leads to the failure of tapetum function, consequently failure of pollen ... destroys the tapetum and thereby induces male sterility in transformed plants.
23 Male Sterility in Vegetables SANJEET KUMAR
Male sterility is characterized by nonfunctional pollen grains, while female gametes function normally. It occurs in nature sporadically, perhaps due to mutation. The phenomenon of male sterility is of great significance to produce cost effective hybrid seeds. Academically, it is of significance for the developmental geneticists/molecular biologists to study the differential/spatial expression of male sterility genes and interaction between the nuclear and cytoplasmic genome, for evolutionary biologists to study the endosymbiotic hypothesis, which focusses on tracing the transmission mechanism of mitochondrical genes into the nuclear genome during the evolution process. Onion crop provided early recognition of male sterility by Jones and Emsweller in 1936 and its use in hybrid seed production by Jones and Clarke in 1943. Since then male sterility has been reported in fairly large number of crop plants including vegetables. These male sterile plants were either isolated in natural populations or were artificially induced through mutagenesis. Now male sterility is also induced through genetic engineering and protoplast fusion. Classification of male sterility On the basis genetic control of male sterility Kaul (1988) classified male sterility into two groups (i) genetic (male sterility under the control of genes) and (ii) non-genetic (male sterility temporally induced by stresses). Since non-genetic male sterility has not been utilized in vegetable crops, only various types of genetic male sterility and its mechanisms and uses in selected vegetable crops will be described in this chapter. On phenotypic basis, genetic male sterility is classified into three classes i.e. sporogenous, structural and functional. Similarly, non-genetic male sterility has been classified as chemical, physiological and ecological male sterility. Further, on genotypic basis genetic male sterility was grouped as genic, cytoplasmic and gene-cytoplasmic male sterility (Table 1). On the basis of location of gene(s) responsible for the male sterility, spontaneously isolated, artificially induced through mutagenesis, artificially incorporated through protoplast fusion or genetically engineered male sterility systems (all are inherited) can be classified as (i) genic male sterility (GMS; more
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appropriately nuclear male sterility) and (ii) cytoplasmic male sterility (cms; more appropreatly nuclear-cytoplasmic male sterility) (Table 1). Pollen fertility in many of the cytoplasmic male sterile plants is usually restored by certain dominant nuclear allele (called fertility restorer gene; Rf allele). Hence, the expression of male sterility in such cases is the result of incompatibility between recessive nuclear allele (called maintainer gene; rf) and cytoplasmic genome. Therefore, those cytoplasmic male sterile lines for which restorer allele (Rf) are discovered and known, are widely known as cytoplasmic-genic male sterility (c-gms) and more often treated as a separate class of male sterility system. However, it is more appropriate to treat cytoplasmic (CMS) and cytoplasmic-genic male sterility (g-cms) under common head (i.e. cms) because of the fact that in both these systems, location of male sterility causing gene is mitochondrial genome (mtgenome) and nuclear dominant restorer allele (Rf) if identified could restore male fertility of sterile cytoplasm (Table 1). TABLE 1 Classification of male sterility in plants (i) Inherited (genetic) male sterility Phenotypic basis (i) Sporogenous male sterility. Pollen formation is completely disrupted. (ii) Structural sterility or postional sterility. Floral organs are modified in such a way that selfing does not occur. (iii) Functional sterility. Pollens are produced but they are unable to self fertilize due to the nondehisence nature of anthers. Genetic basis
(i) Genetic male sterility (gms). Male sterility is caused by the gene(s) from nuclear compartment (nuclear gene) (ii) Cytoplasmic male sterility (cms). Male sterility is caused by the gene(s) from cytoplasm (mitrochondrial genes) (iii) Cytoplasmic-genic male sterility (c-gms). Male sterility is caused by mitochonridal genes and restored by the nuclear genes. (ii) Non-inherited (non-genetic) This kind of male sterility is temproraly induced by ceratin male sterility environmental stresses e.g., temperature, etc.
Genic male sterility (gms) or nuclear male sterility GMS has been reported in about 175 plant species (Kaul, 1988) including important vegetable crops (Table 2). As the name suggests, this type of male sterility is controlled by the gene(s) from the nuclear compartment. Most of the naturally occurring or induced male sterile mutants are recessive in nature with few exceptions in cole crops e.g., cabbage and broccoli and genetically transformed male sterile lines. The occurrence of predominantly recessive male sterility clearly demonstrate that gms is a loss of function mutation and is the result of mutation in any gene(s) eitheir controlling microsporogenesis (pollen development process), stamen development or microgametogenesis (male gamete development process). All the transgenic male sterile lines developed till date are GMS because they have been developed through transformation of male sterility causing gene construct(s) inside the nuclear genome.
