au C.N.R.S.
No. 241, 118 Route de Narbonne, 31077
Tou1ouse Cedex, France
Fallot, J. Eco1e Nationale Superieure, 145 Avenue De
Muret, 31076 Tou1ouse, Cedex, France
Feys, M. D. Laboratory of Food Preservation de Croy1aan
42, Catho1ic University of Leuven, B 3030
Hever1ee, Be1gium
Fountas, D. 2 Sarantaporou, Ka11ithea, Athens, Greece
Fuchs, Y. Division of Fruit & Vegetab1e Storage, The
Vo1cani Center, Agricu1tura1 Research
Organization, P.O.B. 6, Bet Dagan, Israel
Goodenough, P. W. Department of Agricu1ture & Horticu1ture,
University of Bristo1, Long Ashton Research
Station, Long Ashton Bristo1 BS 189AF
Eng land
Gorini, D. F. Techno1ogica Dei Prodotti Agrico1i, Via G
Venezian, 26 - 20133, Mi1ano, Ita1y
Gree1ey, M. The Institute of Developmenta1 Studies,
University of Sussex, Brighton, Sussex
BN1 9RE, England
Grierson, D. Department of Physio1ogy & Environment Studies,
Schoo1 of Agricu1ture, Sutton Bonington,
Loughborough, LE12 5RD, University of
Nottingham, England
Haard, N. Department of Biochemistry, Memorial
University, St. Johns, Newfound1and,
Canada
Ha1der-Doll, H. University Hohenheim, Institute fur Obst-
Gemuse and Weinbau, 370, Post Fach 700562
7000 Stuttgart, Federa1 Repub1ic of
Germany
Hartmann, C. Laboratoire De Physiologie De La Maturation,
Universite Orleans, 45017 Orleans Cedex
France
PARTICIPANTS 549

Herregods, M. Tiense Vest 136, B01300 Leuven, Be1gium

Hof tun, H. Department of Vegetab1e Crops, The
Agricu1ture University of Norway, P.O.B.22
N1432 Aas-NLH, Norway
Hussein, A. Department of liorticulture, College of
Agricu1ture, Alexandria University,
Shatbi Alexandria, Egypt
Kader, A. Department of Pomo1ogy, University of
Ca1ifornia, Davis, CA 95616, U.S.A.
Kaminiaris, D. 34 Nikiforou Ouranou Street, Athens, Greece
Karaca1i, I. Ege University, Bornova, Izmir, Turkey
Karaou1anis, G. Food Techno1ogy Institute, Lykourissi
Amaroussion, Athens, Greece
Krogh, P. Department of Microbio1ogy, Royal Dental
College, Ju1iane Maries Vej 30, DK-2100,
Copenhagen, Denmark
Kuru, A. Department of General Botany, University
of Istanbu1, Su1eymaniye, Istanbu1,
Turkey
Kyriakidis, I. Agiou Me1etiou 100, Patisia, Athens, Greece
Larsen, F. Department of Horticu1ture, Ro1ighedsvej 23
The Royal Veterinary & Agricu1tura1
University, DK-1958 Copenhagen V, Denmark
LeNard, M. D. Station D'Ame1ioration de 1a Pomme de terre
et des P1antes a Bu1bes BP 5, 29207
Landerneau Cedex, France
Lieb erman , M. Postharvest Physio1ogy Laboratory, U. S.
Department of Agricu1ture. BARC-W,
Be1tsvi11e, Mary1and, 20705, U.S.A.
Lurie, S. Fruit & Vegetab1e Storage Division, The
Vo1cani Institute, P.O.B. 6, Bet-Dagen
20-500, Israel
Marcellin, P. Laboratoire de Physiologie, Des Organes
Vegetaux. CNRS, 4 ter Route Des Gardes,
92190 Meudon, France
Maz1iak, P. Universite Pierre et Marie Curie (Paris VI),
Physiologie Ce11uaire VER 59, 4 P1ace
Jussieu, 75230 Paris Cedex 05, France
Meghir, H. Pennwa1t France, 1 Rue Freres Lumiers,
78370 P1aiser, France
Monning, A. Institut für Obstbau und GemUsebau. der
University Bonn. 5300 Bonn, Federa1
Repub1ic of Germany
Norris, Kar1 Instrumentation Research Laboratory, U. S.
Department of Agricu1ture, BARC-W,
Be1tsvi11e, MD, 20705, U.S.A.
Papadatou, P. Department of Pomo1ogy. Agricu1tura1 College
of Athens. Votanikos 75, Athens 301,
Greece
550 PARTICIPANTS

Pappelis, A. Department of Botany, Southern Illinois

University, Carbondale, Illinois 62901,
U.S.A.
Passan, H. C. Tropical Products Institute, 56-62 Gray's
Inn Road, London WC2X8LV, England
Pech, J. C. Ecole National Superiure Agronomique, 145,
Avenue De Muret, 31076 Toulouse, Cedex,
France
Petropoulos, A. 15 Ithakis, kypseli, Athens 801, Greece
Petropoulou, S. Eratosthenous 31, Hiliodorou 2-4, Sq; Plastira
Pograti, Athens, Greece
Pontikis, K. A. Department of Pomology, Agricultural College
of Athens, Votanikos 75, Athens 301, Greece
Pratt, H. Department of Vegetable Crops, University of
California, Davis, CA 95616, U.S.A.
Rhodes, M.J.C. Food Research Institute, Coloney Lane, Norwich,
NOR 26G, Uni ted Kingdom
Richardson, D. G. Department of Horticulture, Oregon State
University, Corvallis, Oregon, 97331, U.S.A.
Rokkas, D. Argous 6, Nafplion Argolidas, Greece
Saltveit, M. E. Department of Horticultural Science, School
of Agriculture & Life Science, North
Carolina State University, Box 5216,
Raleigh, NC 27650, U.S.A.
Sapoutzaki, E. Sevastoupolis l50-B, Erythros Stavros,
Athens, Greece
Shou-chun, Q. Shansi Fruit Institute, Taigu Shanxi Province,
Peoples Republic of China
Simon, P. J. Research Station of Gorsen, Brede Akker 3,
B 3800 Sint-Truiden, Belgium
Skytt-Anderson, A. Royal Veterinary & Agricultural University
Dept. of Plant Physiology & Anatomy,
Thorvaldsensvej 40 DK 1871, Copenhagen
V, Denmark
Solomos, T. Department of Horticulture, University of
Maryland, College Park, Md. 20742, U.S.A.
Sovatzoglou, G. Palados 9, Kato Petralona, Athens, Greece
Stoll, K. Station Federale de Recherches, 8820
Waedenswid, Switzerland
Streif, J. Verchusstation Fur Intensivkulturen, 7980
Ravensburg, Federal Republic of Germany
Tobback, P. Laboratory of Food Preservation, Catholic
University of Leuven, De Croylaan 42,
B 303, Heverlee, Belgium
Vlitos, A. J. Tate and Lyle ReSearch Laboratory, P. O. Box
68, Reading RG 5 2BX, United Kingdom
Weichmann, J. Lehrstuhl Für Gemüsebau, Technische
Universität München, 8050 Freising,
Weihenstephan, Federal Republic of Germany
PARTICIPANTS 551

