INTRODUCTION

Amazingly diverse reproductive systems of flowering plants have been

fascinating biologists since the time of Charles Darwin (Barrett, 2002). Reproductive

biology of flowering plants is significant in determining the barriers of fruit and seed­

set, effecting plant conservation and understanding pollination as well as breeding

systems. Biological processes such as flowering, functioning and behaviour of

gametes, embryogenesis and seed developments have been the focus of scientific

pursuit in the field of plant reproduction.

Sexual reproduction in angiosperms is highly selective. It is the only natural

process that incorporates variability and ensures survival of species under adverse

conditions. One of the essential features of plant sexual reproduction is the ability of

the organism to recognize and select suitable gametes for fertilization. This ability of

recognition and rejection enables the plant to fulfill two primary functions:

maintaining stability of the species and permitting genetic variation within the species

(Stebbins, 1950; Grant, 1981). This unique feature has made angiosperms the most

efficient outbreeding systems (Grant, 1981; Rieseberg, 1997; Arnold, 2006).

Reproductive characteristics such as seed dispersal, germination capacity,

survival rate of seedlings and adults, age at flowering, reproductive lifespan and

number of flowers and seeds refer to a set of responses that allow a species to adapt to

a particular environment. Besides these, gametogenesis, pollination, endosperm and

embryo development and other reproductive features can provide important clues

regarding the reproductive constraints ofplants.

Botanists around the world have been concentrating on comparative and

descriptive embryology and have generated a pool of information regarding various

reproductive features and anomalies in a large number of plants. Sexual reproduction

is based on the phenomenon of syngamy and double-fertilization. Successful

fertilization is dependent on effective pollination. Pollination studies alone can

provide a gamut of information about the loss of many species because pollination is

the fundamental step in plant reproduction. Successful pollination is an essential pre­

requisite for survival of plants in natural communities and is dependent on many

biotic and abiotic factors.

Understanding of reproductive biology is essential for mass propagation,

multiplication, hybridization and conservation of endangered and threatened plants.

Most of the researchers are now revolving around the functional aspects of

reproductive processes. Starting from morphological studies of reproductive organs,

the field of plant reproductive biology is advancing rapidly and has entered into an

area of experimental, molecular and genetic approaches.

1.1 Structure and role of reproductive parts

Reproductive structures of flowering plants are some of the most

evolutionarily diverse structures varying widely across plant families. Among all

living organisms, flowers, the reproductive structures of angiosperms, show the

greatest diversity (Barrett, 2002). A flower is an extremely efficient reproductive unit

because it can promote male and female functions at the same time.

1.1.1 Pollen

Pollen, the male gametophyte of higher plants, is a biological system playing a

central role in plant sexual reproduction (Cresti et al., 1992; Moscatelli and Cresti,

2001). They are multicellular in structure composed of two or three cells at maturity:

in certain cases, a large vegetative cell that contains two smaller sperm cells, while in

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some other cases a single generative cell within the vegetative cell which divides to

two sperm cells within the pollen cytoplasm that constitutes the mature male

gametophyte (McCormick, 2004; Borg et aI., 2009). Pollen walls are multilayered and

include the intine, constructed primarily of cellulose, and the exine, an intricately

ornamented structure composed of the resilient biopolymer sporopollenin (Dobson,

1989; Bedinger et aI., 1994; Thorn et aI., 1998). The sculptured exine wall anchors the

protein and lipid-rich pollen, which is vital for interactions with dry stigmas (Luu et

aI., 1999; Doughty et aI., 2000; Takayama et aI., 2000a). Pollen is dispersed biotically

or abiotically and comes in contact with the stigma.

