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POLLINATION STUDIES IN WALNUT (JUGLANS REGIA L.)

Research · October 2016

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Shaziya Hassan Khalid Bhat

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Save Nature to Survive
11(4): 2683-2686, 2016 (Supplement on Agronomy)
www.thebioscan.com

POLLINATION STUDIES IN W
POLLINATION ALNUT (JUGLANS REGIA L
WALNUT .)
L.)

K. M. BHAT1*, SHAZIYA HASSAN1, M. A. BHAT2, M. A. MIR1 AND S. N. KIRMANI*1

1
Division of Fruit Sciences,
2
Department of Plant Biotechnology,
Sher-e-Kashmir University of Agricultural Sciences and Technology- Kashmir, Shalimar - 190 025 (J&k) INDIA
e-mail: [email protected]

KEYWORDS ABSTRACT
Germination The present investigation entitled “Pollination Studies in Walnut (Juglans regia L.)” was carried out on the walnut
Pollination selections available at Horticultural Farm, Division of Fruit Science, Sher-e-Kashmir University of Agricultural
Pistillate flower Sciences and Technology, Shalimar Srinagar. Pollen viability of all investigated genotypes was greater than 87 %.
abscission Highest viability of 91.40 % was observed in selection SKAU-W-0020 and the lowest (87.40 %) in SKAU-W-
Viability 0022. Maximum pollen germination was recorded in SKAU-W-0020 (51.51 %) on media containing agar agar
Walnut powder (1%) + Sucrose (15%) + boric acid (100 ppm) + CaCl2 (1 mM). There was a significant effect of
genotype and pollination method on pistillate flower abscission and was maximum (42.40 %) in SKAU-W-0015
Received on : through open pollination and minimum (15.77 %) in SKAU-W-0025 through cross pollination. Fruit retention
03.03.2016 also varied significantly with maximum (90.61 %) in SKAU-W-0025 X SKAU-W-0022 combination and minimum
(81.49 %) in SKAU-W-0024 under open pollination. Taking into consideration the pollen viability and germination
Accepted on : values, investigation revealed that these selecyed selections of walnut can be used as pollinizers for walnut
14.10.2016 genotypes that bloom in the same period and the level of pistillate flower abortion was significantly reduced
under controlled pollination when compared with open pollination.
*Corresponding
author

INTRODUCTION 1965). Some studies have been carried out on walnut pollen
production, viability and amount of pollen for different
The Persian walnut (Juglans regia L.) also known as English cultivars (Saglam and Gulcan, 1995; Sutyhemez and Eti, 2005)
walnut belongs to family Juglandaceae. Walnut is one of the but such pollen tests have not been carried out in walnuts
most important nuts grown in India. In India, walnut grows in grown in Kashmir. Pistillate flower abortion (PFA) is
North-western Himalayan belt, expanding to Darjeeling and understood as the loss of flowers early in the season due to
Sikkim. Jammu and Kashmir state is the major producer of excess of pollen. This loss is stimulated by an excess of pollen
walnut covering an area of 93,642 ha and production of about on stigmas due to high rate of ethylene biosynthesis which
2, 09,051 metric tonnes (Anonymous, 2012). Pollen activates preformed abscission zone, resulting in flower
functional quality is important for crop improvement and abscission which can seriously reduce yield (Gonzalez et al.,
fertility studies. One of the important factors for fertilization 2008) and pollen donor has its effect on other paramerters of
success is pollen viability and germination. Pollen viability is nut and kernel. (Golzari et al., 2010). Many observations made
an ability of a pollen grain to germinate and develop as a in countries like France, USA, Spain, Chile demonstrated that
pollen tube (Prajapati and Jain, 2011). In vitro germination PFA exists nearly in all walnut cultivars (Polito, 1998).
studies of pollen has been used as powerful tool for genetical, However, such studies regarding pollen performance and
physiological, biochemical and cytochemical processes for a effect of pistillate flower abortion on walnut production have
wide range of plant species belonging to different families not been undertaken under Kashmir condition. The paper
(Sarika Gupta and Mary Varkey Boswal, 2012). The rate of deals with the objective to determine viability, germination,
pollen germination of fruit and nut species and cultivars varies pollination ability of different walnut varieties and to determine
depending on the medium or chemical concentration. For efficient mode of pollination on pistillate flower abscission
this reason the suitable germination medium should be and fruit retention.
obtained for each species. Germination percentage is affected
by many factors, including the concentration of calcium, MATERIALS AND METHODS
hydrogen and borate in the germination media (Holdaway-
Clarke et al., 2003). The addition of boron and calcium to the Plant material
germination media increases germination percentage and The present study was carried out to determine the viability,
length of pollen tube growth in many fruit species. Sucrose germination, and pollen production capacity of walnut
primarily serves to control the osmotic potential of the varieties and also to determine efficien mode of pollination in
germination media but may also provide a base for walnut (Juglans regia L) during the year 2012 at the Horticulture
polysaccharide, synthesis and metabolic energy (Kwack, farm, Division of Fruit Science, Sher-e-Kashmir University of