Williams, P. Grain Research Laboratory, Canadian Grain

Commission, 303 Main Street, Winnipeg,
Manitoba, Canada REC 3G9
Woo1house, H. Director, John Innes Institute, Co1ney Lane
Norwich NR4 7UH, England
Xirouhakis, S. Department of Pomo1ogy, Agricu1tura1 College
of Athens, Votanikos 75, Athens 301, Greece
 INDEX

Abiotic elicitors, 303-305 Agriculture (continued)

Abscisic acid environmental impacts of, 540
in corm dormancy breaking, 221 percent of population employed
in fruit ripening, 152 in, 545
Abscisins, in plant senescence, Alar
144 ethylene production and, 387
ACC, see l-Aminocyclopropane-l- fruit quality and, 341-343, 385
carboxylic acid as growth regulator, 335, 341,
ACC synthase, in fruit ripening, 385
46 Alcohol concentration, 233-236
Acer platanoides, 130 in Coxes apples, 241
Acetaldehyde metabolism, in in fruits following alcohol
fruit tissues, 240-243 feeding in air, 243
Adenine nucleotide changes, in metabolism of, 241-243
senescing fruits and Allometric growth, mechanical or
leaves, 74 physiological instability
Aflatoxins in, 4
adverse effects of, 325 Allosteric inhibition, of phospho-
chemical characteristics of, fructokinase, 107
316 Allosteric regulation, by plant
in foodstuffs, 318 enzymes, 103-104
in groundnuts, 319 Alternaria spp., 249, 269, 272,
in maize, 319 280
in milk, 320 Alternaria citri, 251
production of, 315 AZternaria decay, in stored leaves,
Aging, see also Plant senescence 357
as artificially induced plant Ambient pressure, respiration rate
senescence, 141 and, 408
defined, 1 Amino acid-derived phytohormones,
plant cell membrane lipid 145
changes during, 123-137 l-Aminocyclopropane-2-carboxylic
Agricultural practice, levels acid, 144
of, 545 Aminoethoxyvinylglycine
Agricultural wastes, utilization in apple treatment, 346-348
of, 537-546 in ethylene evolution from Trades-
Agriculture catia, 149-150
energy inputs in, 545 in ethylene inhibition, 144

553
554 INDEX

Aminoethoxyvinylglycine (con- Aspergillus parasitiaus, 315

tinued) Atmosphere pollution, effects of,
in postharvest treatment of 541
fruits and vegetables, Atmospheric pressure, hydrostatic
366-373 pressure and, 401-402
a-Aminooxyacetic acid, 144 Autophagic vacuole concept, 32
Anaerobic enzymatic products, Auxins
formation of, 232-233 fruit ,quality and, 338
Animal enzymes, covalent modifi- in plant senescence retardation,
cation of, 105 144-146
Anthranilic acid, in banana in postharvest treatment, 356-
leachate, 252 358
Antimicrobial agents, in posthar- synthetic, 336
vest disease control, AVG, see Aminoethoxyvinylglycine
265-281 Avocados
Antimycin-A, 78 carbon isotope values in ripen-
AOA, see a-Aminooxyacetic acid ing of, 70
APF-l protein, 333 cellulase activity in, 154
Apical pud, of bulbous iris, 208 glycolytic changes in, 72
Apical bulbing, in tulip bulbs, respiratory drift of, 62
205
Apples Bacteria, in postharvest food
alcohol concentration changes losses, 497
in, 233-236, 241-243 Bacterial enzymes, covalent modi-
bitter pit in, 384 fication of, 105
chemicals in ripening of, 386- BA-ethepon treatment, fruit qual i-
388 ty and, 339
decreased acidity of, 256 Bananas
ethyl alcohol and acetaldehyde CO 2 output changes in, 77
formation in, 233 enzyme changes in, 77
fruit thinning effects in, 336- glycolysis in, 72
337 pathogens in, 254-255
growth regulator effects in, starch conversion to sucrose in,
346 89
internal atmosphere changes in SRAM effect on respiration rate
during senescence, 239- of, 78-80
240 Bangladesh, technical change and
linolenic acid breakdown in, food losses in, 525
130-131 Bartlett pears, ethylene recepti-
long-term controlled atmosphere vity in, 389-390
storage of, 393 Benomyl, 274-275, 281
pathogens of, 254 Benzimidazole fungicides, 273-276-
red anthocyanin pigments in 277
skin of, 117 Peniaillium resistance to, 281
respiration patterns and ethy- Benzyladenine fungicides, 338-341
lene production by, 61- Betagarin, 302
62, 343 Betarulgarin, 302
shrivel in, 384 Biological deterioration, factors
Artemisia absinthum, 143 in, 460-463
Aspergillus flavus, 315, 318
INDEX 555

Bio1ogy of seneseenee, genetie Bu1bs (eontinued)

problems in, 1-34, see physio1ogy and storage of, 191-
also Plant seneseenee 224
Biomass of interest, 546 storage temperature effeets in,
Bioregulators, effeet of posthar- 191-223
vest treatment on longe- water eontent of, 191
vity of fruits and vege- sec-Buty1amine, 269, 272-273, 277
tab1es, 355-373
Biotie e1ieitors, 302-303 C.A. 4 H enzyme, P.A.L. protein and,
B1aek buttons, in stored 1emons, 113-116
357 Capsicum brutesceus~ 301
B1ighted potatoes, diterpene Carbendazim, 274-275
stress metabo1ites in, Carbohydrate wastes, eonversion to
306 mierobia1 proteins, 546
B1ueberries, daminozide and AVG Carbon dioxide, skin resistanee
treatment of, 368-373 to, 414
Botrytis spp., 248-249, 256-257, Carbon dioxide eoneentrations, in
269 eontro11ed atmosphere
Botrytis cinerea~ 254, 258, 300 storage, 404-405
Botrysphaeria ribis~ 258 Carbon dioxide zymosis, in fruit
Breakfast eerea1s, speetra of, injury, 236-237
477 Carnations, eoo1down of, 420
Brown rot, in eitrus fruits, Carotenoids, in peaeh treatment,
249, 266 367
Bu1b dormaney, 199-224, see also Caryopsis strueture, after-ripen-
Embryo dormaney ing germination and, 177-
abseisie aeid in breaking of, 179
221 Cassava, e1ona1ly propagated varie-
bioehemiea1 basis of, 220-223 ties of, 5
breaking of, 220-223 CA storage, see Contro11ed atmo-
eoneept of, 216-220 sphere storage
eorm evolution and, 217-218 Ce11-membrane lipids, see Plant
eytokinins in breaking of, 221 ee11-membrane lipids
organogenesis in, 219 Ce1l rejuvenation
Bu1bous iris, 208-212 meiosis in relation to, 13-15
apiea1 bud of, 208 in mitosis, 10-12
bu1bing .initiation in, 210 Ce11 surfaee interaetion, in HRGP
dormaney in, 217-218 biosynthesis, 294-296
high-temperature storage of, Cellular level, seneseenee at,
211 6-15
physiologieal state of, 212 Cellular organization, deereased
sprouting aeeeieration in, 222 resistanee of in respira-
temperature requirements for, tion ehanges, 87
208-209 Ce11u1ar seneseenee, 6-15, see
Bu1bs, see also Bu1bous iris; also Plant seneseenee
G1adio1us; Iris bu1bs; genetie damage aeeumu1ation in,
Tulip bu1bs 6
diversity of, 191 in postmitotie ee11s, 6-9
dormaney in, 191-224 Centrospora acerini~ 256
environmenta1 faetors affeet- 2-CEPA, in iris bulb sprouting,
ing, 192 222
556 INDEX

