RESULTS

Hamelia patens Jacq. belong to the family Rubiaceae was collected from

different parts of Kerala. Plant collected from Parassala has remarkable

morphological differences (posses dark green leaves and small flowers) compared to

all other accessions (posses light green leaves and large flowers). In the present study

Acc. No. 1 (from Kariavattom, Fig. 1) and Acc. No. 3 (from Parassala, Fig. 2) were

selected, since they were morphologically dissimilar (Fig. 3).

4.1 Phenology

The flowering phenology showed uniformity throughout the year. The

inflorescence is a terminal cyme of about three inches long bearing flowers in a

helicoid cyme. The flower open around 1-2 am and last for one day. The petals,

stamens and stigma drop off while the calyx is persistent with ovary. Flowers are

reddish orange in colour, pedicellate, bisexual, actinomorphic, complete, regular,

pentamerous, and epigynous. Sepals are five in number and gamosepalous. Corolla

consists of five gamopetalous petals with cylindrical corolla tube. Androecium has

five stamens, in epipetalous condition and anther dehisce around 11-12 pm. Style is

long with linear shaped stigma, in which sticky excudates were seen from very early

stages onwards. Ovary is pentacarpellary, syncarpous, pentalocular with numerous

ovules on axile placentation. Mature fruits (drup) persist for 2-3 weeks with sterile

seeds (Figs. 4-6) and then drop off.

4.2 Structure of reproductive parts

4.2.1 Anther

Fully matured anther is bithecous and tetrasporangiate (Fig. 7).

Microsporangium consists of a mass of haploid pollen enclosed within the

microsporangial wall. Outermost layer of the anther is epidermis, followed by a single

layer of endothecium, two or three middle layers and a single layered tapetum

(Figs. 8, 9). When pollen matures, the tapetum disintegrates and the dehiscence of

anther takes place longitudinally at the time ofanthesis.

4.2.2 Pistil

Stigma is wet, papillate and linear in shape (Fig. 10). Unicellular and rod

shaped papillae are arranged uniformly throughout the surface of stigma (Fig. 11).

The cryotome sections of stigma revealed two zones, an outer zone and inner zone.

The outer zone consists of epidermis and 1-3 layers of laterally extended cells

constituting the secretory tissue. The inner zone consists of parenchymatous ground

tissue with central transmitting zone and vascular strands on either side (Fig. 12).

SEM studies revealed that the linear shaped stigma is covered with unicellular

and rod shaped papillae. From the very early stage onwards to the stage of maturity,

the morphology of stigma surface was found to be similar (Figs. 13-20). The surface

of stigma is irregular and posses copious exudate from the very early stage of the bud

which accumulates all around the stigmatic surface. Below the papillar cells, the

subepidermal cells of the stigma converge into a short solid style made up of a central

core of transmitting tissue surrounded by parenchymatous cortex and vascular tissues.

SEM showed numerous sterile ovules on axile placentation (Figs. 21-23).

Ovary wall consists of a single layered parenchymatous epidermis. Vascular tissues

are clearly visible intermittently.

57

4.3 Cytology

4.3.1 Meiosis

Meiotic chromosome studies showed the presence of 12 bivalents at

metaphase (Fig. 24) and subsequent stages ofmeiosis were normal. The chromosomes

were small in size.

4.3.2 Pollen cytology

Pollen grains were kept in lactopropionic orcein and observed after 24 hrs.

The protoplast completely extruded out of the pollen exine and the presence of two

intensely stained nuclei was distinctly seen (Fig. 25).

4.4 Pollen viability studies

4.4.1 Fluorochromatic reaction test.

When pollen grains were mounted in FDA solution and observed under UV

light, about 90% of pollen grains showed a bright green or yellowish green

fluorescence which revealed their viability status (Fig. 26).

4.4.2 In vitro germination

In order to assess the percentage of viable pollen grains, in vitro test was

conducted. Pollen germination commenced after 3 hrs in the medium supplemented

with 5% sucrose and attained a maximum germination of 72.5% was observed the

medium with 25% sucrose (Fig. 27, Table 4, Fig. I). Above 25%, there was a gradual

decrease in the rate of pollen germination, the content of the pollen cytoplasm

extruded out through the germ pore.