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Environmental sensitive genic male sterility (EGMS). Certain GMS plants, are conditional mutants, meaning thereby, in particular environment male sterile plants turn into male fertile. After determination of critical environment (usually temperature or photoperiod) for male sterility/ fertility expression, such GMS mutants are classified under environmental sensitive genic male sterile (egms) lines. The EGMS lines (mostly temperature sensitive) have been reported in several vegetable crops like cabbage, Brussels sprout, broccoli, peppers (chilli and sweet pepper), tomato and carrot. A majority of these, however, were previously identified as normal genic male sterile lines. Utilization of GMS and EGMS First step in the utilization of GMS system is seed multiplication of male sterile line, which is not possible through selfing or sibing, since pollen is either not viable or not produced. Commonly utilized monogenic recessive GMS (msms) are maintained by back crossing it with heterozygous isogenic line (msms × Msms) for male sterility. Therefore, in hybrid seed production field, 50% male fertile segregants (Msms) need to be identified and removed before they shed pollen (Figure 1). Hence the stage of identification of male sterile/fertile plants in hybrid seed production field is very important. In general, male sterile plants are morphologically not distinguishable from the sister fertile plants. However, in certain GMS lines, ms gene has been found to be tightly linked with the recessive phenotypic marker gene. Such marker genes, especially those that express at seedling stage are good proposition for the identification of sterile/fertile plants at seedling stage. Thus labour and time required for identification and removal of fertile plants in hybrid seed production field could be avoided. In muskmelon and chilli GMS lines are commericially utilized to produce hybrid seeds (Table 2). Hybrid seed production through EGMS line is more attractive because of the ease in seed multiplication of male sterile line. Seeds of EGMS line can be multiplied in an environment where it expresses male fertility trait while hybrid seeds can be produced in other environment, where it expresses male sterility. Male (pollen) fertility in hybrid crop is not affected, as male parent contributes normal (wild) allele of the environmental sensitive mutant gene. Since only two parental lines are involved, breeding method involving EGMS is more popularly termed as “Two Line Hybrid Breeding” as against “Three Line Hybrid Breeding” in case of CMS system. In CMS, apart from female (male sterile; A line) and male (restorer; R or C line) parents, an additional line (B line) is required for the maintenance of male sterile seeds. Limitations of GMS. Because of more tedious maintenance process and non-availability of suitable marker gene among the vegetable crops, GMS are utilized commercially only in chilli and muskmelon in India. Cytoplasmic/cytoplasmic-genic male sterility or nuclear-cytoplasmic male sterility (CMS)
CMS has been reported in 150 species and is presently most extensively utilised to produce hybrid seeds at commercial scale in several vegetables (Table 3). Cytoplasmic male sterility is a maternally inherited trait, because mt-genome is responsible for the expression of male sterility and the mitochondria are usually excluded from the pollen during fertilization. Once dominant restorer gene (Rf) located in nuclear genome for pollen fertility of a cytoplasmic male sterile line is identified, it is commonly known as cytoplasmic-genic male sterility (CMS). Hence male sterility in CMS is expressed under the presence of sterile mt-genome located in cytoplasm (Scytoplasm) and recessive allele of restorer (maintainer allele; rf) is located in the nuclear genome.
