MATERIALS AND METHODS

3.1 Material

Hamelia patens Jacq. plants were collected from different parts ofKerala and

maintained in the Botanical Garden, Department of Botany, University of Kerala,

Kariavattom.

3.2 Methods

3.2.1 Phenology

Day today assessments of flowering patterns were observed in plants for one

year.

3.3 Structure of reproductive parts

3.3.1 Anther

For anatomical studies, cross sections of anther with 10 - 12 Ilm thickness

were taken using cryostat (Leica eM, 1100) and stained with safranin. The stained

sections were analysed and photomicrographed using a trinocular research

microscope (Olympus Bx5I).

3.3.2. Pistil

Morphological and anatomical characters of the pistil were studied using

cryotome sections stained with safranin under the microscope.

To analyse the fine morphological changes in the stigma during different

developmental stages, SEM studies were carried through the following steps:

Fixation

Fresh materials were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer for

one to seven hrs. The fixed materials were washed four times thoroughly in cold

phosphate buffer for 15 min at 4°C.

Dehydration and drying

The serial dehydration of fixed materials in acetone (25, 50, 75 and 100%)

was done for 10 min and stored in 70% acetone. The tissues were transferred to 100%

acetone for 30 min and later in isoamyl acetate for 15 min at room temperature. The

dehydrated stigmas were dried in a critical point drier (H.C.P. 2-Hitachi).

Mounting

The dried specimens were mounted on stubs using double adhesive tape and

sputter coated with gold (Model-E-101-Hitachi). The coated specimens were observed

under SEM (S-2400, Hitachi) and photomicrographs were taken.

3.4 Cytology

3.4.1 Meiosis

For meiotic studies, young flower buds were fixed in Carnoy's fluid (3 ethyl

alcohol: 1 glacial acetic acid), temporary smear preparations were made in 2%

acetocarmine and photomicrographed.

3.4.2 Pollen cytology

Pollen grains collected from flower buds prior to anthesis were mounted in a

drop of lactopropionic orceine and kept undisturbed for two hrs, and observed at

30 min interval until the internal content of pollen extrude out of the membrane. This

enabled the clear identity of the nucleus. Photomicrographs were taken under

appropriate magnification.

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3.5 Pollen viability studies

3.5.1 Flurochromatic reaction (FeR) test

Fl:urochromatic reaction test was carried out according to Heslop-Harrison and

Heslop-Harrison (1970). For this, fluorescein diacetate (FDA) was prepared in

acetone (2mg/ml). Sucrose solution (20%) was prepared separately to prevent

bursting of pollen grains. In a glass vial, 5 ml of sucrose solution was taken and FDA

was added drop by drop until it became turbid. A drop of this mixture was taken on a

micro slide and sufficient quantity of pollen grains were placed and uniformly spread.

The preparation was incubated in a humidity chamber for 5-10 min and observed

using fluorescece microscope under UV excitation. The viable pollen grains showing

intense fluorescence were counted and the percentage ofviability was calculated.

3.5.2 In vitro germination studies

In vitro germination of the pollen grains was carried out in Brewbaker and

Kwack's medium (1963) consisting of boric acid (10mg), potassium nitrate (lOmg),

magnesium nitrate (20mg), calcium nitrate (30 mg) in 100mi of distilled water. Along

with these standard ingredients, 5-50% sucrose was supplemented separately to the

medium. Pollen grains collected from mature buds, just before anther dehiscence,

were dusted on a drop of culture medium placed on a clean slide and incubated in a

germination chamber. The chamber used in the study was a pair of petridish lined

with moist cotton. Two match sticks were placed parallel within the bottom half on

which the slide containing culture medium with pollen grains was placed. For each

concentration of sucrose five slides were prepared. These slides were kept undisturbed

at room temperature. After three hrs, total number of pollen grains from 20 fields of

each slide was analysed and recorded. Germination percentage was calculated.

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3.6 Study of stigma receptivity

To find stigma receptivity, buds at vanous developmental stages were

pollinated with mature pollen and the rate of pollen adhesion, germination and tube

growth were observed.

3.6.1 In vivo pollen germination

Stigma receptivity was assessed under ultraviolet fluorescece microscope

according to the ABF (Aniline Blue Fluorescence) method (Martin, 1959). For

microscopic observation, flower bud at different developmental stages were collected

and pollinated with mature pollen grain and fixed in Carnoy's fluid and stored for

further studies. The fixed pistils were transferred to 0.8 N NaOH for 6 hrs to make the

tissue soft. Then the pistils were washed thoroughly in distilled water and stained with

1% aniline blue in O.IM K2HP04 solution for over night. Stained pistils were mounted

in a drop of glycerine. Percentage of pollen germination was estimated using

fluorescece microscope and photomicrographs were taken.