CEPHA, see Ethre1 Cut f10wers (continued)

Ceratoaystis fimbriata, 273, 302 fading of, 148-150
Chenopodium album, 167 postharvest treatment of, in
Chenopodium amarantiaolor, 10-12, Common Market countries,
176 450-451
Chlamydomonas reinhardi, 9 respiration in, 63-66
Chlorophenoxyaaetia aaid, 356 senescence phenomena in, 148-
Chloroplast protein synthesis, in 150
1eaf senescence, 21-24 storage of, 445-446, 450
Chloroplast protein turnover, 21 wet-coo1er system for, 446
Citrate, decrease of in anaerobic Cytochrome pith, e1ectron f1ux
pea seeds, 70-72 through, 82
Citrate synthetase, 76 Cytokinins
Citrus fruits, see also Oranges in corm dormancy breaking, 221
brown rot of, 249, 266 in de1ay of fruit ripening,
co1oring of, 362 151
ethy1ene effect on, 253, 361 in de1ay of plant aging, 143,
gibbere11ins in color maturity 146
of, 358 fruit qua1ity and, 338
P. digitatum on, 256, 274 in 1eaf aging, 147
Citrus sinensis, 361 in postharvest treatment, 360
Cladosporium fulvum, 302
CLPA, see Ch1orophenoxyacetic 2,4-D, see 2,4-Dich1orophenoxyace-
acid tic acid
Colletotriahum spp., 248, 251- Daminozide-ethephon interaction,
252, 280 363, 368
Colletotriahum gloeosporioides, Daminozide treatment, for b1ue-
258 berries, 364
Colletotriahum lagenarium, 293, Dluaus aarota, 10
295 Detached avocados, respiratory
CoZZeotriahum musae, 252, 254, dri~t of, 62-63, see also
273 Avocados
Contro11ed atmosphere storage, Detached f1owers, respiratory
383-397 behavior in, 63, see also
for app1es, 393 Cut f10wers
C02 concentrations in, 405 Detached fruit
pressure-induced changes in, respiration of, 61-62
404 ripening and qua1ity of, 363-
in Western Europe and United 366
Kingdom, 446 Detached plant organs, respiratory
Convective coup1ings, 409-412 drifts of, 61-63
Cooldown process, 420 Deve10ping countries, see also
Corms, dormancy in, 216 Less deve10ped countries
Corm storage and evolution, in food shornages. in~ 515
gladio1us, 214-216 inadequate transportation faci1i-
p-Coumary1quinic acid, 252 ties in, 464-465
CUaurbita maxima, 129-130 ma1function of faci1ities in,
Cut f10wers 465-466
cooldown of, 420 maturity and qua1ity indices in,
CO 2 output of, 65 457-459
INDEX 557

Deve10ping countries (continued) Energy crop concept, for period

postharvest 10ss of perishab1e 1980-2000, 544
foods in, 485-510 Energy demands, increase in, 540
postharvest qua1ity maintenance Energy metabolism, in senescing
in, 455-469 plant tissues, 61-90
postharvest techno10gy in, 466- Enzymatic products, formation of
468 during fruit growth and
qua1ity components in, 455-457 storage, 231-243
qua1ity deterioration los ses Enzyme activity in vivo
in, 460-463 factors affecting, 100-106
qua1ity standardization and postharvest change and, 99-119
inspection in, 466 Enzyme protein, catalytic activi-
Diaporthe pernieiosa, 252 ty and, 103
2,4-Dich10rophenoxyacetic acid, Enzymes, see also Animal enzymes;
356-357 Plant enzymes
Dic1oram, as fungicide, 273-274 cova1ent modification of, 105
Diffuse reflectance spectropho- in protein turnover, 16
tometry, of grains and proteo1ytic breakdown of, 101
oi1seeds, 471-472, 479 Enzyme synthesis
Dipheny1amine, 384 glycosy1ation in, 102-103
Diplodia spp., 248-249 structural modifications in, 101-
Disease development, see also 102
Plant disease Erwinia spp., 248, 276
pathogen enzymes in, 249-251 Erwinia earatovora, 250-251, 254,
susceptibi1ity to, 251-252 257
DNA repair, 1ifespan and, 6-8 Eseheriehia eoli, soluble protein
DNA synthesis, 6-8 factors from, 22
Dormancy concepts, for bu1bs, Ethanol metabolism, in fruit
216-220 tissues, 240-243, see also
Dry rot, of potatoes, 274 Alcoho1 concentration
Dry storage, of seeds, 176-178 Ethephon
Durable crop products, character- in chlorophyll breakdown in
istics of, 491 fruits, 362
Dutch iris, see Bu1bous iris; daminozide and, 363
Iris bu1bs in fruit growth and ripening,
385-388
E1ectron transport, in senescing as replacement for ethylene gas,
fruits, 78-85 361
E1icitor response, mechanism of, in tomate ripening, 345
302-305 Ethoxyquin, 384
Embryo dormancy, seed generation Ethrel, in fruit ripening, 335,
and, 176, 178, 180-183, 344-345
see also Bu1b dormancy Ethy1ene
Empire app1es, ethy1ene levels anthracnose and, 253
in, 392-394 autocata1ytic production of, 65
Endoeonidiophora, 258 in Bartlett pear ripening, 390-
Endopectate 1yases, in posthar- 391
vest diseases, 249-250 and C02 output of sweet potato
Endopolyga1acturonases, in post- roots, 84
harvest diseases, 249- in degreening of citrus fruit,
250 361
558 INDEX

