Discipline: Botany Paper: Reproductive biology of angiosperms Lesson: Self-incompatibility Lesson Developer: Yash Mangla1 and Charu Khosla Gupta2 Department/College: Plant Reproductive Biology Laboratory, Department of Botany, University of Delhi; 2

Department of Botany, Acharya Narendra Dev College, University of Delhi

Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL

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Introduction All flowering plants are sessile and unable to reach their partners for reproduction. Nature has devised diverse means of pollination by which the fertile pollen grains are able to reach the receptive stigmas. Landing of pollen grains on the female stigma initiates the process of fertilization. But the whole gamut of pollen landing on the stigma is not able to effect fertilization. Pistil is equipped with devices to allow only the right type of pollen grains to effect fertilization and all others are rejected. This process of recognition and also rejection of pollen grains is called the pollen-pistil interaction, where the pistil decides whether the pollen will germinate or not. In nature there are many incidences where pistil carrying functional female gametes are unable to set fruits and seeds after being pollinated by viable and fertile pollen grains. This inability is called incompatibility. If the pistil is pollinated by viable and fertile pollen grains of different species but no seed set occurs, the incompatibility is termed as inter-specific incompatibility. The second possibility arises when viable and fertile pollen grains of the same species are lodged on the stigma (by autogamy or geitonogamy) but are unable to effect fertilization. This phenomenon of rejection of self pollen grains is termed as intra-specific incompatibility or more elaborately, self-incompatibility (SI). Self-incompatibility (SI) is a general term used for several genetic mechanisms in flowering plants, which prevent selffertilization and promote out-crossing and allogamy. SI is considered as one of the principal means to promote new genotypes in plants and is also one of the primary reasons for the spread and success of the angiosperms on the earth. In plants with SI, when a pollen grain reaches a stigma of the same plant or another plant with a same genotype, the process of pollen germination, pollen tube growth, syngamy or embryogenesis is terminated at one of these stages resulting in failure of seed-set. There are incidences where the pollen tubes are able to reach the ovules but fertilization fails or even if it occurs, the embryo gets aborted after syngamy. The abortion of the embryo can be attributed to inadequate development of the endosperm or because of incompatibility between embryo and endosperm. Thus, self-incompatibility involves an exchange of information or biomolecules (especially proteins) between the haploid pollen and the diploid tissues of the female

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reproductive organs to distinguish „self‟ and „non-self” pollen grains. It has been variously investigated that the authority of stigma to accept or reject particular type of pollen grains is provided by the genetic make-up, physiology and the type of biomolecules present in the species. The Inter-specific incompatibility is controlled by several genes at different loci on the chromosomes, hence referred to as heterogenic incompatibility. This prevents inter-species hybridization and also provides a barrier towards formation of many new races and species. In this chapter, we will chiefly focus on various aspects of SI including classification and genetics. Subsequent sections will provide information of the physiology, biochemistry and genetics of recognition and rejection reactions of self pollen in/on pistil. A concise note on the methods to overcome the SI is also given at the end of the chapter as SI can prove a major hurdle in breeding and hybridization experiments.

Self-Incompatibility: Classification and Types Out-breeding promotes the variation in genetic make-up of progeny and it is learnt that about 30% of angiosperms are out-breeders. They employ a variety of devices and mechanisms to promote cross-pollination like dichogamy, herkogamy, dioecy etc. Self- incompatibility refers to inability of a fertile hermaphrodite plant to set seeds when pollinated by self pollen grains. Thus, self-incompatible flowers provide guarantee for crosspollination or out breeding even if the plant produces functional male and female gametes. Selfincompatibility has been reported in a total of 116 families. Most of the perennial grasses (Poaceae), legumes (Fabaceae), members of Brassicaceae, Asteraceae, and Solanaceae are known to have various types of SI. Based on morphology, SI has been categorized as Heteromorphic and Homomorphic. As the name suggests, in heteromorphic SI, there is occurrence of more than one mating types within a species (distylous or tristylous condition, Figure 1) and homomorphic SI refers to species where all individuals of a species produce only one type of flowers which are similar in morphology. In heteromorphic species, the morphological differences in the length of stigma, style and stamens are assessed for finding SI without undergoing any breeding experiments as the differences are quite pronounced e.g. Primula, Oxalis, Lythrum etc. In Primula, (a dimorphic

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species) the incompatibility is controlled by a single gene with two alleles; where as in Lythrum, (a trimorphic, tristylous species) incompatibility is controlled by two genes with two alleles. Figure 1: A: Primula sp. a dimorphic system. Left side: Thrum morph; Right side: Pin morph B. Oxalis sp. a trimorphic system. Levels of stigma in each morph are marked with arrow. „a‟ depicts the level of anthers.