1.1.2 Pistil

The pistil, the plant female reproductive organ, has a dual function: the

production of the female gametophytes in the ovary and the discrimination of the

pollen grains that land on the stigma surface. Different pollen grains are carried to the

stigma by insects, wind, water, or by direct contact between the open anther and the

stigma, initiating the programmic phase. During this phase, intensive pollen-pistil

interactions occur, including important recognition events that determine the fate of

the pollen. Ultimately, the success of the pollination depends on a congruous and

compatible recognition of the pollen and the appropriate conditions for pollen

hydration, pollen tube germination, and directional growth towards the ovules, all

taking place at the specialized tissues of the stigma/style. When landing on the stigma,

the pollen grain hydrates and germinates. The pollen tube then penetrates the

specialized tissues of the pistil, growing into the stigma and the style to reach the

ovules in the ovary. Pistil, the pollen accepting organ that occupies the central

position in a flower, is composed of one or more free or fused carpels that bear the

ovules. Pistil is essential for flowering plant reproduction, even when asexual

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mechanisms of reproduction are available in some cases (Bicknell and Koltunow,

2004). The pistil must maintain the right balance between two important functions;

pollen carries the sperm cells to the embryo sac and selects the fittest pollen tube to

ensure the production of vigorous progeny. Therefore, it should not be surprising that

many checkpoints exist in pollen tube growth that is regulated by finely tuned

mechanisms.

1.1.3 Stigma

Stigma is the first site where the recognition events lead to the acceptance of

compatible pollen or the rejection of incompatible pollen. The position of stigma

within the flower is a key aspect of floral morphology, which influences the efficiency

ofpollen transfer (Campbell et aI., 1996; Karron et aI., 1997; Cresswell, 2000; Motten

and Stone, 2000; Nishihiro et aI., 2000; Elle and Hare, 2002; Medrano et aI., 2005).

The recognition events are extremely complex and are not yet fully understood

(Holdaway-Clarke and Hepler, 2003; Edlund et aI., 2004; Sanchez et aI., 2004;

Swanson et aI., 2004; Takayama and Isogai, 2005; Malho et aI., 2006; McClure and

Franklin-Tong, 2006; Wilsen and Hepler, 2007; Sandaklie-Nikolova et aI., 2007;

Sauter, 2009).

The developmental anatomy of stigma (Konar and Linskens, 1966a) showed

that it could be separated into two zones; an upper zone consisting of the epidermis

and 1-3 layers of secretory zone, and the lower zone of parenchymatous ground

tissue. In continuum with the sub-epidermal region ofthe upper zone, a central core of

transmitting tissue usually occurs. Two provascular tissues transverse through the

ground tissue. Important correlation between stigma type and pollen morphology also

exist, most notably the general occurrence of trinucleate pollen in species with dry

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stigma and binucleate pollen in species with wet stigma (Heslop-Harrison and

Shivanna, 1977; Hiscock and Allen., 2008) implying a strict co-evolution of pollen

and stigma structure (Edlund et aI., 2004).

Angiosperm stigma can be classified into two categories, wet and dry,

depending on whether or not they posses a surface secretion (Heslop-Harrison and

Shivanna, 1977; Hiscock et aI., 2002a; Edlund et aI., 2004; Hiscock, 2004). Wet

stigma produces a copious surface secretion that may be either predominantly

aqueous or lipidic. In contrast, dry stigmas have no surface secretion; instead the

cuticle is overlaid by a condensed surface layer of protein, the proteinaceous pellicle

(Heslop-Harrison et aI., 1975a; Hiscock, 2004). In species with wet stigma, pollen

capture by the stigmatic secretion is non-specific, and hydration of pollen within the

secretion appears to be passive and largely unregulated (Swanson et aI., 2004),

whereas in species with dry stigma, pollen capture and adhesion to the stigma exhibit

a degree of species specificity (Luu et aI., 1997a; Zinkl et aI., 1999; Heizmann et aI.,

2000; Zinkl and Preuss, 2000) and hydration of pollen on the stigma is a highly

regulated process (Heslop-Harrison, 1979; Dumas et aI., 1984; Dickinson, 1995;

Dickinson and Elleman, 1995). Epidermal cells of wet stigma often lack a continuous

cuticle, so penetration of pollen tubes into the stigma is relatively unimpeded

compared with the same event in species with dry stigma where pollen tube secretes

hydrolytic enzymes such as cutinase to break a continuous cuticle (Hiscock et aI.,

1994, 2002b).