Ethy1ene (continued) Ethy1ene production, see also

in fruit and f10wer respira- Ethy1ene biosynthesis;
tion, 65-66, 87 Ethy1ene synthesis
in fruit growth and deve1op- AVG inhibition of, 368
ment, 385-395 inhibition of ~n app1e tissue
in fruit ripening, 152, 158, slices, 347
390-391 in morning glory f1owers, 131-
in HRGP biosynthesis, 294-296 132
as index of fruit maturity and rhizobitoxine inhibition and,
storability,. 389-395 366
in 1eaf aging and cut f10wer slowing of in contro11ed atmo-
fading, 147-149 sphere, 387
in plant senescence, 90, 144 Ethy1ene scrubber, 395-396
in plant tissue respiration, 66 Ethy1ene synthesis, see also
as primary hormone in fruit Ethy1ene production
ripening, 158 in fruit ripening, 46, 154
reduction in level of, 346 in tomatoes, 56-57
respiration and, 65-66, 87 Eukaryotes, trans1ationa1 contro1
in senescence acce1eration, of, 22
247-248 European Economic Community, post-
skin resistance to, 415 harvest treatments in,
in stress metabo1ite accumu1a- 450-451, see also United
tion, 305 Kingdom; Western Europe
in vo1ati1e f1avor substance Evaporative coup1ings, heat trans-
production, 344 fer and, 412-413
Ethy1ene activity
e1evated C02 in suppression of, F6P, see Fructose-6-phosphate
404 FAO/UNEP Expert Consu1tation, 486,
in tomato ripening, 156-157 489, 491, 500
Ethy1ene biosynthesis, see also Fatty acid metabolism, in soybean
Ethylene production; coty1edons, 131
Ethy1ene synthesis Ficus domestiaa, 5
abscisic acid in, 152 Fisheries, growth in, 539
inhibition of with AVG or etha~ F1avor, qua1ity components of, 457
no1, 149 F10wer differentiation, tempera-
Ethy1ene evolution ture requirements for,
in harvested cucumber fruit, 208-209
154 F1owers, see also Bu1bs; Seeds
in tomato ripening, 155 cut, see Cut f10wers
Ethy1ene level respiratory behavior of, 63
in contro11ed atmosphere Food avai1abi1ity
storage, 394 increase of through postharvest
po1yga1acturonase activity in techno1ogy, 518-526
tomatoes and, 153 population increase and, 485-486
regulation of by preharvest Food crops, postharvest atmosphere
app1ication of growth manipulation for, 383-397
regulators, 343-348 Food losses, see Postharvest food
Ethy1ene measurement, "Snoopy" losses
instrument for, 392 Foods, perishab1e, 485-510
INDEX 559

Food shortages, in deve10ping Fruit ce11s, C02 zymosis damage

countries, 515 in, 236
Forest losses, environmental fac- Fruit deterioration, bio1ogica1
tors in, 541 and environmenta1 factors
Forest resources, estimated, 539 in, 460-463
France, PRAC storage system in, Fruit ethy1ene levels, as guide
448 for harvest and storage
Fraxinuß excelsior~ 176 decisions, 393, see also
Freesia corms, bu1b dormancy and, Ethy1ene
219 Fruit growth, RNA synthesis dur-
Fructose-6-phosphage, phosphory- ing, 49-53
1ation of, 106 Fruit maturation, see Fruit ripen-
Fruit(s) ing
aerobic and anerobic respira- Fruit qua1ity maintenance, in
tion in, 232 deve10ping countries,
assimilate partitioning with 455-469
vegetative tissue, 335- Fruit ripening
337 bio1ogica1 oxidation in, 231
biological processes in, 231 chemica1s in contro1 of, 366,
chemicals in maturation and 385-388
ripening of, 385-388 climacteric rise in respiration
classification of, 63 during, 63-68
detached, 363-366 ethylene in, 152, 158, 390-391
enzymatic product formation in, gene expression in, 46-47
during growth and stor- glycolic and respiratory acti-
age, 231-243 vities in, 231-232
growth regulation and, 335 hormonal regulation in, 150-158
harvested, 99-100, 384-385 metabo1ic pathways associated
interna1 atmospheric changes with, 99-100
during senescence, 238- po1yga1acturonase synthesis
240 during, 53-56
low temperature or freezing in- RNA in, 45-56
juries to, 237 senescence and, 150-151
maturity and quality indices total cell RNA breakdown in,
for, 457-459 47-51
qua1ity components for, 455- variable factors in, 392
457 Fruit senescence, interna1 atmo-
respiratory substrates in, 231 sphere changes during, 238,
ripening of, 45-56, 63-68, 99- see also Plant senescence
100, 152, 158, 231, 335, Fruit storage, see also Storage
344-345, 385-388, 390- enzymatic product formation
392 during, 233, 237-238
senescence of, 150-158, 238- under high C02 concentrations,
240 234-236
skin resistance measurements Fruit thinning
for, 413-415 in Golden De1icious app1es, 336-
storage ö·f, see Fruit storage; 337
Storage postharvest qua1ity and, 333-
vitamin los ses in, 455 334
water status of host tissue in,
255-256
560 INDEX

Fruit tissues, acetaldehyde and Gibbere11ins (continued)

ethanol metabo1ism in, in postharvest treatment of
240.,..243 fruits and vegetab1es,
Fumigants, in grain storage, 338-339
440-442 reduced senescence and, 360
Fungicides in retardation of fruit ripen-
decay contro1 by, 273 ing, 151-152
in grain storage, 441-442 G1adio1us
pathogen resistance to, 276- corme1 sprouting acce1eration
277 in, 221
in postharvest disease contro1, corm evolution in, 214-218
266-267, 270-276 corm growth acce1eration in,
Fungus damage, in grain storage, 221
434, see also Fusarium cyc1e of, 212-213
fungi high temperature storage of,
Furanocoumarin, 315 213-214
Furanoterpenes, 301 Gladiolus gPandifloris hort., 193
Furanoterpenoids, 306 Global population, growth of, 485,
Fusarium fungi, 250, 257, 315- 538, 543
316 Gloesporium spp., 248, 250
Fusarium graminearum, 316 Gloesporium fructigena, 254
Fusarium moni Zi forme, 316 G1ycoa1ka1oids, toxicity of, 306
Fusarium so lani, 302 G1ycoproteins, as defense mechanism
in diseased plant ce11
GA3-ethephon treatment, fruit wall, 287-299, see also
qua1ity and, 339, see Hydroxypro1ine-rich gly-
also Gibbere11ins pro teins
Ga1actosy1 serine, 291 G1yco1ysis, in plant respiration
GAs, see Gibbere11ins regulation, 69-74
Gene expression, contro1 points G1yco1ytic enzymes, mass action
in, 47-49 ratios of, for pea seeds,
Geotrichum spp., 248, 250, 269, 71
271-272, 276, 280 Glycosy1ation, of plant glycopro-
Germinabi1ity of seeds, after teins, 102
harvesting, 174-183 Golden De1icious app1es
Germination AVG treatment for, 348
after dry storage, 176 fruit thinning effects on, 336-
embryo dormancy and, 176, 178, 337
180-183 Grain(s)
externa1 conditions in, 174- marketing of, 429-430
176 moisture content of, 430
hard-seed treatments in, 180- NIR spectra of, 472-479
183 nutritiona1 qua1ity of, 427-428
special treatment in, 178-183 processing quality of, 429
Gibbere11ins, see also Growth Grain crops, qua1ity maintenance
regulators in, 425-443
in aging retardation, 144 Grain damage
in bu1b or corm dormancy causa1 agencies in, 431-438
breaking, 221-222 c1imate and, 436
in fading of cut f1owers, 148- containers as cause of, 432
149 dockage in, 433
INDEX 561