58

Table 4. Results of in vitro pollen germination ofH. patens in growth medium at

various concentrations of sucrose

Concentration of Pollen germination Average pollen tubesucrose v/v in % (%)±SE length (~m)±SE

5 42.5±0.9 249.1±28.3

10 45.6±1.1 429.2±46.7

15 50.8±1.5 729.8±37.9

20 64.8±13 803.5±29.1

25 72.5±2.1 946.1±20.1

30 49.1±2.0 696.7±28.6

35 30.8±1.1 587.8±50.5

40 15.3±0.8 344.6±27.1

45 8.4±0.5 260.1±16.8

50 2.4±0.2 184.8±13.9

Fig. I. Pattern of pollen germination and tube length ofH. patens in growth

medium at various concentrations of sucrose

SO

70

60l:.g 50IIIC

's 40~~

Q 30~

20

10

00 5 10 15 20 25 30 35 40

Sucrose {w/v)

1000950900850800750 E700 a650WO

,t;"6l550 c:

500 .!450 CI)

400 .til:::I

350 ...3()O250200150100500

45 50 55

-II- gerrrination (%) -+- tube length (IJm)

59

4.5 Study of stigma receptivity

4.5.1 In vivo germination

To find out the receptive period of stigma, buds at various developmental

stages (Fig. 28) were pollinated with mature pollen and the rate of pollen adhesion,

germination and tube growth was observed. The rate of pollen adhesion, pollen

germination, and tube growth increases with the age of the flower bud and found

maximum (75.5±2.l) on the day ofanthesis (Table 5).

Table 5. Result of pollen germination and tube length at various developmental

stages of bud in H. patens.

Developmental stage Pollen germination Average pollen tube

(%)±SE length(llm)±SE

I stage 75.5±2.1 757.7±25.1

II stage 56.1±1.8 555.1±45.9

III stage 52.8±2.5 397.5±20.5

Iv stage 45.1±3.2 226.7±19.0

4.5.2 Cytochemical localization of esterases on stigma surface

The receptive surface of wet stigma invariably contains extracellular proteins

as a component of the exudates. 0.- napthyl acetate is colourless and forms a reddish

insoluble complex with coupling reagent fast blue B. Activity of esterase is maximum

in stigmas on the day of anthesis and minimum in bud on three days before anthesis

(Figs. 29-33).

4.6 Pollination experiments

Pollination studies were carried out in open pollinated and self-pollinated

flowers. Pollinated stigmas were fixd in carrnoy's fluid, 8hrs hrs after pollination.

Some of the pollinated flowers were also left for seed-setting.

60

4.6.1 Open pollination

Pollination in H patens is entomophilous (Figs. 34,35). In natural pollination,

the number of pollen grains deposited on the surface of the stigma varied and the

percentage of pollen germination ranged from 57.24 to 79.24%. The open pollinated

flowers withered away 24 hrs after pollination. The persistent ovary developed into

fruit without viable seeds. These fruits remained for 2-3 weeks in the plant.

4.6.2 Self-pollination

The pollen germination and tube length after self-pollination are given in

Table 6. The pistils stained with aniline blue showed very high percentage of pollen

grains that were adhered and germinated on the surface of the stigma (Figs. 36,37).

But pollen tubes never penetrated into it. Pollen tube growth was inhibited on the

stigma surface with various abnormalities such as curling of pollen tube, irregular

callose deposition and bulging at the tip of the tube (Figs. 38-41). These pollen tubes

grew parallel instead of penetrating into the stigma. When compared to open

pollination, no significant difference in pollen adhesion, germination and tube growth.

Table 6. Pollen germination and tube length on self-pollinated flowers.

Serial No. Pollen germination (%)±SE Average pollen tubeLength (j.lm)±SE

1 70.8±1.8 731.5±42.1

2 71.0±1.2 750.1±51.0

3 68.7±2.0 846.2±37.7

4 66.5±3.7 733.3±41.9

5 63.5±4.6 782.8±40.8

6 63.0±3.0 857.3±14.9

7 63.6±2.9 857.3±50.7

8 61.2±3.2 834.7±39.6

9 65.3±1.7 809.3±67.1

10 65.2±2.8 837.2±49.1

61

4.7 Confirmation of self-incompatibility by in vitro bioassay

In order to assess the role of stigmatic / stylar leachate on pollen tube growth

in vitro bioassay was done. Brewbaker and Kwack's medium supplemented with 25%

sucrose showed maximum germination of 72.5%. When stigmatic leachate was added

along with 25% medium significant reduction in the pollen germination and tube

growth was observed. Stigma leachate alone was used as growth medium, only 22.5%

pollen germination was noticed, when stylar leachate was added instead of stigma

leachate, reduction in the pollen tube growth was observed. But when compared to the

stigma leachate, reduction in the pollen tube growth was not so significant in stylar

leachate (Table 7).

