4. Plant Reproductive Systems

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by Rattandeep Gill, Clark MacAllister, Tiantian Zhang; Institution of Plant Breeding, Genetics and Genomics, University of Georgia

Sexual Reproduction

Reproduction is a biological process by which living organisms produce more individuals of their own kind. There are two modes of plant reproduction: asexual reproduction and sexual reproduction. Sexual reproduction in plants consists of alternating, multicellular haploid and diploid generations. In angiosperms, the female gametophyte is the embryo sac and the male gametophyte is the pollen. The haploid egg and sperm fuse to form diploid zygotes, from which new sporophytes develop. In asexual reproduction, offspring are produced without meiosis or fusion of gametes and the plant multiplies through tubers, bulbs, corms and other vegetative parts. Sometimes a third mode of reproduction, apomixis, may be distinguished. Apomixis is the formation of new individuals from the sexual organs of a plant, without fertilization (Fryxell, 1957).

Knowledge of the mode of reproduction of a given species is essential for a plant breeder to accomplish crop improvement in that species. A plant breeder needs to know both how the plant reproduces naturally, and which possible methods of reproduction can be employed for artificial breeding (Fryxell, 1957). Knowledge of the natural mode of reproduction of a species helps the breeder to predict its behavior in field conditions, and knowledge of the possible methods helps to determine the potential manipulation available to accomplish crop improvement (Fryxell, 1957). For hybrid development in a naturally self-pollinated plant, the breeder needs to emasculate the female parent and artificially pollinate it with desired pollen to obtain a particular cross. For obtaining successful emasculation and artificial pollination, prior knowledge of floral biology, that includes time of anthesis and period of stigma receptivity of the species is required. The choice of selection method in breeding also depends on the natural mode of reproduction of a species. Mass selection, pure line selection, pedigree method, bulk population breeding and backcross breeding methods are all commonly used in self-pollinated crops whereas mass selection for intra-population improvement, and recurrent selection methods for inter-population improvement, are used in cross pollinated crops (Chahal and Gosal, 2002).

Flower Morphology

Flowers are the reproductive organs of a plant and the knowledge of various parts of a typical flower is necessary to understand plant sexual reproduction. A flower consists of different floral whorls, each with a different function. The outermost whorl is called calyx and consists of sepals. Sepals are usually green and they enclose and protect the developing bud. The whorl next to calyx is the corolla, which consists of petals, which usually help to attract the pollinators. In some species such as tulips, the sepals and petals look very much alike and act together to provide the color attracting pollinators. Together, the calyx and corolla make up the perianth. The whorl next to the corolla is androecium, which consists of male organs called stamens. Each stamen typically consists of a slender stalk or filament attached to the flower at its base and carrying on its free, upper end, a structure called an anther, which contains the pollen. Finally, the innermost whorl of the flower, the gynoecium, consists of the female organs called carpels. Each carpel consists of a basal ovary containing the ovules, a slender column-shaped structure, the style, and on the end of the style the stigma, the function of which is to receive the pollen grains. Additional whorls, such as the epicalyx, consisting of bracts, which occur outside the calyx, may be present in some flowers such as Cotton.


Fig.1. Longitudinal section of a typical flower. The four main floral whorls, calyx of sepals, corolla of petals, androecium of stamens, and gynoecium of pistils of a typical flower have been labeled in the figure.


The floral morphology discussed above is very typical, but in the real world this logical regular pattern of flower parts is not always so obvious. In atypical flowers, some parts may appear similar or some parts may be missing or some parts or groups of whorls may be coalesced. The most common instance of similarity of parts is resemblance between the sepals and the petals, which has already been mentioned as occurring in tulips. Similarly, brightly colored leaves and bracts surrounding the flower may also be confused with the petals (e.g. Bougainvillea). Many species have evolutionarily lost some parts of the flowers. The most obvious situation is that in which a plant or a species has different male and female flowers. In this case, flowers have lost one sexual function, allowing them to specialize in the other. Cohesion and fusion are common both within and among flowers. The petals may be fused to make a tube, as in a petunia flower. Flowers may combine to form what is called an inflorescence as in Brassicas.