Source: de vos et. al. 2014 (needs permission)

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Source:http://brilliantbotany.com/post/12810432544/oxalis-stricta-and-tristyly In homomorphic species, all mating types are morphologically indistinguishable and hence require proper breeding tests for recognition of different mating types. Petunia, Brassica and Nicotiana are the common examples of homomorphic incompatibility. Unlike heteromorphic incompatibility, this type of incompatibility is controlled by multiple alleles of the S gene. Pollen tube carrying a particular s allele is inhibited in the style if the style has a similar s allele. Homomorphic SI is classified into gametophytic and sporophytic types. In both systems, incompatible pollinations leads to a match between the S-alleles present on the pollen and in the style. Products of the S locus are known from several families with gametophytic SI, including the Solanaceae, Papaveraceae, Poaceae and Rosaceae. Sporophytic self-incompatibility: Incompatibility is controlled by diploid genotype of sporophytic tissue of plant from which pollen is derived. In other words, incompatibility is controlled by the anther. Example: Brassicaceae. Gametophytic self-incompatibility: Incompatibility process is determined by the genotype of the male gamete. In other words, incompatibility is controlled by the genotype of the pollen grains. GSI is determined by a single gene. Examples: Solanaceae, lilies, grasses Institute of Lifelong Learning, University of Delhi

Genetics of Self-incompatibility We have understood that the process of SI is under a complex machinery of genes. The first and most plausible hypothesis about the genetics of homomorphic incompatibility was postulated by East and Mangelsdorf (1925) for Nicotiana which is popularly known as the S-allele hypothesis. According to this hypothesis, the SI is regulated by a single gene „S’ with multiple alleles. The alleles of this gene (or locus) can be written as S1, S2, S3 …….Sn. Pollen grains with an S-allele identical to one or both the alleles present in the pistil are inhibited. Thus this hypothesis is also accepted as Opposition S-alleles hypothesis. The number of alleles could be 40 (S1 to S40) as reported in Oenothera organensis, Papaver rhoeas and Lolium perenne. Even in Brassicaceae e.g. in Raphanus sativus, Brassica campestris, SI determined by multiple alleles (~ 60) has been reported. It has been described that two types of SI can be distinguished on the basis of expression of S-gene. In sporophytic systems, the S-gene is activated before completion of meiosis and products of both the alleles are distributed in all the pollen grains (Figure 2 A). In gametophytic systems, there is delayed activation of S-gene until the completion of meiosis. Thus, in a tetrad the products of one of the alleles are present in only two microspores, while the products of another allele are present in the other two pollen grains of the tetrad (Figure 2 B). Figure 2: The S-gene specific protein accumulation in the pollen grains during microsporogenesis. A. In sporophytic system

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B. In gametophytic system

Source: Authors Homomorphic Self-incompatibility Gametophytic Self-incompatibility (GSI): Pollen grains that carry the alleles similar or common to any of the alleles present in pistil are rejected and are non functional on that particular pistil. Let‟s consider a plant carrying alleles S1S2. During meiosis in such plant, half the pollen grains will receive the S1 allele and half the pollen grains will receive the S2 allele. If the pistil of such plant gets self pollen grains (with alleles S1S2) pollen tube inhibition will occur in the style and such pollen would not be able to effect fertilization (Figure 3A). In next case, if plant carrying alleles S1S2, gets pollinated with the pollen carrying S1S3 genotype; only 50% of the pollen grains carrying S3 allele would be functional and could bring about the fertilization (Figure 3B) as S3 is not common between the pollen and the pistil. However, if this female plant (with S1S2 genotype) receives the pollen grains of S3S4 genotype, 100% pollen grains could lead to fertilization (Figure 3C). Here, it is important to notice that S-allele of the pollen grains or male gametophyte determines the GSI. Figure 3: Manifestation of gametophytic self-incompatibility (GSI) response in S1S2 female plant. Refer to the text for details.

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Source: Authors Sporophytic self-Incompatibility (SSI): In the SSI, there is a dominance interaction and all the pollen grains behave in the same manner irrespective of the S-allele they carry. For example, a plant of genotype S1S2 would produce pollen grains with S1 and S2 allele products. But, all the pollen would behave as either S1 or S2; depending on the dominance of S1 or S2 respectively. Consider a condition where S1 is dominant , then pollen grains with S1 or S2 would not be able to effect fertilization in pistil with genotype S1S2; S1S3; S1S4 and so on (Figure 4A and 4B). However, such pollen grains (with S1 dominant) will carry effective fertilization with pistils of genotype S2S3; S3S4 etc. In other words such pollen grains would be 100% compatible (Figure 4C).

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Figure 4: Manifestation of sporophytic self-incompatibility (SSI) response in S1S2 female plant. Refer to the text for details.

Source: Authors GSI in Grasses: Two-locus or bi-factorial System The two locus, or bifactorial GSI system was first explained in grasses. Most grasses exhibit SI of this type except rye which is self compatible. The two loci involved in the SI are named as S and Z. Both the loci are polyallelic. It has been suggested by McCubbin and Dickinson (1997) that there is cooperation between S and Z in the pollen but they perform their actions

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independently in the pistil. Each combination gives rise to a distinct specificity in the pollen and rejection occurs when this specificity is matched by one of the four possible combinations of S and Z alleles in the diploid stigma suggesting that this two locus or the bi-factorial GSI system is more stringent. Heteromorphic Self-incompatibility It is quite evident that heteromorphic SI manifests mostly in two types of mating classes popularly known as dimorphic systems and trimorphic systems.