Recent investigations have revealed the presence of extracellular protein on

the surface of stigma, irrespective of the morphological variation of the stigma and

style (Tang et aI., 2002; Hiscock, 2004; Quiapim et aI., 2009). The receptive surface

contains extracellular protein either in the form of a pellicle or as a component of

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exudates (Heslop-Harrison and Shivanna, 1977; Heslop-Harrison, 1981; Shivanna and

Johri, 1985). Esterases are important components of the stigma surface protein and its

presence is related to stigma receptivity (Bhattacharya and Mandal, 2003). However,

the entire exudates are not proteinaceous; it may contain carbohydrates and lipoidal

components as well. The wet type stigma secretes exdudates containing lipids,

phenolic compounds, carbohydrates, proteins, phosphatases, lectins and aminoacids as

well as esterases (Konar and Linskens, 1966b; Baker et aI., 1974; Vasil, 1974; Cresti

et aI., 1986). According to Heslop-Harrison et ai. (l975a), wet stigma is correlated

with gametophytic incompatibility while the dry stigma is with sporophytic

incompatibility.

1.1.4 Style

During ovary closure, the carpel walls extend vertically to form one or more

solidlhollow cylinders, the styles. The length, number and structure of the styles vary

within and among the species. Stylar extension facilitates pollen capture, and the wide

variety of pistil morphologies reflects the different pollination mechanisms among the

angiosperms (Barrett et aI., 2000). The style is differentiated into a specialized

secretory zone on top and transmitting tissue within.

Angiosperm styles are of two types - hollow and solid (Shivanna, 1982). In

hollow-styled species, stigmatic secretions are continuous with secretions lining the

style, and pollen tubes grow within these secretions in close contact with stylar

epidermal cells, following what has been described as a 'facilitating mechanical

pathway' (Dumas et aI., 1984; Gaude and Dumas, 1987). In species with solid stigmas

and styles, pollen tubes grow into the stigma through the intercellular spaces

(extracellular matrix, ECM) between the cells of the secretory zone (de Nettancourt

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et aI., 1974; Heslop-Harrison, 1976; Herrero and Dickinson, 1979, 1981). Once pollen

tubes enter the stigma, they continue to grow within the ECM of the transmitting

tissue, which is continuous with the style and enter into the ovary.

1.1.5 Ovary

Ovary, part of the carpel which holds the ovule(s), is located above or below

or at the point of connection with the base of the petals and sepals. After pollen tubes

emerge from the transmitting tissue and enter the ovary, they elongate into the

placental surface within the ovarian locule. As pollen tubes travel along the placental

surface towards the proximal end of the ovary, individual pollen tubes diverge from

the masses by 90° before elongating through the last several micrometers to enter the

ovular micropyle. In ovaries with a single ovule, subsequent pollen tubes stop

growing when one pollen tube grows into the ovule. However, when multiovulated

ovaries are penetrated by pollen tubes, arriving tubes simply bypass the penetrated

ovules to grow towards the receptive ovules.

Carpel provides a location for selective mechanisms that operate on pollen,

such as self-incompatibility, which promotes out-breeding. Following pollination,

compatible pollen tubes are guided with meticulous accuracy through the tissues of

the carpel, specifically towards unfertilized ovules. After fertilization, the carpel

tissues undergo further developmental changes to become the fruit, which in turn

protects the developing seeds and later contributes to the dissemination of these by a

wide variety ofmechanisms in different species.

The proportion of ovules that develop into a mature seed is an important factor

in determining the reproductive potential of most plant species. Surveys indicated that

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in outcrossing perennial plant species, approximately 50% of ovules do not form seed

(Wiens, 1984).

1.2 Self-incompatibility systems in plants

Self-incompatibility (SI) is an intraspecific reproductive barrier inherent in

many flowering plants to prevent self-fertilization and thereby generate and maintain

genetic diversity within a species (de Nettancourt, 2001). A generalized definition of

self-incompatibility is "the inability of a fertile hermaphrodite seed plant to produce

zygotes after self-pollination" (de Nettancourt, 1977). A cell-cell recognition system

allows the pistil to recognize and to reject genetically identical pollen and to allow

"non-self' (compatible) pollen to grow. This recognition inhibits the germination of

self-pollen on the stigmatic surface or the growth of self- pollen tube in the style.