3.6.2 Cytochemical localization of esterases on stigma surface

Cytochemical localization of non specific esterases was done based on

hydrolysis of the substrate a-naphthyl acetate. Fresh unpollinated and pollinated

flowers were collected and carefully excised the pistil without damage and kept them

on a clean micro slide in a humid chamber.

In a cavity slide, 2 ml of the substrate solution was taken and applied directly

on the stigmatic surface at different developmental stages and incubated the

preparation at 25-35°C in a humid chamber for 10-20 min. After the incubation

period, the stigma was rinsed thoroughly with the phosphate buffer in a petridish, and

41

the whole mount preparations were observed under microscope and

photomicrographed.

3.7 Pollination experiments

In order to analyse the pollen tube growth, behaviour and the rate of seed set,

self-pollination was done and compared it with open pollination.

3.7.1 Open pollination

For open pollination, naturally pollinated flowers were collected after 6 hrs of

anthesis.

3.7.2 Self-pollination

For self-pollination, flower buds were bagged a day before anthesis. On the

day of anthesis flowers were hand-pollinated with pollen grains from the same flower,

bagged and labelled for further observations.

Open and self-pollinated pistils were collected after 8 hrs of pollinations and

fixed in Carnoy's fluid. Fixed pistils were transferred to lactophenol solution in which

a few drops of anline blue stain was added and incubated in an oven at 60°C for

20 min. The stained pistils were mounted in a drop of glycerine on a microslide. A

cover glass was placed over it, pressed gently and photomicrographed. Gennination

percentage and tube length were estimated.

Pollen tube growth and behaviour after open and self-pollinations were also

assessed under ultraviolet fluorescence microscope according to ABF method

(Martin, 1959).

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3.8 Confirmation of self-incompatibility by in vitro bioassay

In vitro bioassay was conducted to find out the presence of incompatibility

factors in the pistil. For this leachate of the stigma and style were incorporated into

the culture medium to find out its effect on pollen germination.

3.8.1 Effect of stigmal stylar leachate on pollen germination

For the preparation of stigmatic leachate, pistils from 50 flowers were taken

and carefully tied together by a thin thread in such a way that the stigmatic heads were

at the same level. The tied pistils were placed in a culture tube containing 1ml of

extracting solvent (water) so as the stigmatic surface touched the solvent to elute the

leachate. The solvent was allowed to remain in contact with stigma for 2 hrs and

removed. The stylar leachate was also prepared after removing stigma. The leachate

was added to the medium for in vitro germination in 1:1 ratio (medium: leachate) on a

slide in the following manner:

1. Brewbaker's medium + 25% sucrose + pollen (control)

2. Brewbaker's medium + 25% sucrose + self stigmatic leachate + pollen.

3. Brewbaker's medium + 25% sucrose + self stylar leachate + pollen

4. Stigmatic leachate + pollen

5. Stylar leachate + pollen

3.9 Methods to overcome incompatibility

3.9.1 Temperature treatment

3.9.1.1 Heat treatment

Stigma and the stylar portions were subjected to treatment in hot water for

3 min at 50, 55 and 60°Cand pollinated.

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3.9.1.2 Cold treatment

In order to study the effect of low temperature on incompatibility reaction, the

flower buds were kept at low temperatures viz 4, 10, 15 and 20°C and pollinated.

3.9.2 Stump pollination

A. Stigma region: a portion of the stigma was removed from the emasculated

flowers using a sharp blade and self-pollination was performed on the stump.

B. Stylar portion: the stigmatic head was removed and the amputated style was then

pollinated by applying pollen directly to the cut end of the style.

C. About 0.5 cm below the stigma: style was removed using a sharp blade, and

pollinated on the cut surface.

D. About lcm below the stigma: style was removed, coated with the stigmatic

exudates and pollinated on the surface.

E. Total stylar portion was removed just above the ovary and pollinated.

3.9.3 Bud pollination

For bud pollination, mature pollen grains were transferred to the stigma of bud

at four developmental stages and estimated the percentage of pollen germination and

length of pollen tubes (Table 1).

