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UNIT 11 PLANT TISSUE AND ORGAN

CULTURE
Structure: Page No.
11.1 Introduction 85
Objectives .

11.2 Techniques of Plant Tissue Culture

Initiation of Aseptic Cultures
Culture Medium
Culture Conditions

11.3 Establishment of Tissue Cultures 90

I Suspension C u l t u ~ s
Single Cell Culture

11.4 Cellular Totipotency

Organogenesis
Somatic Embryogenesis

11.5 Applications
Production of Rare Hybrids
Somatic Hybridization and Cybridization
Haploid Production
Clonal Propagation
Production of Diseasefree Plan&
Other Applications

11.6 Summary
11.7 Terminal Questions

r 11.8 Answers. I08

11.1 INTRODUCTION
I In the preceeding units you have studied that cells grow and divide in a
selfregulating manner, and differentiate into diverse tissues and organs. The concept
that the individual cells of an organism are totipotent is implicit in the cell theory
put forth bySchleiden and Schwann. A totipotent cell is capable of regenerating into
a whole plant. In this unit you will learn how the fundamental tenets of the cell
theory have been experimentally verified by the techniques of plant tissue culture.
In plant tissue culture isolated protoplasts (i.e., cells without cell walls), cells,
tissues or organs are grown aseptically in an artificial nutrient medium, under
controlled temperature and light. Gottlieb Haberlandt, a German plant anatomist
and physiologist, was the first to attempt the cultivation of isolated mesophyll cells
of several flowering plants. The cells increased in size, synthesised starch and
survived for a month but failed to divide. His failure could be attributed to two main .
factors: (i) selection of highly differentiated cells as the experimental material and
(ii) the lack of knowled~eof plant growth substpnces, that promote cell division.
The progress of plant tissue culture has since been fast, aided by some important
discoveries, notably: (i) recognition of the importance of B-Vitamins in plant
growth, (ii) identification of auxin as a natural growth regulator and (iii) discovery
of cytokinins. White was the first scientist to succeed in establishing continuously
growing cultures of tomato roots by the addition of B-Vitamins in the culture medium.
Plant Development-ll Initially the main concern i n the plant tissue culture studies was to induce division
in cultured cells and organs and to optimise the nutritional and hormonal
requirements to establish continuously growing tissue cultures. Later, plant tissue
culture was employed as a research tool to study basic problems of physiology and
biochemistry and the complex process of differentiation. However, the discovery
that whole plants can be regenerated from any living cell raised the technique of
plant tissue culture to the status 'a major technology' in making important
contributions to agriculture and plant biotechnology.

Objectives:
After studying this unit you should be able to:
describe the methods employed in aseptic manipulation of plant cell and tissue
culture,
apply the methods of plant tissue and organ culture to culture tissues and single
cells,
just@ the role of plant growth regulators in the growth and differentiation of
plant tissues,
differentiate between in-Vitro embryogenesis and organogenesis, and
discuss the applications of plant tissue culture in agriculture, horticulture and
industry. . ,

11.2 TECHNIQUES OF PLANT TISSUE CULTURE

- -
A standard tissue culture laboratory should provide facilities for washing and
storage of glass ware, preparation and storage of nutrient media, aseptic
manipulation of plant material, maintenance of cultures under controlled
temperature, light and humidity. At least two separate laboratories or rooms should
be available one for glassware washing storage and media prepration and another to
store cultures. Also it is very essential to maintain a sterile environment during plant
tissue culture. By following a few simple precautions against microbial
contamination you will save valuable laboratoly time in repeating experiments. In
the following subsections we will discuss (i) Initiation of aseptic techniques (ii)
Culture media and (iii) Culture conditions.

11.2.1 Initiation of Aseptic Cultures

The nutrient medium (basal medium) used for plant tissue culture would promote
luxuriant growth of micro-organisms such as bacteria and fungi. Widely used basal
medium for tissue and culture studies is that of Murashige and Skoog (1962).
Reaching the medium, these microbes grow much faster than the plant cells
cover the tissue surfaces impeding its growth and finally killing it. The microbes
may be present in the medium right from the beginning. To destroy them the culture
vessels containing the medium are properly plugged and autoclaved (steam heating
under pressure) at 121' C for 15-20 minutes. Also you can use pressure cooker for
sterilising small volumes of media. If you use presterilised, nonautoclavable culture
vessels then the medium is autoclaved in 100-1000 ml. coming or pyrex flasks or
bottles and the sterilised medium poured into the culture vessels under aseptlc
conditions. You should know that most of the media constituents are heat stable and
can be added to the medium before autoclaving. Growth regulators such as
Gibberellic acid, Abscisic acid, Zeatin and some vitamins are thermolabile i.e. they
are rapidly degraded by elevated temperatures. The solutions of such compounds
are sterilised by passing them through a bacteria proof filter membrane (Pore size
0.2 um-0.45 um) and then added to the autoclaved medium cooled to 60° C.
The pieces of tissue used to start a culture referred to as the 'Explant' are the
principal source of contamination of the cultures. To eliminate the micro organisms
canied on the surface of the explant, the latter are properly surface sterilised before Plant Tissue And Organ
planting them on the medium (inoculation). The most commonly used surface Culture
sterilising agents are sodium hypochlorite and mercuric chloride. Sodium-
hypochlorite is generally used at a concentration of 2 per cent for 5 to 30 minutes.
While hard tissues such as stem pieces and seeds are directly exposed to the
sterilants, soft and delicate tissues such as embryos and shoot tips are dissected
from surface sterilised plant parts. Addition of a few drops of a surfactant such a&
triton-X or tween-80, to the sterilant soiution enhances the efficiency of the
sterilising agent. After treating with a sterilant solution, the plant material is rinsed
several times in sterile distilled water to remove all traces of the sterilant.
All manipulations after surface sterilisation.of the tissue are ,carried out in aseptic
conditions. Presently a laminar air flow cabinet is used for this purpose (Fig. 11.1).
Air is forced into the cabinet through a bacterial filter. It flows outward and forward

Fig. 11.1: Laminar airflow cabinet ready for use.

over the bench at a uniform speed. The work bench in the cabinet is cleaned in
cotton soaked in ethanol (70-80 %) before starting the work. Instruments such as
forceps, needles, and steel knives used fo; preparing materials for inoculation are
sterilised by dipping in ethanol and flaming. This is done at the start of the work
and several times during the op&ration. Even the hands of the operator are sterilised
by dipping in alcohol and drying in air.