According to S-allele

hypothesis, in dimorphic systems, distyly is controlled by a single gene complex, with two alleles S and s (diallelic SI). The allele for short style; S (Thrum morph) is dominant over the allele for long style; s (Pin morph). Long style morphs/plants are homozygous i.e. ss while the short style morphs/plants are heterozygous i.e. Ss. A cross between short style morph and long style morph (Ss X ss), produces equal number of progeny of short style morph and long style morphs (Figure 5). Figure 5: Red solid lines indicate the compatible crosses while black dotted line shows the incompatible cross.

Source: Authors It is important to understand here that in heteromorphic systems incompatibility manifested in the pollen is sporophytically determined. The reason being that sporophytic pollen

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with recessive s allele from a short-styled morph (ss) is compatible on a long-styled morph (ss) but incompatible on a short-styled morph (Ss). The S-gene in such systems has been considered a supergene comprising of several closely linked genes. These genes correspond for stigma surface, pollen sculpturing, pollen size etc. Thus, these characters often vary in the two morphs. In trimoprhic systems, tristyly is controlled by two genes namely: S and M, with two alleles each. In this case S is epistatic over M. The long style morphs are homozygous recessive for both the genes and four alleles namel. mmss, while mid style morphs are homozygous recessive for S gene and heterozygous or homozygous for the alleles of M gene (MMss/Mmss). Short style morphs are heterozygous for alleles of gene S and homozygous or heterozygous for alleles of gene M i.e. MMSs/MmSs/mmSs (Figure 6). Figure 6: Red solid lines indicate the compatible crosses while black dotted lines show the incompatible cross among morphs.

Source: Authors

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Pollen and Pollen tube rejection in Homomorphic systems: Evidences from Molecular Biology, Biochemistry, Physiology and Cytology Earlier studies with respect to biochemistry and physiology on inter and intra specific incompatibility has found the role of proteins, present in the intine and the exine of the pollen grains. These studies suggest that (1) these proteins have hydrolytic activity and (2) the only pollen wall extracted could onset the incompatible reaction. The proteins incorporated in intine are contributed by pollen cytoplasm (gametophytic origin) while proteins present in the exine are donated by tapetum (sporophytic origin) during microsporogenesis. The intine held proteins or proteins of gametophytic origin will be released only after hydration of pollen on the stigma. So, it takes longer for pollen recognition and rejection. The proteins present on sporophyte are readily available after landing of pollen on the stigma. Thus the rejection reaction in SSI is faster and immediate. These proteins are characterized as S-locus proteins. The further details are provided in the following paragraphs. In the last few decades it has been found that SI reaction involves highly ordered, specific and molecular recognition between the pollen and the pistil. In maximum incidences it could also be manifested by cytological or by histochemical means. The stages or sequential reactions for effective SI are as following (Source-Shivanna, 2002): 1. Production of S-allele specific products in the pistil and the pollen 2. Interaction of S-allele specific products in the pistil with those of pollen grains during pollen-pistil interaction to recognize the pollen 3. Inhibition of incompatible pollen following pollen recognition These steps clearly indicate that for

better understanding of SI recognition and rejection

reaction, isolation and characterization of S-allele specific genes and proteins is the foremost thing by employing molecular tools e.g. SDS gel electrophoresis, chromatography, immunodiffusion, cDNA isolation etc.

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Sporophytic Self-incompatibility System Cytology and Histology In SSI and some GSI (Poaceae, Oenothera) the recognition and rejection reaction occurs on stigma. In SSI incompatible pollinations result in abnormal behavior of pollen grains (male gametophyte) in several ways listed below: 1. pollen grains fail to germinate 2. small protuberance like pollen tube emerges 3. malformed pollen tubes are unable to penetrate the stigmatic tissue 4. emergence of multiple pollen tubes 5. swelling of the tip of pollen tube The most characteristic feature of SSI is the development of callose plugs after the landing of incompatible pollen on stigma. The callose plug formation occurs at the point of contact of pollen grain and stigma as well as in the pollen tube tip. The formation of callose plugs is a reliable histological and biochemical feature which could be easily assessed using decolorized Aniline Blue (Figure 7 and 8). Another characteristic feature of SSI is concurrence of dry type of stigma (Figure 9). Dry type of stigmas possesses a hydrated layer „pellicle‟ over the lipidic cuticle layer. Soon after hydration of pollen grains on stigma, proteins held in exine get released and interact with proteins of pellicle. This interaction is responsible for recognition of pollen grains of the right type. In Arabis and Brassica, it has been suggested that in case of compatible pollinations an enzyme „cutinase‟ becomes activated after the interaction (proteins held in exine and proteins held in stigma papillae cells) which erodes the cuticle layer, thus allowing the pollen tubes to penetrate the stigmatic tissue. Figure 7: Cross pollen grains deliver pollen tube which is able to penetrate the cuticle-pellicle layer of stigmatic papilla (A) while callose gets deposited at the germ pore and in stigmatic papilla (rejection reaction) after pollination with self pollen grain (B).