Table 1. Buds at different developmental stages were pollinated using mature and

viable pollen grains

Bud stage Age ofbud Average bud length(cm)

Stage I On the day of anthesis 2.1± 2-6

Stage II 24 hrs before anthesis 1.84± 3.2

Stage III 48 hrs before anthesis 1.45 ±3.1

Stage IV 72 hrs before anthesis 0.9 ±2.6

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3.10 Molecular characterization of accessions using RAPD

For RAPD (Random Amplified Polymorfic DNA) analysis, 14 accessions of

H patens from different regions of Kerala were collected (Table 2). The leaves were

thoroughly rinsed with autoc1aved distilled water and samples of 2 to 3 gm fresh

weight were wrapped in aluminum foil, frozen in liquid nitrogen and stored at -80°C

until DNA extraction.

Table 2. Showing places of collection of accession of H. patens

Acc. No Place of collection

1 Kariavattom

2 Palode

3 Parassala

4 Kollam

5 Ranni

6 Thodupuzha

7 Chengannoor

8 Emakulam

9 Kodakara

10 Ottappalam

11 Vandoor

12 Koduvalli

13 Thalasseri

14 Ambalavayal

Isolation and purification of DNA

Genomic DNA was isolated and purified by cetyltrimethylammonium bromide

(CTAB) method (Dellaporta et aI., 1983), with minor modifications. Leaf tissue

(200mg) was grind to fine powder in a pre-chilled mortar and pestle using liquid

45

nitrogen. The powdered tissue was made into a slurry using 2ml of extraction buffer

containing 1M Tris pH 8.0, 0.5M EDTA (ethylenediaminetetra acetic acid) pH 8.0,

5M NaCI, 5% (w/v) CTAB, 0.05% (v/v) ~-mercaptoethanol and 1%

polyvinylpyrrolidine (PVP) and incubated at 70°C in a shaking water bath (Superfit,

India) for Ihr. The slurry was returned to room temperature slowly. An equal volume

of chloroform: isoamyl alcohol (24: 1v/v) was added, mixed well by gentle shaking

and centrifuged (Eppendorf, Germany) at 12,000 rpm for 10 min at 4°C. The upper

aqueous layer was collected in a fresh centrifuge tube and resultant supernatant was

carefully transferred to a new microfuge tube. This was repeated twice in order to

eliminate the last traces of PVP. The upper layer was collected and 1/lOth volume of

3M sodium acetate and equal volume of chilled isopropanol was added to it, shaken

gently, and kept overnight at -70°C. The precipitate was collected by centrifuging at

13,000 rpm for 15 min at 4°C. Precipitated DNA was washed by adding 200lli ice­

cold 80% (v/v) ethanol and centrifuged. The collected pellet was air dried for 15 min

at room temperature and dissolved in 250111 autoclaved distilled water. The extracted

DNA was purified further by adding 1111 RNase A and kept the tube at room

temperature for 30 min. Equal volume of saturated phenol and chloroform: isoamyl

alcohol (24:1) was added and vortexed (Spinix, India) for 15 seconds and centrifuged

at 10,000 rpm for 10min. The upper layer was collected and equal volume of

chloroform: isoamyl alcohol was added, mixed well and centrifuged at 10,000 rpm for

10 min. The resultant supernatant was carefully transferred to a new microfuge tube

and DNA was precipitated overnight by adding 1/10th volume of 3M-sodium acetate

and equal volume of isopropanol. This was left overnight at -70°C. The mixture was

centrifuged for 15 min at 13,000 rpm. The DNA pellets were washed with 80%

ethanol and vortexed. It was then centrifuged at 10,000 rpm for 10 min and the pellet

46

was collected. These pellets were air-dried for 15 min and dissolved in a minimum

volume of autoclaved distilled water as per quantity of the pellet, and stored at -20°C

until use.

The DNA quality was checked in agarose gel by using 1% TE buffer, and

DNA quantification was done by using a UV-VIS spectrophotometer (UV1700,

Shimadzu, Japan).

Arbitrary primers

A total of twenty 10-mer oligo nucleotide primers (Integrated DNA

Technologies, USA) were initially screened using a sample from each of the fourteen

populations. Out of them ten primers were selected for final analysis based on

reproducibility, clarity of bands and minimum interspecific polymorphism (Table 3).