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K. M. BHAT et al.,

Agricultural Sciences and Technology of Kashmir, Shalimar SKAU-W-0022 and SKAU-W-0020 was collected by shaking
Campus , Srinagar. The present investigation was conducted the catkins in glass flasks. The pollen was then diluted with
on two promising pollen parents of walnut (SKAU-W-0022 talcum powder in order to obtain 5 per cent concentration of
and SKAU-W-0020) crossed with following female parents of pollen (w/w) to carry out manual pollination test. Each replicate
walnut (SKAU-W-0024, SKAU-W-0025, SKAU-W-0008 and of female genotypes, were labeled and bagged with muslin
SKAU-0015). cloth bags. At the bi-fid stage which is the stage of stigma
Pollen germination and viability receptivity with an angle of 45î between two stigmatic lobes,
the bags were removed and the pollen of the SKAU-W-0022
Pollen germination and pollen viability tests were performed
was applied on pistillate flowers of SKAU-W-0025 and SKAU-
according to Eti (1990). The freshly dehisced pollen grains
W-0024 and pollen of the SKAU-W-0020 was applied on
were dusted on sterilized petri plates containing media agar
pistillate flowers of SKAU-W-0008 and SKAU-W-0015.
agar powder (1%) solidifying against different concentrations
Pollination was performed by depositing a prepared load of
of sucrose (10, 15, 20 , 25%) alone and in combination with
pollen on the stigmas of each flower using the eraser end of
100 ppm boric acid and 1mM calcium chloride. The petri
pencil , considered as the contact surface of a known area as
plates were incubated at 27±1ºC for 24 hours. The pollen
earlier used by (Gonzalez et al., 2008) while performing
grains were considered as germinated when length of pollen
controlled pollination in walnut. The degree of covering was
tube was equal or exceeded pollen diameter. Pollen viability
superficial without agglomeration of pollen. After pollen
was estimated using two staining techniques, TTC (2,3,5-
application the stigmas were covered with cotton to avoid
triphenyl tetrazolium chloride) and Fluorescence diacetate.
contamination. Fig. 1. Shows the steps involved for artificial
TTC and FDA solutions were prepared according to (Norton,
pollen application and state of development of pistillate flowers.
1966) and Heslop and Harrison and Heslop and Harrison
Then the pollinated flowers were again bagged. The bags
(1970) respectively. Pollen grains were scattered onto TTC
covering the pollinated flowers were removed 3 weeks after
and FDA solutions, and stained pollen grains were counted
pollination. Percent of pistillate flowers showing PFA
after 2 hours and fifteen minutes respectively. Under TTC test,
characteristics were recorded. The final fruit retention was
the pollen grains that stained orange or bright red colour were
determined on the basis of number of fruits retained at the
counted as viable. In the second staining method, pollen grains
time of harvest on the selected branches.
that fluorescence were counted as alive. The experiments were
designed as completely randomized blocks with three
replications. Randomly selected visual areas, including about RESULTS AND DISCUSSION
100 pollen grains were counted in each replicate. All Pollen germination and viability
observations of germination and viability were made at x100
magnification using a light microscope. Different media using different sucrose were used to test the
germination of walnut pollen. The in vitro germination was
Modes of pollination highest in the pollen parents (51.51% in SKAU-W-0020 and
In open pollination four different branches on four sides of a 50.41 per cent in SKAU-W-0022) in medium containing Agar
tree in each genotype were selected and number of healthy Agar 1% + Sucrose 15 % + boric acid 100ppm + CaCl2
female flowers were counted and marked for open pollination. 1Mm and lowest germination of 34.83 per cent was recorded
In cross pollination pollen from two (male) genotypes viz., in medium containing Agar Agar 1Mm + Sucrose 25% (Table

Table 1: Effect of different sucrose concentrations alone and in combination with Boron and Calcium Chloride on pollen germination
percentage in pollen parents

Treatments Pollen germination (%)

SKAU-W-0022 SKAU-W-0020
1% Agar + 10% Sucrose 39.46 37.88
1% Agar + 15% Sucrose 43.48 40.89
1% Agar + 20% Sucrose 37.43 35.29
1% Agar + 25% Sucrose 38.93 34.83
1% Agar + 10% Sucrose + 100ppm boric acid + 1mM CaCl2 48.09 49.68
1% Agar + 15% Sucrose + 100ppm boric acid + 1mM CaCl2 50.41 51.51
1% Agar + 20% Sucrose + 100ppm boric acid + 1mM CaCl2 46.65 45.69
1% Agar + 25% Sucrose + 100ppm boric acid + 1mM CaCl2 42.67 43.17
CD0.05 3.88 2.73

Table 2: Determination of pollen viability of pollen parent genotypes in walnut using T.T.C. and FDA stain tests.