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TABLE 2 Genic male sterility or nuclear male sterility (GMS) in selected vegetable crops Crop Tomato†
Brinjal Pepper†
Cauliflower Cabbage
Brussels sprout Broccoli Watermelon Muskmelon†
Cucumber Summer squash Onion Carrot Radish
Salient features of gms More than 55 recessive genes have been reported sl-2, ms-13 and ms-15 are temperature sensitive; ps-2 gene has been exploited at commercial scale in some Eurpopian countries; Yeast artificial chromosome (YAC) containing ms-14 gene has been cloned. Monogenic recessive gene has been reported; monogenic recessive functional sterility available in India and utilized for developing experimental crosses. More than 12 recessive genes have been reported; MS-12 (ms-509/ms-10) and ms3 genes are commercially utilized in India and Hungary, respectively. In India farmers in Punjab are producing seeds of CH-1 (MS-12 × LLS) and CH-3 (MS12 × 2025) hybrids. The ms-10 gene of MS-12 line is linked with taller plant height, erect growth and dark purple anther. Both recessive and dominant genes have been reported. Both recessive and dominant genes have been reported; RAPD marker linked to dominant gene has been identified and its use in hybrid seed production has been proposed. Recessive male sterile mutant has been reported but not utilized due to availability and utilization of CMS system. Six recessive non-allelic genes have been reported; linkage of ms gene with bright green hypocotyl. Recessive mutants have been reported; linkage of ms gene with delayed-green (dg) seedling marker gene. Five recessive non-allelic genes have been reported; ms-1 is commercially utilized by the farmers of Punjab in India to produce seeds of Punjab Hybrid (ms-1 × Hara Madhu). Monogenic recessive gene has been reported; limited scope of utilization because of the availability and utilization of of gynoceous lines. Monogenic recessive gene has been reported; very limited scope of utilization because of the availability of sex regulating mechanism using certain chemicals. Monogenic recessive genes are known but not utilized at commerial scale due to availability and utilization of CMS system. Recessive male sterile genes have been reported; not utilized at commercial scale because of the availability of CMS lines as an alternative method. Three recessive mutants have been reported; commercially not utilized because of the availability of CMS lines as an alternative method.
†
Exploited at commercial scale
Based on mode of action of the pollen fertility restorer (Rf) and mainainer (rf) alleles, CMS are of two types, viz., gametophytic and sporophytic. Cytoplasmic male sterility may originate from inter-generic or inter-specific crosses and may be artificially induced through mutagenesis or antibiotic effects on cytoplasmic genes. Cytoplasmic male sterile plants have also been developed
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in several vegetables through protoplast fusion. In near future, genetically engineered cytoplasmic male sterility may be available after standardization of transformation technique for mt-genome. Utilization of CMS The CMS is the most commonly utilized male sterility to produce commercial hybrid seeds. Three Line Hybrid Breeding involving A line (male sterile; S-rfrf), B line (maintainer; N-rfrf) and C or R line (restorer; S or N-RfRf), is explained in figure 2. A line is developed by back crossing of selected B line on to a already available A line for six to seven times. This generates a pair of A and B line in the new genetic background. Restorer gene (Rf) is either introgressed into identified male parent or male parent is directly used for hybrid seed production on CMS, if Rf gene in homozygous state is available in male parent. Cytoplasmic male sterility without restorer gene cannot be utilized in fruit vegetables e.g., tomato, chilli, melon etc., but it can be utilised in vegetables where vegetative part is of economic value e.g., onion, carrot, radish, leafy vegetables, etc (Table 3). TABLE 3 Cytoplasmic-nuclear male sterility (CMS) in vegetable crops Crops Tomato Pepper†
Cole crops
Onion† Carrot†
Radish†
Salient features of cms Sterile cytoplasm has been derived from the distinct species through protoplast fusion; restorer gene (Rf) is not available. First reported in an Indian accession; most of the CMS lines are temperature sensitive; occurrence of Rf allele is common in small fruited (usually hot pepper) and rf in large fruited (usually sweet pepper) lines; RAPD markers linked to Rf gene have been identified. Sterile cytoplasm is derived from B. nigra and Raphanus sativus, Ogura type (Ogura, 1968); problem of seedling yellowing (at low temperature) associated with Ogura based cms lines of broccoli, cauliflower, cabbage, Brussels sprout has been solved using protoplast fusion. Seed companies in France are utilizing Cybrid CMS lines of cabbage and cauliflower; protoplast fusion has been utilized to transfer Ogura cytoplasm from brocolli into cabbage. Two types of sterile cytoplasms, viz., S and T have been reported; S-cytoplasm is most widely exploited. Two types (petaloid and brown anther) of male sterile lines are available; genetics of fertility restoration is complex because structural variants of mt DNA are numerous. Sterile cytoplasm is widely distributed in wild radish; occurrence of Rf allele is frequent in European and Chinese cultivars and rf in Japanese cultivars.