Hermaphrodity and Unisexuality

In a flower, androecium and gynoecium are called the essential floral parts as they are directly involved in reproduction. All the other floral parts are known as the non-essential whorls as they contribute indirectly to reproduction, i.e. by protecting the developing bud or attracting pollinators etc. The flowers, in which one of the essential parts is missing, are called unisexual flowers. Unisexual flowers are subcategorized as pistillate/ female flowers, when only gynoecium is present or staminate/ male flowers when only androecium is present. Different families have different types of flowers, legumes have bisexual flowers with petals modified into banner petal, wing petals, and keel (Fig.1), cucurbits usually have unisexual flowers but may sometimes have bisexual flowers.Fig.2 shows a female watermelon flower.


Fig.2. Longitudinal section of a legume flower. Legumes have perfect flowers. A typical legume flower has a corolla modified into banner, wing petals and keel.


Fig.3. Longitudinal section of a female Watermelon flower. This is an example of unisexual flowers in cucurbits. Among the essential floral whorls, only gynoecium is present, so it is a pistillate/female flower.


Fig.4.1.Terminology. Pink flower = female flower; Blue flower = male flower; Bicolored flower = bisexual/hermaphrodite flower. The first two plants show the dioecious condition in which male and female flowers are borne on separate plants while the fourth plant exhibits monoecy, where male and female flowers are borne on the same plant. The third plant shows the bisexual/hermaphroditic condition with male and female parts in the same flower on the plant.


There exists a specific terminology for plants based on what type of flowers they bear and which type of flowers exist on each plant. The plant is called bisexual/hermaphrodite if it bears only bisexual/hermaphrodite flowers. The plants bearing unisexual flowers are further subcategorized as monoecious if both the male and female flowers occur on the same plant and dioecious if male and female flowers occur on different plants.

Another condition called subdioecy may sometimes occur. Under subdioecy, the plants are subcategorized as andromonoecious if both the male and hermaphrodite flowers; gynomonoecious if both female and hermaphrodite flowers; trimonoecious if female, male and hermaphrodite flowers are borne on the same plant.


Fig.4.2. Subdioecy. Pink flower = female flower; Blue flower = male flower; Bicolored flower = bisexual/hermaphrodite flower. The first plant is gynomonoecious i.e. it bears both the hermaphrodite and female flowers. The second plant is andromonoecious i.e. it bears both the male and hermaphrodite flowers and the third plant is trimonoecious i.e. it bears all three types of flowers, male, female and hermaphrodite.


Self-pollination and Cross-pollination

Sexually reproducing plants can be subcategorized based on the source of the pollen that pollinates the plant. Self-pollination occurs when the pollen from a flower pollinates the stigma of the same flower or another flower on the same plant. A species is said to be cross-pollinated if the pollen from a flower on one plant pollinates the stigma of a flower on another plant. Stebbins (1950) observed that there is a relationship between the length of lifecycle of a plant and its reproduction mode. Since annual plants have fewer opportunities for genetic recombination in their short life span, self-pollination is the key to reproductive assurance (Duvick, 1966). On the other hand, perennials mostly tend to outcross because they have more opportunities to genetically recombine in a life span spread over many years (Duvick, 1966). The terms self-pollinating and cross pollinating crops just mean that one method of pollination is more predominant than the other in that crop because some amount of outcrossing in self-pollinating crops and selfing in cross-pollinating crops commonly occurs.Table1 gives information about the common agricultural crops and their mode of pollination.


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Mechanisms Influencing the Mode of Pollination

a. Morphological mechanisms

i. Mechanisms promoting self-pollination

Monoecy, the presence of male and female organs in the same flower or on the same plant, facilitates self-pollination (e.g. wheat). Cleistogamy, or flowers opening only after pollination has occurred, is also called bud pollination, as the pollination takes place when the flower is still unopened. In this case there is some chance of cross-pollination, as the flower finally opens. However, cleistogamy ensures self-pollination as the flower never opens (e.g. basal inflorescences of California oat grass). Sometimes the morphology of the flower is such that the pistil is enclosed in the staminal cone (e.g. tomato). In such flowers, as soon as the male and female organs reach sexual maturity self-pollination occurs.

ii. Mechanisms promoting cross pollination

In dioecious species, those with different male and female plants, the only possibility is cross pollination (e.g. papaya). Sometimes, in a perfect flower, stamens and pistils attain maturity at different times, such condition is called dichogamy. Dichogamy ensures cross pollination due to lack of synchronization of maturity in the reproductive parts of a flower. Protandry is the condition of a flower if male matures first (e.g. maize), and protogyny if female matures first (e.g. pearl millet).

b. Genetically controlled pollination systems

Male sterility is a condition that occurs when a plant produces non-functional pollen whereas self-incompatibility is a condition in which the plant produces functional pollen that cannot fertilize the female gamete of the same genotype. Self-pollination cannot occur in any of these systems, so the default mode of pollination is cross-pollination. The male sterility and self-incompatibility systems are explained in detail later in this chapter.