Table 7. Effect of incorporation of aqueous stigma and stylar leachate into

culture medium

Type of germination Pollen germination Average pollen tube

medium (%)±SE length (Jlm)±SE

Medium+25%sucrose (control) 71.5±O.6 946.8±20.1

Medium+25%sucrose +stigma leachate 24.5±1.2 239.7±12.1

Medium+25%sucrose +stylar leachate 67.05±O.6 775.7±12.2

Stigma leachate+ pollen 14.5±1.2 124.8±12.1

stylar leachate+pollen 57.3±1.2 621.2 ±12.1

4.8 Methods to overcome self-incompatibility

In order to overcome incompatibility in H patens, various methods such as

temperature treatment, stump pollination and bud pollination were done.

62

4.8.1Temperature treatment

4.8.1.1 Heat treatment

To find out the effect of temperature in overcoming incompatibility barriers

located in the pistil, three different hot-water treatments at 50,55 and 60°C were done

and the results were showed in the Table 8.

Table 8. Results of heat treatment before self-pollination in H. patens

Type ofpollination Temperature Pollen germination Average pollen tube

(OC) (%)±SE length (J..Lm)±SE

50 23.7±2.6 118.8±11.8

Self- pollination 55 9.9±0.8 93.2±7.9

60 00 00.00

Control 71.3±65.2 864.6±25.7

4.8.1.2 Cold treatment

For this, mature flower buds were kept at low temperatures viz 4, 10, 15 and

20°C. At 4°C, the pollen adhesion and germination was very low and observed only

8.2% germination. At 10, 15 and 20°C significant increase in pollen tube length were

recorded. When compared to the control the cold water treatment showed reduction in

pollen germination and tube growth (Table 9).

63

Table 9. Percentage of pollen germination and average tube length after coldtreatment

Type of Temperature (OC) Pollen germination Average pollen ube

pollination (%)±SE length (Jlm)±SE

4 8.2±3.6 23.3±9.7

10 48.2±2.6 346.5±23.0

Self-pollination 15 53.7±3.l 476.2±39.1

20 65.9±2.2 653.6±28.2

Control 72.7±2.1 820.5±33.6

4.8.2 Stump pollination

Pollination was carried out on the stylar stumps generated by excising the

pistil at various lengths. None of the experiments were found to be successful to

overcome incompatibility barrier in H patens and the results are shown in Table 10.

Table 10. Results of Stump pollination in H. patens

Type of Treatment % of germination

pollination

Stigma region 42% germination

Stylar portion No pollen germination

Self-pollination About 0.5 cm below the stigma No pollen germination

About 1 cm below the stigma No pollen germination

Style removed just above the No pollen germinationovary

64

4.8.3 Bud pollination

Bud pollinations at 4 different stages viz, on the day of anthesis, 24, 48 and

72 hrs before anthesis were tried and found to be not effective to overcome the

incompatibility barrier in H patens (Table 11).

Table 11. Percentage of pollen germination and pollen tube length after bud

pollination.

Type of Stage of bud Length of Pollen Average tubepollination style + stigma germination length(llm)±SE

(%)±SE

Stage I 1.5 75.5±2.1 757.7±25.1

Stage II1.4 56.1±1.8 555.1±45.9

Self-pollination

Stage III1.3 52.8±2.5 397.5±20.5

Stage IV1.0 45.1±3.2 226.7±19.0

4.9 Molecular characterization by RAPD

The RAPD analysis has been found to be a valuable DNA marker system to

evaluate genetic diversity. It has been done using fourteen accessions of H patens

collected from different parts of Kerala. Among the 20 random primers used for the

initial screening, ten gave optimum RAPD profiles with all the accessions studied.

Majority of the primers detected a high level ofpolymorphism.

A total of 65 bands were generated from the ten chosen primers. The number

of bands ranged from 4 to 9 per accession, when data from all the ten primers were

pooled. The amplified products varied between 150 and 3350 kDa (Figs. 42-51).

65

The RAPD generated dendrogram based on Euclidean distance using UPGMA

method was presented in the Fig. II. It revealed two major clusters. The cluster I

consisted of only one accession (Ace. No.3) and cluster II consisted of 13

accessions (Ace. No. 1,2,4,5,6,7,8,9,10,11,12,13,14). The dendrogram showed

clusters similar to the grouping based on morphology, ie, the morphologically similar

accessions in one cluster and the rest is in the other.

Fig. II. Showing UPGMA dendrogram of fourteen accessions ofH. patens

I4.8

I3.2

Euclidean

Ace. NO.3

Ace. NO.2

Ace. No. 13

Ace. No. 12

Ace. No. 14

Ace. NO.8

Ace. NO.4

Ace. NO.7

Ace. NO.9

Ace. NO.6

Ace. NO.11

Ace. NO.5

Ace. NO.10

Ace. NO.1

d

4.10 Infraspecific pollination

Infraspecific pollinations between Ace. No. 1 and 3 were found to overcome

incompatibility in H patens. Crossing between morphologically similar plants from

various localities failed to set-seeds and these crosses were referred to as incompatible

combinations. Crosses between morphologically dissimilar plants (Ace No.1 and 3)

always set-seeds and these crosses were referred as compatible combination

(Table 12, Fig. III).

