DISCUSSION

Self-incompatibility mechanism in angIOsperms IS widespread and has

received considerable attention in recent years (Takayama and Isogai, 2005; McClure

and Franklin-Tong, 2006). Hamelia patens of the family Rubiaceae is grown chiefly

for its attractive flowers throughout the year. The plant is propagated mainly by

vegetative means due to failure of seed-set. In order to study the reproductve failure,

detailed analyses ware carried out.

According to Brewbaker (1959) and Vuilleumier (1967), there are six families

VIZ. Primulaceae, Saxifragaceae, Rubiaceae, Oleaceae, Apocynaceae and

Polygonaceae displaying both homomorphic and heteromorphic systems of self­

incompatibility. The family Rubiaceae shows diversity in sexual systems. Florally

homomorphic and heteromorphic as well as sporophytic and gametophytic

incompatibility systems have been reported (Bir Bahadur, 1968; Bawa and Beach,

1983; Faivre, 2002). In Rubiaceae, self-incompatibility studies have been done in

Coffea arabica and C. canephora. In C. canephora, the self-incompatibility system is

of the gametophytic type and is found controlled by a single locus with multiple

alleles (Berthaud, 1980). H patens is homomorphic because it does not show any

morphological differences in the floral characters.

Fertilization in flowering plants requires remarkable cellular coordination to

carry sperm cells to the ovules through stylar tissues. The study of factors that control

the reproductive process in higher plants necessitates a thorough knowledge of the

pollen and gynoecium. While considerable information is available regarding the

pollen and the ovary, not much is known about the structure of stigma (Maheswari,

1950; Hulskamp et aI., 1997; Spielman et aI., 1997; Kuzoff et aI., 1999,2001; Soltis

and Hufford, 2002; Yang et aI., 2003).

Anther has been a matter of interest for investigators not only as the male

reproductive organ of the flower but also as a system to facilitate breeding

manipulations in crop plants. In H patens, the fully developed microsporangium

consists of a mass of haploid pollen enclosed within the sporangial wall. Mature

anther is bithecous and tetrasporangiate. The development of anther corresponds to

the reduced type. The outermost layer is epidermis. The layer of cells immediately

beneath the epidermis is endothecium. The cells of the endothecium became radially

elongated and decorated with fibrous bands that run upward from the inner tangential

walls, ending near the outer wall of each cell as an incomplete ring. Fibrous

thickening of endothecium was reported by Periasamy and Kandasamy (1981) in

Annona squamosa, Strittmatter and Galati (2001) in Mysotis azorica and M laxa and

Oliveira and Mariath (2001) in Anacardium occidentale. The tapetum is a specialized

layer of cells whose development is very much in harmony with the events occurring

in the sporogenous cells. At maturity, the tapetal cells collapse. According to

Raghavan (1999) tapetal cells attain the maximum development when the microspores

are in the tetrad stage, after which the cells collapse. Functional as well as molecular

aspects of tapetum have been studied by Mariani et al. (1990) and Staiger et al.

(1994). In H patens anther dehiscence occur between 11-12 pm. Zavada (1984)

revealed that in angiosperm pollen, the reticulate perforate exine sculpturing and

imperforate and microperforate exine sculpturings are associated with the

gametophytic self-incompatibility. However, perforate-matted exine sculpturing was

noticed in H patens, but the plant showed many characters of gametophytic

incompatibility.

78

The relationship between stigma characteristics and the self-incompatibility

was studied by Heslop-Harrison et ai. (l975a). Stigmas not only discriminate between

different pollen grain but also promote out crossing or self-fertilization by the

coordinated timing of their maturation with pollen release. Angiosperm stigmas is

classified into two broad categories, wet and dry, depending on whether or not they

possess a surface secretion (Heslop-Harrison and Shivanna, 1977; Heslop-Harrison,

1981). Stigma of H patens is wet and papillate with sticky exudate. Rupture of cuticle

layers during development of wet stigma is associated with exudate production

originating from epidermal and adjacent cell layers that accumulates in the

intercellular spaces of the stigmatic tissue and below the cuticle-pellicle layer of

epidermal cells. The cuticle-pellicle layer is tom away with continued secretion of

exudates (Konar and Linskens, 1966b; Dumas et aI., 1978). Degeneration of stigmatic

surface cells was accompanied by the profuse accumulation of surface exudate in

older flowers. Copious stigmatic secretions could enhance pollen hydration,

germination and tube growth. The papillae are modified epidermal cells that cover the

stigma and are responsible for most of the secretions on the stigma surface (Heslop­

Harrison and Shivanna, 1977; Considine and Knox, 1979; Owens and Horsefield,

1982). In H patens, from the early bud stage (4 days before anthesis) onwards, the

stigma surface appears as an irregular mass due to the presence of sticky exudate.

Taking into account all the features described by Heslop-Harrison and Shivanna

(1977), it seems fairly justifiable to categorize the stigma of H patens under group III

of wet type.

The stigma is reported to be receptive at the time of anthesis in many tree

crops such as peach, apricot, sweet cherry, apple and kiwi (Sanzol and Herrero, 2001).

Herrero (1983) reported that stigma of pear was not receptive at anthesis and pollen

79

germination increased after four days. Likewise, stigma in apricot was not mature at

the balloon stage, but attained higher receptivity in two and four days after anthesis

(Egea et aI., 1991). Contrary to this, H patens stigma is receptive from the very early

stages of bud development.

According to Heslop-Harrison and Shivanna (1977), receptivity in wet

stigma often coincides with the accumulation of stigmatic secretion. In vivo pollen

germination revealed that even in the IV stage of the bud development, 45% pollen

germination was noticed. Stigma receptivity of H patens was also confirmed by the

activity of esterase enzyme.

Stigmatic secretions play a key role in pollen capture and adhesion (Dumas

and Gaude, 1983; Dumas et aI., 1984). In solid-styled sporophytic self-incompatible

species, the pollen tube was arrested on the stigma. In yellow passion fruit, two

inhibition sites ofpollen-tube growth, in stigma and style, were identified (Rego et aI.,

2000). In the case of dry stigma, the pellicle acts as the first site of molecular contact

for the pollen grains (Mattsson et aI., 1974; Heslop-Harrison, 1978; Dumas et aI.,

1984). The pellicle serves two functions, one is to confine the exudate to the stigma

and the other is to localize and expose pollen recognition molecules in a prominant

and accessible position.