†
Exploited at commercial scale
Limitations of CMS. CMS system though is the most commonly utilised male sterility, its utilisation is restricted in specific species, because of certain limitations. These are non-availability of CMS in many crops and their wild relatives; need of fertility restorer allele in fruit producing vegetables; undesirable pleiotropic effect of sterile cytoplasm on horticultural qualities; highly unstable sterile cytoplasm in several cases; poor cross pollination ability of flowers of plants with
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Fig. 1. General scheme of hybrid seed production utilizing monogenic recessive GMS (Table 2)
sterile cytoplasm due to altered morphology and technical complexity involved in seed production and maintenance of parental lines. Beside these, vulnerability of sterile cytoplasm to specific diseases is a major risk due to monopolistic cultivation of hybrids derived from single source of sterile cytoplasm. The devastation of corn hybrids derived from T-cytoplasm by Helminthosporium blight in USA during 1970’s, is well known example of such risk. Mechanisms of GMS and CMS: some common features In both GMS and CMS systems, male sterility is the consequence of breakdown of tightly regulated pollen development and fertilization processes at any of the pre- or post-meiotic stages during the formation of tetrad, during the release of tetrad, at the vacuolate microspore stage or at pollen dehiscence stage. Expression of male sterility trait is associated with a large number of morphological, physiological, histological, cytological, biochemical and molecular changes in male reproductive tissues at various stages of microsporogenesis and microgametogenesis. Role of tapetum. Tapetum cells are innermost layer of the anther wall that surrounds anther locule possessing sporogenous cells (developing pollen). These cells are involved in the transmission of nutrients/energy to the sporogenous cells and associated with synthesis of callase enzyme. The most striking histological feature associated with the majority of male sterile plants (GMS and CMS) is persistence or premature breakdown of tapetum. Aberration in tapetum development leads to the failure of tapetum function, consequently failure of pollen development and, therefore, expression of male sterility. Behaviour of some genetically transformed male sterile plants, supports failure of tapetum development and its function leading to male sterility. In fact, knowledge on importance of tapetum, has actually been utilised to develop gene construct that selectively destroys the tapetum and thereby induces male sterility in transformed plants. Role of callase. Callase is an enzyme required for breakdown of the callose that surrounds the pollen mother cells (PMCs), thus helps in release of microspores (pollen) from tetrad after meiosis. Early or delayed callase activities have been found to be associated with male sterility. Mistiming of callase activity leads to the pre mature or delayed release of meiocytes and microspores, resulting in male sterility. Role of esterase. Esterase isozymes are believed to play role in the hydrolysis of sporopollenin, the polymer required for pollen formation. Decreased activity of esterase in male sterile plants has been observed in petunia, tomato and in radish and has been proposed that decreased activity of esterase has adverse effect on pollen development. In contrast, it has also
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been demonstrated that decreased esterase activity and composition in cms petunia plants are the result rather than cause of male sterility. Role of PGSs. Endogenous plant growth substances (PGSs) play very important role in stamen and pollen development. Male sterility has been reported to be associated with changes in a number of PGSs, rather than any specific substance and perhaps it is the altered balance of PGSs that affects the pollen development process. Reduced level of cytokinins and increased level of abscisic acid associated with rape seed GMS and CMS plants, indicates that both kind of male sterility system probably involve some common pathways. On one hand exogenous supply of reduced substances in several male sterile lines has been found to restore fertility e.g., tomato, on the other, in several cases expected fertility restoration could not be obtained. Plant growth substances like GA3 (gibberellic acid), NAA (naphthalene acetic acid), MH (maleic hydrazide), ethrel etc. induce male sterility. These chemicals are called male gametcides since they lead to pollen abortion and thereby cause male sterility: they are also called chemical hybridizing agents (CHA) (Table 4). TABLE 4 A list of chemicals used to produce male sterility Chemicals
Crop
Ethrel FW 450 Gibberellic acid (GA3) Maleic hydrazide (MH) Naphthalene acetic acid (NAA)
Beet Beet, tomato Lettuce, onion, maize Cucurbits, onion, tomato Cucurbits