11.3.2 Culture Medium

In nature green plants are capable of synthesising organic compounds necessary for
their growth and development from the mineral nutrients and they can absorb water
from the soil and obtain C 0 2 from the atmosphere. However, in tissue cultures the
normal biosynthetic potentiality of the cells is weakened, therefore, it is necessary
Plant Development-11 to provide all the essential organic and inorganic nutrients (including sucrose) and
growth regulators, particularly an auxin and a cytokinin.
Nutritional requirements for optimal growth of tissue cultures may vary with the
source (plant). They are also affected by the age of the explant and the stage of
development. For example very young embryos require a more elaborate-
component of the medium as compared to mature embryos. Similarly culture
requirements of single cells are more complex than shoots.
Composition: A standard plant tissue culture medium (Basal medium) contains all
the essential macroelements (Carb~n~hydrogen, oxygen nitrogen, phosphorus,
sulphar, calcium,potassium and magnesium) and iron and microelements(iron,
manganese, copper, zinc, boron and molybdenum) but the concentration of their
salts in different formulations vary. In addition some vitamins and sucrose (2-3%)
are universal constituents of plant tissue culture media. The composition of Ms
medium (Murashige and Skoog's) medium found satisfactory for a wide range of
monocotyledonous and dicotyledons species is given in table 11.1.
Table, 11.l: Composition of Murashige and Skoog 's
(MS) basal medium, widely used in plant tissue culture
studies (1960)
Constituents Concentration (mgll)
Inorganic constituents
A. Macronutrients

MgSo4.7H20
KH2 Po4
B. Iron
FeS04.7H20
Na2 EDTA. 2H2 0
C. Micronub-ien&
MnS04.4H20
ZnS04.7H20
H3B03
KI
Na2 Mo04.2H20
CuS04.5H20
CoC12.6HzO
Organic constituents
Myo-inositol
Glycine
Nicotinic acid
Pyridoxine-HCL
Pyridoxine-HCL 0.10
Sucrose 30,000.00
Agar . 8,000.00
Besides the nutrients, one or more plant growth regulators (PGR7S) are generally Plant Tissue And Organ
Culture
required for supporting good growth of the cultured material. The PGR'S most
widely used in tissue culture media are auxins (2,4-D, IAA, NAA, IBA) and
cytokinins (BAP, Klnetin). The PGR7sare particularly important for the growth of
Callus tissues and organogenicl embryogenic differentiation. Usually the medium is
gelled with 0.8% bacteriological agar.
Preparation: Now that you are familiar with the constituents the preparation of
medium is quite simple. Several plant tissue culture media are now available
commercially in the form of dry powders, containing all ing~edientsexcept growth
regulators, sucrose and agar. They are very convenient to prapare media for routine
maintenance of cultures. Generally concentrated stock solutions of major inorganic
nutrients (200 times concentrated expressed as 20 x) micro inorganic nutrients
( 200 x concentrated ) iron ( 200 x concentrated) and organic nutrients except
sucrose are prepared and stored in a refrigeratsr at 4" c. Separate stock solutions are
prepared and stored for each growth regulator by dissolving it in a minimal quantity
of appropriate solvent and adjusting the final volume with distilled water.
A general protocol for media preparation is as follows:
1. Prepare stock solutions one day before the medium is to be made.
2. Weigh the required quantities of agar and sucrose and dissolove them in
water (about 314th the final volume of the medium) by heating in a-waterbath
or autoclaving at low pressure.
3. Pipette the required volumes of each of the stock solutions into the above
solution kept on a stirrer.
4. Make up the final volume of the medium by addition of distilled water.
5. Adjust the pH to 5.8 with 0.1-0.5 N NaoH or Hcl.

11.2.3 Culture Conditions

The culture vessels placed in suitable trays or racks, are stored on the shelves in a
hygenically maintained culture room, under controlled conditions of light and
temperature. If possible the relative humidity should not fall below 50% to prevent
rapid desiccation of the medium. To minimise the infection of cultures during
incubation, the culture room should be dust free. It is often provided with double
doors and entry of people is restricted.
The requirement for light and temperature varies considerably. The cultures of high
altitude plants as well as those of desert species grow well at 25O c-28"c. However,
induction of pollen embryogenesis require a high temperature treatment (35" C). As
mentioned earlier, in cultures even green tissues and shoots do not exhtbit active
photosynthesis and are largely heterotrophics. The differentiation of shoots from
callus or explants and axillary shoot proliferation are favoured by light. A callus is
defined as an unrecognised mass of tissue varying widely in texture, appearance
and growth rate. Such cultures should be maintained in continuous diffuse light
(1-3K lux) provided by fluorescent tubes. You should know that unorganised callus
and freshly isolated protoplast cultures are often sensitive to light and are incubated
in darkness. Total darkness is also favourable for rooting and for the initial stages of
pollen and somatic embryogenesis.

SAQ 1:
a) Why is it extremely important to sterilise the nutrient media used for plant
tissue culture?Answer in about 40 words
Plant Development-11 b Choose the Correct statement from among the following:
Many undesirable microorganisnis may find the culture media suitable for
their growth and compete with plant tissue for various nutrients.
The main aim of surface sterilisation is to remove all of the microorganisms
with a minimum of damage to the plant system to be cultured.
iii) Plant tissues often require rich media for their growth, the presence of
m i c ~ ~ o r ~ a n i sasmcontaminants
s may hamper the interpretation of results.
It is in fashion these days to autoclave the media before inoculation.
Sterilised media, any way give a better look.
Some compounds are thermolabile and should be added separately to a
sterilised medium, after passing themthrough a bacteria-proof filter. What
does the term thermolabile mean?
State which of the following statements is true:
Thermolabile compounds are those compounds which retain their chemical
structure and activity when exposed to heat.
The compounds which can pass through a bacteria proof filter membrane are
called thermolabile compounds.
iii) Some compounds become active only after passing through a bacteria proof
filter membrane, such compounds are called thermolabile compounds.
iv) Compounds subject to destruction or loss of characteristic properties by the
action of moderate heat.
You have used the term 'Explant" in this section. Say which of the following
statements best defines this term.
1) The plant which acts as a source of inoculum for tissue culture.
ii) The plants obtained from tissue culture grown on an artificial medium.
iii) The tissue pieces used to initiate cultures.
iv) The medium which provides nutrients to plant parts under culture.
e) Fill in the blank spaces with appropriate words from the text:
i) Besides the nutrients, one or more . . . . . . . . . . . . . . . . . .: . . . . . . . . . . . . . are
required for callus growth and organogenic/embryogenic differentiation.
ii) Culture requirements of callus tissue are. . . . . . . . . . . . . . . . . . . . . .
elaborate than single cell.
iii) Media are sterilised by autoclaving at. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv) pH of the culture medium is adjusted at.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v) Plant tissue cultures require light for.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11.3 ESTABLISHMENT OF TISSUE CULTURES

By now you are familiar with the term "Explant". After a few days in culture the
explant becomes slightly rough in texture and its surface glistens in reflected light.
This is a sign of the beginning of callus formation. A "callus is defined as an
irregular tissue mass varying considerably in texture, appearance and growth rate.
In nature plants produce callus as a result of mechanical injury (wound tissue),
invasion by microorganisms or by insect feeding. Callus formation has been
observed in angiosperms, gymnosperms, ferns, mosses and liverworts. Infection by
Agrobacterium tumefaciens causes the production of tumours or 'Crown gall' in
dicotyledonous plants. The stimulus for cell proliferation in these cases is provided
by endogenous hormones, auxins and cytokinins. Plant material typically cultured
included vascular cambia, storage parenchyma, pericycle of roots, cotyledons, leaf Plant Tissue And Organ
Culture
mesophyll and provascular tissue. You should know that all multicellular plants are
potential sources o f explants for callus initiation. In the laboratory you can easily
establish callus cultures from the stem pith tissues of tobacco or slices of carrot root
on MS medium supplemented with 2,4-D (Fig. 11.2). Olhertissuesmayrecluirea
cytokinin for &induction. In such cases a hgh 2,4-D. Cytokinin ratio favours callus formalion.