In H patens, the style is solid with a multi-layered stigmoid tissue. The

stigmoid tissue has been named differently by different authors: conducting tissue

(Hanf, 1935) and transmitting tissue (Vasil and Johri, 1964). Most dicotyledons have

a closed style in which the pollen tubes grow through the transmitting tissue, whereas

their longitudinal walls are separated by the intercellular matrix (1M). Chemical

analysis has shown that the 1M contains free sugars, polysaccharides, free amino

80

acids, proteins, glycoproteins, proteoglycans and phenolic compounds, usually

thought to function in pollen tube nutrition, recognition, and guidance (Cheung,

1996b). According to Maheswari (1950), the transmitting tissues serve an easy

passage for the pollen grains and provides nutrients for the growth of pollen tubes

towards the ovary. In H patens, the transmitting tissue is made up of elongated,

parenchymatous cells connected end to end, which is rich in cytoplasm. According to

Garg and Bhatnagar (1988), in Withania, the transmitting tissue is in continuity with

the placental epidermis. The transmitting tissue provides continuity, through or over

which pollen tubes grow en route the ovary (Cresti et. aI., 1979; Tilton and Homer,

1980; Clifford and Owens 1990). In Acer saccarum, the path from stigma to

micropyle is open (stylar canal) but pollen tube path is a continuous tract of mucilage

from the stigma surface through the solid style to the ovule micropyle (Webb and

Williams, 1988; Peck and Lersten, 1991). In soybean stigma, the cells in the stylar

transmitting tissues are secretory, producing exudate, detectable in the intracellular

spaces (Tilton et. aI., 1984). It is quite obvious that the pollen tubes while growing

through the intracellular spaces initially will have to exert pressure on the spaces to

widen. The normal occurrence of longitudinal separation of transmitting tissues

ensured that an axial array of channels filled with exudate, through which the pollen

tube can grow.

In Prunus avium and in Petunia hybrida the stylar vascular elements are found

around the transmitting tissue (Konar and Linskens, 1966a; Uwate and Lin, 1981). In

Ornithogalum, ground tissue consists of elongated parenchyma cells and vascular

tissues (Tilton and Homer, 1980).Vascular element in H patens are seen outside the

centrally placed transmitting tissue as distinct patches.

81

The ovary bears ovules on a specialized ridge on the ovary wall termed

placentum. The ovule generally develops by periclinal divisions of cells of either L2

or L3 or both these layers of the placenta (Raghavan ,1999). The mature ovule is

raised on a stalk and consists of a mass of homogenous cells of the nucellus, protected

by one or two multilayered covering, the integument. The degree of curvature of the

ovule and the number of integuments covering the nucellus add to the variation in the

morphology of embryosac developed on the placenta. Extensive studies have been

carried out on the structural details of the ovule and embryosac (Maheswari, 1950,

1963; Tilton, 1981a, b; Tilton and Lersten, 1981; Cresti et aI., 1992). The ovary in H

patens is pentacarpellary, syncarpus, pentalocular with centrally placed axile

placentum.

In H patens, meiotic chromosome analysis was carried out in order to analyse

the possibilities of any meiotic abnormalities that may lead to the sterile pollen grains.

However microsporogenesis was normal and showed 12 bivalents at metaphase-l

which is first in the species. Futher stages of meiosis were found normal and pollen

sterility was only 10%.

Mature angiosperm pollen grains contain vegetative and generative nuclei,

complete with cell wall and plasma membrane (Raghavan,1999). This arrangement is

accomplished soon after meiosis. Subsequently, the generative cell undergoes a

second mitosis to form two sperm cells required for double fertilization.

"Tricellular" pollen completes this division before it is released from the anther,

whereas "bicellular" pollen undergoes this division only later, within the elongating

pollen tube. Brewbaker (1957) pointed out a correlation between incompatibility

systems and the number of nuclei in the pollen. In general, species showing

gametophytic and sporophytic incompatibility systems have two or three nuclei

82

respectively. Most dry stigmatic SI systems were historically associated with

tricellular pollen grains and bicellular pollen grains with wet stigmas and stylar SI.

Stigmatic SI in association with bicellular pollen and a dry or wet stigma was

considered anomalous (Brewbaker, 1957; Heslop-Harrison, 1975; de Nettancourt,

1977). Recognition and rejection of bicellular pollen on wet stigma was reported in

Rubiaceae (Brewbaker, 1957; de Nettancourt, 1977, 1997; Franklin et aI., 1995). Wet

stigmas with binucleate pollen grains were observed in H patens. Since SI in all taxa

with bicellular pollen has been demonstrated to be under gametophytic control,

regardless of whether recognition and rejection takes place on a dry/wet stigma or in

the style, it is assumed that the SI in Rubiaceae is under gametophytic control.

Pollen viability is considered as an important parameter of pollen quality

(Dafui and Firmage, 2000). In physical method, pollen viability is generally tested by

FCR which has been suggested by Heslop-Harrison and Heslop-Harrison (1970). FeR

test was found satisfactory for a range of pollen species (Shivanna and Heslop­

Harrison, 1981; Heslop-Harrison et aI., 1984; Jain and Shivanna, 1988). Most of the

pollen grains are metabolically quiescent and highly desiccated, ranging from 15 to

35% water content, when released from anthers (Heslop-Harrison, 1979; Buitink et

aI., 2000). Shivanna and Heslop-Harrison (1981) reported good correlation between

FCR and in vitro germination. In H. patens, 90% of pollen grains were intensely

stained with FDA showing its viability status.

In biological method, germinability is used to test the viability of pollen

grains. In vitro pollen germination method is rapid, reasonably simple and the most

commonly used for assessing pollen viability. Pollen germination generally requires a

carbohydrate source. Sucrose is the most commonly used carbohydrate source in

culture medium which serves the maintenance of osmotic pressure of the medium

83

and as carbon source for metabolism which is essential for pollen germination (Johri

and Shivanna, 1985). The optimum sugar concentration required for the pollen

gennination differs considerably with species (Johri and Vasil, 1961). Besides the

carbohydrate source, boron and calcium also play an important role in pollen

gennination and tube growth (Brewbaker and Kwack, 1963; Kwack, 1967; Balatkova

et aI., 1980; Steer and Steer. 1989). A culture solution containing sucrose and

minerals suggested by Brewbaker and Kwack (1963) is widely used for in vitro pollen

gennination. By manipulating individual components, it has been possible to achieve

satisfactory in vitro germination in a large number of taxa with binucleate pollen. In

the present study, Brewbaker and Kwack's medium supplemented with 25% sucrose

showed optimum germination of 72.5%. Since the pollen sterility is less (10%) and

pollen germination is more, pollen sterility cannot be attributed as the reason for lack

of seed- set in this plant. In many species in vitro pollen germination showed positive

correlation with fruit and seed-set (Janssen and Hermsen, 1976; Akihama et aI.,

1978). But in H patens, a negative correlation was found between pollen germination

and seed-set.

