~ 32 ~
International Journal of Chemical Studies 2018; 6(6): 32-40
P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2018; 6(6): 32-40
© 2018 IJCS
Received: 15-09-2018
Accepted: 20-10-2018
Vijayakumar Rathod
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
TK Behera
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
AD Munshi,
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
Kumar Durgesh,
Divsion of Genetics, ICARIndian
Agricultural Research
Institute, New Delhi, India
GS Jat,
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
Boopala Krishnan G
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
Neetu Sharma
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
Correspondence
Vijayakumar Rathod
Divsion of Vegetable Science,
ICAR-Indian Agricultural
Research Institute, New Delhi,
India
Pollen viability and in vitro pollen germination
studies in Momordica species and their intra and
interspecific hybrids
Vijayakumar Rathod, TK Behera, AD Munshi, Kumar Durgesh, GS Jat,
Boopala Krishnan G and Neetu Sharma
Abstract
The present work aimed to compare five staining techniques for pollen viability and three in vitro pollen
germination media in Momordica spp and their interspecific hybrids. The freshly opened male flower
were collected (7.30am to 8.00am) and evaluated for pollen viability and in vitro pollen germination. For
pollen viability studies five different stains were used viz., Tetrazolium stain, I2KI stain, Aceto-carmine
stain, Aniline blue stain and Modified Alexander stain. In vitro pollen germination were estimated using
three pollen germination solutions viz., 10% sucrose, 10% Sucrose + 100 ppm Boric acid and sucrose
@15 % + Boric acid @0.25% + Calcium nitrite @300mg solution. Off the pollen viability stains,
Modified Alexander stain differentiate viable and non-viable pollen more accurately than other stains.
Among the in vitro pollen germination media, sucrose @15 % + Boric acid @0.25% + Calcium nitrite
@300mg solution showed maximum per cent of pollen germination and pollen tube growth. All five
staining methods overestimated the pollen viability.
Keywords: Momordica charantia, M. c. var muricata, M. balsamina, Tetrazolium stain, I2KI stain,
Aceto-carmine stain, Aniline blue stain and Modified Alexander stain, In vitro pollen germination
Introduction
Quality of pollen are crucial for pollen storage studies, pollen-pistil interaction studies,
understanding incompatibility and fertility studies, for breeding and crop improvement and for
seed industry (Shivanna and Rangaswamy., 1992) [29]
. Pollen viability and vigor decide the
quality of pollen (Ottaviano and Mulcahy., 1989) [18]
. Pollen fertility and viability have a
paramount importance in hybridization programme. Successful pollination is a prerequisite for
fertilization and seed set in most plants, and the insight knowledge on pollen biology,
including pollen viability, pollen germination and pollen tube growth, is required for any
rational approach to increase productivity (Bolat and Pirlak, 1999; Shivanna, 2003) [6, 27]
. To
differentiate aborted and non-aborted pollens, a simple and hasty technique is required for high
throughput. An unambiguous procedure involves pollen deposition on receptive stigma
followed by evaluation of seed set. These methods, however, are indirect and sluggish
methods, associated with physical and physiological features of pollen with its ability to
fertilize the ovule, are universally preferred (Rodriguez-Riano & Dafni 2000) [7, 22]
. Pollen
germination assays frequently require optimization as well as being time consuming and
difficult to reproduce (Boavida, 2007) [5]
.
Reproduction is a critical step in the life cycle of all organisms and pollination is one of the
decisive stage (Piechowski et al., 2009; Jha and Dick, 2010) [20, 13]
of reproduction process.
The male gamete, pollen is surrounded by sporoderm, which consists of the inner layer intine
(which is composed of pectin, cellulose and protein) and outer layer exine (synthesized and
secreted by the tapetal tissue of the anther and is composed mainly by sporopol-protein). The
glycoprotein present on the pistil generally interact with pollen coat protein (Zhang et al.,
2008) [32]
. During the pollination process pollen grain deposited on and adhere to the stigma,
the pollen hydrates and germinate by emitting pollen tube, which penetrate the cuticle of the
stigma cells and grows through the extracellular matrix of the style. The process culminates in
the discharge of male gametes into the embryo sac. In bitter gourd, the pollen tube penetrate
papillae tissue within 1 hour of pollination and reaches to the ovule about 6 hour after
pollination and fertilization is completed within 18- 24 hours after pollination.