(el
0.5 cm.
C,

-a

Fig. 11.2: Fig. 11.2: Diagram sbowing the preparation ofexplants from the cambial region of
carrot (Daucus carota) taproot. (a,b) A segnent approximately 0.5 cm in thickness is
removed from the taproot. (c) The segments are surface sterilized and subsequently
rinsed repeatedly in sterile DDHzO (not shown). (d,e) Short cylinders of tissue (0.5 em
O.D.) are removed with a stcrile cork borer from the cambial zone of the taproot.
( f ) After trimming the ends oftissue tbat may have been injured by the sterilization,
tbe explants are placed on the surface of the agar-solidified medium. (g)Following
incubation at a suitable temperature, callus will arise from repeated divisions of the
cultured cells.
The most important characteristic of callus from a functional view point is that
abnormal growth has the potential to develop normal roots, shoots and embryoids
that can form plants. Callus cultures become brown and necrotic if allowed to grow
, on the same medium for an extended period because of the depletion of essential
nutrients, gradual desiccation of the agar due to water loss and accumulation of
toxic metabolites in the medium the tissue may eventually die. The callus is cut into
two or more pieces and transferred to a fresh medium. Such cultures are referred to
as "sub-cultures". After a few subcultures some tissue systems do not require
exogenous auxin for callus proliferation. Such cultures are said to have become
'8abituated to auxin". However, this phenomenon is not a genetic change but a
selective gene expression as habituated cultures are known to revert to auxin
requirement with time.
In the following subsections we will discuss the establishment of suspension
cultures and single cell cultures.

11.3.1 Establishment of Suspension Cultures

A callus crumbles into smaller clumps and single cells in liqu~d~medium by gentle
agitation (100-120rPM) on a shaker. Shaking the cultures also helps
-
to aerate
P
the
Plant Development-11 cells. Such suspension cultures however rarely comprise single cells alone because
cells tend to aggregate in clusters of 2-100. Suspension cultures can be maintained
indefinitely by inoculations of known aliquot5 of cells to a fresh medium. This
process is termed as "batch cultures". Alternatively, the medium is replenished at
regular intervals. This process is termed as "continuous culture". In the continuous
culture process at the time of replenishing the medium, cells are also harvested
(open continuous system) or the biomass is allowed t~*increase(close continuous
system). Suspension cultures are useful in studying problems related to cell biology
including cell cycle and production of secondary metabolites like alkaloids,
steroids, glycosides, napthaquinones, flavones etc. which find medicinal and
industrial application. Pharmaceutical industries use large bioreactors for
suspension cultures to obtain valuable bioorganic compounds. A bioreactor is a
vessel of glass or steel in which cells are cultured aseptically and culture conditions
are closely monitored. This results in higher yield of metabolites. In a bioreactor
there is provision for adding fresh medium, for harvesting cells, for the aeration of
products, for mixing and sampling, for controlling pH, 0 2 content and temperature
Plant cells are immobilised in alginate, agarose, polyacrylamide beads.
Immobilisation of cells enables i) re-use of biomass by rotation of cells ii)
separation of cells from the medium and iii) leaching of metabolites in it .
Immobilised cells are cultured in column reactors. Column reactors are of different
types with different agitation and flow systems. Such reactors may be i) stirred tank
type ii) air lift type iii) bubble column type and iv) rotating drum type.

11.3.2 Single Cell Culture

This is an important invitro technique which enables the cloning of selected cells.
Single cells can be obtained directly from plant organs by treatment with enzymes
that dissolve middle lamellae. The separate cells can sieve into liquid medium to
start a suspension culture. The most widely used technique for single cell culture is
the Bergmann's method of Cell Plating and. Microchamber technique.

Bergmann's Method of Cell Plating:

/ -
flltrate
molten
Wh~te's agar
with single medlum
cells and
suspenr
cell suspension
' c e l l aggregates

aqar medium
+I
cell colony

top vlew s ~ d eview

Figure. 11.3: A Summary diagram of Bergmann's method of cell Plating

In this method free cells are suspended in a liquid medium at a density twice the Plant Tissue And Organ
Culture
finally desired plating density. Melted agarcontaining medium of otherwise the
same composition as the liquid medium is maintained at 35Oc in water bath. Equal
volumes of the two media are mixed and rapidly spread out in petri dishes in such a
manner that the cells are evenly distributed and fixed in a thin layer (about 1 mm
thick) of the medium after it has cooled and solidified. The dishes are sealed with
parafilm. The cells to be followed are marked on the outside of the plate and before
the colonies derived from individual cells grow large enough to merge with each
other. They are transferred to.separate plates. (Fig. 11.3).
Another popular method for single cell culture is the microchamber technique,
developed by Jones et al. (1960). In this method mechanically isolated single cells
are cultured in separate droplets of liquid medium. While Jones et al. used sterile
microslides and three coverglasses to make microchamber, it is now possible to buy
pre-sterilised plastic plates with several microwells (Cuprak dishes). Individual
cells are cultured in separate wells each containing 0.25 ml of the liquid medium.
The culture requirement of single cells increases with decrease in the plating cell
density, and the cell cultured in complete isolation require a very complex culture
medium. A simple medium conditioned by growing cell suspension for some time
rlso fulfils the requirements of single cell culture at low density.(Fig. 11.4)

Fig. 11.4: Development of a tobacco plant from a single cell A callus is raised from a small piece
of tissue excised from the pith (A). By transferring it to a liquid medium and shaking
the culture flasks (B) the callus is dissociated into single cells. A ceU (C) is mechanically re-
moved from the flask and placed in a drop of culture medium in a microchamber (D).
A small tissue (E), derived from the cell through repeated divisions is then transferred
to a semi-solid medium where it grows into a large callus (F),and eventually differen-
tiates plants (G,H). When transferred to soil (1,J) these plants grow to maturity flower
and set seeds.