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Source: http://www.intechopen.com/source/html/44293/media/image7.jpeg Figure 8: Part of pollinated stigma with incompatible pollen grains (stained with Aniline-blue: a callose specific fluorochrome) showing deposition of callose (in red) in pollen tubes (arrows).

Source: Vikas and Tandon, 2011 (need permission)

Figure 9: Cross section of dry stigma of Hippophae rhamnoides stained with fluorochrome Auramine-O specific to cuticle-pellicle layer. Arrows indicate the presence of cuticle-pellicle layer over stigmatic cells.

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Source: Authors

Molecular Biology and Biochemistry First study to understand the molecules and biomolecules involved in SI was conducted by Nasrallah and associates in Brassica oleracea. They showed that each S-allele produces a specific protein and each S-allele specific protein is heritable. In B. oleracea, the SI attainment coincides with the appearance of S-allele specific proteins. The molecular weight of all S-allele specific glycoproteins in B. oleracea is estimated approximately ~ 55 KD. Further studies with molecular tools established that S-gene in Brassica is a complex multigene family. The two important genes linked with S-locus have been identified as SLG gene (S-locus specific glycoprotein gene) and SRK (S-locus receptor kinase gene). Other genes which are not linked to S-locus are called the SLR genes (S-locus related genes). However, SRK is the chief determinant of SI in the pistil. Similar genes have also been identified in family Papaveraceae. Gametophytic Self-incompatibility System Cytology and Histology Generally in GSI, the germination of pollen on the stigma is not inhibited but the growth of pollen tubes is inhibited in the style, as the S-locus specific proteins are secreted in the stylar

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canal. However, in grasses the zone of inhibition is variable. In family Papaveraceae zone of inhibition is stigma itself. The genetic studies have concluded that the recognition factor on the male side is contributed by the pollen cytoplasm and not by the tapetum. Comparable to the SSI, in GSI callose also plays a major role in inhibition of pollen tube growth. Deposition of pectic material at the tip of the pollen tubes growing in the incompatible style was also observed. There is excessive deposition of callose in the tips of tube which generally show swelling and/or bursting in the stylar region. In Lycopersicon peruvianum, pollen tubes in the compatible style have two layered wall -outer being pectocellulosic made of loose fibrils and inner being homogenous containing callose. Pollen tubes in the incompatible styles also display similar wall structure during initial stages of growth. After traversing about two-third of the style, inner wall gradually becomes thin and numerous particles accumulate in the tube cytoplasm. Eventually, the inner wall disappears and pollen tube bursts. This suggests the incompatibility reaction in GSI is an active process. Molecular Biology and Biochemistry A difference in the metabolism between self and cross pollinated pistils has been observed in Nicotiana alata. Style with incompatible tubes showed higher activity of Peroxidease-10. However, the activity of Peroxidease-10 was very low in style with compatible pollen tubes hence suggesting the probable role of Peroxidease-10 in rejection reaction. Several S-allele specific glycoproteins have been identified from systems like Solanaceae (Petunia, Lycopersicon, Nicotiana), Rosaceae, Scrophulariaceae, Plantaginaceae exhibiting GSI. The molecular weight of all the isolated S-proteins ranges from 27-33 KD. Various studies have shown that these proteins are Ribonucleases which at times are referred to as S-RNases. Several studies in Lilium longiflorum, Petunia hybrid and Nicotiana; have shown that the production of S-allele specific molecules is not a pollination induced response.

Molecular mechanism of self/non-self discrimination in angiosperms The SI is taxonomically widely distributed amongst angiosperms and several incompatibility genes (as mentioned earlier) have been identified. However, the interaction and full molecular

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mechanisms have been understood only in some families like Solanaceae, Brassicaceae, Rosaceae, Scrophulariaceae and Papavaraceae. It is quite evident that SI is controlled by single locus S, with multiple alleles which are inherited as single segregating unit. The variants of this gene complex are termed as S-haplotypes. Each haplotype encodes both male specific and female specific determinants (S-determinants). The male specific determinants are carried by pollen grains and female specific determinants are present in the pistil. The interaction between these S-determinants discriminate the self pollen (incompatible reaction) as they are encoded by same haplotype (Figure 10). Figure 10: S-locus with male and female determinant allele. The interaction of male and female determinant derived from same haplotype results in incompatibility (black broken line) and the interaction of male and female determinant derived from different haplotype accomplished compatibility (red solid line).

Source: Adopted and modified from Takayama and Isogai, 2005.