Grain damage (continued) Heat transfer

fungi in, 434, 437 convective vs. radiative, 409-
insects in, 433-435 413
moisture and, 435-437 by evaporation, 412-413
prevention of, 438-444 noda1 model of, 406-409
from rodents, 437-438 skin resistance and, 413-415
in storage, 431-434 water 10ss and, 415-419
temperature and, 436-437 Helminthosporium carbonum, 300
during transportation, 438 Host-pathogen interactions, in
Grain qua1ity, maintenance of, postharvest diseases, 247-
425-443 259
Grain storage, see also Storage Host tissues
aeration during, 439 hypersensitivity to pathogens
damage in, 431-434 in, 255
design of faci1ities for, 439- pH and nutritiona1 level of,
440 256-257
fumigation in, 440-442 HRGP, see Hydroxyproline-rich gly-
10w temperatures in, 442 coproteins
moisture and, 435-437, 442-444 Hyacinth bu1bs, dormancy of, 217
nutritional and processing qua- Hydrocyanic acid, in c1imacteric
lity during, 425-443 respiration rise, 78
rodents and, 437-438 Hydrolase regulation
Grain transportation systems, by compartmentation, 31
426-427 in protein turnover, 29
Gray mold of grapes, 249 Hydrolytic enzyme activity
Greenhouse effect, 541 regulation of, 30
Greening, of citrus fruits, 361- in senescing 1eaves, 26-29
362 Hydroxypro1ine
Green mo1d, ethylene effect on, glucosamine and, 293
253 serine and, 291
Green Revolution, 425 Hydroxypro1ine-containing com-
Growth regulators, see also Gib- ponents, 288-290
berellins Hydroxyproline-rich glycoproteins
ethylene level regulation by accumulation of in diseased
preharvest app1ications plants, 292-294
of, 343-348 amino acids and sugars of, 289
fruit quality and, 341-343 biosynthesis of, 290
fruit $ize and, 359 as defense mechanism in plant
postharvest treatment effects disease, 287-299
on longevity, 355-373 inso1ubility of, 292
in plant-pathogen interactions,
Harvested fruits and vegetab1es, 291
see also Postharvest Hydroxypro1ine-rich glycoprotein
(adj. ) biosynthesis
metabolie adjustments in, 99- ce11 surface interaction in,
100 294-296
qua1ity maintenance for, 384- ethy1ene e1icitation of, 294-
385 296
Heat loads, at optimal storage Hyp, see Hydroxypro1ine
pressure and temperature, Hypobaric conditions
407
562 INDEX

Hypobaric conditions (continued) Krebs cyc1e

freezing point elevation in, metabolites in activity of en-
403 zymes in, 76
heat loads under, 406 in plant respiratory regimes,
metabolism and water loss, 399- 75-76
420 residual oxidase, 85
microbe growth inhibtion in,
403 Leaf drop, 2,4-D treatment for,
pressure-induced changes in, 357-358
404-506 Leaf senescence, see also Plant
Hypobaric storage, 395-397 senescence
elements of,399-40l ethy1ene and, 147-149
hormonal effects in, 147-148
Imazalil, 278-279 phases of, 62-64
Imidazole fungicides, 278 Leafy vegetables, 2,4-D treatment
Immunity, phytoalexin theory of, for, 357
299, see also Plant im- Leaves
munity; Stress metabo- protein turnover in, 16
lites respiration changes during senes-
Induced parthenocapic fruit set, cence of, 62
332-334 sequential senescence of, 4
Inherent storage life, concept Legume-Rhizobium symbiososi, HRGP
of, 491, 494, see also in, 292
Storage Lemna minop, 9-10
Injured tissue, pH of, 270 Less developed countries, see also
Insect damage, to stored grain, Developing count ries
433-435 cost reduction in, 529-531
International Centers for Agri- increased food output in, 531-
cultural Research, 425 532
Ipomea batatas, 5, 301 low cost innovation in, 527-
Ipomeamarone, 301 529
Ipomoea tPicolop Cav., 87, 131, mi11ing los ses in, 524-526
149, 151 population increase and 1iving
Iris bulbs, see also Bulbous iris standards in, 485
10w-temperature treatment for, postharvest equipment manufac-
209 turers and, 517
storage temperature vs. bu1b postharvest food losses in, 485-
behavior for, 209-211 510
temperature requirements for, rain damage in, 528
208-209 threshing losses in, 531
Isocitrate dehydrogenase, 76 vegetable and fruit crops in,
Isocoumarins, 301 488
Isoflavanoids, 300, 306 Lifespan, DNA synthesis and, 6
Isoprenoid phytohormones, 145 LiUum spp., 14
Ivy leaves, respiration rate of, Lilium longijtorum, 14
67 Lily bulbs
gibbere11in in sprouting of, 222
Jerusa1em artichoke tuber, as rest and dormant periods in, 222
chi11ing-to1erant organ, Limes
129 degreening of, 360
INDEX 563

Limes (continued) Metabo1ic interactions, in phytoa-

postharvest treatment of, 360 1exin theory of plant im-
Lino1eic acid, oxidation by aque- munity, 299
ous extracts of edib1e Metabo1ic pathways, in fruit ripen-
p1ants, 136 ing, 99-100
Lino1enic acid Metabolism, pressure effects in,
degradation of in app1e fruits, 401-404
130-131 Metabo1ite transport systems, in
reduction of, 129 plant mitochondria, 76-78
Lino1enoy1-ga1acto1ipids, 126 Michae1is~Menten kinetics, 73
Lino1eoy1-phosphatidy1cho1ine, Microbia1 growth inhibitors, in
126 plant tissues, 253-254
Lipid autoxidation, 135 Microbia1 proteins, carbohydrage
Lipid metabolism, 131 conversion to, 546
Lipid mo1ecu1es, in plant ce11 Microorganisms, plant invasion by,
membranes, 123-137 247
Lipid peroxidation, see also Mitosis, ce11 rejuvenation in, 10-
Membrane peroxidation; 12
Plant ce11 membrane Moisture content
mechanism of, 132-134 in diffuse ref1ectance spectropho-
as step toward sensecence, 136 tometry, 476
Lipid peroxides, scavenging of, grain damage and, 435-437
134 Monilinia fruotioola, 248, 273,
Lotus seeds, longevity of, 167 278, 280
Lupinus aroticus, 167 Monilinia rot, of stone fruits,
Lyoopersioon esoulentum, 361 274
Lyophi1ization of seeds, germi- Monocarpic senescence, 146-147
nability fo110wing, 175 Morning glory f10wers, phospho-
lipid metabo1ism and ethy-
Mclntosh apples, Alar in ripen- 1ene production in, 131-
ing of, 386-388, see al- 132
so App1es mRNA
Malate-oxaloacetate shuttle, 76 contro1 at level of, 58-59
Malic acid dehydrogenase, 76 in fruit ripening, 50-52
Malic enzyme, 76 in vitro translation products
Manihot usitatissima, 5 of, 54
Meiosis, in ce11 rej uvenation , Muoors spp., 280
13-15 MYoophaerella pinodes, 303
Me10n seed1ings, HRGP in, 291- Mycotoxic porcine nephropathy,
293 326-327
Membrane changes, in plant ce11 Mycotoxins
senescence, 18 adverse effects of, 320-323
Membrane functions., liquid turn- assessment of 10ss due to, 323
over in, 34 as deteriorating factor in
Membrane lipid peroxidation, stored crops, 315-327
senescence and, 132-137 formation of, 316-320
Membrane systems, constituent myco1ogica1 and chemica1 natur.e
proteins of, 19 of, 315-316
Messenger RNA, see mRNA
Metabo1ic adjustments, in har- 1-Naphtha1eneacetic acid, 357
vested crops, 99-100
564 INDEX