SAQ 2:
Choose the correct word from the bracket and fill in the blanks:
i) An undifferentiated mass of cells, irregular in outline, produced in cultures, is
known as. . . . . . . . . . . . . . . . . . . . . . . . . . . . .(Callus/Crown gall).
Plant Development-I1 ii) Callus formation can be induced in nature as a result of mechanical injury.
Such a callus is called. . . . . . . . . . . . . . . . . . . . . . . . . . . ..(wound tissue or
proliferated tissue).
iii) Callus formation is stimulated by a synthetic auxin, such as. . . . . . . . . . . . .
. . . . . . . . . . . . .., (2,4D/lAA)
iv) Periodic division of a callus tissue and transfer of each segment to fresh
medium is called. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(Inoculation/subculture).
b) Match the items given in column A with their respective explanation
given in Column B and compare your answer with those at the end of
this unit.
Column A Column B

i) Single cell culture i) A large vessel used for

mass in-Vitro Propagation
of cells in a liquid medium
and monitoring their growth
ii) Batch culture ii) Cells growing singly or in
small clumps, in a liquid
medium, gently agitated on
a gyratory or rotatory shaker
for better aeration and fast
growth.
iii) Bioreactor iii) An unorganised, undifferen .
tiated mass of cells grown
on an agar medium; usually
having an irregular boundarq
iv) Suspension culture iv) Suspension cultures in which
the medium is replenished
periodically, the cells being
simultaneously harvested or
the biomass is allowed to
accumulate.
v) Continuous culture ' v) The practice of growing
selected cells obtained
either from suspension
cultures or maceration of
plant parts.
vi) Callus vi) Suspension cultures in
which aliquots of cells are
periodically transferred to
fresh medium.

C. Choose the false statement among the following:

i) Wound callus is initiated by auxin and cytokinin.
ii) Callus formation can be induced in numerous plant tissues and organs that do
not usually develop callus in response to an injury.
iii) In the laboratory we can easily establish callus cultures from the stem pith
tissues of tobacco or slices of carrot root.
iv.) Initiation of callus is restricted to some specialised cells
4. Callus cultures become brown and necrotic if they are left too long on the Plant Tissue And Organ
Culture
same medidm why?

11.4 CELIiULAR TOTIPOTENCY

In the preceding units of this course you have read that innumerable cells which
constitute the body of a higher plant or animal and containing identical genetic
material can be traced to a single cell-the zygote. During development cells
undergo diverse structural and functional specialisation depending upon their
position in the body. Leaf cells bear chloroplasts and act as the site of
photosynthesis. The colourless root hairs perform the function of absorbing
nutrients and water from the soil and some other cells become part of the colourful
petals. Normally fully differentiated cells do not revert back to a meristematic: state,
which suggests that the cells have undergone a permanent change. In earlier
sections of this unit you have read that the regenerative capacity is retained by all
living cells of a plant. Several horticultural plants regenerate whole plant from root,
leafiand stem cuttings. Highly differentiated and mature cells such as those of pith
and cortex and highly specialised cells as those of microspores and
endosperm,retain full potential to give rise to full plants under suitable culture
conditions. G. Haberlandt was the first to test this idea experimentally. This
endowment called "cellular totipotency" is unique to plants. Animal cells possibly
because of their higher degree of specialisation do not exhibit totipotency. Whole
plant regeneration from cultured cells may occur in one of the two pathways:
;)shoot bud differentiation, (organogenesis) and ii) embryo formation
(Embryogenesis). The Embryos are bipolar structures with no organic connection
with the parent tissue and can germinate directly into a complete plant. On the other
hand, shoots are monopolar. They need to be removed from the parent tissue and
rooted to establish a plantlet. Often the same tissue can be induced to form shoots o r
embryos by manipulating the components of the culture conditions.
In the following sub sections we will discuss organogenesis and embryogenesis in
detail.

11.4.1 Organogenesis
Organogenesis refers to the differentiation of organs such as roots, shoots or
flowers. Shoot bud differentiation may occur directly from the explant or from the
callus. The stimulus for organogenesis may come from the medium, from the
endogenous compounds produced by the cultured tissue or substances carried over
from the original explant. Organogenesis is chemically controlled by growth
regulators. Skoog while working with tobacco pith callus, observed that the addition
of an auxin Indole Acetic Acid (IAA) enhanced formation o f roots and suppressed
shoot differentiation. He further observed that adenine sulphate, (Cytokinin)
reversed the inhibition of auxin and promoted the formation of shoots. You should
know that:

1) Organogenesis is contolled by a balance between cytokinin and auxin

concentration i.e. it is their relative rather than the absolute concentration that
determines the nature of differentiation.
2. A relatively high auxin: Cytokinin ration induces root formation, whereas a
high cytokinin: auxin ratio favours shoot bud differentiation.
3. Differential response to exogenously applied growth regulators may be due to
differences in the endogenous levels of the hormones within the tissue.
Organogenesis is a complex process. Whereas in the cultured tissues of many
species organogeiiesis can be demonstrated in this pattern, some plants,
notably the monocots, are exceptions. Plant tissues respond differently to
Plant Development-Il exogenously applied PGR's because of differences in the levels of
endogenous Plant Growth Regulators (Fig. 11.5 A,B).
I A A Concentratfon in mg !-.?

Fig. 11.5: Organogenesis in tobacco callus cultures as affected by IAA and kinetin at different
concentrations, individually and in various combinations. Note that root formation
has occurred only in the absence of kinetin (and in the presence 010.18-3.0 m@ of
IAA) (A) and shoot differentiation in the presence of kinetin, particularly with 0.005-
0.18 m@ (k).
A

11.4.2: Somatic Embryogenesis

The process of embryo development is called embryogenesis. It is not the
monopoly of the egg to form an embryo. Any cell of the female gametophyte
(Embryo sac) or even of the sporophytic tissues around the embryo sac may give
rise to an embryo. Thus we can say that 'The phenomenon of embryogenesis is not
necessarily confined to the reproductive cycle". In this subsection we will discuss -,-
some examples of "embeos formed in culture", also referred to as "somatic -
embryos".
The first observation of somatic embryos were made m Dacus Carota. Other plants
in which the phenomenon has been studied in some detail are Ranunculus scleratus,
citrus and coflea spp.
In Rarrunculus scleratus somatic as well as various floral tissues, including anthers
proliferated to form callus which, after limited unorganised growth differentiated
several embryos. These embryos germinated in situ and a fresh crop of embryos
appeared on the surface of the seedling. The embryos were derived from individual
epidern~alcells of the hypocotyl.(Fig. 11.6)
Citrus is commonly cited as an example of natural polyembryony.
In the preceding units of this course (Block I) you have read about polyembryony
and parthenocarpy. The nucellus cultures of monoembryonate as well as
polyembryonate cultures of citrus could be promoted if malt is added to the basal,
 l j E CELL

Q GLOBULAR STAGE

8
pm-o
YOUNG
HEARTS A G E

TORPI30 STAGE

Fig. 11.7: Stages of somatic embryogenesis. Following repeated cell divisions, cell aggregates
progressively develop and pass through globular, heart, and torpedo stages before ul-
timately forming plantlets.

11.5 APPLICATIONS

Plant tissue culture is an essential component of plant biotechnology. The

possibility to regenerate whole plants from protoplasts, single cells, tissues and
organs, in-vitro, has opened out entirely new approaches to plant improvement, and
has considerably enhanced the efficiency of the conventional methods of plant
breeding and plant propagation. This section highlights some of the applications of
plant tissue culture in agriculture, horticulture and industry.