Table 3. List of selected random primers used for molecular characterization

Primer No. Primer sequence

1 5'-TGC CGA GCT G-3'

2 5'-GGT GAC GCA G-3'

3 5'-GTA GAC CCG T-3'

4 5'-CCT TGA CGC A-3'

5 5'-GGA GGG TGT T-3'

6 5'GGT GAA CGC T-3'

7 5'·TGG GTC CCT C-3'

8 5'-GTC AGT GCG G-3'

9 5'·ACA GCC CCC A-3'

10 5'-GGT GCG GGA A-3'

47

PCRmixture

The total reaction volume was 25 Ill, consisted of 2.5 III of lOX reaction buffer

with MgCh, 2.01l1 of lOmM of dNTP, 2.0 III of 0.2 pM of a single lO-mer

oligonucleotide, 0.5 III of 0.04 unit Taq polymerase, 14 III of water and 4 III of 20ng

template DNA. All the chemicals except primers were obtained from Genei

(Bangalore, India).

PCR reaction

The polymerise chain reaction was carried out in a Gradient Palm Cycler

(Corbet, Australia). All programs had 1 initial cycle with melting at 94°C for 2 min,

annealing at 40°C for 30 seconds and extension at 72°C for 1 min followed by 40

cycles of 94°C for 20 seconds, annealing at 40°C for 30 seconds and extension at

72°C for 1 min.

Agarose gel electrophoresis and documentation

Agarose gel was casted in a mini model horizontal gel electrophoresis unit

(Scie Plas, UK) by melting agarose in IX TE buffer at pH 8.0. Ethidium bromide

(Ill!) was added to the buffer after sufficient cooling, which fluoresce DNA. Agarose

solution (0.7%) was poured to gel casting tray after positioning the comb, carefully

avoiding air bubbles. The mold was allowed to solidify at room temperature. After

20-30 min, the comb was carefully removed. TE buffer (IX) was poured into the tank

and the level of buffer was maintained up to lmm above the gel. Five III of each of

reaction products and molecular weight marker (Genei, Bangalore, India) were mixed

with 2111 of 6x gel loading dye (0.25% bromophenol blue and 40% sucrose in distilled

water). After loading, the apparatus was connected to an electric field (IOOV) of

constant voltage and direction. During the experiment, the negatively charged DNA

48

migrated towards anode at neutral pH. The gel was allowed to electrophoresis, until

the dye migrated up to more than half of the gel. Electric current was turned off and

electrodes were removed. Gel was viewed in a UV-Transluminator to detect DNA

bands and photographed by using Gel Documentation System (Alpha Innotech, USA).

Cluster analysis

Only clear and evenly distributed bands were selected and scored as present (1)

or absent (0) in a matrix that was manually constructed. The reproducibility of the

patterns observed was assessed by conducting the whole RAPD protocol twice.

Data analysis

Relative molecular weight of polypeptide bands was calculated by comparing

the resolved bands of marker using the gel documentation software (Alpha Innotech,

USA). Euclidean distance was calculated among pairs and the dissimilarity matrix

was prepared and analyzed using UPGMA (Unweighted Pair Group Method with

Arithmetic average) method.

3.11 Infraspecific pollination

Based on the RAPD analyses, morphologically distinct Acc. No. 1 and 3, were

used for infra-specific pollinations. For this, flower buds prior to anthesis were

emasculated and bagged one day before anthesis. On the day of anthesis, pollen grains

from Acc. No.1 were transferred to Acc. No. 3 and reciprocal cross were also done.

Each pollination was repeated in 50 flowers. Few flowers were retained for observing

seed-set and rest of the pollinated flowers were used to study the developmental

anatomy of ovary at various intervals. Cryotome sections of ovary at different

developmental stages were studied under a microscope. Few pollinated stigmas were

collected after 24 hrs for analyzing pollen tube growth and behaviour.

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3.12 General histochemistry

Cytochemical analyses were carried out in unpollinated, self-pollinated and

cross-pollinated pistils after 24 hrs to study the biochemical changes and the

distribution of primary metabolites like starch, protein and lipids. For general

histochemistry and enzyme cytochemistry, sections of 8-10 !J.m thickness were taken

using cryostat.

3.12.1 Starch, I-KI reaction (Johansen, 1940)

For qualitative analysis of starch, sections were placed in iodine potassium

iodide (I-KI) solution for 2-3 min, rinsed in distilled water and mounted in glycerine

on a clean micro slide. Starch deposition appeared as bluish black.

3.12.2 Protein (Durrum, 1950)

For detection of proteins, sections of pistils were stained in 1% mercuric

chloride and 0.05% bromophenol blue in 2 % acetic acid for 10-15 min. They were

then washed in tap water and mounted in glycerine. Protein appeared blue in colour.