Homozygosity and Heterozygosity

The genetic structure of a plant species is largely determined by its reproductive system. In asexually reproducing species, offspring are genetically identical to their parents. Any variation in the asexual progeny is attributable to the environmental effects or a rare genetic mutation. Vegetatively reproducing plants are heterozygous and their heterozygosity is fixed through clonal propagation because no recombination occurs and all the progeny essentially arise from the same plant. In sexually reproducing species, two kinds of mating are possible: self-pollination and cross-pollination. There is no opportunity for gene recombination in self-pollination, except the occasional events of outcrossing. In self-pollinating species, variation is more common among populations than within populations. This trend has been reported in Leavenworthia of the Brassicaceae family (Charlesworth, 1998; Liu et al., 1999). This variation among populations in a self-pollinating species is greater than that observed in a cross-pollinating species. The genetic structure of a species further influences the adaptability of that species. The wider genetic base of the cross-pollinating species gives them better buffering capacity to survive various biotic and abiotic stresses as compared to the self-pollinating species. This idea is supported by the experiment conducted by Stevens (1948) when he estimated the crop losses due to disease in different reproduction systems. The results suggested that maximum disease loss occurs in asexually propagating species, followed by self-pollinating, and finally the cross-pollinating species. However, in cross pollinated crops, continuous (artificial) self-pollination has an adverse effect in the form of inbreeding depression. This occurs due to the accumulation of deleterious recessive alleles, which express in the homozygous state in the selfed plants of a cross pollinated species. The self-pollinated plants do not face this problem because due to continuous selfing over many generations, the deleterious recessive alleles get purged.

The mode of reproduction also influences the genetic structure of the population. Self-pollination increases homozygosity due to accumulation of similar alleles resulting from selfing over several generations, whereas cross-pollination increases heterozygosity due to frequent recombination and segregation. So the genetic structure of a self-pollinated population is heterogeneous with homozygous individuals, and that of a cross pollinated population is homogeneous with heterozygous individuals. The influence of selfing on heterozygisity is demonstrated in Fig. 5.1, 5.2 and 5.3.


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Fig.5.1 & 5.2. Effect of self-pollination on heterozygosity. Fig.5.1 and fig. 5.2 are different ways of demonstrating the effect of self-pollination on heterozygosity. Aa is a single locus with two alleles in a F1 hybrid between two inbred lines with genotypes AA and aa. After the first generation of selfing, the proportion of homozygotes and heterozygotes in the population is same i.e. 50%. But on further selfing, the proportion of heterozygotes decreases while that of homozygotes increases.


Fig.5.3. Effect of self-pollinating on heterozygosity. This figure provides a graphical representation of the effect of self-pollination on heterozygosity. The starting population S0 is 100% heterozygous and after 1 generation of selfing, in S1 the level of heterozygosity falls to 50%. In subsequent generations of selfing, from S2 to S5 , the heterozygosity goes on decreasing and the homozygosity keeps on increasing. Thus, the general trend being: selfing increases homozygosity and decreases heterozygosity.


a. Inbreeding

Self-pollinated crops and cross-pollinated crops response differently to inbreeding. In general, inbreeding is the natural mode of breeding in self-pollinated crops and it produces desirable results by increasing homozygosity and uniformity of the plants. There is no adverse effect of inbreeding on the self-pollinated plants because due to continuous selfing over the generations, the population has been purged of the recessive deleterious alleles. However, enforced inbreeding in naturally cross-pollinating species may lead to drastic consequences. The adverse effects of inbreeding can be illustrated by the results of independent inbreeding experiments in corn, conducted by East (1908) and Shull (1909). Allard (1960) summarized the most important effects of continued inbreeding reported by these investigators as follows: It starts with the appearance of several lethal and subvital types in early generations of selfing, followed by separation of population into distinct lines, which become increasingly uniform within and increasingly distinct from other lines over the generations of selfing. Many of these lines show general decrease in their vigor and fertility and are difficult to maintain even in the most favorable cultural conditions. In the end, even the lines that survive exhibit decreased size and vigor.