5.1 Pollen-pistil interactions

During pollination, numerous cell to cell interaction events occur between the

cells of the sporophyte and the male gametophyte. The timing of pollination is

critical, because the stigmatic surface of the pistil is only receptive to pollen for a

relatively short period. Pollination outside this window of female receptivity will

result in reduced seed set or no seed set (Herrero, 2003). The recognition events

appear extremely complex and are not fully understood (Clarke and Gleeson, 1981;

de Nettancourt 1984; Dumas et aI., 1984; Knox, 1984). The influence of the stigma

and style on pollen germination and pollen tube growth was studied by Heslop-

84

Harrison (1983). According to him, the pollen tube growth terminates when pollen

falls on the pistil of the same genotype. Self-incompatible stigma rejects self-pollen

by inhibiting pollen hydration, germination and tube invasion into the stigma. These

processes have been reviewed extensively (Nasrallah, 2000; Silva and Goring, 2001;

Edlund et aI., 2004).

Most reliable test for pollen viability, stigma receptivity and seed setting is the

in vivo germination method. Aniline blue staining method is commonly used to study

in vivo germination. The fluorescence microscopic method has been used extensively

for tracing pollen tube growth and behaviour in pistil in a wide range of species (Kho

and Baer, 1968; Stout, 1972; Remming et aI., 1978; Vishnyakova.1991; Sniezko and

Winiarezyk, 1995). In H patens, 70-75% pollen germination was noticed on stigma

surface of open and self-pollinated flowers. But the pollen tubes never penetrated into

the stigma and they showed various abnormalities like curling of the tubes, swelling

of tube tip and irregular callus deposition followed by pollen tube growth inhibition. It

clearly indicates stigmatic surface recognition which leads to the inhibition of pollen

tube growth.

In general, the pollen tubes develop and deposit a considerable amount of

callose along their wall during elongation, which is a pre-programmed step in the

development of pollen tubes (Shivanna and lohri, 1985). In the germinating pollen of

angiosperms, callose plugs are formed in the pollen tube as it grows. According to

Hepler et al. (2001) callose plug formation helps to maintain a constant amount of

cytoplasm containing the germ units in the pollen tube tip region without division of

the tube cell, although it has been indicated that callose plug synthesis is not

dependent on the movement of germ units into the tube. The formation and function

of callose plug have been studied by Laitiainen et ai. (2002). Although callose was

85

originally believed to play a role in prevention of pollen tube penetration into the

stigmatic papilla, evidence indicated that callose is unlikely to play an essential part of

self recognition and rejection in other species (Singh and Paolillo, 1990; Franklin

et aI., 1995; Sulaman et aI., 1997; Radhamany, 2002). In H patens, incompatible

pollen tube growth was arrested on the surface of stigma and showed various

abnormalities like callose deposition at irregular intervals and swollen tube tips.

However, deposition of callose cannot be considered as the cause of pollen tube

inhibition as it is observed in the case of compatible pollination also.

Quite often, callose is synthesized in the cell as a response to some stress

conditions. According to Vishnyakova (1991), strong anomalous callose occlusions

occur in pollen grains and pollen tubes as a result of their incompatible interactions

with the pistil. Such non-programmed callose occlusions, as a rule, are linked with

metabolic changes. Hence in H patens, there was an incompatible interaction

between pollen tube and components on the surface of the stigma after self-pollination

and cause metabolic changes and irregular callose deposition, ultimately leading to

pollen tube growth inhibition.

According to Knox et al. (1976), the receptors on the stigma surface consist of

numerous components, some of which are involved in pollen germination and others

help in the entry of tubes. Previous reports suggested that phenolic compounds in the

surface deposits were involved in pollen tube growth inhibition (Martin, 1969; Martin

and Ruberte, 1972). Various enzymes were reported to play an important role in

pollen tube interaction on the stigma surface (Knox and Heslop-Harrison, 1970;

Heslop-Harrison and Heslop-Harrison, 1975a; de Nettancourt, 1977; Heslop-Harrison,

1977; Heslop-Harrison and Shivanna, 1977). Pollen- pistil interaction was reported to

be based on the interaction of protein of the stigmatic surface and the pollen wall

86

(Shivanna, 1978). The rejection response in stigma can be interpreted as an evidence

for a mechanism controlling normal tube growth of the pollen which is responding to

molecular signals from the pistil (Trognitz, 1995). According to Taylor and Hepler

(1997) pollen tube growth requires highly active and polarized secretion at the tube

apex. Hydrolysis of pectin in the stigma cell wall is also necessary for pollen tube

growth (Kim et aI., 1996; Wu et aI., 1996). According to Hiscock et ai. (2002a),

cutinase inhibitors significantly reduce the ability of pollen tube to penetrate the

stigma in Brassica. A pollen specific Brassica polygalacturonase have been detected

at the tip of the pollen tube as they enter the stigmatic papillar cell walls (Dearnaley

and Daggard, 2001). In H patens, as it has wet stigma, the compounds on the surface

deposits may playa role in the inhibition ofpollen tube growth.

5.2 Methods to overcome self-incompatibility

Various methods such as temperature treatment, bud pollination and excised

style pollination were employed to eliminate the incompatibility barriers in H patens.

Extreme conditions such as end-of season and high temperature were reported

to reduce incompatibility. The effect of temperature in incompatibility response was

studied by Lewis (1942) and Radhamany (2002). Leffel (1963) showed plants grown

at relatively high temperature produced more seeds after self-pollination than plants

of the same clones at low temperature. Temperature has been found to affect the

incompatibility response of Oenothera rhombipetala, 0. organensis, Lilium

longiflorum and Trifolium (Bali, 1963; Hecht, 1964; Ascher and Peloquin, 1970).

Townsend (1968) found that style exposed to 32°C for one or two days changed self­

incompatibility in alsike clover (Trifolium hybridum ) to self-compatibility. Exposure

of the pistil above 34°C at the time of pollen germination caused decrease in the

87

germination percentage and the rate of pollen tube growth in this plant. Two species

of Primula as well as Prunus avium and Pyrus malus also showed retarding effect of

high temperature on t~ growth of incompatible pollen tube (Lewis, 1942;

Modlibowska, 1945). In H patens, treatment of style with extreme temperature was

tried. However exposure to 45, 50 and 55°C for 3 minutes caused reduction in the

pollen tube growth. Low temperature treatment at 20°C caused 65.9 % pollen

germination on the surface of stigma where as very low temperature (4°C) showed

only 8.2% pollen germination. In gametophytically controlled systems, overcoming

self-incompatibility by heat treatment seems to be caused by the denaturation of

metabolites (Hecht, 1960). Most probable molecules for the metabolite mediated heat

treatment are proteins including glycoprotein. The absence of fruit-set at high

temperature is not considered as outcome of a single malfunctioning factor, but

triggering a series of factors simultaneously impaired by high temperature. Lower

temperature (12°C) and higher humidity (90%) positively affected bud pollination, in

Brassica oleracea L. var. capitata (Zur et aI., 2003).