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International Journal of Chemical Studies
In spite of taking proper care during pollination, breeder fail
to get fertile seed during artificial pollination. Unless sterility
is the main cause the failure of seed setting may be the result
of slow growth of the pollen tube or its early degeneration in
the style. To overcome these difficulties it is necessary to
study the pollen viability, physiology of pollen germination
and pollen tube growth.
There are numerous techniques are available to study pollen
viability and the method choice depends on crop (species) and
on establishing relationship between test and the fertility
(Hanna and Towill 1995) [10]
. The most accurate method of
pollen viability is the ability of pollen to effect fertilization
and seed set (Smith-Huerta and Vasek, 1984; Shivanna and
Joshi 1989) [30]
. There are different methods to evaluate pollen
viability; some noteworthy tests are dye and in vitro
germination. The dye test have advantages as indicator of
pollen viability because they are faster and easier compared
with pollen germination, but they do tend to overestimate the
viability and real germination of pollen grains. On the other
hand, in vitro pollen germination depends on the genotype,
environmental conditions, pollen maturity, composition and
pH of the medium; thus it is necessary to determine optimum
conditions for pollen germination. It is recommended
simultaneously use several tests to reflect pollen performance.
The literatures on different tests of pollen viability was
reviewed by Dafni and Firmage (2000) [7]
and they explained
the advantage and disadvantages of each test. The ability to
use chemical staining to discriminate aborted from nonaborted
pollen grains has well-known practical applications in
agriculture. Pollen staining method some time not reliable for
most of the species (Barrow, 1981) [4]
, hence in vitro pollen
germination is more practical used to determine the pollen
viability. The ability of pollen germination play an important
role not only on fruit set but also the flower-flower and
flower-pollinator interaction. Pollen germination and pollen
tube growth very crucial for fertilization and seed
development. Due to involvement of the pistillate tissue in the
nature, physiological and biochemical investigations on
pollen germination and pollen tube growth in vivo are rather
difficult. Hence in vitro germination techniques generally
been used extensively on a variety of pollen systems. Such
studies have provided considerable information on the
physiology and biochemistry of pollen germination and pollen
tube growth (Shivana and Johri 1989; Heslop-Harrison 1987;
Steer and Steer 1989) [28, 12]
.
To reach pollen tube to micropyle region, it passes through
four stages, a) imbibition phage, b) Lag phage, c) tube
initiation phage and d) rapid tube elongation phage. Pollen
tube growth proceeds through tip extension and can be
affected by many factors, including temperature, medium
osmolarity and the availability of sucrose, calcium, zinc and
boron (Sawidis and Reiss, 1995; Tylor and Hepler, 1997.) [25,
23]
.
Momordica genus is one of the major cucurbitaceous
vegetable cum medicinal crop and it is grown in most of the
Asian countries. The genus comprises 57 species, of which 12
species are distributed in Asian continents (Schaefer and
Renner 2010); Out of 12, nine species are presently found in
India include cultivated, semi-cultivated and wild species.
Momordica charantia var charantia, M. c. var muricata and
M. balsamina are predominantly grown monoecious species
in India. The pollen viability of Momordica species lost very
rapidly (Desai and Musmade 1998) [8]
and till the date very
limited systematic studies has been carried out in Momordica
on pollen physiology studies. Hence, the present investigation
carried out to compare different staining method to study
pollen viability and in-vitro pollen germination studies in
these three species of Momordica.
Materials and Methods
Study species
The three species of Momordica i.e. Momordica charantia,
M. c. var muricata and M. balsamina were used for pollen
viability and pollen germination test. These three species are
grouped in to gene pool I (Bharathi et al., 2012) [3]
and they
are highly cross pollinated due to their monoecious sex form
where male and female flower borne on same plant but at
different node position. The inflorescence is solitary and male
flower nectary is closed in M. charantia whereas, partially
closed by corolla scale in M. balsamina. The anthesis occurs
between 3.30 and 7.30 a.m., when flowers are completely
open (Miniraj et al., 1993) [17]
. The pollen viability is lost
relatively rapidly (Desai and Musmade 1998) [8]
. The stigma
is receptive for 24 hours before and after flower opening, after
which it dries and turns brown (Rasco and Castillo, 1990) [21]
.