b. Hybrid vigor/Heterosis

It is the phenomenon of increased vigor in the hybrids as compared to both of its parents. This phenomenon came to light in the 20th century in corn F1 hybrids. The resulting plant had a higher growth rate, was phenotypically superior and had increased yield as compared to the parents. Because one hybrid could not adapt to the whole country, different hybrids had to be developed in order to adapt to specific areas. The basic mechanisms proposed to be involved in the heterosis are dominance and overdominance. Dominance means that the dominant allele masks the effect of recessive allele. Overdominance means that the combination of genotypes from two different parents leads to supplementing the effect of each other; therefore, the effects lead to increased vigor (Chahal and Gosal, 2002). Most heterosis studies have been done on corn as heterosis has been exploited commercially in corn more than any other crop.

Self Incompatibility

Self-incompatibility (SI) in flowering plants is thought to be an evolutionary advantage due to its effectiveness in avoiding inbreeding and encouraging outcrossing. In recent years, many vegetable and fruit hybrid cultivars have been created by means of SI. One advantage is the possibility to produce F1 hybrids using two SI lines as parental components in order to eliminate laborious emasculation of the female parent. In modern plant breeding, F1 hybrids are one of the most important objectives of breeders. The first F1 hybrid breeding system was established in corn, as ‘hybrid corn’ in the USA in 1921. One problem of this system was high seed costs due to laborious emasculation of the male flower (tassel) (Franklin-Tong, 2008). Self-Incompatibility (SI) is the mechanism utilized by flowering plants (angiosperms) to prevent self-pollination (Silva and Goring, 2001). Therefore, breeders started to utilize SI to achieve a more efficient F1 hybrid breeding system. Because SI prevents self-fertilization and promotes outcrossing, F1 hybrid seeds can be produced readily from two parental SI lines with the help of pollinators.

Mechanisms of Self-Incompatibility

Flowering plants have evolved several unique mechanisms for SI. Some species of flowering plants produce unisexual flowers, which are either male/staminate or female/pistillate thereby acting as natural barrier to self-fertilization (McCubbin and Kao, 2000). However, the vast majority of flowering plants have perfect/hermaphrodite/bisexual flowers, containing both male and female reproductive organs within close proximity on the same flower (Kao and McCubbin, 1996).

SI is broadly categorized into heteromorphic SI and homomorphic SI. Heteromorphic self-incompatibility refers to SI due to some morphological barriers in flowers which occurs in some hermaphroditic flowering plants that produce structurally distinct reproductive organs, for instance: thrum flowers with long stamens and a short style or pin flowers with short stamens and a long style (e.g Primula, Oxalis). Relative positions of reproductive organs pose a topological barrier to self-fertilization (Ebert et al., 1989). Another form of SI is homomorphic SI, in which avoidance of self-fertilization depends on genetic mechanisms. Based on the type of genetic mechanisms involved, it is subcategorized into gametophytic SI (GSI) and sporophytic SI (SSI).

Gametophytically controlled SI is the most widespread SI system. SI is usually controlled by a single S locus that has multiple S-alleles (Franklin-Tong and Franklin, 2003). In some species (e.g. the grasses), there are two loci, S and Z (Franklin-Tong and Franklin, 2003). In the GSI system, the SI phenotype of the pollen is determined by its own haploid (gametophytic) genome. In the SSI system, the pollen SI phenotype is determined by the diploid genome of its parent (sporophyte) (Silva and Goring, 2001) (Fig.6).