Thus SI is a prezygotic reproductive barrier by which incompatible pollen/pollen

tubes are prevented from delivering the sperm cells to the ovary.

As a prelude to fertilization, pollen must first establish molecular

congruity/compatibility with the stigma and then germinate to produce a pollen tube

that penetrates the stigma and grows through the transmitting tissue of the style to

locate an ovule within the ovary. Initiation and successful completion of this sequence

of events depends upon the stigma and style providing the exact requirements for

pollen germination and sustained growth and guidance of the pollen tube through the

pistil and ovary (Heslop-Harrison, 2000; Herrero, 2003; Swanson et aI., 2004). The

mechanism of self-incompatibility involves a number of stages which begin with the

production of the pollen and pistil component(s) of self-incompatibility (Takayama

and Isogai, 2005; McClure and Franklin-Tong, 2006). These components are involved

in the recognition process leading to the inhibition of self-incompatible pollen (Luu et

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aI., 1999; Schopfer et aI., 1999; Dickinson et al., 2000; Kachroo et aI., 2001; Shiba et

aI., 2001; Johnson and Preuss, 2002; Hiscock and McInnis, 2003; Swanson et al.,

2004). The site of inhibition differs depending on the self-incompatibility system and

can lead to the failure of pollen grain to adhere, hydrate, or germinate on the stigma

surface, or if pollen germination occurs, pollen tube growth can be inhibited by either

the stigma or the style (Heslop-Harrison and Shivanna, 1977).

A variety of self-incompatibility systems have evolved in flowering plants.

Based on whether it is associated with floral polymorphism, these systems have been

classically divided into two - homomorphic and heteromorphic (Trick and Heizmann,

1992; Hinata et aI., 1993; Nasrallah and Nasrallah, 1993). The flowers of

homomorphic species have the reproductive organs in close proximity to each other.

In contrast, heteromorphic species have morphologically distinct flowers with the

reproductive organs in different positions. Self-incompatibility systems have also

been classified as being either gametophytic or sporophytic, based on the genetic

control of pollen behaviour. In gametophytic self-incompatibility (GSI), the

phenotype of the pollen is determined by its own haploid genotype while in

sporophytic self-incompatibility (SSI), the pollen phenotype is determined by the

genotype of the pollen parent.

1.3 Genetics of incompatibility

In homomorphic incompatibility, the flowers produced by different plants do

not show morphological variation. Incompatibility is governed by multiple alleles,

termed S-alleles at a single locus. Pollen grains carrying an S-allele identical with one

or both the S-alleles present in the pistil are incompatible. Lewis (1956) and Pandey

(1958, 1970a) explained two types of incompatibility on the basis of the difference in

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time of S-gene action. In sporophytic incompatibility systems, the S-genes are

activated in meiocytes before the completion of meiosis, is the result that the product

of both the genes are under sporophytic control. In this case also most of the species

show one locus with multiple alleles (Hughes and Babcock, 1950; Crowe, 1954a;

Bateman, 1955). Control of incompatibility by one locus multi allelic sporophytic

system has been clearly demonstrated in Crucifers (Bateman, 1954; Thompson, 1957;

Sampson, 1957). In Brassica, at least 50 different alleles at the S-locus control self­

incompatibility (Ockendon, 1975; Cui et aI., 1999; Suzuki et aI., 1999; Casselman et

aI., 2000). The two alleles of a heterozygous plant interact with expression of

dominance, partial dominance or mutual weakening (Thompson and Taylor, 1966;

Nasrallah, 1974; Ockendon, 1975). The sporophytic nature of this self-incompatibility

system has been hypothesized to occur by the deposition of the pollen S protein in the

outer coat of the pollen grain during pollen development by the diploid anther

tapetum (de Nettancourt, 1977).