11.5.1: Production of Rare Hybrids

Hybridization is a well established plant breeding procedure to obtain superior
plants by combining useful characters distributed in different plants. However,
cross-pollination does not always result in the formation of viable seeds. The cross
may fail due to a pre-zygotic barrier to crossability, which prevents fertilization, or
the hybrid embryo may abort due to post-zygotic incompatibility. It is now possible
to overcome both types of incompatibility bamers.
Hybrid embryo normally aborts on account of the failure of endosperm
development or due to embryo endosperm incompatibility, In such instances it can
be excised from the young seed and cultured in-vitro.
This technique was first used by Laibach to raise viable interspecific hybrids in
Linum. Since then embryo culture also called embryo rescue has been successfully
applied to several sexually incompatible crosses (e.g., Hordeum vulgare x H.
Bulbosum, Lycopersicon esculentum
.- - x L. Peruvianum).
 r l a n t I wsue And urgan
Culture

Fig. 11.6: Stages in the differentiation of somatic embryos from stem epidermal cells of Ranun-
culus. A Oncmonth old seedling bearing embryos all over the stem. B. A portion of
the seedling in A enlarged to show several embryos hanging from the surface of the
stem. C-F. Sectional views of globular (C), heart shape @), and cotyledonary @,F)
embryos differentiated from epidermal cells of the stem.

medium. You must be knowing that the seedless grapes are the result of
parthenogenetic embryos (embryos formed by the unfertilized egg. In nucellus
cultures of vitis embryo formation occurred in the presence of B. Naphthoxyacetic
acid and BAP (Benzyl Amino Purine). However, minimal level of endogenous or
exogenous auxln is necessary for in-vitro somatic embryogenesis. Supplementing
the medium with activated charcoal facilitated embryogenesis in Daucus carola and
Hedera helix (English ivy). Somatic embryos have been formed directly from leaf
mesophyll cells of Dacfylis glomerala L. (Orchard grass), without an intervening
callus. Though regeneration of whole plants by embryogenesis has been relatively
rare in Gramineae. (Fig. 11.7).
 In several interspeciflc crosses of Brassica abortion of hybrid embryo occurs at Plant Tissue And Organ
Culture
such an early stage that it is not possible to excise and culture the embryo. A hybrid
can be obtained by culturing the ovules (Ovule culture) or ovaries (Ovary culture)
enclosing the hybrid embryo. Embryo culture has also been used to raise hyrbids
between the sexually incompatible parents, Gassypium hirsutum and G.arbareum.
The pre-fertilization barriers to incompatibility include: i) failure of pollen to
germinate on an alien stigma, ii) inability of pollen tube to reach the ovule due to its
iI inherent short length or slow growth of pollen tube so that the ovary abscises before
i the pollen tube reaches the ovary, or iii) Pollen-pistil incompatibi1ity:In such cases
techniques of in vitro pollination or test-tube polliakion (TTP),developed by Kanta
et al. (1962) at the University of Delhi, hold promise, In TTP the ovules attached to
a piece of placental tissue are excised one day before anthesis and planted on a
suitable culture medium. The following day pollen grains are collected from the
desired male parent and applied aseptically to the cultured ovules. Under suitable
culture conditions the pollen grains germinate on the susface o f the ovules, the
pollen tubes find their way into the ovules and enable fertilization to take place.
'I'TP has been successfully applied to obtain hybrids-between sexually incompatible
species of Nicotiana and to overcome self-incompatibility in Petunia axillaris.
Very recently, Kranz and Lorz (1990) have succeeded in fusing, in vitro, the
excised male and female gametes of maize: the fusion product divided to form a .
small amount of callus. Regeneration of plants from the in vitro fertilized egg or the
callus derived from it would open out an entirely new approach to overcome
pre-fertilization barriers to incompatibility.

11.5.2 Somatic Hybridization and Cybridization

In the early 1970's an altogether new approach to raise hybrids which could not be
produced through the conventional method of hybridization was proposed. It .
involves the fusion of somatic cells and regeneration of plants from the fusion
i products (somatic hybridization).

Fig, 11.8: A. Freshly isolated protoplasts ofwhite clover. B,C. Stages in the fusion of a nonchlo-
rophyllous protoptast from ,suspension culture of Petunia hybrida with a green meso-
phyll protoplast of P. parodii. First the fusion body apears dumbhell shaped which
eventually becomes spherical. Even in C the chloroplasts are confined to one side of
the heterkaryon. With the mixing of cytoplasm of the two protoplasts the chloroplasts
would become evenly distributed.
Plant Development-11 Plant cells are bounded by a rigid cellulose wall and are cemented together by a
pectin-rich matrix to forni tissues. An essential step in fusion of plant cells is to
bring together the plasma membrane by degrading the cellulosic wall. Thus, the
first step in sonlatic hybridization is tlie isolation of plant protoplasts (spherical
naked cells which hawe bcen str~ppedoff their cell wall). (Fig. 11.8A)
Of the several kinds of materials tested for protoplast isolation mesophyll (leaf
Parenchyma) and rapidly fast growing cell cultures have been most useful. Several
potent and fairly purified enzymes (e.g., Pectolyase y-23, Onozuka R-10,
Macerozyme R-10, Driesalase) obtained from fungi are available. These can
convert plant tissues into a large number of protoplasts. In practice, small pieces of
leaves or cells from actively growing cell cultures are iiicubated in a mixture of a
cellulase and a niacerozyme, at 30°c, in dark, for 3-12 hr (the duration of treatment
varies with the tissue) to digest the cell wall and middle lamella respectively. The
e n q m e solution also contains a suitable osmoticuni (Sucrose or mannitol) as the
freshly isolated protoplasts may burst or shrink. After incubation the protoplast are
cleaned by repeated washing in salt solution or culture medium. The protoplasts arc
directly cultured as single cells or used in fusion experiments. Freshly isolated
protoplasts are also useful for genetic transformation as they behave like animal
cells. They can rcadily take up macromolecules, such as purified DNA.
The protoplasts are cultured either in liquid medium or on agar plates. The
protoplasts readily regenerate a cellulose cell wall, and under suitable culture
conditions, the cells undergo divisions to form a totipotent callus. Complete plants
have been regenerated from isolated protoplasts of several plant such as rice, cotton,
potato. tonlato and mustard.
The freshly isolated protoplasts readily fuse (Fig. 1 1.8 b,c) with each other when
brought in intimate contact, irrespective of their taxonomic relationship. Several
chemical substances tliat facilitate fusion of protoplasts (fusogens) have been used.
Of these the high niolecular weight (1,500-1,600) polyethylene glycol (PEG)
applied in tlie presence of high pH (8-10) and high cat+ has been niost effective. In
recent years electrofusion of proto~laslshas become popular because of the control,
efficiency and versatility of this method.
A highly significant application of protoplast fusion is the production of asynimetric
hybrids by partlal genome transfer from an irradiated donor protoplast to an
acceptor protoplast and the selective transfer of cytoplasmic genes. Many important
agronomic traits, such as herbicide resistance and cytoplasniic male sterility, are
often controlled by extra nuclear genes. Selective transfer of cytoplasmic traits is
achieved by (lie fusion of norilia1 protoplasts of the recipient parent' with the
donor's protoplasts in which the nucleus has been rendered lnactlve by ~rradiation
or with its enucleated sub protoplasts or niin~plasts.Such hybrids are called
Cybrids. Medgyesy et al. (1 980) transferred streptomycin resistance (controlled by
chloroplast DNA) fro~nNicotlono robac~rrnto N sylvestr;~by fusing iodoacetate
treated. non-dividing protoplasts of streptomvcin-resistant N. taboczlrn with nonnal
protoplasts of streptomycin sensitij~eN.sylr1estr.i~.
Alloplasniic male sterilc lines of Rrassrco nnpzls and Rrosslco oleracco produced
by substituting their cytoplasm by the ogura cytoplasm of male sterile Raphanlts
sativus could not be util~sedfor hybrid seed production because of their yellowing
of leaves at low temperature (Jourdan et al., 1985). By fusing the protoplasts of
these lines with thosc containing nor~iialcytoplasm of respective species,
rcsearclicrs working with pelletier (1983),Robertson (1985) and Menzel (1987)
replaced the sensitive chloroplasts by insensitive ones. The new alloplasm~clines
retained the uselul male sterility while acquiring functional chloroplasts.
Fusion of dissimilar protoplasts (from different parents) results in tlie formation of
heterokaryons. After the fusion treatment the fusion mixture contains, besides
heterokaryons, the unfused protoplasts and homokaryons (the fusion product of
siniilar protoplasts from the parents). It has been possible in some cases to isolate
the heterokaryons mechanically, using micropipettes or by using a cell sorting Plant Tissue And Organ
Culture
machine. Generally, however, a suitable selection pressure is applied which permits
the growth of only hybrid cells by selectively suppressing the division of the other
types of cells. The nuclei in a heterokaryon fuse to form a hybrid cell. The latter
may divide and produce a callus mass and eventually whole plants, thus may be
differentated.
Several interspecific and intergeneric somatic hybrids have been produced. Fusion
between potato and tomato created 'Pomato' (Melchers et al. 1978) and fusion
between Arabidopsis and Brassica resulted in Arabidobrassica. However, such
distant hybrids are generally sterile and do not produce viable seeds. Therefore, it
has been now realised that somatic hybridization is likely to be successful only
when closely related but sexually incompatible parents are involved. For example,
Solanum breviciense, a wild species, is resistant to potato leaf roll virus (PLRV) and
potato virus y (PVY) but it can not be directly crossed with Solanum .tuberosum
(potato). Some of the somatic hybrids between these two species, showing
resistance to PLRV and PVY, are cross compatible with S. tuberosum allowing
introgression of virus resistance gene in potato cultivars.