The sex ratio (staminate to pistilate flower) in monoecious
line throughout the flowering period is typically 50:1 (Rasco
and Castillo, 1990) [21]
but ratio can vary from 9:1 to 48: 1
(Dey et al., 2005) [9]
. The ovary of bitter gourd is inferior and
three carpels with 14 to 18 ovules (Pillai et al., 1978). In
India, Bees are important pollinator vectors (Behera et al.,
2004) [2]
.
Study site and plant materials
Seeds of M. charantia (DBGS-2), M.c. var muricata (DBGS-
34) and M. balsamina were procured from division of
Vegetable Science, Indian Agricultural Research Institute,
New Delhi. The seeds were sown in portrays in the month of
January-2016 in portrays with media composition of coco
peat and vermicompost (1:1), seedling were transplanted at 2-
3 leaf stage during spring-summer (April-July, 2016-17) and
grown under the insect proof net house condition
(Temperature-35-370
C, RH 60-70%). Freshly opened male
flowers were used for pollen viability and in vitro pollen
germination studies.
Pollen viability tests
The literature was reviewed to determine the types of tests
used in the past for pollen viability and to determine what
problems had been reported for these tests (Dafni and
Firmage, 2000) [7]
for a list of tests reviewed and the
advantages and disadvantages of each test. Base on the
literature and facility availability at laboratory condition, we
choose five staining methods for our pollen viability
experiment.
Tetrazolium Test: Tetrazolium (2, 3, 5 triphenyl tetrazolium
chloride) test, commonly known as the TZ test. All living
tissues, which respire, are capable of reducing a colour less
chemical 2, 3, 5 triphenyl tetrazolium chloride) into a red
colored compound formazan by H transfer reactions catalysed
by the enzyme dehydrogenases (Norton, 1966). This method
provide accurate results for many taxa (Shivanna and Johri,
1989) [28]
. However this method has some drawbacks like it
tends to overestimate viability (Sedgley and Harbard, 1993)
[26]
, sometime it stains non-viable pollen (Khatum and
Flowers 1995) [14]
. The concentration of tetrazolium salt,
temperature and period of incubation needs to be standardized
to get optimal results in various pollen samples (Shivanna and
Johri 1989) [28]
.
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International Journal of Chemical Studies
Procedure: 1% TTC was prepared by adding 0.2 g. TTC and
12 g sucrose dissolved in 20 mL distilled water. Two drop of
the mixture was dropped on a microscope slide and the pollen
were dusted over it and covered with a coverslip. Pollen
viability counts were made after 30-40 min incubation at 400
C under dark place. Pollen grains stained that orange or bright
red color were counted as viable.
Iodine- potassium iodide Test (I2KI): The technique
described indicates viability and starch content of pollengrains.
Iodine broke up in a watery arrangement of potassium
iodide the tri-iodide-anion edifices with starch, creating blueblack
color.
Procedure: Dissolve 1 g potassium iodide and 0.5 g iodine in
distilled water to make a final volume of 100 ml. Put 1 or 2
drops of the dye over pollen and mix thoroughly. Place a
cover slip and after 5-10min count the number of darkly
stained (viable) pollen grains under the microscope.
Aceto-carmine test (2%): Carmine show the presence of
cytoplasm. The pollen nucleus is rich in chromatin material
and viable pollen stains pink to deep red with aceto-carmine,
while sterile (mostly shriveled) pollen does not take any stain
and thus remains almost white and transparent (McKellar and
Quesenberry, 1992; Marutani, et al., 1993) [16, 15]
Procedure: Weigh 2 g of carmine powder, dissolve it in 95ml
of glacial acetic. Add distilled water to make a total of 100ml
solution. Boil it, cool and filtered and stored in a refrigerator.
Two to three drop of stain was placed on slide and pollen
grains were dusted on it followed by covered with coverslip
and pollen viability was recorded after 5-10 min. The dark red
colored grains are counted as viable pollens.
Aniline blue (Cotton blue) test: Aniline blue stain detect the
callose in the pollen walls and pollen tubes (Hauser and
Morrison, 1964) [11]
. The solution is prepared by adding
200mg/lit of aniline blue in a mixture of 10ml each of phenol,
lactic acid, glycerol and distilled water. The viable pollen
stained dark blue color, while dead pollen are unstained.