GSI is widespread and is estimated to occur in around half of the flowering

plant species (Brewbaker, 1959; Richards, 1986; Weller et aI., 1995). In GSI, the SI

phenotype of the pollen is determined by its own genotype (Wheeler et aI., 2001). In

most GSI systems, incompatible pollen germinates successfully on the stigma surface,

penetrates the stigma, and grows into the style, where, the tube grows between the

longitudinal files of cells of the central transmitting tract and at some point, pollen

tube growth through the transmitting tract towards the ovary is arrested.

Two different single S-locus GSI systems have been investigated at molecular

level. The first of these is the S-RNase system originally identified and extensively

characterized in members of the Solanaceae, Rosaceae, Scrophulariaceae and

Campanulaceae (de Nettancourt 2001; Franklin-Tong and Franklin, 2003; Hiscock,

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and McInnis, 2003; Kao and Tsukamoto, 2004). The second is found in Papaveraceae

(Hiscock and Mcinnis, 2003; Franklin-Tong and Franklin, 2003). SI in these families

is controlled by a single locus, but there are more complex systems, those in some

grasses, in which SI is controlled by two loci, S and Z (Lundqvist, 1964; Hayman and

Richter, 1992). Four loci controlled SI was also reported in Beta vulgaris (Lundqvist

et aI., 1973; Larsen, 1977) and Ranunculus acris (Osterbye, 1975).

Pandey (1962) proposed that the physiology of S gene is controlled by four

components, viz., growth substance, protective substance, primary specificity and

secondary specificity, each with corresponding pollen and stylar units attached in that

order, the components of secondary specificity being added last. Lewis (1965) and

Ascher (1966) have proposed gene action models to explain gametophytic

incompatibility. The dimer hypothesis of Lewis suggests that an identical polypeptide

molecule is produced in the pollen and style by the S-gene complex and that each

allele has its own characteristic monomers. These two monomers combined to form a

dimer protein in both pollen and style. Following an incompatible mating a tetramer is

formed by the polymerization of identical dimers. Pollen and style with the aid of an

allosteric molecule, and the tetramer act as a genetic regulator either to induce the

synthesis of an inhibitor or to repress the synthesis of an auxin needed for pollen tube

growth. According to Ascher's model (1966), the S allele act as a regulator gene

governing two sets of operons. In the pollen tube, which control pollen metabolism,

one set controls the mechanism utilizing reserves available in the pollen itself. The

other controls a different metabolic pathway, which enables the pollen tube to utilize

stylar nutrients. Several hypotheses exist concerning the control of mechanisms

prevailing pollen tube growth after an incompatible pollination (Lewis, 1965;

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Nasrallah et al., 1970; Pandey, 1970a; Kroes, 1973; Heslop-Harrison et aI., 1975b;

Vander-Donk, 1975). However no hypothesis has gained universal acceptance.

1.4 Molecular basis of incompatibility

During the past two decades, much progress has been made in identifying and

characterizing the S-locus genes that control the specificity of the SI interaction in the

five families- Solanaceae, Rosaceae, Scrophulariaceae, Papaveraceae and

Brassicaceae (Broothaerts et aI., 1995; Xue et aI., 1996), Comparisons of the S-locus

genes expressed in the pistil among the different families have revealed three

biochemically distinct mechanisms. Solanaceae, Rosaceae and Scrophulariaceae have

the same mechanism (Sassa et aI., 1996; Xue et aI., 1996; Ishimizu et aI., 1998),

Papaveraceae has another (Franklin-Tong et aI., 1988; Thomas et aI., 2003), and

Brassicaceae (Casselman et aI., 2000; Kachroo et aI., 2002) exhibited the third. For

Solanaceae and Papaveraceae mechanisms, the gene that controls female specificity

has been identified (Bredemeijer and Blass, 1981; Ai et aI., 1990; Clark et aI., 1990).