3.12.3 Lipids (Jensen, 1962)

Thin sections were kept in Sudan black (0.5% in ethyl alcohol) for 3-5 min.

The tissues were differentiated in 50% ethyl alcohol before and after staining and

mounted in glycerine. Lipids were stained black or blue.

3.12.4 Periodic acid Schiff (PAS) reaction for polysaccharides (Hotchkiss, 1948)

Sections were treated with 70% alcohol for five min and immersed in periodic

acid sodium acetate solution for 10 min. Subsequently they were rinsed in 70%

alcohol and transferred to reducing bath for three min and the stained sections were

washed three times in S02 water and mounted in glycerine jelly.

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3.13 Enzyme cytochemistry

Enzyme reactions were carried out at specific temperature and pH. Controlled

reactions were performed in parallel by incubating sections in test solutions lacking

the substrate, heating the sections in distilled water at 90°C for 10-15 min in order to

inactivate the enzymes before performing the reactions.

3.13.1 Succinic dehydrogenase (SDH) (Lillie, 1965)

SDH reaction was carried out using sodium succinate as the substrate in a

coupling reaction with nitroblue-tetrazolium (NBT) at pH 7.6. Fresh sections were

kept in the incubation medium for 15-40 min at 37°C, washed thoroughly and

mounted in a micro slide without air bubbles and observed under microscope and

photomicrographed.

3.13.2 Acid phosphatase (APase) (Gomori, 1952)

Samples for enzyme reactions were incubated in test solution containing 3%

aqueous sodium tetra glycerol phosphate and 0.12 gm lead nitrate in acetate buffer at

pH 5 for 10 min at 37°C. After washing the samples in distilled water, they were

placed in an aqueous solution of 1% acetic acid. The sections were rinsed in distilled

water and differentiated in freshly prepared lead sulphide.

3.13.3 Adenosine tri phosphate (ATPase) (Wachstein and Meisel, 1957)

Fresh sections were incubated at room temperature in test solutions containing

2 mM adenosine triphosphate, 2 mM calcium nitrate and 3.6 mM lead nitrate in Tris

malate buffer at pH 7.0 for 30 min. Incubated sections were transfered to freshly

prepared hydrogen sulphide for 2 min. Sections were rinsed in glycerine and observed

under microscope.

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3.13.4 Peroxidase (PRX) (Graham and Karnovsky, 1966)

Sections were incubated at room temperature in medium containing 10 ml of

0.05 M Tris HCI buffer of pH 7.6 containing 5 mg of 3-3 diaminobenzidine tetra

hydrochloride (DAB) and freshly prepared 1% hydrogen peroxide. Sections were

washed in buffer, mounted in glycerine and photomicrographed immediately.

3.13.5 Esterase (Burstone, 1958)

Sections were incubated at 37°C in test solution containing 5 mg napthol

acetate in 0.2 M Tris buffer at pH 6.5 and 20-40 mg Fast Blue BB salt. Sections were

mounted in glycerine and observed photomicrographed immediately.

3.14 Electrophoresis

For the electrophoretic separation of protein, unpollinated, self- and cross­

pollinated pistils were selected.

Protein extraction

Stigma protein of unpollinated, self- and cross-pollinated pistil of different

accessions and stigmatic leachate of self-pollinated stigmas were analysed by

sodiumdodecylsulphate - polyacrylamidegel electrophoresis (SDS-PAGE). The

extraction was done in 0.2 M Tris HCI (pH 7.4) containing 1% PVP, 0.9% NaCI and

0.05% ~-mercaptoethanol. Fresh stigma (lOOmg) was grinded with 1 ml of extraction

buffer in a pre-cooled mortar and pestle. The extract was centrifuged at 10,000 rpm

for 10 min at 4°C in a refrigerated table top centrifuge (Eppendorf, Germany). The

supernatant was collected and stored at -20°C in a deep freezer.

52

Protein preparation and electrophoresis

The sample preparation and SDS-PAGE were carried out according to the

procedure described by Laemmli (1970). Protein sample (10 JlI) was diluted to 30 JlI

with 20JlI of the sample buffer (SDS reducing buffer) and heated in a water bath

(Superfit, India) at 95°C for 4 min. For loading, 12JlI of this diluted protein sample

was used. Electrophoresis was performed in 10% polyacrylamide gel having Imm

thickness, in a vertical gel tank (Scie, UK) at a constant voltage at 100V for stacking

gel and 75V, for the separating gel.