Modified Alexander method: It is a modification of
Alexander’s stain (Alexander, 1969) [1]
, which clearly
differentiate aborted and non-aborted pollen. Acid fuchsin,
present in this dye, stains the protoplasm and malachite green
stains cellulose in walls of pollen; dark purple pollen was
scored as viable and green as aborted. The non-aborted pollen
grains stained magenta-red and aborted pollen grains stained
blue-green (Peterson et al., 2010) [19]
.
Protocol: The stain is prepared by adding following
constituents in the order given. 10ml of 95% ethanol, 1 ml of
Malachite green (1% solution in 95% alcohol), 50 mL
Distilled water, 25 mL Glycerol, 5 mL Acid fuchsin (1%
solution in water), 0.5 mL Orange G (1% solution in water), 4
mL Glacial acetic acid and Add distilled water (4.5 mL) to a
total of 100 ml. Flower bud are fixed in 2 hour before
Carnoy’s fixative (6 alcohol:3 chloroform:1 acetic acid)
before anthesis which sufficiently removes oils and sticky
materials and clear the pollen and anther walls. The flower
buds or pollen grains are dried and apply 2-4 drops of stain,
once the sample is stained, slowly heat the slide over an
alcohol burner until the stain solution is near boiling (~30
seconds). A more moderate rate of heating allows better
penetration of the dye into the cellulose and protoplasm of the
pollen. Slides are covered with cover slip and images are
captured under light microscope. The per cent pollen viability
was calculated using formula
Pollen Viability (%) =
Number of stained pollen grains
Total number of pollen grains on slide
× 100
In vitro pollen germination test: In vitro pollen germination
test were carried out using hanging drop method (Shivanna
and Rangaswamy, 1992) [29]
with three different germination
media suggested by Saoji, (1975) [24]
in bitter gourd with
media composition of 10 per cent sucrose with 100 ppm boric
acid, Kielkowska and Havey (2012) media suggested for
cucumber with media composition of 15 % sucrose, 0.025%
boric acid, 300 mg of Calcium Nitrate and and a control
treatment with 10 per cent sucrose. The culture was
maintained in a humidity chamber to prevent evaporation.
The freshly opened male flower are collected between 7.30-
8.00 a.m. and used for pollen viability and in vitro pollen
germination studies. Germination of pollen from open male
flowers was counted after incubation for 24 h at 250
C on
germination medium. Per cent pollen germination was
computed using formula given bellow.
Pollen Germination (%) =
Number of germinated pollen grains
Total number of pollen grains on slide
× 100
Results
Pollen viability using different staining techniques
According to the analysis of variance, all the test showed the
significance difference at 1% level of significance except
treatment I2KI, whereas, at 5 % level of significance all the
treatment showed significance different (Table 1).
Tetrazolium staining method (Fig 1)showed highest number
of stained pollen in M. c. var charantia × M. c. var muricata
(95.33 %) followed by M. charantia (94.31%) and lowest was
recorded in M. charantia × M. balsamina (89.66%). In the
I2KI staining (Fig 2) method, non-significant difference was
observed among the species and crosses at 1 per cent level of
significance. However, it had shown significant value at 5 %
level of significance. Among the species and their crosses, the
aceto-carmine staining method (Fig 3) showed significance
difference at both 1% and 5 % level of significance. M.
charantia showed highest number of stained pollen (96.42 %)
followed by M. c. var muricata (93.66 %), while M. charantia
× M. balsamina showed lowest per cent of pollen stainability
(90.21%). Aniline blue staining method (Fig 4) also showed
significant difference among the Momordica species and their
crosses at both 1 % and 5 % level of significance. The highest
per cent of pollen were stained in M. charantia × M.
balsamina (94.00 %) followed by M. balsamina (92.71%) and
lowest was recorded in M. c. var muricata × M. c. var
charantia (85.33 %). In modified alexander staining (Fig 5)
method, highest percent of pollen viability was recorded in M.
charantia (86.00 %) followed by M. balsamina (84.53 %) and
lowest was recorded in M. charantia × M. balsamina (77.33
%).
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International Journal of Chemical Studies
Table 1: Pollen viability test in Momordica species and its intra and interspecific hybrids using different staining methods.