These genes were named the S-RNase gene and the S-gene, respectively. SI has been

studied at the molecular level in four genera of Solanaceae (Lycopersicon, Nicotiana,

Petunia and Solanum), three genera of Rosaceae (Malus, Prunus and Pyrus) and one

genus of the Scophulariaceae (Antirrhinum). The Papaveraceae mechanism is

mediated by a signal transduction cascade in pollen that involves a number of known

components of signal transduction. For the SSI mechanism found in Brassicaceae,

both the genes that control male specificity, S-locus cysteine-rich protein (SCR)/S­

locus protein-II (SPll), and the gene that controls female specificity, S-locus

receptor kinase (SRK), have been identified (Bower et aI., 1996; Gu et aI., 1998). The

SI response is mediated via a signal transduction cascade in the stigmatic papilla,

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which is elicited by the interaction of a pollen-borne ligand, SCR/SPll, and SRK, a

receptor kinase in the stigmatic papilla (Cabrillac et aI., 2001).

1.4.1 Female specificity determinant: the S-RNase gene

The biochemical nature of S-proteins was revealed when the sequence of

RNase T2 of Aspergillus oryzae was determined (Kawata et aI., 1988) and found to

share sequence similarity with S-proteins (McClure et aI., 1989). This finding led to

the subsequent confirmation that S-proteins have RNase activity in vitro (McClure et

aI., 1989; Singh et aI., 1991). RNase T2 and S-RNases have been placed in a large

family of RNases, named the T2/S-RNase family, which also includes S-like RNases

and relic S-RNases (Green, 1994; Golz et aI., 1998). S-like RNases do not exhibit

allelic sequence diversity, and they have been identified from both self-incompatible

and self-compatible species of Solanaceae, Rosaceae and Scrophulariaceae as well as

from self-compatible species of several other families (McCubbin and Kao, 2000).

Relic S-RNases have been identified from both self-incompatible and self-compatible

species of Solanaceae, Rosaceae, and Scrophulariaceae, and they are more similar to

S-RNases than S-like RNases (Kuroda et aI., 1994; Golz et aI., 1998; Katoh et aI.,

2002; Kondo et ai. 2002a; Liang et aI., 2003). S-like RNase belong to the S-RNase

lineage but are no longer involved in SI and have been referred to as relic S-RNases

(Golz et aI., 1998). Golz et ai. (1998) have also proposed that relic S-RNases may be

associated with the partial duplication of an S allele in a self-incompatible species or

may arise during the transition from SI to self-compatibility in a self-incompatible

speCIes.

The S-RNases contain five highly conserved regions designated as CI-C5

(Joerger et aI., 1991). C1, C4 and C5 contain mostly hydrophobic amino acids and

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may be involved in forming the core structure of the S protein (Kao and McCubbin,

1996). Analysis of S allele sequences from Rosaceae revealed that all the features and

characteristics of the predicted protein are similar to Solanaceae S-RNase except that

the C4 region was lacking (Sassa et aI., 1993, 1994; Broothaerts et al., 1995). The C4

region was also missing in the Antirrhinum (Scrophulariaceae) S-RNase sequences

(Xue et aI., 1996). The C2 and C3 regions show sequence similarity to the fungal T2

RNase (Kawata et aI., 1988). There are also two histidine residues in the active site of

the T2 RNase which are required for the RNase activity. These histidines are

conserved in the S-RNases, one in the C2 region and the other in the C3 region

(Kawata et aI., 1988; McClure et aI., 1989; Royo et aI., 1994; Ishimizu et aI., 1995;

Parry et aI., 1997). All S-RNases are glycoproteins with up to five potential N­

glycosylation sites. The N-glycosylation site closest to the C2 region is conserved in

all S-RNases identified to-date (Oxley et aI., 1996; Ford et aI., 2000; Carrera et al.,

2009).

Outside the conserved regions, the S alleles show a high level of variability,

and potentially important variable domains which may play a role in S allele

specificity have been identified by different authors (Kao and McCubbin, 1996;

Matton et aI., 1997; Zurek et aI., 1997). Ioerger et ai. (1991) identified two areas

exhibiting the highest levels of variability and were referred to as "hypervariable

domains" - HVa and HVb. Due to the high sequence diversity and the hydrophilic

nature of these regions; they are thought to be involved in determining S allele

specificity (Ioerger et aI., 1991; Sims, 1993). They may possibly constitute the

recognition regions of the S-gene in GSI systems.