Fig.6. Self-incompatibility systems. (A) Gametophytic self-incompatibility: The SI phenotype of the pollen is determined by its own haploid (gametophytic) genome. (B) Sporophytic self-incompatibility: The pollen SI phenotype is determined by the diploid genome of its parent (sporophyte) (Adapted from Silva and Goring, 2001)


Self-Incompatibility as an Important Trait in Plant Breeding

The advantage of SI in plant breeding is that heterozygosity is promoted by outcrossing. In modern plant breeding, F1-hybrids are of great economic importance in a number of crops on account of their uniformity and hybrid vigor (heterosis) (Franklin-Tong, 2008). One efficient method of producing F1-hybrid seed on a large scale is utilizing SI. The SI trait is essential to avoid contamination by self-pollinated seeds (Franklin-Tong, 2008). The main problem of utilizing SI is establishment of two pure line parents in SI plants. To resolve this problem, breeders choose the SI lines from a large number of seed stocks and use physiological or genetic breakdown of SI to produce pure lines because SI is not always stable (Franklin-Tong, 2008). SI can be easily overcome under various external and physiological conditions such as treatment with CO2 gas, irradiation and pistil grafting (Nettancourt, 2001).

In the SSI system, receptor protein S-receptor kinase (SRK) covers the stigmatic surface just prior to anthesis and acts as a barrier for penetration of the stigma by germinating pollen grains (Silva and Goring, 2001). Seed sets of pure line parents can be obtained if pollen is applied after buds opening and before the SRK protein barrier formed (Franklin-Tong, 2008). This procedure is called bud pollination. By using bud pollination, SI can be overcome. Thus, self-pollinated seeds can be produced and SI parental lines can be maintained (Franklin-Tong, 2008). However, efficacy of production of a large number of parental seeds of F1-hybrid by bud pollination needs to be enhanced (Franklin-Tong, 2008). Unfortunately, none of the major field crop plants has the SSI system. Brassica SSI is the most extensively studied SI system (Franklin-Tong, 2008). The well-known genetic and molecular mechanisms of Brassica SSI make Brassica an important subject in plant breeding. The first successful F1-hybrid variety of cabbage (cv. Suteki kanran) by employing the SI trait was produced in a Japanese seed company, Sakata Seed Co. in 1940 (Franklin-Tong, 2008). Later, in 1950, another Japanese seed company, Takii & Co. Ltd., released F1-hybrid varieties of cabbage (cv. Choko-1c) and Chinese cabbage (cv. Choko-1cc) (Franklin-Tong, 2008).

Breakdown of Self-Incompatibility

When hybridization involves species that have SI, this barrier to self-pollination must be overcome or lost in order to establish parental pure lines. Different types of modifications can lead to breakdown of SI. The physiological modifications are often temporary and cannot be transmitted from one generation to the next (Nettancourt, 2001). Genetic modifications may or may not be permanent and result in various effects (Nettancourt, 2001). Recently, hybrid self-fertility resulting from epigenetic changes in expression of the S-locus genes has been demonstrated (Nasrallah et al., 2007). Epigenetic mechanism for breakdown of SI in hybrids is reversible and noteworthy in facilitating hybrid in the future.

a. Physiological breakdown of self-incompatibility

There are various physiological factors and environmental circumstances that can prevent SI. Because SI phenotype of the pollen and pistil is fully determined in the mature flower, selfing can be induced by using immature material in which the S phenotype is not yet expressed (bud pollination) or by using old flowers and aged pollen, in which SI are getting weaker (Mable, 2008). Several studies also point out that heat treatment at a temperature ranging from 32°C to 60°C can lead to breakdown of SI in the pistil during the first two days following pollination in many plant genera (Lilium, Trifolium, Lycopersicum, Raphanus and Brassica) (Nettancourt, 2001). The mechanism of heat treatment is still not clear (Nettancourt, 2001). Another applicable method for breakdown of SI is treatment with CO2 gas. The most active concentrations of CO2 gas range between 3% and 5% (Franklin-Tong, 2008). The timing of the CO2 gas treatment is critical, which is usually right after pollen grains are germinated on the stigma papilla cells (Nettancourt, 2001). Although the mechanism of the CO2 gas effect has not been elucidated and genetic variations exist in the reaction to treatment of CO2 gas, CO2 gas treatment has replaced bud pollination with honey bees on large-scale propagation of the parental seeds of F1-hybrid in Brassica (Nettancourt, 2001). A special method of breakdown of SI is the “mentor effect”, which results from pollination with mixtures of compatible (mentor) and incompatible pollen (Nettancourt, 2001). The mentor effect can be enhanced by using inactivated or dead pollen (Nettancourt, 2001). The mentor effect has been applied successfully to a wide range of different plant genera, such as Citrus, Cola, Crocus, Lotus, Paspalum and Theobroma (Nettancourt, 2001). However, the understanding of the effects of mentor pollen is limited and mentor effects are not always reproducible (Nettancourt, 2001). Other methods of physiological breakdown include irradiation, hormones, pistil grafting (Nettancourt, 2001).