Excised style culture technique might be helpful not only in studying the

interaction between style and pollen tubes during the tube growth but also in detecting

the number of tubes at a distance from the stigma. The application of pollen to the cut

surface of the style after removing the stigma in parents with strong incompatibility

was effective in overcoming cross-incompatibility (Gardella, 1950). Stigma and style

grafts have been employed to overcome incompatibility barriers in Petunia and

Oenothera (Hecht, 1960, 1964; Herrero and Dickinson, 1979). Style shortening and

stigma regrafting have been successfully applied in Oenothera (Gardella, 1950).

Pollination on decapitated stigma has been used successfully in Solanum

(Swaminathan, 1955). In the present study, application of the pollen to the cut surface

88

of the stigma and style has been found not successful to overcome incompatibility

barriers.

Eventhough many techniques have been standardized to overcome self-·

incompatibility, bud pollination and delayed pollinations were found to be the

simplest and most commonly used methods (Shivanna, 1982; Shivanna and Johri,

1985). It could be utilized in species with gametophytic as well as sporophytic

incompatibility. Bud pollination was found to be very successful in Brassica,

Raphanus and Brunelsia (Haruta, 1966; Radhamany, 2002). In 1964, Linskens

suggested that the principle of self-compatibility following bud pollination is based on

the fact that the incompatibility substances are absent or are not fully effective in

developing styles. According to Shivanna (1978), bud self-compatibility is due to the

stigma factors inhibiting the pollen in incompatible combinations which are not

present or are inactive in buds of the age accepting self pollen. In the present study

bud pollination was not successful.

5.3 Molecular characterization by RAPD

Random amplified polymorphic DNA markers have been used to analyze the

genetic diversity at low taxonomic levels (Mehrnia et aI., 2005; Rajasekar et aI., 2006;

Das et aI., 2007). RAPD is simple and easy to perform and can be applied to a large

number of genotypes without adding much to the cost of experiment (Srivastava et al.,

2007). It has been successfully employed in the evaluation of genetic relationship in

various plant species (Gunter et al.,1996; Gielis et aI., 1997; Nair et aI., 1999; Nayak

et aI., 2003; Rout, 2006)

The self-incompatibility recognition systems of angiosperms are a classical

object of interest to geneticists interested in the spectacular polymorphism found in

89

natural populations, and cell biologists interested in recognition functions. The

maintenance of multiallelic polymorphism has been thoroughly investigated by

classical evolutionary and population genetic approaches (Lawrence and Franklin­

Tong, 1994; Lawrence et aI., 1994; Vekemans and Slatkin, 1994; Schierup, 1998). In

all the species studied with homomorphic SI systems, (gametophytic as well as

sporophytic) the S-loci have spectacular numbers of S-allele within the populations

(Lane and Lawrence, 1993). It has long been understood that, if two different loci are

involved, the self-incompatibility genes must recombine rarely or not at all, since

recombination would generate genotype having the pollen type of one allele with the

pistil specificity of a different allele, and these would presumably be self-compatible.

Outcrossing species tend to have higher levels of variability within populations but

smaller degrees of differentiation among populations than selfing species (Hamrick

and Godt, 1990; Schoen and Brown, 1991).

Recently, biochemical and molecular tools have determined the S-genotype of

cultivars in various species. PCR based S-allele typing system could be useful for

determining pollen compatibility groups of commercially important new cultivars to

elucidate their incompatibility relationships. The molecular typing system of

genotypes based on PCR is useful and has been used as a rapid method for indicating

new S-allele and incompatibility groups in sweet cherry in the absence of pollination

tests (Choi et aI., 2002).

For RAPD marker analysis, 14 different accessions of H patens collected

from various parts of Kerala have been used. Field observations showed that

morphological characters are quiet consistent within 13 accessions, but accession

No. 3 shared some unique morphological features and also form compatible

combination when used as either male or female parent. For the RAPD analysis,

90

10 among 20 pnmers used were selected from the clear, reproducible, and

polymorphic banding profile. Genotypic relationship based on RAPD phenotypes of

the 14 accessions were analysed and constructed a UPGMA phenogram. The topology

of the phenogram shows expected grouping with relation to the morphological and

compatibility difference found among the accessions.

RAPD and sdf-incompatibility (SI) typing techniques were applied to find out

the genetic integrity of the two varieties of Raphanus sativus (Kwak et aI., 2009).

Bulk segregant analysis (BSA) and RAPD were employed to identify molecular

markers linked to a self-incompatibility gene in self-incompatible and self-compatible

near-isogenic lines of non-heading chinese cabbage (Brassica campestris L. ssp.

chinensis Makino var. communis), (Shi and Hou, 2004). PCR analysis with primers

designed on the conserved sequences of sweet cherry S-RNases has been used to

characterize the S-genotype of 71 sweet cherry cultivars, including 26 cultivars whose

S-allele constitution had not been previously described (Wunsch and Hormaza, 2004).

A total of 17 pollen incompatibility groups in sweet cherry (Prunus avium L.) were

identified among 46 accessions by PCR based S-allele typing analysis and by

controlled test pollinations (Choi et aI., 2002). cDNA sequences corresponding to

five self-incompatibility alleles (S-alleles) of the apple cv 'Golden Delicious' have

been reported by Janssens et aI. (1995). Based on the nucleotide sequences of these

S-allele cDNAs, a molecular technique has been developed for the diagnosis of the

five different S-alleles in apple cultivars.

Tamura et aI. (2000) developed a method for identifying S-RNase using the

cDNA sequence, a PCR-based identification system using genomic DNA. PCR

amplification of genomic DNA followed by restriction analysis is used for the

identification of S alleles involved in self-incompatibility in Brassica oleracea (Brace

91

et aI., 1993). In H patens, RAPD analysis of 14 accessions revealed two different

groups with variation in the specific bands, clearly evident that allelic variation may

exist in Acc. No.3. In the present analysis Acc. No.3 is distinctly seperated from the

rest. It has same reproductive significance in the sense that it is compatible when used

as either male or female parent. So it can be concluded that Acc. No. 3 with this

reproductive significance may have a different genotype.