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1.5 Methods to overcome incompatibility

Though the incompatibility reaction occurs as a natural phenomenon in

angiosperms, many researchers have adopted a range of techniques to overcome self­

incompatibility in different taxa (de Nettancourt, 1977; Shivanna and JoOO, 1985) and

to get seeds from self- and cross-incompatible combinations. Treatment of stigma of

Brassica with cyclohexamide, sugars and cytokinin (Ferrari and Wallace, 1977a;

Sharma and Shivanna, 1983; Matsubara, 1973), mentor pollen technique (Dayton,

1974; Williams and Church, 1975; Den Nijs and Oost, 1980; Visser, 1983) and high

temperature treatment (Hecht, 1964; Hopper et aI., 1967; Ronald and Ascher, 1975;

Matsubara, 1980; Campbell and Linskens, 1984; Okazaki and Hinata, 1987) have

been found to overcome self- incompatibility. Intraovarian pollination enables one to

eliminate stigma/style totally and success has been obtained by this technique in the

cross between Argemone mexicana and A. oehrdenes (Kanta and Maheswari, 1963a;

Maheswari and Kanta, 1964). Other techniques used in overcoming incompatibility

include stump pollination (Swaminathan, 1955), bud pollination, delayed pollination

(Bredmeijer, 1976) and treatment of incompatible stigma with the extract obtained

from the compatible pistil (Pandey, 1963; Shivanna and Rangaswamy, 1969).

Regarding the incompatibility reaction, Linskens (1975) and Van der Donk

(1975) have argued that the rejection of pollen tubes is the reaction for which the style

is prepared, whereas unaffected pollen tube growth is related to the synthesis of

special enzymes that break down the incompatibility barrier.

Self-incompatibility (SI) in Brassica has been extensively studied, but

information on SI in Eruca sativa is limited. Of the six chemicals used to treat the

stigmas to overcome SI in five lines of E. sativa, gibberellin was the most effective.

15

As gibberellin is well known for its ability to break donnancy and to promote cell

elongation, its effectiveness may help to understand the mechanisms of SI. Urea and

ammonium sulphate were also effective. These two chemicals are known to affect

protein stability, which may help explain their effects on SI. Although table salt has

been reported as being effective in overcoming SI in B. rapa, B. oleracea and B.

napu, it was not effective in E. sativa. Sucrose and alcohol also had negligible effect

(Sun et aI., 2005).

1.6 Future Perspectives

Since the discovery of the S-RNase gene almost two decades ago, much of

what we have learned about the Solanaceae type of SI is limited to this female

detenninant of the SI interaction (de Nettancourt, 2001). Recent identification of the

SLF/SFB gene very likely will change the landscape of research in this type of SI

(Lai et aI., 2002; McClure, 2004; Qiao et aI., 2004a; Sijacic et aI., 2004; Ushijima et

al., 2004; Ikeda et aI., 2004; Sonneveld et aI., 2005a; Tsukamoto et al., 2006; Hua et

aI., 2007; Sassa et aI., 2007). The most urgent task, in the short run, is to detennine,

by in vivo approaches, whether SLF/SFB encodes the male-specificity detenninant of

SI. If SLFI SFB are confinned to be the pollen S-gene, this will open new avenues of

research and bring us closer to an understanding of the mechanism of S-haplotype­

specific inhibition of pollen tube growth. Questions can be asked regarding whether

SLF/SFB functions as a conventional F-box protein in mediating the specific

degradation of all non-self S-RNases or whether it functions in some unexpected

manner. With the genes that encode both the male and female detenninants in hand,

we could also address one of the most perplexing questions about any type of SI

systems: how did the male and female specificity genes coevolved to maintain SI?

The fact that multiple F-box genes are linked to the S-RNase gene in all the three

16

families (Solanaceae, Rosaceae and Scrophulariaceae) raises questions about the

physiological functions of the non-SI-related F-box genes that are linked to the S­

locus and about the evolutionary relationships among the various S-linked F-box

genes.

Although it is important to focus on how S-haplotype specificity is

determined. We also should keep in mind that additional proteins are required for the

full manifestation of the SI response. Because most of the candidate proteins

identified to date do not appear to be specific to the SI system, understanding how

they function in SI will likely have implications for other developmental processes.