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Recent molecular and biochemical approaches for understanding the SI have directed that self/non-self discrimination mechanisms of SI can be classified into two primary systems: Self recognition and non-self recognition. For Self recognition and non-self recognition systems both male and female determinants have been identified in Brassicaceae, Rosaceae, Scrophulariaceae and Solanaceae while in Papaveraceae only female determinants have been identified (Table 1).

Self-recognition system prevalent in Brassicaceae and Papaveraceae

depends upon the specific interaction of male-determinants and female determinants encoded by same S-haplotype. Non-self recognition system is present in family Solanaceae. The mechanism of SI in three families Brassicaceae, Papaveraceae and Solanaceae is discussed in detail in subsequent paragraphs.

Table 1: List of male-determinants and female determinants identified in different families (SP11: S-locus protein 11, SCR: S-locus cysteine rich protein, SLF: S-locus F box, PrpS: Papaver rhoeas Pollen S, PrsS: Papaver rhoeas Stigma)

Family

Type of SI

Male determinant

Female determinant

Brassicaeae

SSI

SP11/SCR

SRK

Solanaceae,

Rosaceae, GSI

SLF

S-RNase

GSI

PrpS

PrsS

Scrophulariaceae Papaveraceae

Source: Adopted and modified from Takayama and Isogai, 2005. Brassicaceae Type SSI: Self Recognition System In Brassicaceae, the S-locus has three polymorphic genes: SLG, SP11, SRK (Figure) which encode proteins which are highly polymorphic. These genes are tightly linked and inherited together like a single Mendelian gene. SRK is the membrane-spanning receptor kinase and is localized in plasma membrane of cells of stigmatic papillae (act as female-determinant, Figure 11). SP11 is a small basic protein secreted from anther tapetum and localizes in the protein coat. Upon pollination SP11-SCR encoded protein penetrates the papilla wall and enters into

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the vicinity of membrane-spanning receptor (female- determinants from SRK). If the SP11-SCR proteins (or male determinants) are from same haplotype (self/incompatible pollination), they immediately bind to each other. There binding initiates the autophosphorylation of SRK and a cascade of signal transduction (yet to be characterized) that rejects the self/incompatible pollen grains (Figure 11). The complete role of SLG is not characterized. However, it is proposed that its protein positively influences the SRK activity. Figure 11: Structure of S-locus: with three genes namely SLG, SP11, SRK. Black dotted line with arrowheads shows the interaction of male and female determinants from same haplotypes. During incompatible pollination, the male-determinant binds to female-determinant. This induces the rejection reaction (SI).

Source: Adopted and modified after Iwano and Takayama, 2012

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Papavaraceae Type of GSI In Papaver rhoeas, the SI is of gametophytic type and the incompatible reaction occurs in the pollen grain. The female-determinant PrsS (P. rhoeas style S) is secreted by stigmatic papilla and is a highly polymorphic protein. However, the presence of male-determinant on plasma membrane of pollen grain is still putative. After incompatible pollination/self pollen landing on stigma, PrsS triggers a series of SI responses: (1) it increases the Ca2+ influx in the cytosol of pollen grain and (2) starts the depolymerization of the actin cytoskeleton. These two actions result in the cessation of pollen tube growth and programmed cell death (Figure 12). Figure 12: Gametophytic SI in Papaveraceae. Red and black double-headed arrows indicate the compatible and incompatible (SI) interaction of male-determinants and female determinants, respectively.

Source: Adopted and modified after Iwano and Takayama, 2012

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Solanaceae Type of GSI: Non-self Recognition System In solanaceous systems the SI is governed by the haploid genotype of the pollen and the SI response occurs in the pollen tube. The female determinants are S-RNases (~ 30 KD proteins) which possess ribonuclease activity. The male determinants are multiple and have been characterized as F-box proteins named as S-locus F-box (SLF or SFB). The female determinant is present in the extracellular matrix of style. In case of incompatible pollen tubes (with matching S-haplotype), S-RNase exhibits cytotoxicity and degrades the RNA present inside the growing pollen tube thus restricted their growth in the style. It is important to note that in the self pollination none of SLF interacts with the self S-RNase (Figure 13). However, in crosspollination SLFs interacts with S-RNase and degrades the S-RNase (Figure 13) thus the pollen tube continues to grow. Figure 13: Self-incompatibility in family Solanaceae. Note there is no interaction of male determinant and female determinant in self/incompatible pollination.