11.5.3 Haploid Production

The higher plants are normally diploid, with two sets of chromosomes in their
somatic cells. Their haploids (with dne set of chromosomes) arise in nature by
parthenogenesis due to malfunction in the normal sexual process. However, such
events are extremely rare and unpredictable.
In 1964, two Indian scientists, Guha and Maheshwari, observed that in cultured
anthers of Datura innoxia some of the microspores, instead of following the normal
gametophytic mode of development, formed sporophytes (Androgenic plants). As
expected, those sporophytes were haploid (Guha and Maheshwari, 1966). This
report caused much excitement because of the considerable importance of haploids
in genetics and plant breeding. To-date androgenic haploids of over 200 species,
including many major crop plants (Cereals, ~ r a s s i c i stomato
~ ~ , and potato), ha;e
been raised through anther and/or isolated pollen culture (Fig. 11.9).

Fig. 11.9: Androgenesis in anther cultures of tobacco. A. A burst anther showing numerous pol-
len embryos. B. The pollen embryos have germinated.

In practice, anthers at the late uninucleate stage of microspore development are

excised from surface-sterilised buds and cultured on a nutrient medium. Often a low
Plant Development-11 temperature (4-5Oc) shock during initial 2-3 days of culture enhances the
androgenic response. However, In Brassica species treatment with higher
temperature (30-35' C) has proved beneficial. Under inductive conditions the

Fig. 11.10: A sumary diagram of haploid production by anther culture and their diploidization
to raise homozygous diploid plants.

microspores undergo repeated divisions to form multicellular structures. Depending

on the plant and the culture medium, such structures directly develop into an
embryo or form a callus from which plants are regenerated via organogenesis or
embryogenesis (Fig. 11.10).
Androgenic haploids of some species, such as wheat, mustard and tobacco, can also
be raised through isolated microspore/pollen culture (Pollen cultures). It must be
realized that in spite of serious efforts androgenic haploids have not been possible
in many other economically important plants.
In-vitro gynogenesis is another approach to produce haploids (Yang and Zhou,
1990). In this technique unfertilized ovules are cultured on media which stimulate
the egg (parthenogenesis) or any other haploid cell of the embryo sac (apogamy) to
undergo embryogenic development without fertilization. Invitro gynogenesis, fust
observed in Hordeum vulgare by San Noeum (1967), has now been reported in at
least 16 species. This technique of haploid production is especially useful in plants
in which the androgenic response is unsatisfactory, a large proportion of pollen
plants are non-haploids or albinos, as in many cereals. .
Haploids are extremely important m genetics and plant breeding. In haploids it is
possible to detect recessive mutants which do not express themselves in diploid
state due to the presence of the dominant allele. In cross pollinated plants and F,
hybrids, with high degree of heterozygosjty, the fixation of a particular trait through Plant Tissue And Organ
Culture
the conventional method of backcrossing takes 7-8 years. By anther or pollen
culture this can be achieved in a single generation. Regeneration of plants from
pollen grains also permits the screening of gametic variations at sporophytic level
and selecting useful variants (gametoclodal variation). The Chinese scientists have
developed and released about 20 new improved varieties of wheat and 61 varieties
of rice through anther culture.

11.5.4 Clonal Propagation

Most cultivars of ornamental and fruit species and forest trees are highly
heterozygous. Consequently, their seed progeny is not true-to-type. To preserve the
unique characters of selected cultivars of horticultural plants nurserymen practise
vegetative propagation, using stem, leaf or root cuttings or propagules such as
tubers, corms, bulbs or bulbils. For plants which do not set seeds, such as edible
bananas,grapes, citrus, petunia, rose and chrysanthemum, vegetative propagation is
the only means of multiplication. A population of plants derived from a single
individual by vegetative propagation is genetically uniform and is called a clone.
The conventional methods of clonal propagation are slow and often not applicable.
For example, the only in-vivo method for clonal multiplication of cultivated
orchids, which are complex hybrids,is "back-bulb" propagation. It involves
separating the oldest pseudobulbil to force the development of dormant buds. This
process allows, at best, doubling the plant number every year. Moreover,

AXILLARV BRANCHIHG

CALLUS
.-
I

CELL SOMATIC GERMINATING

5USFR1510H EMBRIOGENESIS EMBm

Fig.ll.11: Diagrammatic summary of steps involved in aseptic multiplication of plants. Shoot

multiplication is achieved through enhanced axillary branching adventitious budding
from explants directly or after callusing The shoots are rooted individually in a me-
dium containing an auxin. The plantlets so obtained are transferred to well drained
potting mix. After maintaining them under high humidity for3-4 weeks the plants are
transferred to ordinary glasshouse or field conditions Plant multiplication involving
a callus phase may occur via shoot bud differentiation or somatic embryogenesis. In
the latter case the rooting step is eliminated as the embryos possess a pre-formed root
primordiurn.
Plant Development-rT monopodial orchids do not form pseudobulbils and, therefore, cannot be clonally
multiplied. In 1960, a French scientist, G.More1, described an in-vitro method for
rapid clonal multiplication of orchids. This revolutionised the orchid industry, and
today tissue culture is the only economically feasible method for clonal
multiplication of orchids and is being widely used.
In-vitro clonal propagation, popularly called Micropropagation (Fig. 11.12) has
been extended to a large number of species other than orchids and is being practised
on commercial scale for numerous ornamental and fruit bearing plant and some
forest trees. After the initiation of aseptic cultures micropropagation generally
involves three steps: Shoot multiplication, rooting and transplantation (Fig. 11.11).