Staining and gel documentation

After the run, gel was stained with 0.1% Coomassie brilliant blue R-250

overnight and destained with destaining solution (6 water: 3 methanol:1 acetic acid,)

in a destainer (Labnet International, USA). The destained gel was scanned and

photographed with the gel documentation system (Alpha Innotech, USA).

Ready to use protein marker for electrophoresis was obtained from Bangalore

Genei (India). Acrylamide, N, N'- methylene bisacrylamide, Coomassie Brilliant Blue

R-250, Sodiumdodecylsulphate, N,N,N',N'-tetramethylene diamine, PVP, Tris buffer,

glycerol, bromophenol blue, glycine, ammonium per sulphate and ~- mercaptoethanol

were obtained from Sisco Research Laboratory (Mumbai, India).

3.15 RNase activity

3.15.1 Diffusion plate assay (Roiz and Shoseyov, 1995)

RNase activity in the stigma was determined by diffusion plate assay, using

0.1% (w/v) torula yeast RNA (Sigma) in I %( w/v) agarose as a substrate. Styles were

cut at their base, just above the ovary, from flowers at specific ontogenic stages,

placed horizontally on the substrate in petri dishes and incubated at 37°C for 2 hrs.

53

The substrate was then stained with 0.02% (w/v) toluidine blue in water. RNase

activity was indicated by a white halo on a blue background.

3.15.2 Quantitative spectrophotometric assay of RNase activity (Stone et aI.,

2006)

RNase activities in stigma of mature unpollinated, self- and cross-pollinated

flowers were estimated. Three separate extractions were done for all the individuals.

For each extraction, 25 styles per flowers were collected on liquid nitrogen and

processed immediately or stored at -80°C. Styles were ground on liquid nitrogen

using the extraction buffer of Golz et al. (1998). Extraction buffer containing 100 mM

Tris HCI of pH 7.5, 10 mM EDTA, 2 mM CaCh and 14 mM ~-mercaptoethanolto

give 25% solution. Protein concentrations were determined by a colorimetric assay

(Bio-Rad) using BSA as a standard. The supernatant was processed immediately or

stored at -80°C.

A modified spectrophotometric method according to McClure et al. (1989)

and Tsukamoto et al. (1999) was used to monitor RNase activity. Buffer-soluble stylar

protein (20 Ill) was added to 200lli digestion buffer (0.1 M K3P04, pH 7; 0.05 M

KCI) containing 4 mg/ml torula yeast RNA. The control was stopped immediately by

the addition of 55 III of ice-cold 20% trichloroacetic acid and placed on ice. The

reaction tube was incubated at 37°C for 30 min before stopping, and then incubated

together with the control for 10 min. Samples were centrifuged at 13000 rpm for 15

min at 4°C. Twenty-fold dilutions were made into RNase free water for

spectrophotomertic readings. The control cuvette was used as a reference for the

reaction cuvette, and absorbance at 260 nm was recorded. Dilutions were adjusted as

necessary so that the absorbance reading was between 0.1 and 1.0. RNase activity was

54

defined as the increase in absorbance at 260 nm per min of incubation per milligram

of buffer-soluble protein.

3.15.3 In vitro bioassay (Kim et al., 2001)

In order to test pollen germination and pollen tube growth, the pollens were

cultured in growth medium containing 50 mM MES/NaOH buffer (pH. 6.5),

100~g/ml H3B03, 200 ~g/ml MgS04.7H20 and 200 ~g/ml Ca(N03)2.4H20 and 25%

(w/v) sucrose. In each 1.5 ml Eppendorf tube pollen grains were suspended in 100~1

of growth medium. The percentage of germination was examined after 2 hrs of

incubation at 25°C in the dark, and the length of the pollen tube was measured after

incubation for 24 hrs under the same conditions. To compare the effect of RNase on

pollen germination and pollen tube growth in self-pollen (pollen from the same

flower) and cross-pollen (pollen used for cross-pollination), RNase A (25 ~g/ml) and

stigmatic leachate were added to the pollen suspension separately. The growth of the

pollen tube was observed with a light microscope.

3.15.4 Effect of RNase regulators on in vitro pollen growth (Kim et aI., 2001)

The S-RNase regulators such as zinc sulfate, cupric sulfate and calcium

chloride, were added separately to the growth medium to a final concentration of

1mM - 5 mM along with stigmatic protein and cultured at 28°C for 24 hrs to study the

regulatory effect of these chemicals on the inhibitory activity of stigma leachate on

pollen germination. The growth of the pollen tube was observed with a light

microscope.

55