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Source: Adopted and modified after Iwano and Takayama, 2012

Pollen tube inhibition in heteromorphic systems The zone and mechanism of pollen tube inhibition in case of incompatible crosses (pin X pin or thrum X thrum) in heteromorphic system is quite variable. It is distinguished from homomorphic SI as the pollen wall held proteins plays no role in incompatible reaction and there is no callose formation in the stigmatic papillae after autogamy. Zone of Inhibition The variability in zone of pollen tube inhibition may lie between species as well as within morphs of species. In family Plumbaginaceae, inhibition occurs on the stigma surface in both morphs as the pollen tube fails to emerge or penetrate the stigmatic tissue. In Fagopyrum, inhibition occurs inside the stigma in case of thrum X thrum pollination and in pin X pin cross inhibition occurs in the style. Similarly, in thrum morph of Primula obconica pollen rejection occurs on stigma while in pin morph site of incompatible pollen rejection lies in style. In Linum grandiflorum, pin X pin pollination results in failure of pollen grains adhesion and hydration while in thrum X thrum pollination, pollen do germinated but pollen tubes get inhibited on the stigma itself. In trimorphic Lythrum, inhibition zone is different among morphs. In short style morph pollen tube growth is detained at the base of stigma, while in mid and long style morph pollen tube stops in upper part of style. In Primula vulgaris there is no localized zone of pollen tube growth and inhibition. Mechanisms of Inhibition As compared to the homomorphic SI very little information is available in literature pertaining to the mechanism of inhibition in heteromorphic systems. In dimorphic Linum grandiflorum , it has been postulated that the responsible factor for rejection of incompatible pollen grains is osmotic potential and it is a passive mechanism. Lewis observed in 1949, that there was a marked difference between the osmotic potential of style and pollen grains among two morphs i.e. thrum morph style 1.0, pollen 7.0, pin morph style: 1.75, pollen 4.0. In the compatible crosses (pin X thrum and thrum X pin), the ratio of osmotic potential of pollen: style was 4:1. In an Institute of Lifelong Learning, University of Delhi

incompatible cross of pin X pin morph this ratio was 5:2 and in an incompatible cross of thrum X thrum morph this ratio was 7:1. On the basis of these observations it was suggested that 4:1 ratio of osmotic potential of style and pollen grains is optimal for pollen hydration, germination and tube growth. Deviation from this ratio leads to incompatibility as if the ratio is too high (thrum X thrum morph, 7:1) pollen tubes will absorb water and burst and if the ratio is too low (pin X pin morph, 5:2) pollen hydration will be prevented. Later on Ghosh and Shivanna (1982) and Shivanna (2002) suggested that in this species intra-morph incompatibility manifests at three levels: pollen adhesion, pollen hydration and pollen tube growth in stigma. The first two levels shows passive process while the third operates as active process. The same condition has been also observed in Primula.

Methods for overcoming Self-incompatibility Though SI is very important for outbreeding of species, it can be a major hindrance in plant improvement programmes. Homozygous individuals obtained by extensive selfing have a poor survival rate. To ensure production of robust species, nature enforced self-incompatibility. In normal course of events, these barriers imposed by nature for selfing are beneficial but when one wants to produce improved varieties using species which are self-incompatible, major problems are encountered. Hence, it becomes important to overcome these barriers to crossability or selfincompatibility. Listed below are a few methods for overcoming these hurdles: 1. Mixed Pollination: Species in which the pollen grains are unable to germinate on the stigma due to absence of certain exine proteins, technique of mixed pollination is adopted. Here, the stigma is dusted with compatible as well as incompatible pollen together. The compatible pollen grains are generally inactivated by irradiation or are killed by treating them with chemicals like methanol or even can be subjected to repeated freezing and thawing treatments. Such pollen cannot affect fertilization but are capable of germination. Also, there is no change in their exine held proteins, which are released as and when the pollen lands on the compatible stigma. These compatible pollen are known as the mentor pollen or the recognition pollen. The recognition proteins from the wall (from intine and exine both) of mentor pollen help the incompatible pollen to germinate on the stigmas. Mentor pollen also provides a pollen factor or P-factor which interacts with S-factor from the stigma and renders the stigma accessible to

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incompatible pollen. Many a time, even after germinating on the stigma, the pollen tubes are inhibited in the stylar canal. In such cases, mentor pollen provides a pollen growth promoting substance-PGS which allows sustained and extra growth to pollen tubes in the style. Mentor pollen also provide substances critical for sustained growth of ovules, ovary and other fruit tissues, thus indirectly influencing fertilization events and seed maturation. This method has been successful in overcoming incompatibility in lily. 2. Bud Pollination: In Petunia, the incompatibility does not occur on the stigma but occurs in the style. Pollination done at the time of anthesis is unsuccessful. But pollination done two days before anthesis or at the bud stage leads to formation of good number of seeds. It is likely that the incompatibility factor of the pollen is produced by the style at the time of flower maturity or opening. Hence, if the pollinations are made at the bud stage, when the incompatibility factor is yet to be produced, the pollen tubes grow normally in the style and affect fertilization. 3. Stub Pollination: When the incompatibility occurs on the surface of the stigma, removal of stigma and even some portion of the style have proved successful in overcoming incompatibility. In Ipomea trichocarpa, the pollen grains are unable to germinate on the stigma as the incompatibility site occurs on the stigma itself. If the stigma along with the upper portion of the style is cut and removed and remaining portion of style or the stub pollinated, these pollinations are successful and effective in fertilization. This is also referred to as „stump-pollination‟‟. 4. Intra-ovarian Pollination: As the name indicates, the pollinations are made directly inside the ovary by introducing the pollen suspension. This is done in those species where the zone of incompatibility lies on the stigma or in the style. The surface of the ovary is sterilised with ethanol and two punctures are made opposite each other in the ovary. Through one of the punctures, the pollen suspension is added to the ovules with the help of a hypodermic syringe. The second puncture or the opening is for the air to escape. Once the pollination is accomplished, the punctures are sealed with petroleum jelly. Pollen suspension is prepared by mixing the pollen with distilled water. If there is a special requirement of any substance for pollen germination, then that substance is also added to the suspension. This method has