b. Genetic breakdown of self-incompatibility

Genetic breakdown of SI occurs in nature and can be induced by breeders artificially. Tetraploid relatives (or induced tetraploids) derived from GSI diploid progenitors usually display a self-compatible (SC) phenotype (Horandl, 2010). Breakdown of SI occurs in pollen, whereas the pistil maintains the function of identifying incompatible pollen and then rejects it. Self-fertilization results from a loss of the incompatible phenotype in the diploid pollen produced by the tetraploid plant (Nettancourt, 2001). For example, pollen from SC Petunia axillaris tetraploid (S1S1S2S2) can grow in both SI progenitor (S1S2) diploid and SC tetraploid (S1S1S2S2) pistils (Nettancourt, 2001). Remarkably, SI breakdown functions only in diploid heteroallelic pollen (S1S2), which is called competitive interaction (Nettancourt, 2001). The mechanism of a competitive interaction that does not occur between identical alleles but take place between different ones is not known (Nettancourt, 2001). On the other hand, modern biotechnologies including site-directed mutagenesis, the use of anti-sense DNA, exploitation of the silencing effects of certain duplications or the swapping of S-locus DNA sequences can be applied to make irreversible switches from SI to self-compatibility (Horandl, 2010).

Applications of Self-Incompatibility in Plant Breeding

The most important application of SI is in Brassica breeding. Vegetable Brassicas are an important and highly diversified group of crops grown world-wide. In western countries, cultivated Brassica crops include cabbage, broccoli, cauliflower and brussel sprouts (Kole, 2007a). In Asia, chinese cabbage is the most important Brassica crop (Kole, 2007). In recent years, most of the vegetables cultivated over a large part of the world have been F1 hybrid varieties. However, F1 hybrids of Brassica have progressed slowly owing to instability and complex inheritance of the SI (Kole, 2007a). Two major seed production methods employing the SI system are used in Brassica crops. One method is the single cross, in which SI is overcome in pure line parents by treating them with 4-5% CO2 gas (Franklin-Tong, 2008). The other method is a double cross, in which hybrid seeds are produced by two near isogenic lines for each parent (self incompatible, but cross compatible).

Cauliflower is one of the most attractive crops within the Brassica species. The advantages of F1 hybrids in cauliflower have been demonstrated in uniform maturity, early and high yield and biotic and abiotic stresses (Kucera, 2006). An interesting study compared two mechanisms used in commercial hybrid seed production of cauliflower, SI and cytoplasmic male sterility (CMS) (Kucera, 2006). In this study, hybrid seed production of SI lines derived from the cultivar Montano were achieved by spraying with 3% NaCl solution in the evening and using bumblebees as pollinators (Kucera, 2006). Hybridization experiment in two CMS line cultivars (Brilant and Fortuna) achieved seed set via honeybee pollination with their fertile analogues respectively (Kucera, 2006). The conclusion generated from this study was that both SI and CMS lines are suitable for hybrid breeding of cauliflower. In regards to seed production, SI appears to be more effective than CMS (Kucera, 2006).

Male Sterility

Male sterility occurs in plants where pollen or anthers fail to function properly. Male sterility was first described by Kölreuter in 1763 when he observed abortion of anthers in specific hybrids (Vinod, 2005). Male sterility can be caused by many factors, including mutations, diseases, or unfavorable environmental and growth conditions (Budar and Pelletier, 2001). The effects of male sterility can vary greatly depending on the species and environment. Some specific effects include absence of stamens in bisexual species, no male flower production in dioecious species, and failure to produce pollen-forming tissues in anthers (Vinod, 2005). Other effects include viable pollen produced in non-dehiscent anthers, deformed or non-viable pollen grains, and abnormal pollen maturation (Vinod, 2005).