Male genotype TTC FDA

Viable Semi-viable Dead Viable Dead
SKAU-W-0022 87.40 ±2.07 6.80±0.84 5.20±0.83 88.40 ±1.15 11.60 ±0.62
SKAU-W-0020 90.20±0.83 5.20±0.84 3.20±0.83 91.40 ±1.15 8.30 ±0.41
tcal 2.80* 3.02* 3.77** 4.16** 7.68**
p-value 0.035 0.016 0.005 0.003 0.002

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 POLLINATION STUDIES IN WALNUT

Table 3: Effect of open and hand pollination on pistillate flower abscission (%) after 3 weeks of pollination and fruit retention (%) at harvest.
Female genotype Pollen parent Pistillate flower abscission (%) Fruit retention at harvest (%)
SKAU-W-0025 : Open pollination 26.53 (5.24) 86.47 (9.35)
X SKAU-W-0022 15.77 (4.09) 90.61 (9.57)
SKAU-W-0024 : Open pollination 30.74 (5.63) 81.49 (9.08)
X SKAU-W-0022 19.25 (4.49) 87.53 (9.40)
SKAU-W-0008 : Open pollination 40.13 (6.41) 82.14 (9.11)
X SKAU-W-0020 27.75 (5.36) 88.23 (9.44)
SKAU-W-0015 : Open pollination 42.40 (6.58) 86.49 (9.35)
X SKAU-W-0020 26.44 (5.23) 89.78 (9.52)
CD0.05 0.27 0.14
*Figures within parenthesis are the square root transformed values

Bagging of flowers Stage of stigma receptivity Dusting of pollen with help of Stigma covered with cotton to
Pencil rubber end avoid contamination

1). Results showed that pollen germination rates were that the highest (42.40%) pistillate flower abscission (PFA)
improved by addition of boric acid and calcium to sucrose. was recorded under open pollination in SKAU-W-0015 which
Boric acid is generally used as boron source. Boron is believed was reduced to 26.44 % under cross pollination with SKAU-
to promote pollen germination by affecting H+- ATPase activity, W-0020. The lowest pistillate flower abscission (PFA) was
which initiates pollen germination and tube growth (Feijo et recorded under open pollination of SKAU-W-0025 (26.53%)
al., 1995). Calcium is involved with pectin synthesis and the which was further reduced to (15.77%) under cross pollination
control of the osmotic conditions. Similar results were also in SKAU-W-0025 X SKAU-W-0022 (Table 3). The reason for
achieved by (Mert, 2009; Kamrani, 2012; Mondal and Ghanta, this is the excess pollen load on stigmas as in open pollination
2012) in different crops. Highest pollen viability (91.40% and the pollen load can be higher but in case of hand cross
90.20 %) was observed in SKAU-W-0020 selection under pollination we use a particular concentration and quantity of
both FDA and TTC stain tests respectively. Semi-viable pollen. Excessive pollen tubes growing down the style of the
percentage of pollen was highest (6.80 %) in SKAU-W-0022 female flower produce higher amounts of ethylene, which is
and the lowest (5.20%) in SKAU-W-0020 through TTC test associated with organ senescence. Beede and Polito (2003)
while as dead pollens were maximum (11.60 %) in SKAU-W- also reported that elevated ethylene levels are cause of flower
0022 through FDA test and minimum (3.20 %) in SKAU-W- abortion. The degree of losses due to pistillate flower abscission
0020 through TTC test (Table 2). The TTC test is based upon was different in different walnut cultivars. Climatic conditions
dehydrogenase enzyme activity. Upon seed/tissue hydration, may also influence the flower drop level due to PFA in some
the activity of this enzyme increases, resulting in release of H + cultivars (Rovira and Aleta, 2006). Hassani et al. (2006)
ions which reduce the colourless Tetrazolium salt into a red reported that PFA was 11 to 92% among Iranian Genotypes.
compound Formazen which stains living cells with red colour Gun et al. (2010) reported that the level of PFA in Turkish
while dead cells remain colourless. In FDA test the Fluorescein walnut cultivars ranged from 65.4% to 100%. The results of
diacetate, a non-polar and non-fluorescent molecule is the present investigation were supported by results of previous
hydrolysed by enzyme pollen esterase into Fluorescein, a polar work (Gonzalez et al., 2008; Lemus, 2010).
and fluorescent molecule which after accumulation inside
the pollen grain appears fluorescent in blue light. The results The highest fruit retention (90.61%) at harvest was recorded
of the present study are in accordance with previous studies under cross pollination in SKAU-W-0025 crossed with SKAU-
(Cosmulescu et al., 2009; Sutyemez, 2011). W-0022 followed by 89.78 per cent under cross pollination
in selection SKAU-W-0015 crossed with SKAU-W-0020 and
Modes of pollination lowest (81.49%) under open pollination in selection SKAU-
The pistillate flower abscission under different modes of W-0024 (Table 4). Fruit retention is higher as little fruit drop is
pollination is given in Table 3. Present investigation revealed observed in walnut as compared to other fruit and nut species