5.4 Infraspecific pollination

As various methods used have failed to overcome incompatibility in

H patens, infraspecific pollinations carried out between Acc. No.1 and 3 were found

to be successful and resulted in viable seed. The production of viable seeds may be

considered as the ultimate criterion for reproductive success (Wiens et aI., 1987).

In the case of cross-pollinations, large number of pollen grains adhered and

germinated on the surface of the stigma. These germinated pollen tubes penetrated

into the style and reached the ovary and effected fertilization. This was confirmed by

pollen-tube behaviour and by the developmental anatomy of ovary after pollination.

Field pollination of H patens showed significant differences in ovule development

between self and cross-pollinations. A significantly greater proportion of megaspores

in the ovules failed to develop after self-pollination. Developmental anatomy of ovary

after cross pollination showed normal cell differentiation. To decide whether certain

cross combination is compatible or not, fruit set and combinations of fruit and seed­

set have been used as indicators (Cipar et aI., 1964; Grun and Aubertin, 1966;

Abdalla, 1970; Olsder and Hernisen, 1976).

In the present study, significant difference was noticed in the pollen

germination and tube growth pattern during self and cross- pollinations. So it is

92

assumed that self-pollination induced biochemical changes in the stigma/style tissues

that may affect the metabolic status of the tissue, acts as a reproductive barrier in

H patens. However, cross- pollination did not affect the metabolic activity and

normal functioning of the stigma/style tissues which in turn lead to compatible

pollination. In H patens, infraspecific pollination was found to be effective in

producing seeds. Continued development of ovary after compatible and incompatible

pollination also clearly indicated the success of infraspecific pollination in H patens.

5.5 Histochemistry and enzyme cytochemistry

Pollination initiates many biochemical changes in the pistil. In order to find

out the pollination induced changes in the primary metabolites and key enzymes in

the pistil of H patens, histochemical and cytochemical localizations were done and

found significant variation between different pollinations (Table 15).

Table 15. Results of localization of primary metabolites and enzymes in the pistil

of H. patens after various types of pollinations

Primary metabolites Unpollination Self-pollination Cross-pollination

and enzymes

starch ++ ++++ +

protein ++ +++ ++

lipid ++ +++ +

PAS +++ +++ +

SDH ++ ++++ +

APase +++ ++ +

ATPase +++ ++ +

Peroxidase ++ I I I I I +

Esterase + ++++ ++

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Accumulation of insoluble starch and polysaccharide was indicated as

reserves, which acts as the main source of energy for cellular metabolism. In

unpollinated pistils, meagre deposition of starch was noticed. In self-pollinated pistils

of H. patens, intense deposition of starch has been found at secretory zone as well as

in ground tissues. Starch is an energy reserve, and its level decrease when the pollen

tube growth is heterotrophic. In Petunia hybrida, the starch level in the transmitting

tissue drops heavily after pollination (Herrero and Dickinson, 1979). In the normal

course of compatible pollination, carbohydrates produced in the pistil are utilized by

pollen tubes for their growth. Activity of polysaccharides and starch in pollinated

stigma is higher than the unpollinated ones, which indicates that these reserves are

accumulated during pollination and subsequently mobilized for meeting the energy

requirement. As a result, there is reduction in the amount of carbohydrate in the pistil

with an increase in age (Yamada, 1965; Rosen and Thomas, 1970). In H. patens,

intense deposition of starch was seen after self-pollination and it may be due to

immobilizition of starch. Since the pollen tube growth was inhibited on the surface of

the stigma, the reserved food materials may not be used for tube growth and thus their

accumulation. In compatible pollination, starch was utilized for normal pollen tube

growth and therefore transmitting tissue was found to be free of its deposition.

Presence of protein was identified by using mercuric-bromophenol blue.

Protein deposition was less in unpollinated stigma, whereas meagre deposition was

seen in papillar region, ground tissues and transmitting tissues. Proteins playa major

role in the recognition and rejection phenomena on the stigma surface (Nasarallah and

Wallace, 1967). Mattsson et al. (1974) suggested that the proteinaceous pellicle

overlaying the stigmatic papillae may contain the molecules responsible for

recognition of pollen grains. Numerous investigations have demonstrated that proteins

94

(Arabinogalactan-protein) are involved in the process of sexual reproduction of

angiosperms and play multiple roles, by providing surface adhesion, nutrients and

directional cues or signals for pollen tubes (Sommer-Knudsen et aI., 1997; Cheung

and WU,1999; Majewska-Sawka and Nothnagel, 2000; Qin and Zhao, 2004; Qin

et aI., 2007) . They may also participate in gametal recognition, embryo development

and differentiation (Hu et aI., 2006). In H patens, the protein component present on

the stigmatic surface may have a role in pollen adhesion. After pollination, proteins

are localized on the transmitting zone and ground tissue which indicated the role of

proteins in directional signal for pollen tubes.

Lipids are essential factors needed for pollen tube to penetrate the stigma and

directing pollen-tube growth by controlling the flow of water to pollen in species with

wet stigmas, while pollen coat is functionally equivalent to the species with dry

stigma (Wolters-Arts et aI., 1998). Lipids probably regulate the availability of water

to the pollen, and prevent desiccation of stigma (Martin, 1970). The greasy exudates

of the stigma included chiefly lipid compounds which can be separated into eight

principal fatty acids (Konar and Linskens, 1966b). Lipids also playa major role in the

nutrition of the pollen grains (Kuruvila and Shah, 1988; Wolters-Arts et aI., 1998). In

the present study, intense accumulation of lipids was found in the extreme tip of the

stigma and on either sides of vascular tissues. In the stigma of Withania somnifera,

stigmatic exudate is rich in lipids (Philip, 1989). Vasil and JoOO (1964) found that in

Aegle and Pavonia, the stigma secretion showed the maximum concentration of lipids

at the time of pollination. In the present study, presence of lipid was mainly localized

in the region where stigmatic exudate is seen. The deposition of lipids was found to be

maximum on the day of anthesis and reduced after pollination. So it is assumed that

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lipid is a major components of the exudate and plays a key role in the adhesion and

hydration ofpollen grains.

In H patens, self- and unpollinated stigmas stained for PAS showed uniform

distribution of PAS positive bodies. When compared to the self- pollinated stigma,

meagre activity was observed in cross-pollinated pistils. In this case, activity of PAS

was found to be less in the secretory zone and transmitting region. PAS existed in

several forms, viz, soluble sugars, insoluble cell wall, polysaccharides (cellulose,

pectin etc) and even storage starch polysaccharides contribute to the cellular

energetics and to the production of energy rich triphosphates. Polysaccharides are

necessary for pollen tube growth (Ciampolini and Cresti, 1998). During this process

of pollen-pistil interaction, pistil plays an essential role in the production of

compounds necessary for recognition, guidance, protection and nourishment of the

pollen tubes (Kao and Mc Cubbin, 1996). The presence of storage starch indicated

potential energy which would be utilized during different growth and differentiation

processes in plants. In H patens, the reserve food materials were not utilized for

pollen tube growth in self-pollination. Hence PAS was accumulated on the surface of

stigma.