Male sterility systems have been exploited in the plant breeding world to help breeders produce F1 hybrid cultivars. The first system using male sterility was developed in 1943 for onions, and soon after, systems were developed for corn, beet, sorghum, rice, sunflower, carrot, and rapeseed (Budar and Pelletier, 2001). Male sterility also plays an important role in keeping newly-introduced plant species from becoming invasive (Gardner et al., 2009). Many of the plant species categorized as invasive today started out as introduced ornamental plants, and male sterility can help prevent unwanted gene flow between invasive and native species. Ornamental crops can also benefit from male sterility by redirecting plant resources away from seed production, allowing for more vegetative and flower growth (Gardner et al., 2009).

Types of Male Sterility

Three types of male sterility exist: genetic male sterility (GMS), cytoplasmic male sterility (CMS), and cytoplasmic-genetic male sterility (CGMS). Genetic male sterility is controlled by a single recessive gene (ms) in the nucleus (Edwardson, 1970). Effects from this type of sterility include reduced pollen production, reduced anther size, and total pollen abortion (Poehlman and Sleper, 1995). GMS is quite common in nature, but it is not very useful to plant breeders because it cannot be used to maintain a pure line of male-sterile crops (Acquaah, 2007). Because male-sterile plants are homozygous recessive (msms), they must be crossed with a heterozygous (Msms) source of pollen, then the male-sterile offspring can be selected (Acquaah, 2007). Because only half of the seeds that result from crossing the male-sterile with the male-fertile plants can be used for future breeding (Poehlman and Sleper, 1995), genetic sterility is not the most optimal method for inducing male sterility in plant breeding. GMS has been observed in sunflower, tomato, cucurbits, wheat and barley.

CMS is a maternally-inherited sterility caused by expression of a mitochondrial gene which causes the production of non-viable pollen without affecting other functions of the plant (Budar and Pelletier, 2001). CMS plants still maintain normal female fertility and normal vegetative growth (Mihr et al., 2001). Two types of cytoplasm behavior can be seen, normal (N) and sterile (S) (Vinod, 2005). CMS is often induced by inter- or intra-specific crosses which combine different nuclear genes with different cytoplasm (Mihr et al., 2001). This sterility system is quite common in nature and has been observed in over 150 plant species (Schnable and Wise, 1998). Some plants naturally carry recessive male sterile genes in their cytoplasms which can be accidentally maintained in hybrid breeding lines, and these male sterile genes may eventually express as a result of a natural mutation (Schnable and Wise, 1998). Effects of CMS include abnormalities such as production of non-viable pollen, absence of stamens, and abnormal production of the cadastral (boundary) patterns in flowers (Pelletier and Budar, 2007). Anther and pollen development may be inhibited at different stages in plant development, either before or after meiosis (Mihr et al., 2001). CMS has been observed in maize, cotton, rice, and sorghum.

Cytoplasmic-genetic male sterility is influenced by both nucleic and mitochondrial genes and is very common in the plant world (Vinod, 2005). There are three different scenarios for CGMS system (Fig. 7), including the crossing of plants with normal/sterile cytoplasm with the plants carrying different fertility restorer genotypes i.e. RfRf, Rfrf, and rfrf. In the first case, sterile cytoplasm is paired with Rfrf, the outcome of this system will always be restoration of male fertility. In the second case, sterile cytoplasm is paired with restorer genes rfrf or Rfrf. Even though sterile cytoplasm is included, the presence of the heterozygous restorer gene mandates the outcome of this system to be male-fertile while plants with the homozygous recessive nuclear genotype will be sterile. In the third case, sterile cytoplasm is partnered with the homozygous recessive restorer genotype, rfrf. Fertility is not restored to the breeding line in this system because of the presence of both sterile cytoplasm and the homozygous recessive restorer genotype (Edwardson, 1970). CGMS has been used in commercial hybrid seed production of sorghum, maize and pearl millet.


Fig. 7 Cytoplasmic-genetic male sterility system: In case (A) sterile cytoplasm is paired with restorer genes Rfrf and even though sterile cytoplasm is included, the outcome of this system will be male-fertile if the restorer genotype is heterozygous dominant Rfrf and male-sterile if the restorer genotype is homozygous recessive rfrf. In case (B) sterile cytoplasm paired with rfrf restorer genes will yield male sterile plants, while sterile cytoplasm paired with Rfrf will yield male fertile plants. In case (C), sterile cytoplasm is partnered with the homozygous recessive restorer genotype, rfrf, yielding sterile plants (Adapted from Allard ,1960).