Treatment Tetrazolium stain I2KI stain Aceto-carmine stain Aniline blue Alexander stain
Population
Pollen viability
(%)
Pollen viability
(%)
Pollen viability (%) Pollen viability (%)Pollen viability (%)
M M. c. var charantia 94.31±0.88 94.19±0.82 96.42±1.20 91.41±0.87 86.00±0.57
M. c. var muricata 92.42±0.31 92.32±0.34 93.66±0.34 94.00±1.52 84.00±1.52
M. balsamina 91.10±0.56 91.00±0.61 93.34±0.81 92.71±0.68 84.53±0.33
M. charantia ×M. balsamina 89.66±0.33 89.68±0.31 90.21±0.64 87.14±0.33 77.33±0.91
M. charantia × M. c. var muricata 95.33±0.28 93.38±0.42 93.00±0.36 87.66±0.19 82.41±0.88
M. c. var muricata × M. c. var charantia 92.00±0.52 92.00±0.18 91.00±0.48 85.33±1.45 81.00±1.15
CD @1% 2.65 NS 3.65 3.63 4.72
CD @ 5% 1.87 2.43 2.57 2.55 3.32
SE(m)± 0.54 0.54 0.79 1.77 1.01
C.V.(%) 1.02 1.02 1.49 1.56 2.15
Fig 1: Tetrazolium staing methods in Momordica species and inte intra and interspecific hybrids
Fig 2: I2KI Staining method for pollen viability in Momordica species and its intra and interspecific hybrids
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International Journal of Chemical Studies
Fig 4: Aceto-carmine staing methods in Momordica species and inte intra and interspecific hybrids
Fig 4: Analine blue staing method in Momordica species and inte intra and interspecific hybrids
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International Journal of Chemical Studies
Fig 5: Modified Alexander staing method in Momordica species and inte intra and interspecific hybrids
In-vitro pollen germination studies
Three different composition of pollen germination media was
used to study the effect media on pollen germination in
Momordica species and their intra and inter specific hybrids
and the results was presented in Table 2. A germination media
with 10 % sucrose showed that pollen germination was less
than 50 per cent (Fig 6), where as 44.25 % to 55.00% pollen
germination was observed in germinated media with 10 %
sucrose + 100 ppm boric acid (Fig 7). In vitro pollen
germination media with 15 % sucrose + 0.25% boric acid +
300 mg of calcium nitrate showed pollen germination ranged
from 62.50 % to 66.00 % per cent (Fig 8). These three
medium showed significant difference at 1% and 5 % level of
significance.
Among the Momordica species and their intra and
interspecific hybrids, significance difference for pollen
germination was observed only in sucrose 10 % media
composition, whereas other two media composition did not
shown any significance difference. The germination media
with 15 % sucrose + 0.25 % boric acid + 300mg of calcium
nitrite showed higher percent pollen germination followed by
media with sucrose 10 % + 100 ppm boric acid, where as
germination media with 10 % sucrose gave poor response.
Among the three species of Momordica and their intra and
interspecific hybrids, significance difference was observed in
media with 10% sucrose whereas, in other two media did not
showed significance difference.
Table 2: In vitro Pollen germination of Momordica species and their intra and interspecific hybrids using different in vitro media.
Treatments
M. c. var
charantia
M. c. var
muricata
M.
balsamina
M. charantia ×
M. balsamina
M. charantia ×
M. c. var muricata
M. c. var muricata ×
M. c. var charantia
CD
@1%
CD @
5%
C.V.
(%)
Pollen
germination
(%)
Pollen
germinatio
n (%)
Pollen
germination
(%)
Pollen
germination
(%)
Pollen
germination (%)
Pollen germination
(%)
Sucrose@ 10% 41.50±0.87 40.25±0.95 40.75±1.10 36.50±0.65 35.00±0.71 35.25±0.85 3.53 2.58 4.54
Sucrose@ 10% + Boric
acid@100 ppm
55.00±1.29 54.50±1.56 52.75±1.49 44.25±0.48 51.25±1.25 48.25±2.17 NS NS 5.88
Sucrose @ 15%+Boric
acid@ 0.25%+ calcium
nitrite@300mg
66.00±0.41 64.75±0.85 65±0.91 62.75±1.65 64.50±0.65 63.00±0.82 NS NS 2.87
CD @1% 4.09 7.22 5.71 4.88 4.88 5.00
CD @ 5% 2.71 4.76 3.77 3.22 3.22 3.30
SE(m)± 0.93 1.16 1.20 1.06 0.91 1.43
C.V.(%) 2.89 4.37 4.13 3.89 3.68 3.91
Fig 6: In vito pollen germination using 10% sucrose solution.