66

Table 12. Showing results of compatible pollination in H. patens

Type of Age of bud Pollen gennination Tube length

pollination (%)±SE ()lm) ±SE

Stage I 77.4±2.3 1165.5±61.0

Compatible Stage II 70.6±1.9 984.5±41.8pollinationAl xA3 Stage III 59.4±1.5 241.7±20.7

Stage IV 51.9±4.1 191.0±16.5

Stage I 72.27±2.1 742.8±15.6

Compatible Stage II 67.62±2.5 534.13±36.19pollination

A3XA I Stage III 53.49±2.3 192.0±45.28

Stage IV 36.3±1.98 93.12±7.23

Al - Accession No.1 A3 - Accession No.3

In compatible pollinations, large number of pollen grains have adhered and

genninated on the surface of stigma (Fig. 52). The compatible pollen tubes were

penetrated into the stigma (Fig. 53), grow down into the style and finally reach the

ovary (Fig. 55) and the ovule (Figs. 56,57). In incompatible pollination, callose

deposition was irregular whereas in compatible pollination regular callose deposition

was noticed (Fig. 54).

67

Fig. Ill. Results of compatible pollination between morphologically different

accessions of H. patens

90

80

70

60

50

40

30

20

10

O-j-----J-L--L.+-----'-+---"--'-t--------L-t-------'--;~-----L_t_-----'-+_----'----'_+

Stage I

A1 x A3

Stage IV Stage I

A3x A1

_ Length of style +stigma c::J Pollen germination (%)±SE -.- Tube length (IJm) ±SE

In the case of incompatible pollinations, petals of the flowers withered away

one day after pollination but those of compatible pollinations petals persisted for

4-5 days. One week after compatible pollination, ovary showed significant difference

in size compared to incompatible pollinations and mature seeds were collected after

45 days.

The developmental anatomy of ovary after compatible pollinations were also

carried out to confirm the effectiveness of fertilization (Figs. 58,59). In H patens,

ovary showed five locules which are found to be growing inwardly and fuse at the

center. The spindle-shaped inferior ovary possess 10-15 unitegmic anatropous ovules.

The ovules within each locule are arranged in a basipetal order as a vertical row. At

maturity, the septa disintegrate in their outer parts and both walls of the locules get

separated from each other (Fig. 60). The disintegration is restricted to the peripheral

parts of the septa, towards the ovary wall and the massive structure of the septa

68

retained (Fig. 61). One week after incompatible pollination, further development of

ovules was arrested whereas normal development of ovary and ovules was observed

after compatible pollination (Fig. 62). Mature fruits collected from compatible

pollination showed 95% viability (Fig. 63).

4.11 General histochemistry

Cytochemical studies were carried out to find out the biochemical changes in

the amount of primary metabolites such as starch, protein and lipid among the

unpollinated, self- and cross-pollinated stigmas.

4.11.1 Starch

Presence of starch was detected using I-KI stain. In unpollinated pistil intense

deposition of starch was observed in secretory zone (Figs. 64,65). Rest of the region

showed meager deposition. After self-pollination, uniformly intense deposition of

starch was observed in ground tissues especially on either sides of transmitting zones

(Figs. 66,67). When compared to unpollinated and self-pollinated pistil, starch

deposition was more in pollinated pistil. In cross-pollinated pistil more intense

deposition was observed in ground tissue whereas transmitting tissues showed only

meager deposition (Figs. 68,69).

4.11.2 Protein

Presence of protein was identified by usmg mercurIC bromophenol blue.

Protein deposition was high in pollinated stigma than in unpollinated ones. In

unpollinated stigma uniformly meager deposition of protein was noticed except in the

transmitting tissue (Figs. 70,71). After pollination, meagre deposition was noticed in

secretory region whereas ground tissues and transmitting zones were intensely stained

for proteins (Figs. 72,73).

69

When compared, protein deposition was found to be higher in self-pollinated

stigma than in cross-pollinated one. Uniformly stained ground tissues and meagrely

stained transmitting tissues were seen in cross-pollinated stigma (Figs. 74,75). Cross

pollinated pistil showed slight reduction in the localization of protein in transmitting

tissues and ground tissues when compared to self-pollinated pistil.