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proved successful for members of Papaveraceae. The only drawback of this technique is that it is applicable to large ovarian cavities. 5. In vitro Pollination and Fertilization: In this method, all stigmatic, stylar and ovarian tissues are completely removed and the pollen grains are directly dusted on ovules. These ovules are cultured on a nutrient medium which promotes pollen germination and subsequently the fertilization. This technique has proved beneficial in Petunia axillaris. Here, the pollen dusted on ovules, germinate within a few minutes and fertilization is affected within two days. Normal seed set occurs. 6. Modification of Stigmatic Surface: In those species, where the surface of stigma proves a barrier to pollen germination , the surface of stigma is modified using organic solvents like hexane, detergents like Triton X-100 or even by application of chemicals like ConcanavalinA. In Brassica, Concanavalin-A is applied to the surface of the stigma which binds to the surface and masks the pellicle which is responsible for the incompatibility. Triton X-100 and hexane also perform similar function of masking the pellicle. 7. Heat Treatment of Style: Moderately high temperatures have also proved beneficial in overcoming self-incompatibility in certain plants. If the style of Lilium longiflorum is subjected to a temperature of 50 C for 6 minutes prior to pollination, the pollination is rendered compatible. If this temperature is further increases to 55C, it proves injurious to the pistil, whereas lowering of the temperature proves ineffective in overcoming incompatibility. It is thought that the increase in temperature either inactivates or selectively denatures the enzymes responsible for self-incompatibility.

Probably heat-sensitive inhibitors of pollen

tube growth are inactivated. These temperatures are species specific. In Lilium, temperature as high as 50C is effective where as in Rye, temperature as low as 30C is enough for selfing. 8. Irradiation: Treatment of pollen or the style with radiation can induce temporary breakdown of incompatibility reaction by bringing about unstable cytological changes in the pollen or the style or due to mutation in the incompatibility gene. 9. Chemical Treatment: In many species such as Ipomea batatas , pollen germination and the growth of pollen tubes in the style are normal but seed set does not occur due to premature abscission of the flowers. Flowers abscise just a day or two after anthesis. The age of such Institute of Lifelong Learning, University of Delhi

flowers is increased by application of chemicals like 2,4-D (100mg/l) which improves fertilization and enhances the development of embryo. 10. Increased Level of Carbon di-oxide: Percentage of Carbon di-oxide in the atmosphere is 0.03. If we raise this level cent percent or even higher to 4-6% at a relative humidity of 100% for several hours prior to pollination, then those pollinations which would have been selfincompatible under normal conditions behave as compatible pollinations. This method has proved beneficial in breeding of Brassica species. 11. Parasexual Hybridization:

The technique of protoplast fusion or the parasexual

hybridization came after EC Cocking in 1969. He demonstrated that naked cells or the protoplasts can be obtained through the enzymatic degradation of the cell wall. It was a novel approach where one need not bother about the incompatibility at stigma, style or the ovary level. The technique involved the fusion of isolated protoplasts to produce hybrids and was called the “parasexual hybridization” or “somatic hybridization”. Bhojwani and Cocking first showed the possibility of fusion of protoplasts of microspores or the young pollen in 1972. Microspore protoplasts fuse readily as they are rich in cytoplasm and are very important in hybridization as they are haploid. Protoplast fusion occurs in three steps: a. Isolation of protoplasts b. Fusion of these isolated protoplasts c. Culture of these hybrid protoplasts to regenerate whole plants. The somatic cells in plants are bound by a rigid cell wall made up of cellulose. Also, the adjacent cells in a tissue are cemented together by a pectin rich matrix. Thus, to obtain protoplasts from somatic cells, we need to treat them with a mixture of cellulase and pectinase enzymes. Concentration of these enzymes is dependent on the tissue used and varies from tissue to tissue and species to species. Osmotic fragility of these isolated protoplasts is very crucial in the entire sequence of events starting from enzyme solution to washing medium, fusion medium and culture medium. Commonly a metabolically inert sugar like mannitol is used as an osmotic stabiliser. Freshly isolated protoplasts in the presence of suitable osmoticum appear spherical.