Enzyme cytochemistry has developed with great speed and is now being

applied to many diverse areas of plant biology. There were a few studies concerning

changes in the activities of enzymes in stigma-style tissues following compatible and

incompatible pollination (Bredemeijer and Blaas, 1975; Dhaliwal and Malik, 1985).

Most of these were conducted in species characterized by gametophytic self­

incompatibility (Schlosser, 1961; Linskens et aI., 1969; Bredemeijer, 1974, 1979).

Since compatible and incompatible pollinations are affected by alterations in the

metabolic status of stigma and stylar tissues, it would be imperative to study the

96

changes in the enzyme activities of stigma and stylar tissues following compatible and

incompatible pollinations (Dhaliwal and Malik, 1985). Since the enzymes control

biochemical reactions, and their synthesis is controlled by specific gene(s), any

change in the activity of an enzyme would reflect in the pattern of gene expression

and corresponding metabolic events in the cell. Hence, the enzymes can be used as

tools to study the problem of self-incompatibility at the biochemical level. In the

present investigation, changes in the activities of peroxidase, esterase, ATPase, APase

and SDH were studied.

The activity of succinic dehydrogenase is very intense in the papillar region

and vascular tissue after self-pollination. Before pollination, the activity of this

enzyme was meagre. SDH has been associated with mitochondria and was involved in

transfer of electrons to oxygen. This enzyme was also found to be involved in the

process of cellular respiration leading to ATP synthesis (Pearse, 1972; Malik, 1977).

The activity of this enzyme increased with a corresponding increase in the age of

stigma, which shows that the rate of metabolism was high in the stigma matrix. In

Catharanthus roseus and Withania somnifera, activity of SDH was moderate in

younger buds, and in mature buds intense activity was noticed (Philip, 1989). Intense

activity of this enzyme is also noticed in the vascular tissue regions of plants (Shah et

aI., 1980). The activity of SDH is more intense in the sub epidermal regions and

stigmatic papillae of Brunfelsia undulata (Radhamany, 2002). From the present study

it is assumed that pollination may induce excess ATP production, which may lead to

an increase in SDH activity in H patens after pollination.

Among the hydrolases, the most pronounced activity was that of acid

phosphstase. The hydrolytic enzymes are lysosomal in nature (Gahan and Mc Lean,

1969). These enzymes have a role in hydrolyzing the stored reserves like protein,

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sugar and starch. The products of hydrolysis of these reserves are usually utilized for

the metabolic activities of the cell (Chaffey and Harris, 1985). Acid phosphatase

(APase) are limited with the differentiation and maturity of stigma. According to

Simola and Sopanen (1970) APase was connected with lipid synthesis. In H patens,

papillar, epidennal, sub epidennal and upper few layers of ground tissue showed

intense activity. The central core of transmitting tissue was devoid of its activity.

APase activity was maximum in unpollinated stigma and after pollination stigma

showed slight decrease in the activity. In H patens, reduced activity of APase is

related to the reduction in the lipid synthesis as well as sugar transfonnation as a

result of pollen tube growth inhibition on the surface of stigma.

ATPase is an enzyme primarily involved in the breakdown of ATP for the

release of high energy present on the phosphate bond of the molecule required for the

various metabolic and developmental activities in plants (Gahan, 1981). In H patens,

intense activity of ATPase was seen in the papillar region which is evidenced by the

lead sulfide deposition, depicting its activity, and was confined to the cell membrane/

wall regions. The activity of this enzyme was restricted to the cytoplasm and the

reaction was diffuse. The cytoplasmic activity may indicate its role in the metabolic

process in tenns of high protein synthesis, while wall bound activity signifies its role

in the intercellular communication and transport required for the cellular

differentiation and subsequent development. In Brassica campestris, ATPase was

differentially distributed in a zonate pattern in the vegetative apices, showed a

heterogeneous localization with the highest activity in the central zone and the

meristematic zone. At the early transition stage of development, ATPase activity in

the peripheral zone increased slightly and at the transitional stage it was not localized

within the peripheral zone. In the present study, ATPase activity was less in pollinated

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pistils. This observation agrees with Gahan (1984), and that high activity of this

enzyme during morphogenesis could be the indication of cell differentiation. In the

present study, pollen tube growth inhibition on the stigmatic surface causes reduction

in the activity of enzyme. So the activity of ATPase also reduced after self­

pollination.

Angiosperm stigmas have long been known to exhibit high levels of

peroxidase activity when they are mature and most receptive to pollen but the

biological function within the stigma is not known. Bredemeijer and Blaas (1980)

reported that pollination and pollen tube growth caused an increase in total peroxidase

activity. Peroxidases generally catalyse the breakdown of hydrogen peroxide to yield

highly oxidizing intermediates, which oxidize a variety of organic and inorganic

reducing substrates. Understanding peroxidase function in plants has proved

problematic because of their broad range of substrate preferences and lack of tissue

specificity. Peroxidases should now be considered as potentially important

components of signal transduction pathways in plants. Early isozyme studies in

Nicotiana suggested a potential role in pollination processes, particularly SI (Pandey,

1967b). These studies showed that stylar peroxidase activity was higher after

incompatible pollinations compared with compatible pollinations. Unpollinated

pistils, however, showed stylar peroxidase activities comparable to those of selfed

(incompatible) pistils, and hence Bredemeijer (1974) inferred that in the compatible

style, peroxidase activity was somehow inactivated. Closer inspection indicated that

only the peroxidase activity associated with the extra cellular secretion of the stylar

transmitting tissues were reduced following compatible pollinations (Bredemeijer and

Blaas, 1975). Later Carraro et al. (1986) suggested that peroxidase is responsible for

the inhibition of incompatible pollen in Petunia and Nicotiana. However, in

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Nicotiana, Lycopersicon and Petunia a peroxidase gene is linked to each of their

respective S loci (Hoopen et aI., 1998), but these S-linked peroxidase loci have never

been characterized. The first plant peroxidase gene expressed exclusively in stigmas

was recently identified (McInnis et aI., 2005). Importantly, this peroxidase, SSP