4.11.3 Lipids

Presence of lipid was studied using Sudan Black-B. In unpollinated stigma,

localized intense deposition of lipid was observed in the secretory zone of the stigma,

where exudates are seen (Figs. 76,77). Ground tissues below the secretory zone are

free from deposition of lipids but the intense deposition was noticed in ground tissues

on either side of the vascular tissues. After self-pollination, intense deposition was

noticed at the secretory zone of the stigma and vascular region (Figs. 78,79). In cross­

pollinated pistil, meagre deposition was observed in transmitting and ground tissues

whereas intense deposition was seen in secretory region (Figs. 80,81).

4.11.4 Periodic acid Schiff (PAS) reaction for polysaccharides

PAS reaction showed uniform distribution of PAS positive bodies.

Polysaccharides appeared as dark pink coloured bodies. Uniformly less intense

deposition of PAS positive bodies were observed in unpollinated stigma (Figs. 82,83)

where as localized intense deposition was noticed in pollinated one (Figs. 84,85).

When compared to self-pollinated pistil, cross-pollinated pistils showed less

deposition (Figs. 86,87).

4.12 Enzyme cytochemistry

Since the enzymes control biochemical reactions, and their synthesis is under

the control of specific gene (s), any change in the activity of enzyme would reflect in

70

the pattern of gene expression and corresponding metabolic events in the cell. Hence,

the enzymes can be used as a tool to study the problem of self-incompatibility at the

biochemical level. In the present investigation, cytochemical changes in the

localization of different enzymes such as succinic dehydrogenase, acid phosphatase,

ATPase, peroxidase and esterase in unpollinated, self-pollinated and cross-pollinated

stigmas were studied.

4.12.1 Succinic dehydrogenase

SDH activity was localized as blue coloured bodies. In unpollinated pistil, the

activity was uniformly high in the entire region except transmitting zone, where

intense SDH activity was observed (Figs. 88,89). After self-pollination localized

intense activity was observed on either side of the transmitting region and secretory

zone (Figs. 90,91). When compared to self-pollinated pistil, cross-pollinated pistil

showed meagre activity except in the tissue on either side of transmitting tissue

(Figs. 92,93).

4.12.2 Acid phosphatase

Acid phosphatase activity appeared as blackish brown deposits and was very

intense in papillar, epidermal and sub epidermal regions (Fig. 94). The central core of

transmitting tissue showed uniformly less enzyme activity (Fig. 95). Maximum

activity was observed in the unpollinated stigma. Self- and cross-pollinated pistil

showed slight decrease in the activity of APase (Figs. 96-99). Secretory zone and

vascular regions showed some localized intense activity.

4.12.3 Adenosine triphosphatase

The activity site of ATPase was observed as brown black deposits of lead

sulphide distributed as granules. In unpollinated stigma, papillar region showed

71

intense activity compared to the ground tissue (Figs. 100,101). The activity of this

enzyme was high in unpollinated pistil. When compared to self-pollinated pistil

(Figs. 102,103), cross-pollinated pistil showed less intense activity (Figs. 104,105).

4.12.4 Peroxidase

Activity site appeared as reddish brown reaction products. Intense activity was

observed in papillar and secretory zone and the rest of the region showed

comparatively less uniform distribution (Figs. 106,107). In H patens, pollination

stimulated the activity of peroxidase in the pistil (Figs. 108,109). In the papilla region,

uniformly intense deposition was noticed. In cross-pollinated pistil, less intense

deposition was observed (Figs. 110,111).

4.12.5 Esterase

Esterase activity was detected as brick red coloured granules. In unpollinated

stigma, uniformly less activity (Fig. 112) was observed except in the region of

secondary tissues (Fig. 113) when compared to unpollinated stigma self- and cross­

pollinated pistils showed higher activity (Figs. 114-117).

4.13 Electrophoresis

Since infraspecific pollination was found to be successful in H patens, the

protein profiles of stigma homogenate and leachate of unpollinated, self- and cross­

pollinated pistils were analysed using the technique of SDS-PAGE. Stigma

homogenate revealed 15 protein bands with molecular weight ranging from

20-100 kDa (Fig. 118). The protein bands of self- and cross-pollinated pistils were

common except in 36 kDa region. When compared the band with molecular weight

36 kDa was found to be absent in unpollinated pistil and also the intensity of the

bands were less intense.

72

However, the electrophoretic studies of stigma leachate of unpollinated, self­

and cross-pollinated pistil enabled the detection of five common bands with molecular

weights 23, 30, 36, 70 and 99 kDa (Fig. 119), but showed quantitative difference. It is

noteworthy that protein band at 36 kDa region was common in stigma leachate as well

as homogenate of self- and cross-pollinated pistil, while the band is totally absent in

unpollinated pistil.