1.7 Reproductive biology of Rubiaceae

Rubiaceae is the fourth largest family of flowering plants, shows a wide

spectrum of floral mechanisms characterized by different types of gynoecium and

androecium organization. Despite this diversity, Robbrecht (1988) pointed out the

presence of three reproductive strategies common in Rubiaceae, which are related to

certain groups within the family. Distyly, morphologically characterized by the

presence of two inter-compatible floral morphs, is generally observed in species of

Rubioideae (Barrett, 1992). Stylar pollen presentation, which includes protandry and

pollen presentation in the style, is generally recorded in Ixoroideae (Nilsson et aI.,

1990; De Block and Igersheim, 2001). In Cinchonoideae, both distyly and stylar

pollen presentation are seen (Robbrecht, 1988; Castro and Oliveira, 2001).

Both distyly and stylar pollen presentation include herkogamy, or the spatial

separation between anthers and stigma of hermaphrodite flowers are also observed in

this family. In distyly, this separation is observed early in the bud stage (Faivre,

2000), whereas in stylar pollen presentation it is the result of style elongation after

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anther dehiscence (lmbert and Richards, 1993; Ladd, 1994). Herkogamous species

may be homomorphic or heteromorphic, depending on the number of floral types.

Moreover, homomorphic herkogamy may be ordered or unordered. In general,

herkogamy is interpreted as a mechanism that avoids self-pollination and the mutual

interference between sexual functions (Webb and Lloyd, 1986).

Apart from being commonly observed in Ixoroideae, stylar pollen

presentation is also thought to be the main reproductive strategy (Robbrecht, 1988) of

the monophyletic tribe Chiococceae of the Cinchonoidae (Bremer, 1996).

Self-compatibility seems to be widespread in Rubiaceae. Documentation of

homomorphic self-incompatibility is very little in Rubiaceae family. Fagerlind (1937)

reported self-incompatibility in Coffea spp. and Galieae. East (1940) reported self­

incompatibility in one species of Crusea. The presence of a gametophytic SI is

reported in all the species of Coffea except the unique tetraploid species C. arabica

(Bridson and Verdcourt, 1988).The presence of SI system was reported in the diploid

species of C. arabica and C. canephora (Devreux et aI., 1959; Conagin and Mendes,

1961). Recently, the genes belonging to the S-RNase lineage were reported in

Rubiaceae (Vieira et aI., 2008a).

1.8 Objectives of the study

Hamelia patens Jacq. commonly called fire bush, is a fast growing, semI

woody and evergreen perennial shrub of the family Rubiaceae, is a native of central

and southern Florida. It is chiefly grown for the showy bunch of beautiful flowers. In

tropical America, local people make use of the extract of its leaves and stem, for

curing diseases such as skin rashes, sores, insect sting and various fungal diseases of

the skin because of its antibacterial and antifungal properties (Grijalva, 1992; Gupta,

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1995; Martinez et aI., 2001; Segleau, 2001). In India this plant IS particularly

cultivated on a wider scale in home gardens as an ornamental plant.

In H patens, sexual reproduction is beset with many problems which restrict

its propagation to vegetative means. To comprehend thoroughly the reasons for the

reproductive barrier, it is essential to have a deep understanding of the morphology

and developmental anatomy of the plant's reproductive parts. In order to study the

failure of seed-set, a detailed analysis of the reproductive biology of H patens is

proposed which necessitated the following studies:

1. Analyze whether any developmental and structural abnormalities

influence in the reproductive characteristics and seed set in H patens.

2. Unravel development and viability of male gametophyte in H patens.

3. Explore the mechanisms to overcome the self-incompatibility in H patens.

4. Identify the genetic variability in terms of s-alleles, infraspecific pollination

and compatibility within the 14 accessions ofH patens

5. Analyze the histochemical and cytochemical variations after self and cross

polllinations with special emphasis on primary metabolites and activity of

enzymes viz, ATP-ase APase, SDH, peroxidase and esterase.

6. Molecular characterization.· of incompatibility factors in the stigmatic

tissue.

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