(stigma-specific peroxidase), is expressed specifically in the specialized secretory

cells (papillae) of the stigma epidermis and is developmentally regulated. Expression

of SSP in flower buds is developmentally regulated, with maximum level of

expression coinciding with anthesis; when stigmas are most receptive to pollen and

expressed self-incompatibility (Bredemeijer, 1974). Increase in peroxidase content of

the transmitting tissue following self-pollination was reported by many authors

(Bredemeijer and Blaas, 1975; Bredemeijer, 1976). The results indicated the presence

of peroxidases on the cell wall of unpollinated and self-pollinated styles support the

hypothesis of Bredemeijer (1977) that only the fraction of extracellular peroxidases is

responsible for pollen tube growth inhibition. Compatible pollination does not cause

the increase in the number of cells showing peroxidase activity, but does cause the

disappearance of the peroxidase activity observed in unpollinated samples. According

to Galen and Plowright (1987) in Pedicularis canadensis and Clintonia borealis the

stigma peroxidase activity is a reliable indicator of receptivity. In H patens, self­

pollination causes an increase in the activity of peroxidase in the pistil and reduced

activity during cross-pollination, which support the hypothesis that only a fraction of

extra cellular peroxidase is responsible for pollen tube inhibition.

Studies on the esterases have utilized the high enzymatic activity in stigma as

indicators to assess the stigmatic receptivity (Lavithis and Bhalla 1995: Tandon et aI.,

2001). Bhattacharya and MandaI (1997, 2003) correlated the esterase activity and

stigma receptivity. The wet type stigma secretes exudates containing lipids, phenolic

100

compounds, carbohydrates, proteins, phosphatases, lectins and aminoacids including

esterases (Baker et aI., 1974; Vasil, 1974). According to Chaffey and Haris (1985)

this enzyme hydrolyse the stored reserves like starch, protein and sugars thereby

initiating them for the metabolism of cells in morphogenetic events. In H patens, the

activity of esterase increases after self-pollination so as to facilitate the growing

pollen tube. But pollen tube growth inhibition on the stigmatic surface may cause

accumulation of this compound.

In H patens, pollination induced many metabolic changes in stigma; most of

these changes are related to pollen tube growth. Many of the metabolites were

accumulated after self-pollination because pollen tube growth was inhibited on the

surface of the stigma.

5.6 Electrophoresis

Although the functional role of S-proteins in the self-incompatibility

interactions has not been demonstrated in vivo, many lines of evidences strongly

suggested their involvement in the recognition and rejection of self-pollen. The

association between S-glycoproteins and self-incompatibility was further supported

by Herrero and Dickinson (1980) in Petunia by locating these proteins at the site of

pollen tube arrest and their increasing expression during development of flower. Since

infraspecific pollination has been found to be successful in H patens, the protein

composition of the stigma homogenate and leachate of unpollinated, self- and cross-

pollinated (compatible) flowers have been analyzed electrophoretically and revealed

the presence of specific protein bands. Between self- and cross-pollinated pistil,

protein bands were found to be common with in arrange of 22 to 95 kDa but showed

quantitative difference.

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Protein profile of stigma leachate showed five bands at 23, 30, 36, 70 and 99

kDa regions. These bands were also found in the protein profile of stigma

homogenate. As like in the stigma homogenate, protein profile of leachate showed

quantitative difference in bands between self- and cross-pollinated pistils, and band at

36 kDa region was absent in unpollinated stigma leachate. Protein band at 36 kDa

region has been found to be present in stigma leachate as well as homogenate while it

is absent in unpollinated pistil. In H patens tube growth inhibition of self-pollen takes

place on the surface of the stigma as well as in the in vitro assay medium. So the

factor(s) responsible for incompatible interaction is present in leachate as well as

homogenate after self-pollination. So it is assumed that protein band at 36 kDa may

be responsible for incompatible interaction in H patens.

Proteins and glycoproteins involved in the manifestation of self­

incompatibility are proven to be associated with particular S-alleles (Nasrallah et

al.,1985b; Anderson et al.,1986; Cornish et aI., 1987, 1988). Immunological and

electrophoretic data indicated that S- allele specific protein present in the stigma of

Brassica caused strong self-incompatibility (Nasrallah and Wallace, 1967; Nasrallah

et aI., 1970; Sedgley, 1974). The protein composition of the stigma secretion and

stigma cell extracts of different species of plants have been analyzed

electrophoretically with or without SDS (Miki-Hiroshige et aI., 1987; Reger, 1989). In

Lycopersicon peruvianum ,the SI associated proteins have been shown to exhibit a

high degree of polymorphism in the molecular weight range of23-30 kDa (Bematzky,

1993; Lee et aI., 1994). McCubbin et al. (1997) obtained several transgenic plants of

Petunia with an intermediate SI system; their styles produce much less S-protein,

whereas self-compatible style lacks S-proteins. S-allele associated proteins with

RNase activity was detected from the Japanese pears (Sassa et aI., 1992; 1993;

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Hiratsuka et aI., 1995; Ishimizu et aI., 1996). According to Zhang and Hiratsuka

(1999), S-proteins in the style may be one of the determinants of SI in Japanese pear

cultivar.

Bredemeijer and Blaas (1981) was the first to establish the correlation between

the genetic phenomenon of S-allele-specific pollen rejection and specific protein

products in the style. Anderson et ai. (1986) cloned the first S-protein in a

gametophytic SI system, Similar S-proteins were also cloned in Nicotiana,

Lycopersicon, Petunia and Solanum and found a basic glycoprotein of about 30 kDa

was extremely abundant in the pollen transmitting tract matrix (Anderson et aI., 1989;

Ai et aI., 1990,1992; Clark et aI., 1990; Kheyr-Pour et aI., 1990; Xu et aI., 1990; Tsai

et aI., 1992).

In buck wheat, protein profile of stigmatic leachate showed quantitative

difference at 56 kDa region in incompatible and compatible pistil (Miljus-Djukic

et aI., 2004). From the previous reports and present study, it is assumed that a protein

band at 36 kDa region may be involved in the self-incompatibility mechanism in

Hpatens.

5.7 S-RNase activity

The most phylogenetically widespread gametophytic incompatibility relies on

ribonucleases called S-RNases (Igic and Kohn, 2001). S-RNases are basic

glycoproteins of about 30 kDa that are secreted into the extra cellular matrix of the

stigma, transmitting tract, and the inner epidermis of the ovary (Anderson et al,

1986,1989; Cornish et al., 1987; McClure et aI., 1993). According to Jahnen et ai.