Mechanism of GMS: specific features Genic male sterility is a result of mutation in any gene involved in the pathway of pollen development. Like abnormal tapetum development described earlier, several other cytological, biochemical and molecular changes have been reported to be associated with GMS. Comparison of RNA populations in floral and vegetative organs reveals that 10,000 anther mRNA are not detected in mRNA populations of other organ. Hence they are anther specific and are supposed to be involved in the synthesis of polypeptides, which directly or indirectly control pollen development process. Several pollen specific genes (expressed only in pollen) and their promoters have been isolated and characterized. Similarly, sporophytic male sterility genes have also been isolated, cloned and characterized. These genes and their promoters are being utilised to design various strategies to develop transgenic male sterile systems. In several cases, developing anthers of the male sterile plants have been found to be associated with qualitative and quantitative changes in amino acid, protein and enzymes. In comparison to the anthers of the fertile plants, variations in the levels of specific amino acids have been reported in different species. Generally, the level of proline, leucine, isoleucine, phenylalanine and valine is reduced and asparagine, glycine, arginine and aspartic acid is increased in the sterile anthers. It has been shown that the level of proline has been found to be particularly affected and mature male sterile anthers contain one-eighth amount of proline in comparison to the fertile anthers. However, exogenous injection of proline in male sterile anthers did not restore pollen fertility in proline deficient
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Fig. 2. General scheme of hybrid seed production utilizing cms (Table 3)
male sterile anthers of sorghum, pea and tomato. This suggests that perhaps, male sterility is not caused by proline deficiency, rather proline deficiency is the consequences of male sterility. Differences in total protein content and polypeptide bands between sterile and fertile anthers have been worked out. In general, lower protein content with fewer bands has been observed in sterile anthers. Mechanism of CMS : specific features The CMS occurs due to incompatibility between nuclear and mt-genome, because many mitochondrial enzymes are encoded jointly by the nuclear and mitochondrial genes. Such incompatibility leads to the dysfunction of the mitochondria, especially in the pollen producing organs such as tapetum, stamens or anther wall. This causes reduced efficiency of mitochondria to give energy to the tapetum cells, leading to the failure of tapetum and pollen development. Dysfunction of mitochondria specifically in pollen producing tissues has been explained in the light of the fact that such tissues require very high energy in comparison to the other tissues. This is evident from the observation that demand on mitochondria of anthers is highest at the time of pollen development. In corn, there is a 40 fold increase in the number of mitochondria per cell in tapetum and 20 fold in sporogenous cells. Mitochondrial dysfunction may be attributed due to the production of chimeric protein, extensive recombination without creation of Open Reading Frame (ORF), mt DNA deletion, decrease or complete lack of RNA editing process in the sterile cytoplasm. Chimeric protein is synthesised from new ORF created in mt-genome due to the DNA rearrangement. Some examples of production of chimeric genes are T-orf-13 in corn, pcf-s in petunia, orf-B and orf-224 in rapeseed. In a classical experiment, tobacco explants (with normal cytoplasm) were transformed with edited and unedited form of atp-9 gene (linked with the signal peptide) under the control of constitutive promoter. Signal peptide addressed the nuclear coded and cytoplasmically synthesized atp-9 sub unit to the mitochondrial compartment. It was observed that significant amount of transgenic plants with unedited atp-9 expressed CMS phenotype, while all transgenic possessing edited atp-9 were male fertile. This experiment not only provided a strategy to produce male sterile transgenic, but also produced evidence on the crucial role of mRNA editing in the expression of CMS phenotype.
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Transgenic male sterility systems Prospects of most commonly utilised CMS system to produce hybrid seeds at commercial scale are restricted to only few vegetable crops due to several reasons described earlier. From the beginning of 1990’s, new genetic approaches have been proposed and implemented to develop male sterility systems through genetic transformation. The ability to design new molecular strategies and their successful execution has been possible because of the isolation, cloning and characterization of anther or pollen specific genes and promoter sequences. These genes are expressed in pollen themselves (gametophytic expression) or cells and tissues (sporophytic expression) that directly or indirectly support pollen development, such as tapetum, filament, anther wall etc. On the basis of mechanism of male sterility induction and fertility restoration, all transgenic male sterility systems developed so far can be described under five classes, viz., (i) abolition-restoration system, (ii) abolition-reversible system, (iii) constitutive-reversible system, (iv) complementary-gene system and (v) gametocide-targeted system. Although in transgenic(s) developed within one system, mode of action of trans-gene(s) remains the same, there can be variations in trans-gene constructs including promoter, targeted site (depending upon the promoter used) and methodology adopted within one system. Regardless of the crop, all these systems with the same trans-gene construct can be used to develop transgenic male sterility system in any crop species including vegetable crops. SUGGESTED READINGS Horner H.T. and Palmer R.G. (1995). Mechanisms of genic male sterility. Crop Sci. 35 : 1527-1535. Kaul MLH (1988). Male Sterility in Higher Plants. Monographs on Theor. Appl. Genet. 10, Springer-Verlag, Berlin. Mariani C., De Beuckeleer M., Truettner J., Leemans J and Goldberg RB (1990). Induction of male sterility in plant by a chimaeric ribonuclease gene. Nature 347: 737-741.
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