Shoot Multiplication:
This is the most important step with respect to the rate of propagation and genetic
uniformity of the product. The most reliable and, therefore, themost popular method
of shoot multiplication is forced proliferation of axillary shoots. For this, cultures
are initiated from apical or nodal cuttings carrying one or more vegetative buds. In
the presence of a cytokinin alone or in combination with a low concentration of an
auxin, such as IAA or NAA, the pre-existing buds grow and produce 4-6 shoots
(sometimes up to 30-40 shoots) within 3-4 weeks. By periodic removal of
individual shoots and planting them on fresh medium of the original composition,
the shoot multiplication cycle can be repeated almost indefinitely, and a stock of
large number of shoots built up in a short period of time. Treatments with PGRs as
described above can also help in a rapid build up of shoots by inducing adventitious
buds by the explant directly or after callusing.
Somatic embryogenesis, which generally occurs after callusing of the explant, is
another method of micro propagation. Somatic embryogenesis is not only fast, but
may also allow partial automation of micropropagation and the propagules so
produced (somatic embryos) bear both, shoot and root meristems. However,
adventitive differentiation of shoots or somatic embryos, especially from callus
tissue, has the risk of genetic variability in the progeny. Such variation, that
develops in tissue culture called "somaclonal variation" is not desirable for
micropropagation but is being exploited as a novel source of useful variations for
crop improvement.

Rooting:
Shoots produced through axillary branching or adventitious differentiation are
rooted in-vitro on a medium containing a suitable auxin, such as IAA, NAA or IBA.
Alternatively, where possible, the shoots are treated with auxin and directly planted
in potting mixture for in-vivo rooting.

Transplantation:
The shoots or plantlets multiplied on a medium containing organic nutrients, show
poor photosynthetic capability. Moreover, in these plants mechanisms to prevent *

loss of water from leaves are poorly developed. Therefore, they require gradual
acclimatization to the field conditions. In practice, the plants are maintained under
high humidity (80-90%) for 10-15 days after they are removed from culture vessels.
During the next few weeks the h u ~ i d i t yaround the plants is gradually lowered,
before they are transferred to naturtil conditions.
The special merits of micropropagation are: i) it considerably increases the rate of
multiplication 2) high rate of multiplication can be maintained throughout the year,
3) the multiplied plants are maintained in disease-free conditions 4) being free from
microbes and insects valuable genotypes of exotic plants can be multiplied for
export purpose, and 5) small size of the propagules and their ability to proliferate in
a soil-less environment facilitates their convenient storage, handling and rapid
transfer by air across international quarantine baniers.
 11.5.4 Production of Disease-free Plants Plant Tissue And Organ
Culture
Under normal conditions plants are infected by a wide range of pathogens such as
bacteria, fungi, viruses, viroids, and insects like nematodes and insects. Many
perennial plants and those propagated by vegetative means are systematically
infected with one or more pathogens, which reduce yield, vigour and quality of the
plant. If explants for micropropagation are derived from an infected plant, the
pathogens can multiply and spread to a large number of plants. It is, therefore,
i essential to use disease free stock plants for micropropagation. Eradication of
1 viruses and other pathogens is also desirable from the point of view of international
b exchange of plant materials.
Whereas bacteria and fungi present on the surface of the plant material can be easily
eliminated by treatment with surface sterilizing agents, there is no dependable
treatment against viruses. Viruses can multiply within the shoots in-vitro without
symptoms. Traditionally, thermotherapy has been used for virus elimination but it is
not effective against all viruses. Moreover, heat treatment may adversely affect the
plant tissues.
For some reasons viruses are unable to enter or survive in the apical meristems.
Therefore, even in infected plants the apical meristems are generally free of viruses.
Taking advantage of this observation, Morel (1950) developed the technique of
shoot tip culture to raise virus-free plants from infected individuals (Fig. 11.12).
Since then it has become the most effective method of virus elimination. It involves
excision of 0.5-1 mm long shoot tips, including apical meristem and one or two leaf
primordia and their cultivation on a suitable medium to regenerate whole plants.

Plant regeneratton
f r o m meristem tip

1.Sy rnptom
Multiplication of Virus
2 Indicator plant
t e s t e d plants in Irisect-
3.Electron microscopy
proof House
L S e r o l o g ~ c a ltest

Fig. 11.12: Production of virus-free plants through shoot tip culture-A diagrammatic summary.
Plant Development-11 Some times a combination of chemotherapy or thermotherapy with shoot tip culture
has enhanced the efficiency of the latter for virus eradication. However, the plants
regenerated from shoot tips must be thoroughly screened for suspected viruses
before declaring them virus-free.

11.5.5 Other Applications

Plants are source of a wide range of industrial products, including drugs. For
considerable time the Ayurvedic system of medicine has been using dried plant
parts or their crude extracts. However, the large scale, uncontrolled collection of
plant materials from nature and habitat destruction by man have reduced the size of
populations of certain species to such an extent that they are threatened to become
extinct. In this context tissue culture provides two possibilities: (i) in-vitro
conservation of endangered species and (ii) production of useful compounds by
cultured cells.

Germplasm Conservation:
Totipotent plant cells and shoot tips can be freeze-preserved in liquid nitrogen
(-196'~) for long periods, and when required they can be thawed and cultured to
regenerate whole plants. Alternatively, for short term storage, the proliferating
shoot cultures or roots can be incubated at growth-limiting conditions, such as
decrease in temperature (4-12'c) or low nutrients. Such cultures have been shown
to retain viability for 1-3 years, without a subculture. Root cultures have been kept
for 25-30 years.