(1989), S-allele specific pollen rejection requires extremely high level of S- RNase in

the extracellular matrix. It has also been shown that the S-RNase activity is required

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for pollen rejection (Huang et aI., 1994; Karunanandaa et aI., 1994). Thus, S-RNases

are thought to function as highly specific cytotoxin that inhibits the growth of

incompatible pollen.

Hamelia patens showed many of the characters of gametophytic

incompatibility system. After self-pollination, pollen tube growth inhibition takes

place on the surface of the stigma and electrophoresis of stigma protein from

incompatible and compatible stigma found variation in the amount of protein at

36 kDa region. In self-pollinated stigma, the intensity of specific protein band was

found more. To determine activity of RNase, diffusion plate assay was done and

found its activity on the surface of the stigma. When compared, cross-pollinated

stigma showed less activity than self-pollinated one, where as in the style or the cut

end of the style lack RNase activity.

RNase activity has been reported III S-proteins extracted from styles of

Nicotiana a/ata and Pyrus serotina (McClure et ai. 1989; Gray et ai. 1991; Sassa et

al., 1992). In P.serotina, significantly more RNase activity was found in styles of self­

incompatible varieties than in those of a self-compatible mutant. In N a/ata, the

S-RNase, which represented a high percentage of the stylar proteins, caused

degradation of self-pollen mRNA and rRNA, leading to the inhibition of pollen tube

growth (McClure et aI., 1989). Proteins with sequences similar to those of S-RNase

have been found in styles of self-compatible Arabidopsis thaliana (Taylor et al.,

1991) and Petunia hybrida (Clark et aI., 1990) may be involved in a wide range of

cell-cell recognition processes. Gray et ai. (1991) used gold-immunolabeling to show

that stylar RNase penetrates into self-pollen tubes. In self-compatible Lycopersicon,

absence or decreased S-RNase activity was accompanied by loss of production of the

stylar protein HT (Kondo et aI., 2002b), which is necessary for a functional SI

104

response (McClure et aI., 1999). Low stylar RNase production was reported to be

associated with SC in both Nicotiana (Golz et aI., 1998) and Lycopersicon (Kowyama

et al., 1994; Royo et aI., 1994; Kondo et aI., 2002a). Less activity of RNase in

compatible pistil (cross) may be the reason for compatible interaction in H patens.

In H patens, activity of RNase gradually increases in the stigma from five

days before anthesis to the day of anthesis. Eventhough the RNase activity in stigma

five days before anthesis was very low, the buds are not compatible with mature

pollen. In Witheringia solanacea, buds 2 days prior to anthesis produced stylar

RNase at levels equivalent to that of mature flowers, yet bud pollinations still had

reduced SI, (Jahnen et aI., 1989; McClure et aI., 1989). The electrophoretic analysis of

S-RNase proteins of W solanacea showed a marked increase between small and large

buds rather than between large buds and open flowers.

The selective inhibitory action of stigma leachate on the growth of self and

cross pollen tube in vitro and the indispensability of RNase activity in inhibition of

pollen tube growth were studied by in vitro assay. In H patens, stigma leachate

selectively inhibits growth of incompatible pollen than cross-pollen. When pollen

germination was done in stigmatic leachate alone or in Brewbakers medium

supplemented with stigmatic leachate, pollen germination and tube growth of self­

pollen were inhibited when compared to the control. In the case of cross pollen,

germination and tube growth were not significantly inhibited. Germination of

incompatible pollen was inhibited by the stigma leachate whereas that of compatible

pollen was not affected by leachate. RNase isolated from self-incompatible apple was

also found to inhibit pollen tube growth in vitro (Katoh et aI., 2002). In Nicotiana

alata, the S-protein was also cloned and sequenced and was found to share homology

with fungal RNase. The isolated S-protein showed RNase activity and inhibitory

105

activity when added to the in vitro pollen germination medium (McClure et al., 1989;

Haring et aI., 1990; Gray et aI., 1991).

To clarify the relationship between RNase activity and pollen growth

inhibition, Rnase A from Bovine serum was added instead of stigma leachate. When

Bovine serum was added alone or in combination with stigma leachate to the culture

medium, significant reduction in the pollen germination and tube growth was

observed in the case of self-pollen. But presence of RNase A did not affect the

germination and growth of cross-pollen. From the present study it may also be

concluded that the stigma leachate has inhibitory activity similar to that of RNase A.

According to McCubbin and Kao (2000), if the S-RNase arrests the pollen

tube growth of self-pollen in the pistil as well as in the in vitro assay, the S-RNase

should be modified or controlled by any molecules in the cross-pollen tube to

inactivate the inhibitory activity. One hypothesis is that the S-RNase is taken up by

the incompatible pollen tube as it grows through the extracellular matrix. Once taken

into the pollen tube, rRNA genes are not transcribed into the pollen, resulting in

pollen tube death (Mascarenhas, 1990, 1993; McClure et aI., 1990). In this case, the

specificity of the interaction would be controlled either by the uptake of the S-RNase

into the pollen tube or by the presence of an inhibitor that would act in an allele­

specific manner to prevent RNA degradation in compatible pollen tube.

To examine the role of RNase activity in pollen tube inhibition, chemicals

such as zinc sulfate and cupric sulfate, having the property to inactivate stigmatic

RNase, were incorporated in the in vitro pollen germination medium. When these

RNase inhibitors were added to the medium, the rate of pollen germination and tube

length of self pollen significantly increases. In H patens, ZnS04 dramatically reduced

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the inhibitory activity of RNase on pollen tube growth whereas CUS04 cause further

disturbed the pollen tube gennination. The inhibitory activity of ZnS04 is depended

on its concentration. One mM ZnS04 showed maximum inhibition of RNase activity

and high rate of self pollen gennination. Above this concentration, ZnS04 disturbed

the germination and growth of pollen. According to Huang et al. (1994) RNase

activity of stigmatic protein is essential for their function in rejecting self-pollen. The

result may suggest that stigma leachate of H patens is having RNase activity and it is

essential for inhibition of self-pollen gennination and tube growth. In cross-pollen the

RNase may modify or less activity of RNase may leads to compatibility. In the

present study, the inhibition of S-RNases by ZnS04 affect the ability to reject self­

pollen in H patens.

RNase activity III the stigma of H patens is developmentally regulated.

During anthesis, the stigma actively secretes RNase, which inhibits pollen

germination and pollen tube growth in vitro and in vivo. The ability of RNA, as a

competitive inhibitor, to reduce the effect of stigmatic diffusate on pollen tube growth

further indicate that the inhibition is derived directly from the stigmatic RNase.

Analyses concluded that strong RNase based gametophytic incompatibility system is

operating in H patens. Due to strong incompatibility sexual reproduction is beset and

seed-set is absent in H patens.

107