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International Journal of Chemical Studies
Fig 7: In vito pollen germination test using 10% sucrose + 100ppm Boric acid.
Fig 8: In vitro pollen germination test using 15% sucrose+ 0.25% boric acid+300mg Calcium nitrite solution
Correlation between staining methods and in-vitro
germination media
The person’s correlation coefficient was performed among the
species of Momordica and their intra and interspecific hybrids
and between the different sating techniques and germination
media used (Table 3). The person’s correlation results showed
positive and highly significance correlation between modified
Alexander stain and Sucrose @ 10% + 100 ppm Boric acid
media (0.974), sucrose @ 15 % + Boric acid @0.25% +
calcium nitrate @ 300mg (0.871). Aniline blue stain had
positive and significant correlation with all three different
composition of germination media i.e. with sucrose @ 10%
(0.893), Sucrose @10%+100ppm boric acid (0.790) and
sucrose @15 % + Boric acid @0.25% +Calcium nitrite
@300mg (0.753). The Aceto-carmine stain had positive
correlation with Sucrose @10%+100ppm boric acid (0.907)
and sucrose @15 % + Boric acid @0.25% +Calcium nitrite
@300mg (0.977) these results indicated that the pollen
viability using these staining techniques are more reliable than
other stain which were used in the present study.
Table 3: Pearson’s correlation coefficient for different pollen viability stains and pollen germination test in Momordica species and their intra
and interspecific hybrids.
Treatments 1 2 3 4 5 6 7 8
Tetrazolium stain (1) 1 0.067 -0.322 -0.531 -0.479 -0.718 -0.391 -0.381
I2KI stain (2) 1 0.617 0.803* 0.609 0.535 0.711 0.673
Aceto-carmine (3) 1 0.68 0.823* 0.752 0.907** 0.977**
Aniline Blue (4) 1 0.744* 0.893** 0.790* 0.753*
Alexander stain (5) 1 0.659 0.974** 0.871*
Sucrose@10 (6) 1 0.720 0.773*
Sucrose @10%+Boric acid @100ppm (7) 1 0.928**
Sucrose@15%+ Boric acid@0.25%+300mg calcium nitrite (8) 1
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International Journal of Chemical Studies
Discussion
In the present study among the five staining methods,
modified alexander methods could easily differentiate the
viable and non-viable pollen grains (Fig 5). In modified
alexander staining method viable pollen grains, stained
magenta-red stain and non-viable pollen grain stained bluegreen
(Peterson, et al., 2010) [19]
. In other methods of staining
used in the present study, could not differentiate easily
between viable and non-viable pollens. In most of the staining
methods, the per cent of unstained pollen grains were very
few which indicate very high per cent pollen viability but
when the germination test was performed the pollen
germination was not showed more than 70 %, this observation
indicated that the stains used for pollen viability were could
not differentiate between viable and non-viable pollen grains.
Barrow (1981) [4]
suggested that some time staining
techniques could not discriminate between aborted and nonaborted
pollen grains, hence in vitro pollen germination study
has to follow. In bitter gourd, Saoji (1975) [24]
reported in vitro
pollen germination media with sucrose @10 % was best for
pollen germination studies, however in or study sucrose @15
% + Boric acid @0.25% + Calcium nitrite @300mg found to
be superior. Off the staining methods tested, none was very
satisfactory as accusatory predictor of the In vitro pollen
germination studied. The pollen germination results are not
accurately resemble to pollen viability tested with different
method, hence different staining methods could be useful for
estimation of pollen viability accomplished with in vitro
pollen germination. Similar observation was reported by
Sedgle et al., (1993) [26]
in Acacia.
Conclusion
Modified Alexander stain was more efficient for
discrimination of aborted and non-aborted pollen in
Momordica species followed by Aniline blue stain and Acetocarmine
stain. Pollen of Momordica species showed
maximum pollen germination and growth in sucrose @15 %
+ Boric acid @0.25% + Calcium nitrite @300mg solution.
Pollen tube growth was abnormal in 10%s sucrose solution.
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