Production of Industrial Compounds:

Cultured plant cells retain their metabolic potential and synthesise secondary
products of commerce. Cell cultures can also be used as factories for bioconversion
of intermediate compounds into more valuable products. Shikonin, an expensive
compound, obtained from the roots of Lifhospermum erythrorhizon, has been used
by the Japanese traditionally as a vegetable dye and in cosmetics and toiletries.
However, due to over exploitation this plant has become almost extinct in Japan. To
reduce dependence on import of this plant material, the Japanese scientists have
developed a tissue culture method for the commercial production of Shikonin.
Another example in which tissue culture production of an industrial compound has
reached commercial level is Berberine from Coptis Japonica. In tissue cultures the
yield of high value compounds can be enhanced by feeding the cells with
precursors of their biosynthetic pathway (Biotransformation), manipulation of the
culture conditions and selecting high yielding cell lines. The production of
secondary plant products of commerce would considerably reduce the pressure on
shrinking arable land. Besides its practical application, tissue culture systems have
been found ideal to study basic aspects such as alternation of generation,
morphogenesis, growth and differentiation and host-pathogen interaction. In fact,
one important fundamental contribution of plant tissue culture is the discovery of
cytokinins.

SAQ 3:
A) mark the correct statements:
a) Animal cells are totipotent
b) All living cells are totipotent v

c) Plant cells are totipotent

d) Differentiation of shoot bud is referred to as cellular totipotency.
B) Select the right answer:
Cellular totipotency refers to the capacity of cells to produce:
a) Shoots Plant Tissue And Organ
Culture
b) Embryos
c) Full plants
d) Roots
C) Fill in the blanks using appropriate words from the text:
a) Fusion of dissimilar protoplasts results in the formation of. . . . . . . . . . . . . . .
......................
b) A plant cell which has been stripped of its wall is called. . . . . . . . . . . . . . . .
......................
c) . . . . . . . . . . . . . . . . . .is the most popular fusogen.
d) Hybrid embryos which abort at a very early stage can be rescued by. . . . . . .
......................
D) Give the technical term for the following statement:
a) In-vitro prouuction of plant from pollen grains
b) In-vitro production of plants from unfertilized egg cell
c) In-vitro propagation of plants
d) Variation among the plants raised from pollen grains
E) Choose the false statement:
a) Microspores and unfertilized egg are known to form haploid plants in culture
b) Haploids are important in genetic studies as they help to detect recessive
mutants.
c) Haploids production by tissue culture is of academic interest only as such
plants are abnormal and can not be integrated into conventional breeding
programmes.
d) Micropropagation allows the production of a large number of propagules in a
relatively short time throughout the year under aseptic conditions.

11.6 SUMMARY

What we have learnt in this unit can be summarised as follows:

1. Plant tissue culture is the aseptic cultivation of isolated cells or protoplasts in
standard plant tissue culture medium (Basal medium).
2. Tissue cultures can be raised from all living plant cells and multiplied
indefinitely by periodic subculture on fresh medium.
3. All living plant cells are totipotent. By manipulating the composition of
growth regulators in the medium it has been possible to regenerate whole
plants from callus and suspension cultures via organogenesis or somatic
embryogenesis.
4. Some chemicals used in Basal medium degrade on exposure to high
temperature (Thermolabile) and some are stable (Thermostable).
5. Isolated protoplast released from the cell wall by either a mechanical or an
enzymatic process is described as naked protoplast.
6. Tissue culture techniques, such as "hybrid embryo culture", "test-tube
pollination or fertilization" and somatic hybridization are being used to
Plant Development-I1 e hybrids or cybrids which can not be produced by the
breeding methods.
ture has become an important horticulture technique to raise
plants and for rapid clonal multiplication of selected genotypes.

1. Why is it essential to maintain a completely aseptic environment inside the

2.
i
culture v ssels.
What are bajor categories of constituents of plant tissue culture media? Why
is sucrose/an essential constituent of all plant tissue culture media.
by cellular totipotency? Who was the first Scientist to test this

4.
i.
Differenti te between inoculation and subculture.
5.
s
Briefly di cuss the role of plant tissue culture in plant breeding.
of micropropagation over the conventional methods
of plants?
ne major contribution of the following scientists to plant tissue
culture:

a and S.C. Maheshwari

n
Self Assessme t Questions:
ely important to sterilise the nutrient media used for plant tissue.
plant tissue culture media contain a high concentration of
the growth of many microorganisms like bacteria and
fungi. 1
(b). IV

!
(c) Compoun s which can not stand heat are called "thennolabile compounds".
They dec mpose and loose their activity on autoclaving.
(d). C.
(e). i) growth regulators.
ii) less
iii) 120°c
iv) 5.8

SAQ 2:
bi
v) Shoot ud differentiation and shoot multiplication.
 Plant Tissue And Organ
2(a). i) Callus Culture
ii) Wound tissue
iii) 2,4-D
iv) Subculture.
b) i) V
I ii) VI
iii) I
t iv) I1
I
v) IV
vi) I11
c) iii)
d) Callus culture becomes brown and necrotic if they are left too long on the
same medium because of:
i) depletion of essential nutrients
ii) desiccation of the agar due to water loss and
iii) accumulation of toxic metabolites in the medium.

SAQ 3:
3) a)c
b) c
c) i) heterokaryon
ii) Protoplast
iii) Polyethylene glycol
iv) In ovule embryo culture
d) i) androgenesis
ii) parthenogenesis/gynogenesis
.
,
iii) micropropagation
iv) gametoclonal variation.
..
e) 11

TERMINAL QUESTIONS

1). The nutrient media used for plant tissue culture would support luxuriant_
growth of many microorganisms, such as bacteria and fungi. Reaching the
medium these microdrganisms grow much faster than the plant cells and
cover the tissue surface, impeding its growth and finally killing it. It is
therefore extremely important to maintain complete aseptic environment
inside the culture vessels.
2) Any plant tissue culture medium should have the following categories of
constituents: a) Sources of major and minor inorganic elements b) Organic
nutrients, such as vitamins and amino acids c) Sucrose as a source of carbon
and d) Plant growth regulators, such as auxin and cytokinin, use of agar is
optional.
Plant Development-11 did not succeed because: a) he selected highly differentiated leaf cells as the
experimental material and b) he did not use growth promotory substances as
they were unknown at that time.
4) Inoculation is the process of planting fresh explants on culture medium at the
time of initiation of cultures, whereas sub culture refers to dividing the
cultured tissue into pieces and transferring them to fresh medium.
5) Application of plant tissue culture.to plant breeding:
a) Embryo culture for the production of rare hybrids.
b) Anther Pollen and unfertilized ovule culture, to devclop haploids for
rapid production of homozygous diploids.
c) In-vitro pollination and fertilization to overcome prezygotic barriers of
sexual incompatibility
d) Somatic hybridization and cybridization.
6) Micropropagation has many advantages over the conventional methods of
clonal plant propagation:
a) It is often faster than the conventional methods of vegetative propagation.
b) Large number of plants can be multiplied in a short space.
c) Multiplication occurs under disease free conditions.
d) Under controlled conditions rate of multiplication is maintained
throughout the year.
7) a) G.Morel: Shoot tip culture for virus elimination
b) F.Skoog: Chemical control of organogenesis
c) P.R.White: Continuous root culture
d) F.C.Steward: Somatic embryogenesis in carrot
e) F.Laiback: Hybrid embryo culture
f) S.Guha and S.C. Maheshwari: Production of androgenic haploids L ,
anther culture
g ) L.H.San Noem: Production of gynogenic haploids by ovule culture.