4.14 RNase activity

4.14.1 Diffusion plate assay

In H patens, the diffusion plate assay for RNase activity appeared as white

halos in a blue background around the stigmatic surface. In vivo germination studies

using decolourized aniline blue confirmed that the inhibition is only on the stigmatic

surface. From the analyses, it was also observed that the RNase activity in the

stigmatic diffusate is less in early developmental stages and reaches maximum at

anthesis. When compared, the activity was very high in self-pollinated pistil followed

by cross-pollinated and less in unpollinated pistil (Figs. 120-125).

4.14.2 Quantitative spectrophotometric assay of RNase activity

Spectrophotometric assay was carried out to check the RNase activity and

found that the activity was high in self-pollinated than unpollinated and cross­

pollinated stigma. Further analysis was done to find out RNase activity in stigma or

style which confirmed the activity high on the stigma than style (Table 13, Fig. IV).

73

Table 13. Results of RNase activity in H. patens after various pollination

experiments

Type of flower Specific activity(units/~g protein)

Unpollinated 4.9

Self-pollinated 15.8

Cross-pollinated 9.2

Stigma (self-pollinated) 13.6

Style (self-pollinated) 00

Table IV, Showing difference in the RNase activity after various types of

pollination in H. patens

18

s:: 16'(jj-E 14c.Cl 12:::l.-III:!::

10s::,j

~ 8'S;..CJ 6I1lCJ

li= 4'uQ)C.In 2

0"C "CQ) Q)- -m mc :;:::; c

'';:::;IJl0 '5. 0 IJl

Cl. Cl. '5.c .,!.j (ii

IJl

-+-Specific activity (units/1J9protein

.,!. I:;:::; lii "CIJl (ii "C Q)'5. IJl Q) IJl coI

I - IIJl "C m cIJl Q) m c Q)0 co E Z, 0... 0() c OJ Cl.

:;:::; Cl. IJl

0 IJl

Cl.

74

4.14.3 Effect of RNase on in vitro pollen tube growth inhibition

In vitro bioassay was used to analyze the role of stigmatic leachate on pollen

tube growth inhibition. An earlier observation showed that growth medium with 25%

sucrose is effective in pollen germination in H patens (Fig.126,128). When

incompatible pollen (self-pollen) was cultured in the Brewbaker and Kwack's

medium supplemented with 25% sucrose along with leachate, significant reduction in

the percentage of germination and pollen tube length was observed (Figs. 127)

whereas when compatible pollen was used, significantly high rate of pollen

germination and pollen tube length were observed (Fig. 129, Table 14).

In order to confirm the mechanism of pollen tube growth inhibition, 2.5 Ilg/ml

of Bovine Pancreatic RNase A has supplemented to the growth medium along with

leachate and the self- and cross-pollen were cultured separately. Significant

differences in pollen tube growth as well as percentage of germination were observed.

The addition of RNase to the medium did not affect the growth and development of

cross-pollen (Fig. 131) but it affects pollen germination and tube growth of self-pollen

was observed (Fig. 130).

Table 14. Result of in vitro assay of RNase on pollen germination and pollen tube

length

Medium Pollen germination Average pollen tube(%)±SE length (Ilm)±SE

control 71.5±0.6 906.1±12.1

Leachate+selfpollen 22.15±0.9 93.7±7.5

Leachate+cross pollen 62.1±1.2 656.8±20.1

Leachate+self pollen+I1llRNaseA 15.4±1.4 38.3±5.9

Leachate+cross pollen+I1llRNaseA 67.3±2.2 731.2±24.9

75

4.14.4 Effect of RNase regulator on in vitro pollen tube growth

Generally two chemicals namely zinc sulfate and cupric sulfate inhibit the

activity of stigmatic RNase and promote pollen germination. In this study also

different concentrations of these chemicals were applied in the medium having

stigmatic leachate. Zinc sulfate (l mM) was found to be more effective in reducing

the inhibitory activity of RNase on pollen tube growth (Fig. 132). After the addition

of ImM zinc sulfate, the inhibition of pollen tube growth was undetected. But when

the concentration exceeds ImM, the pollen tube exhibited disturbed growth

(Fig. 134,136). Additionally, zinc sulfate was superior to cupric sulfate as a chemical

ofpollen tube growth in vitro by decreasing the RNase activity. The cupric sulfate did

bind to the outer surface of pollen the tissue. This disturbed the pollen tube

germination (Figs. 133,135,137).