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Polyethylene glycol (PEG) is a widely used fusogen because of its high efficiency in heterokaryon formation. PEG has been used for fusion of protoplasts from diverse taxa like soybean, corn, and pea or even fusion of plant cell and animal cell. Equal quantities of dense suspension of the two types of protoplasts are mixed along with 15% PEG solution. After10-30 minutes, PEG solution is diluted with the medium in which PEG is prepared. Finally, the PEG is washed off completely. PEG facilitates the aggregation of the protoplasts. They become closely appressed to each other over major portion of their membranes. These membranes fuse with each other during the dilution of PEG and not during actual treatment with PEG. The cytoplasmic continuities expand and fuse to form broader connections. Eventually the protoplasts fuse completely and attain a spherical shape. Fusion of two dissimilar protoplasts leads to the formation of a heterokaryon. When subjected to culture conditions, the two nuclei of a hetrokaryon fuse to establish a true hybrid cell. Once the fusion is accomplished, it is essential for the fused protoplasts to grow into whole plants or else the purpose of fusion is defeated. However, before attempting this with hybrid protoplasts; it is desirable to test the potentiality of parental protoplasts to be able to form whole plants in culture. When placed in a culture medium, the fused protoplasts synthesise a new wall around them and are reconstituted as cells. These cells divide and redivide to form a callus which ultimately forms a plantlet.

Conclusions Till now we could say that, the chief role of the SI in nature is to maintain the reproductive isolation. Secondly, SI is a mechanism to promote cross-pollination and increases the genetic variability in the species. However, in-spite of its natural necessity, SI sometimes turns into a block in plant breeding exercises especially in production of homozygous lines which is an important requirement. Moreover, it prevents distant hybridization. Hence, combining two genomes becomes very much challenging. In this regard, elucidation of the details of mechanisms governing SI is essential. Presently we are able to understand the molecular mechanics in some systems but definitely more understanding is required because SI is well distributed feature in angiosperms. Thus, the field of self-incompatibility research is highly dynamic and full of opportunities and challenges.

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References/Suggested readings 1. Bhojwani SS and Bhatnagar SP (eds.). 2008. The embryology of angiosperms. Vikas publishing house private limited. Noida, India. 2. Iwano M and Takayama S. 2012. Self/non-self discrimination in angiosperm selfincompatibility. Current Opinion in Plant Biology 15: 78-83. 3. McCubbin A and Dickinson H. 1997. Self-incompatibility in: Pollen biotechnology for crop production and improvement. Cambridge University Press, Cambridge: pp. 199-217. 4. Shivanna KR (ed). 2002. Self-incompatibility in: Pollen biology and biotechnology. pp. 140-166. 5. Takayama S and Isogai A. 2005. Self-incompatibility in plants. Annual Reviews of Plant Biology 56: 467-489. 

Vikas and Tandon R. 2011. Reproductive biology of Azadirachta indica (Meliaceae), a medicinal tree species from arid zones. Plant Species Biology 26: 116-123.

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de Vos Jurriaan M. Hughes CE, Schneeweiss GM, Moore BR and Contil E. Heterostyly accelerates diversification via reduced extinction in primroses. Proceedings

of

Royal

Society:

Biological

Sciences

281:

20140075.

http://dx.doi.org/10.1098/rspb.2014.0075. (These two are references of papers from which figures (1 and 8) are adopted and need permission from the authors. After permission these two could be omitted from the list.)

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Glossary  Viability of pollen grains: The pollen grains which have ability to transfer male gametes to the stigma are known as viable pollen grains.  Fertility of pollen grains: Fertile pollen grains are those which have capability of bringing fertilization in ovules.  Out-breeding: Breeding of distantly related individuals for the purpose of producing offspring of superior quality and new genotypes.  Dichogamy: It is a condition where the stamens and pistils of a flower mature at different times, so that self-fertilization is prevented.  Herkogamy: It is a spatial separation of the anthers and stigma or a barrier which prevents self-pollination. e.g. distylous system.  Cross-pollination: The transfer of pollen from an anther of a flower of one plant to a stigma of a flower of another plant (different genotype) of the same species.  Dioecy: The unisexaul flowers are present on separate plants. Dioecy is the equivalent of the separate sexes of most animals. But it is rather rare. Some examples: Morus alba, Papaya etc.  Fertile plant: Plants which produces functional male and female gametes.  Male and Female determinants: Broadly, the proteins derived from the S-locus genes specific to male and female.  Mating types: The term refers to the morphologically distinguished male and female gametes.  Haplotype: A haplotype is a set of specific alleles in a group of tightly-linked genes on a chromosome that are likely to be inherited together. e.g. S1 haplotype is a set of 2-3 alleles (SRK and SLG) of S-locus.  Allele: The alleles are variant forms of a gene which are located at the same position, or genetic locus, on a chromosome in genome.

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Activity 1. Differentiate between: a Homomophic and Heteromorphic self-incompatibility b Sporophytic and Gametophytic self-incompatibility c Intra-ovarian and In-vitro pollination 2. Define: a Self-incompatibility b GSI in grasses c Mating types 3. Write note on: a Genetics of homomorphic self-incompatibility b Genetics of heteromorphic self-incompatibility c Parasexual hybridization d S-allele hypothesis e Mixed pollination f Stub pollination

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