Narcissus bu1bs, dormancy in, Packaging methods, in Western

217-219 Europe, 451-452
Near-infrared ref1ectance, 471- Paeonia spp., 14
479 Pa1mitic acid, plant ce11 membrane
Near-infrared transmittance, 480- lipids· and, 126
481 P.A.L. protein
Neatria galligenia, 5, 254, 256 anthocyanin and, 117
Nelumbo nuaifera, 167 C.A. 4H enzyme and, 113-116
Nether1ands changes in level of, 118
ethy1ene measur~ment for Gol- increased avai1abi1ity of, 114-
den De1icious app1e stor- 117
age in, 448 P.A.L. protein inactivating sys-
postharvesting techniques in, tem, 117
445-454 Papaya fruit
NIR, see Near-infrared ref1ec- convection from, 409
tance evaporative heat transfer from,
Nitrate reductase activity, in 412-413
PeriZZa 1eaves, 18 heat transfer and water 10ss in,
Nuc1eic acid, see also RNA 406-408, 415-419
in fruit ripening, 47 radiative heat transfer from,
protein turnover and, 15-34 413
Nuc1eus-ch10rop1ast interaction, Parthenocarpic fruit, induced set
regulation of, 25 in, 333-334
Pathogens, plant, see Plant patho-
Ochratoxin A gens
adverse effects from, 325 Peaches
in foods and feeds, 318, 321- Alar treatment of, 342
322 carotenoids in postharvest treat-
Oi1seeds, NIR spectra of, 472- ment of, 367
479 postharvest treatment of, 363-
01eic acid, plant ce11 membrane 367
lipids and, 126 Pears, chemica1s in ripening of,
Oospora, 272 386-388
Oranges Pea seeds
a1coho1 and aldehyde in f1esh g1yco1ysis rate in, 70
of, 234-235, 242 lactate increase in, 72
gibbere11ins .in color treat- sugar phosphate and nuc1eotide
ment of, 358-359 changes in, 71
interna1 atmospheric changes Pectin activity, in tomato ripen-
in during senescence of, ing, 156-157
239-240 Peniaillium spp., 248, 269, 275,
preharvest degreening of, 361 315
regreening of, 358 on citrus fruits, 274
Organogenesis, bu1b dormancy temperature factor in, 258
and, 219 PenaiZZium digitatum, 247, 250,
a-Oxyg1uturate dehydrogenase, 76 256, 259, 271, 276-277
Oxygen tensions, plant tissue Peniaillium expansum, 247, 251,
respiration rates and, 254, 272, 278, 280-281
80 PeniailZium infestans, 303
PeniaiZZium itaZiaum, 271-272, 277
INDEX 565

Penicillium viridicatum, 316-318 Phosphofructokinase

PEP, see Phosphoeno1pyruvate in ripe and unripe tomatoes,
Perilla frutescens, 16-17 108
Perilla 1eaves, respiration and Phospho1ipids
photosynthesis changes time course of change in, 151
in, 64 turnover of, in soybean tissue
Perilla vulgaris, 21 cultures, 128
Perishab1e crop products, charac- Phytoa1exins, in plant immunity,
teristics of, 491 299
Perishab1e foods, see also Post- Physio10gica1 maturity, of tu1ip
harvest food los ses bu1b, 202, 207
postharvest 10ss of in deve10p- Phytohormones
ing countries, 485-510 amino acid-derived, 145
types of, 487-489 groups of, 142
Pezicula spp., 272 isoprenoid, 145
PFK, see Phosphofructokinase in plant senescence, 141-146
pH Phytophthora spp., 266, 276, 280-
of injured plant tissue, 270 281, see also Brown rot
in plant disease, 256-257 Phytophthora infestans, 299
Phaseo11in, 306 Phytophthora megasperma, 302
Phaseolus lunatus, 301 Pinus aristata, 9
Phaseolus vulgaris, 24, 300 Pisatin, 306
Phenylalanine pisum sativum, 300
conversion to p-coumaric acid, P1ant(s)
113 gas exchange in, 416
pheny1propanoid compounds from, hydroxypro1ine-containing com-
111 ponents in, 288-290
o-Pheny1pheno1, as fungicide, microorganism invasion of, 247
270-271, 278-279 pheny1propanoid metabo1ism re-
Pheny1propanoid compounds, 111- gulation in, 111
112 Plant ce1l membranes, see also
Pheny1propanoid metabolism, re- Plant membrane lipids
gulation of in p1ants, lipid breakdown in, during
111-119 senescence, 129-132
Phoenix dactylifera, 5 lipid composition of, 123-126
Phoma spp., 257, 272 lipid peroxidation in, 132-
Phomopsis spp., 249 137
Phosphoeno1pyruvate lipid turnover and, 127
as a110steric inhibotor of PFK, unsaturated fatty acids in,
107 128
in plant repsiration regula- Plant ce1ls, hydrostatic pres-
tion, 70, 73 sure in, 401-402
Phosphofructokinase Plant ce11 wall
Pi in inhibitory activity of, as barrier for protoblast,
109-110 287
PEP as a110steric inhibitor glycoproteins as defense mecha-
of, 107 nism in, 287-299
in plant glycolysis regula- Plant disease, see also Host
tion, 106-111 tissues; Plant pathogens;
in plant respiration regula- Postharvest disease; Post-
tion, 69-70 harvest food losses
566 INDEX

Plant disease (continued) Plant organs

host tissue hypersensitivity membrane lipid turnover in, 127
to, 255 senescence of, 4, 141-142
hydroxyproline-rich glycopro- Plant pathogen enzymes, host tis-
teins as defense mecha- sue resistance to, 258-
nism in, 287-299 259
pathogen enzyme role in, 249- Plant pathogen growth, inhibition
251 of by host constituents,
pathogen growth restriction due 253-255
to morphological barriers Plant pathogens, see aZso Plant
in, 257-258 disease; Postharvest
pR and nutritional level of disease
host tissues in, 256-257 enzymes of, 248-251
in postharvest food losses, growth restriction in, due to
497-498 host morpho1ogical bar-
storage factors in control of, riers, 257-258
259 host tissue hypersensitivity
susceptibility of host tissues to, 255
to, 251-252, 255 hydroxypro1ine-rich glycopro-
temperature management in, 259 teins and, 291
water status of host in, 255- resistance of to fungicides,
256 276-277
Plant enzymes, see aZso Enzymes ro1e of in disease deve1opment,
allosteric regulation by, 103- 249-252
104 stimulation of by host con-
proteolytic breakdown of, 101 stituents, 252-253
Plant glycolysis strategies for contro1 of, 266
phosphofructokinase in, 106- type and sites of infection
111 for, 248
plant respiration regulation Plant proteins, breakdown of,
and, 69-74 15, see also Protein
Plant glycoproteins, glycosyla- Plant respiration, see aZso
tion of, 102 Respiration rate
Plant growth regulators, see aZ- ethy1ene action on, 66-68
so Growth regulators; glycolysis in, 29-74
Phytohormones physio1ogical significance of
physiological effects of, 146 rise in, 88-90
residues of, 359-360 regulation of, 69-88
Plant hormones, defined, 142, Plant senescence, see also Senes-
see also Phytohormones cence
Plant immunity, phytoa1exin aging and, 1, 141
theory of, 299 bio1ogy of, 1-34
Plant life cyc1e, environment lipid breakdown during, 129-
and, 147 132
Plant membrane lipids lipid peroxidation in, 134-137
half-lives of, 127 membrane lipid changes during,
as potential danger for life, 126-137
137 monocarpic, 146
Plant mitochondria, metabo1ite as result of biomembrane dam-
transport in, 76-78 age, 137
INDEX 567