2685
K. M. BHAT et al.,

after fruit set. McGranahan and Leslie (1990) reported that by AVG. 2nd International Symposium on Sustainable Development
there is sporadic fruit drop in walnut which may continue (ISSD’ 10). Proceedings Science and Technology, Sarajevo. 3 : 29-34.
throughout the growing season. The results of our study are Hassani, D., Eskandari, S. and Jarrahi, K. 2006. Pistillate flower
supported by Kumar et al. (2005) who reported high values of abscission of walnut genotypes. Acta Hort. 705: 257-260.
fruit retention in walnuts (52.93 %- natural pollination; 68.02% Heslop-Harrison, J. and Heslop-Harrison, V. 1970. Evaluation on
- self pollination and 65.37% under cross pollination). Lower pollen viability of enzymaticaliy induced fluorescence intracullular
values of fruit set and fruit retention may be attributed to hydrolysis of fluorescein diacetate, Stain Technol. 45: 115-120.
younger age of plants, genetic makeup of the varieties/selections Holdaway-Clarke, T., Weddle, N., Kim, S ., Robi, A., Parris, C.,
and also to the climatic and environmental factors (Pandey Kunkel, J. and Helper, P. 2003. Effect of extracellular calcium, pH
and Tomar, 2012). and borate on growth oscillations in lilium formosanum pollen tubes.
J. Expt. Bot. 54: 65-72.
From the above observations it is concluded that both staining
Kamrani, R. 2012. Study on pollen germination and pollen tube
methods gives good response in determination of pollen
growth of five Iranian apricot cultivars on in vitro condition.
viability. The in vitro germination assey was highest in the International Conference on Applied Life Sciences. pp. 299-302.
pollen parents (51.51% in SKAU-W-0020 and 50.41 per cent
Kumar, A., Kumar, K. and Sharma, S. D. 2005. Extent of fruit set and
in SKAU-W-0022) in medium containing agar agar 1% + retention under different modes of pollination in Persian walnut
Sucrose 15 % + boric acid 100ppm + CaCl2 1Mm. Highest (Juglans regia L.). Acta Hort. 696: 327-330.
pollen viability (91.40% and 90.20 %) was observed in SKAU-
Kwack, B. 1965. The effect of calcium on pollen germination. Proc.
W-0020 selection under both FDA and TTC stain tests Amer. Soc. Hort. Sci. 86: 818-823
respectively. On the basis of pollen viability and germination
Lemus, G. 2010. PFA control in ‘Serr’ in Chile. Acta Hort. 861: 263-
walnut genotypes tested in this study have the characteristics 266.
of good pollinizers and can be used as pollinizers for those
McGranahan, G. H. and Leslie, C. 1990. Walnuts (Juglans). In:
walnut genotypes that bloom in the same period. Pistillate
Ballington J. R and J.N. Moore (Eds.), Genetic Resources of Temperate
flower abscission under hand pollination reduced significantly Fruit and Nut Crops, Vol. II. ISHS Secretariat, The Netherlands, pp.
when compared to the open pollination. Results showed that 907-951.
the degree of Pistillate Flower Abscission varied with the variety. Mert, C. 2009. Temperature responses of pollen germination in
SKAU-W-0022 proved to be effective pollinizer for SKAU-W- walnut (Juglans regia L.). J. Biol. Environ. Sci. 3(8): 37-43.
0024 & SKAU-W-0025; and SKAU-W-0020 for SKAU-W-0008
Mondal, S. and Ghanta, R. 2012. Effect of sucrose and boric acid on
& SKAU-W-0015. The studies showed that the level of pistillate in vitro pollen germination of Solanum macranthium dunal. Ind. J.
flower abortion in walnut genotypes was significantly reduced Fund. App. life Sci. 2(2): 202-206.
under controlled pollination when compared with open Norton, J.D. 1966. Testing of plum pollen viability with tetrazolium
pollination. salts. Am. Soc. Hort. Sci. 89: 132-134.
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