76

Figures 1- 6

I. H patens Accession No.l- Habit

2. H patens Accession No.2- Habit

3. Morphological variation between the two accessions

4. Fruit and L.S offruit

5. C.S of the ovary with sterile ovules x 4

6. Stereomicrograph ofC.S ofthe fruit

Figures 7-12

7. Stamen from a single flower

8. C.S of anther - Tetrasporangiate showing fibrous endothecium and stomiumx4

9. C.S of anther- A portion enlarged showing fibrous endothecium and pollengrains x 40

10. Stereomicrograph of mature stigma and style

11. L.S ofthe stigma showing papillae x 10

12. L.S of the stigma showing vascular tissues and central transmitting tissue x4.(t-transmitting tissue, v- vascular tissue)

Figures 13- 20. Scanning Electron Micrographs of stigma at different developmental

stages

13. Four days before anthesis

14. Irregular stigmatic surface four days before anthesis - a portion enlarged

15. Three days before anthesis

16. Stigma surface three days before anthesis - a portion enlarged

17. Two days before anthesis

18. Fully developed papillae covering the stigmatic surface - a portionenlarged

19. One day before anthesis

20. Stigma surface with pollen grains one day before anthesis

Figures 21-26

21. SEM of L.S of the ovary showing sterile ovules (ov-ovule)

22,23. SEM ofL.S ovary - a portion enlarged (ov-ovule)

24. Meiotic metaphase n = 12 x 40

25. Pollen grain showing two nuclei at the time of shedding x 40

26. Pollen grains showing intense fluorescence by FeR test x 10

Figures 27-35

27. In vitro pollen germination in Brewbaker and Kwack's medium supplementedwith 25% sucrose x 10

28. Buds at various developmental stages

29-32. Stereomicrograph of localization of esterase on the stigma surface atdifferent developmental stages

29. Bud at 3 days before anthesis

30. Bud at 2 days before anthesis

31. Bud at one day before anthesis

32. Bud on the day of anthesis

33. L.S of the stigma and style showing the activity of esterase x 40

34-35. Various floral visitors

Figures 64-69

64. Unpollinated pistil showing localized intense deposition of starch x 4

65. Unpollinated pistil showing intense deposition of starch in the secretoryregion x 10

66,67. Self-pollinated pistil showing uniformly intense deposition in the vascularregion x 10

68,69. Cross-pollinated pistil showing intense deposition of starch x 4, x 10

Figures 70-75

70. Meagre deposition ofprotein before pollination x 4

71. Uniformly meagre deposition ofprotein in the secretory zone x 40

72,73. Meagre deposition ofprotein in secretory region and intensely stainedtransmitting zones in self-pollinated stigma x 4, x 10

74,75. Uniformly stained ground and transmitting tissue and intenselystained vascular tissues in cross-pollinated stigma x 4, x 10

Figures 76-81

76.

77.

78,79.

80.

81.

Localized intense deposition of lipid in the secretory zone ofunpollinated stigma x 4

Intense deposition of lipids in ground tissues on either side of thevascular tissues in unpollinated stigma x 10

Intense deposition of lipids in stigma after self-pollination x 4, x 10

Meagre deposition of lipid in the transmitting zone of cross-pollinatedstigma x 10

Intense deposition of lipid in the secretory region after cross-pollination x 40

Figure 82-87

82,83. Uniformly less intense deposition ofPAS positive bodies in maturestigma x 10

84. Localized intense deposition ofPAS positive bodies in self-pollinatedpistil x 4

85. Deposition of PAS bodies in papillar region x 40

86. Cross-pollinated pistils showed less PAS deposition x 4

87. Uniformly meagre deposition of PAS positive bodies x 40

Figures 88-93

88. Unpollinated pistil showing intense activity ofSDH at transmittingregion and vascular tissues x 10

89. Intense activity of SDH at transmitting region x 40

90. Localized intense activity of SDH on either side of the transmittingregion after self-pollination x 10

91. Intense activity ofSDH in secretory zone and vascular tissue afterself-pollination x 10

92. Cross-pollinated pistil showing meagre activity of SDH x 4

93. Localised intense activity of SDH after cross-pollination x 10

Figures 94-99

194. Intense activity of acid phosphatase in papillar, epidermal and subepidermalregions ofunpollinated stigma x 4

95. The central core oftransmitting tissue showing uniformly less enzymeactivity x 10

96,97. Activity ofAPase in self-pollinated stigma x 4, x 10

98,99. Activity ofAPase in cross-pollinated stigma x 40

Figures 100-105

100,101. High activity of ATPase in unpollinated stigma x 4, x 10

102,103. Intense activity ofATPase in self-pollinated stigma x 4, x 10

104,105. Activity ofATPase in cross-pollinated stigma x 10, x 10

100

102

101

Figures 76-81

76.

77.

78,79.

80.

81.