Plant tissues Postharvest disease control

acetaldehyde and ethanol meta- (continued)
bolism in, 240-243 strategies and methods of,
ethylene effect on respiration 265-270
rate in, 66 successes and challenges in,
host-pathogen interactions in, 280-281
247-248 Postharvest food, defined, 489
microbial growth inhibitors in, Postharvest food losses, see
253-254 also Postharvest disease
pR of, 270 bacteria in, 497
protein cycle in, 15-16 causative factors in, 490-499
Plasmodesma chemical treatments in pre-
of C amaranticolor~ 12 vention of, 506-507
cell rejuvenation, 10 "cold chain" system and, 508
structure and dimensions of, 11 curing practices and, 504-505
Polygalacturonase defined, 489-490
ethylene synthesis and, 56-58 in developing countries, 460-
mRNA measurement in, 58 463, 485-510
in tomato ripening, 153, 156 disease in, 497-498
Polygalacturonase synthesis environment control in reduc-
in fruit ripening, 53-56 tion of, 507-509
in tomatoes, 57 at farm and village level,
Polypeptide chains, synthesis of, 520-521
102 field supervision problems in,
Population growth, 1980-2030, 522
485, 538, 543 food availability and, 486-487
Postharvest atmosphere, manipu- harvesting and field handling
lation of, 383-397 factors in, 503-504
Postharvest change, enzyme acti- high-yielding varieties and,
vities in, 99-119 516
Postharvest deterioration, bio- literature on, 515
logical and environmental magnitude of, 499-501
factors in, 460-463 milling practices and, 524-526
Postharvest disease, see also nontechnical factors in, 498-
Plant disease; Plant 499
pathogens physical deterioration factors
in developing countries, 497- in, 492-493
498 physiological considerations
development of, 248-251 in, 493-496
endopolygalacturonases pro- phytopathological considera-
duced in, 249 tions in, 496-498
host-pathogen interactions in, predators in, 492-493
247-259 preharvest consideration of,
practical treatments in con- 502-503
trol of, 268 rain damage in, 528
time and site of infection in, reduction of, 501-509
248 storage structures and, 506
Postharvest disease control storage techniques and, 523-
antimicrobial agents in, 265- 524
281 technical causes of, 491-498
568 INDEX

J:>ostharvest food losses (con- Premature ripening, inhibitors

tinued) of, 366, see aZso Fruit
temperature factors in, 496 ripening
threshing losses and, 531 Prochloraz fungicide, 278-279
"top-icing" process in reduc- Prokaryotes, translational con-
tion of, 508 trol of, 22
transportation and packaging Pro tein
facilities relating to, carbohydrate waste conversion
505-506 to, 546
in tropical world, 500-501 energy-dependent breakdown of,
in United States, 500 34
Postharvest fruit, see Fruit(s); scatter plot of, 478
Harvested fruit Pro tein cycle
Postharvest fungicides, 266-267, in leaf cells, 33
see aZso Fungicides in plant tissues, 15
current developments in, 278- Protein degradation, control of,
279 26-34
Postharvest planning, in less de- Protein errors, for whole-grain
veloped countries, 517 wheat, 480
Postharvest quality, factors in- Protein glycosylation, tunicamy-
fluencing, 458-466 cin in inhibition of,
Postharvest storage, see Con- 102
trolled atmosphere stor- Protein kinases, in plants, 104-
age; Hypobaric storage; 105
Storage Protein synthesis
Postharvest techniques, in Wes- chloroplasts in, 21
tern Europe, 445-454 climacteric enhancement of, 63
Postharvest technology, food control of, 20-26
availability increase Protein turnover
and, 518-526 degradative side of, 26
Postharvest treatments, in Wes- enzymes in, 16
tern Europe, 450-451 nucleic acid in, l5~34
Postmitotic cells regulation of, 19-34
lifespan of, 6 in ripening bananas, 89
mitotic division in, 9 Proteolytic breakdown, of plant
"scrubbing" of, 13 enzymes, 101
Potato blight, diterpene stress ~as domestica, 5
metabolites in, 306 Pyruvate dyhydrogenase, 76
Potatoes
dry rot in, 274 Quality deterioration
Phoma gangrene of, 257 biological and environmental
Potato tubers, cyanide-resistant factors in, 460-463
respiration in, 87 nonbiological or socioeconomic
PRAC (precooling under vacuum factors in, 464-466
and controlled atmosphere Quality measurement, instrumental
storage) system, France, techniques in, 471-483
448 Quality standardization, in de-
Precooling methods, in Western veloping countries, 466
Europe, 448-449 Quercus seeds, desiccation of,
172
INDEX 569

Rabbit reticulocytes, soluble Rotenone, a ....oxidation of fiatty

protein factors for, 23 acids and, 76
Radiative coup1ings, heat trans- RuBP carboxy1ase, in pro tein turn-
fer and, 413 over, 16
Rain damage, in postharvest food Rubus idaeus'~ 5
losses, 528 Rwnex spp., 144
Rainfall, pR value of, 541
Ref1ectance measurements, in SADR, see Daminozide
color evaluation and Sclerotinia spp., 250
defect detection, 481- Secondary foods, in deve10ping
482 countries, 487
Refrigeration subsystem, in hy- Seed death, origin and causes
pobaric storage, 401-402 of, 167-174
Regu1atory metabolites, ce1lu1ar Seed germination, 174-183, see
distribution of, 87 also Germination
Residual oxidase, 85-87 hard-seed treatment and, 182-183
Respiration temperature and, 180-183
c1imacteric rise in, 63-68 Seed 10ngevity, water content and,
in senescing plant tissues, 173
61-90 Seed p1ants, sequence processes
Respiration rate in, 3
ambient pressure and, 408 Seed qua1ity, evaluation of, 167-
ce11u1ar organization decrease 169
in, 87 Seeds
Respiration rise, physio10gical after-ripening of in dry stor-
significance of, 88-90 age, 176
Respiratory heat loads, at opti- bio10gica1 pecu1iarities of
mal storage pressure and after harvesting, 165-167
temperature, 407 dehydration of, 166, 171-172
Rhoeo discolor, 14, 31 desiccation sensitivities of,
Rhizobitoxine, 366 166-167, 171-172
Rhizopus spp., 250, 256, 258, dormancy of, 176
269, 271, 276 germinability of, 174-183
Rhizopus stolonifer~ 254, 273 10ngevity of, 167-168, 173
Ribonuc1ear acid, see RNA mesobiotic and microbiotic,
Ripening, see Fruit ripening 167
Ripening bananas, starch-sucrose postharvest physio10gy of,
conversion in, 89 165-184
Rishitin, 306 qua1ity and germinability of,
RNAse activity, in postmitotic 165-184
ce11s, 10 storage of, 171-178
RNA contro1, in fruit ripening, water content of, 166, 171-174
45-46 Seed viability
RNA synthesis 10ss of, 169-171
cessation of in P. vulgaris~ prolongation of, 173-174
21 storage problems and, 171-174
during fruit growth and ripen- tests for, 167-179
ing, 47-53 theories of, 170
measurements of, 49
in premeiotic phase, 14
570 INDEX

Senescence, see also Aging; Sorting and grading practices, in

Plant senescence Western Europe, 449-450
bio10gy of, 1-34 Soybean tissue cu1tures, phospho1i-
at ce11u1ar level, 6-15 pid turnover in, 128
clonal, 4-5 Spectrophotometry, diffuse ref1ec-
cytokinins in de1aying of, 360 tance, 471-479
defined, 1 Spergula arvensis, 167
deve10pmenta1 pro gram and, 2 Staple foods, in deve10ping coun-
direct or positive1y pro- tries, 487
grammed, 3 Stem end rot, ethy1ene and, 253
hormonal regu1qtion of, 141- Stop-drop agents, for app1es and
15.0 peaches, 344
indirect programming of, 4 Storage, see also Seed storage
and interna1 atmosphere of contro11ed atmosphere, see Con-
fruits, 238 tro11ed atmosphere storage
lipid breakdown during, 129- hypobaric, 399-420
132 postharvest atmosphere manipu-
occurrence of, 1-2 lation in, 383-397
plant ce11 membrane lipid weight 10ss in, 495
changes during, 126-137 in Western Europe, 445-454
protein turnover in contro1 Storage 1ife
of, 1-34 inherent, 491, 494
as "ridd1e," 90 of perishab1e crop products,
in seed p1ants, 3 491
Senescing fruits, see also Storage losses, see also Post-
Fruit(s); Fruit ripening harvest food los ses
adenine nuc1eotide level avoidab1e vs. unavoidab1e, 493-
changes in, 74 497
~lectron transport in, 78-85 in 1ess deve10ped countries,
pentose pathway in, 74-75 523-524
Senescing 1eaves range of, 519
hydro1ytic activity in, 26-28 Storage temperature effects, for
respiratory drift of, 62-63 tu1ip bu1bs, 193-203
Senescing plant tissues Stored crops, mycotoxins as deteri-
metabolie energy sinks in, 89 orating factor in, 315-
respiration and energy meta- 327
bo1ism in, 61-90 Strawberry ripening, respiration
SRAM, residual oxidase and, 85 rate in, 90
Skin resistance Stress metabolites, 299-307
to C02 and water vapor, 413- chemica1 nature and distribution
415 of, 300-302
heat transfer and water 10ss e1icitor·response in, 302-305
in relation to, 417-418 ethy1ene in accumu1ation of,
"Snoopy" device, in ethy1ene 305
measurements. 392 toxicity of, 300, 305-307
Sodium-o-pheny1phnate, 269-273, Succinate dehydrogenase, 76
276 Succinic acid,-2,2-dimethy1hydra-
Solanaceae fami1y, 301 zide. see Daminozide
Solanum tuberosum, 301 Sugar cane, Third Wor1d scenario
SOPP, see Sodium o-pheny1phenate for, 542-544
INDEX 571

Sweet potato Transport practices in Western

ethy1ene effect on C02 output Europe, 452-453
of, 84 Tree, as cora1 co1ony, 5
02 concentration effect on, 83, Tricarboxy1ic acid cyc1e (Krebs
86 cyc1e), 75-76
SHAM effect on respiration rate Trichothecenes, 320, 324, 326
of, 81 Trillium spp., 14
Sweet potato furanoterpenoids, Tu1ip bu1bs, 193-207
toxicity of, 306 apica1 bu1bing in, 200-202, 205
bu1bing induction vs. tempera-
Taraxacum spp., 144 ture in, 199
Temperature effects, in ripening dormancy in, 217-218
and storage, 180-183, ear1y f10wering of, 197-198
191-223, 237, 259, 407, elongation and bu1bing in, 197-
436-437, 442, 496 198
Tetrazo1ium test, of seed viabi- growth processes vs. storage
lity, 168-169 temperature for, 195-203
Thiabenzado1e, 274, 281 high-temperature storage effects
Thielaviopsis spp., 248 • in, 195-197, 203-207
Thiophnate methyl, 274-275 incomp1ete bu1bing of, 205
Third \t\for1d, see also Deve10ping low-temperature storage of, 197-
countries; Less deve10ped 203
countries periodic lifting and storage
carbohydrate crop cu1tivation of, 195-200
in, 542 "physio1ogica1 maturity" stage
economic problems of, 537-541 in, 201-202, 204, 207,
financia1 investment in, 542 222
food problems of, 515-532 storage temperature vs. growth
food requirements of, 1980- process in, 195-203
2000, 541-542 Tu1ip cyc1e, main phases of, 205-
Tobacco-pith ca11us, soluble 207
auxin receptor in, 143
Tobacco plant 1eaves, regreening Ulmus spp., 5
of, 146 Ulmus americana seeds, 172
Tomatoes U1travio1et radiation, genetic
ethephon effect in ripening ce11 damage from, 8-9
of, 345 Umbe11iferae fami1y, 301
ethy1ene evolution in, 57 United Kingdom, contro11ed atmo-
ethy1ene synthesis in, 56-58 sphere storage in, 446-
growth and ripening of, 47-51, 447
54 United States, perishab1e food
pathogens in, 254 losses in, 500
phosphofructokinase from, 107-
108 Vaccinium ashei 3 363
po1yga1acturonase synthesis in, Vacuum coo1ing, 420
56-58 Vacuum/water subsystem, in hypo-
visible absorption spectra of, baric system, 399-401
481-482 Vegetab1es
Tradescantia spp., 14, 149
572 INDEX

Vegetab1es (corttinued) Wor1d population, expected in-

growth regulators and biore- crease in, 538
gulators in postharvest Wor1d forest resources, 1978-2000,
treatment of, 355-373 539
maturity and qua1ity indices Wor1d population, expected in-
for, 457-459 crease in, 485, 538, 543
qua1ity maintenance of in ~e­ Wyerone, as stress metabolite,
ve10ping countries, 455- 306
469
water status of host tissues Yam tubers, storage weight 10ss
in, 255-256 in, 495
Vegetative tissue, assimilate
partitioning with fruits, Zea mays, 14
235-237 Zeara1enone, 320, 325-326
vertiaiZZium fungus, 250 Zymosis, in fruit injury, 236-237
Viaia faba, 301
Visible absorption spectra, of
tomatoes, 481-482, see
aZso Spectrophotometry
Vitamin C losses, in postharvest
period, 455-457
Vo1ati1e f1avor substances,
ethy1ene in production
of, 344-345

Water activity, water potential

and, 401
Water-so1ub1e pectin, in tomate
ripening, 156-157
Water vapor, skin resistance
for, 414-415
Western Europe
packing methods in, 451-452
postharvesting techniques in,
445-454
precoo1ing methods in, 449
qua1ity and grading regula-
tions in, 453-454
sorting and grading in, 449-
450
transport of produce in, 452-
453
Wet-coo1er system, for cut-
f10wer storage, 446
Wheat
moisture levels in, 476
protein errors for, 480
Wor1d food production, percent
increase in, 538
Wor1d forest resources, 1978-
2000, 539