Localized intense deposition of lipid in the secretory zone ofunpollinated stigma x 4

Intense deposition of lipids in ground tissues on either side of thevascular tissues in unpollinated stigma x 10

Intense deposition of lipids in stigma after self-pollination x 4, x 10

Meagre deposition of lipid in the transmitting zone of cross-pollinatedstigma x 10

Intense deposition of lipid in the secretory region after cross-pollination x 40

Figure 82-87

82,83. Uniformly less intense deposition ofPAS positive bodies in maturestigma x 10

84. Localized intense deposition ofPAS positive bodies in self-pollinatedpistil x 4

85. Deposition of PAS bodies in papillar region x 40

86. Cross-pollinated pistils showed less PAS deposition x 4

87. Uniformly meagre deposition of PAS positive bodies x 40

Figures 88-93

88. Unpollinated pistil showing intense activity ofSDH at transmittingregion and vascular tissues x 10

89. Intense activity of SDH at transmitting region x 40

90. Localized intense activity of SDH on either side of the transmittingregion after self-pollination x 10

91. Intense activity ofSDH in secretory zone and vascular tissue afterself-pollination x 10

92. Cross-pollinated pistil showing meagre activity of SDH x 4

93. Localised intense activity of SDH after cross-pollination x 10

Figures 94-99

94. Intense activity of acid phosphatase in papillar, epidermal and subepidermalregions ofunpollinated stigma x 4

95. The central core oftransmitting tissue showing uniformly less enzymeactivity x 10

96,97. Activity ofAPase in self-pollinated stigma x 4, x 10

98,99. Activity ofAPase in cross-pollinated stigma x 40

Figures 100-105

100,101. High activity of ATPase in unpollinated stigma x 4, x 10

102,103. Intense activity ofATPase in self-pollinated stigma x 4, x 10

104,105. Activity ofATPase in cross-pollinated stigma x 10, x 10

100

102

101

Figures 106-111

106. Activity ofperoxidase in unpollinated stigma x 4

107. Intense activity ofperoxidase in papillar and secretory region ofunpollinated stigma x 10

108. Activity ofperoxidase on stigma after self-pollination x 4

109. Intense activity ofperoxidase after cross-pollination x 10

110. Intense activity ofperoxidase after cross-pollination x 10

111. Intense activity ofperoxidase at papillar region after cross-pollination x 40

Figures 112 -120.

112. Meagre activity of esterase in unpollinated stigma x 10

113. Intense activity ofesterase in secondary tissues x 10

114,115. Self-pollinated stigma showing increase in the activity of esterase x 10

116,117. Intense activity of esterase on the surface ofcross-pollinated stigma x 10

Figure 118. Protein profile of stigma homogenate ofunpollinated, self- and cross­pollinated stigma

Figure 119. Protein profile of stigma leachate ofunpollinated, self- and cross­pollinated stigma

M - Molecular marker, up - unpollinated, sp - self-pollinated,

cp - cross- pollinated pistil

U.8

Figure 120-125. In H patens, diffusion plate assay showing RNase activity appeared

as white halos on blue background

120. Mature unpollinated stigma showing less RNase activity x 4

121. RNase activity ofunpollinated stigma at different developmental stages x4

122. Mature self-pollinated stigma showing high RNase activity x 4

123. RNase activity of self-pollinated stigma at different developmental stages x4

124. Cross-pollinated stigma with less RNase activity x 4

125. RNase activity on stigma at different developmental stages aftercross-pollination x 4

____ll

Figure 126-131. In vitro pollen tube growth inhibition by self stigma leachate of

Hpatens

126. Pollen germination at 25% sucrose supplemented in Brewbacker'smedium (control) x 4

127. Pollen germination in 25% sucrose medium with self stigma leachate x 4

128. Self-pollen cultured with cross stigma leachate containing sucrosemedium x 4

130. Self-pollen cultured with self stigma leachate containing medium withRNaseA x4

129,131. Self-pollen cultured with cross stigma leachate containing mediumwith RNaseA x 4

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Figure 132-137. In vitro pollen tube growth by self stigma leachate ofH patens

containing RNase inhibitor (ZnS04 and CUS04)

132. Self-pollen cultured with self stigma leachate containing 1mM ZnS04 x 4

133. Self-pollen cultured with self stigma leachate containing 1mM CUS04 x 4

134. Self-pollen cultured with self stigma leachate containing 2mM ZnS04 x 4

135. Self-pollen cultured with self stigma leachate containing 2mM CUS04 x 4

136. Self-pollen cultured with self stigma leachate containing 5mM ZnS04 x 4

137. Self-pollen cultured with self stigma leachate containing 5mM CUS04 x 4