Mechanism of Fertility Restoration in CGMS hybrid lines

Plant breeders use CMS systems to produce hybrid seeds by developing female lines carrying CMS cytoplasm and by breeding male lines that carry the restorer genes (Schnable and Wise, 1998). Crossing these lines yields fertile progeny because the male maintainer lines contribute nuclear restoring genes (Schnable and Wise, 1998). The first crops that used the CMS fertility-restoring gene system as a method for commercially producing hybrid seed were sorghum and corn (Poehlman and Sleper, 1995).

The main CMS model used for hybrid lines utilizes a system with three lines. The A-line consists of lines that are seed-bearing and are used as the female parent line. A-lines contain the homozygous recessive non-restorer genes (rf1, rf2). They are then backcrossed with inbred lines which do not contain restorer genes and are a source of CMS. Five to seven backcrosses are usually necessary until the wanted genotype from the A-line is recovered in the male-sterile cytoplasm. A male-fertile line, the B-line, is used to pollinate the A-line and maintain its male-sterility. The B-line has the same genotype as the A-line and the same non-restorer genes (rf1, rf2), but with normal cytoplasm (Poehlman and Sleper, 1995).

A third line, called the R-line, is used as the fertile pollen-giving parent in a hybrid cross. R-lines are also used to restore fertility to progeny of the hybrid cross (Poehlman and Sleper, 1995). R-lines are chosen based on their ability to produce large amounts of pollen and anthers that are suitable for proper pollen dissemination .The R-lines are able to restore fertility to the hybrid progeny because they contain the nuclear restorer genes Rf1, Rf2. They may have either normal or sterile cytoplasm, but it is advantageous for breeders to use R-lines with sterile cytoplasm because presence of restorer genes can be more easily conferred (Poehlman and Sleper, 1995). R-lines are also expected to cross with A-lines to produce very vigorous hybrid progeny (Poehlman and Sleper, 1995). This superior combining ability is an important aspect of R-lines that plant breeders look for when trying to obtain higher yielding crops (Poehlman and Sleper, 1995). One study showed that R-lines with higher ability to restore fertility can produce hybrid cotton plants with higher heterosis levels (Zhang et al., 2010).

The usable hybrid seed is produced from the A-line after it has been crossed with the R-line. In commercial production, where large amounts of seeds need to be produced, R-lines are planted in alternating rows between A-lines. Pollen from the R-line is blown by wind and naturally pollinates the A-line plants. This method is a much easier and more efficient tool for pollinating hybrid lines than some other, more labor-intensive methods, such as hand emasculation and hand pollination (Poehlman and Sleper, 1995).

A genetic-cytoplasmic male sterility model was discovered in the 1940s in corn (Zea mays) and utilized until the 1970s (Vinod, 2005). This system, called Texas cytoplasmic male sterility (CMS-T), was used in 85% of all US corn in the 1970s (Vinod, 2005). During the peak of CMS-T use, a new strain of the southern corn leaf blight pathogen, Bipolaris maydis, developed and wiped out all of the CMS-T corn in the Eastern US (Vinod, 2005). The CMS-T corn carried a specific gene, T-urf13, which caused chimeric sequences to be expressed in mitochondria, thus causing male sterility (Budar and Pelletier, 2001). However, this gene also caused the corn crop to be highly susceptible to two fungal pathogens, Bipolaris maydis and Phylostica maydis (Budar and Pelletier, 2001). The T-urf13 gene was highly susceptible to a specific toxin (T-toxin) produced by the T-strain of Bipolaris maydis (Vinod, 2005).

Cytoplasmic male sterility is an important area of plant breeding and crop research. The ability to control the fertility of hybrid progeny has many advantages to farmers and breeders. Male-sterile plants make the breeder’s job easier by eliminating the risk of unwanted cross-pollination and gene flow. Eliminating risk of gene flow also greatly assists farmers. They expect all hybrid crops to turn out similar in size and quality because that is what the consumer market demands. Male sterility also allows F1 hybrid seed production companies to obtain more consistent profit because farmers cannot save seed. The current research and future outlook of engineered male-sterile crops looks promising. More research needs to be done on finding applicable systems within each specific crop, but the end benefits should be worth the effort.

Interspecific Hybridization

Interspecific hybridization is also called wide hybridization as it involves crossing distantly related or sometimes unrelated species. Many problems are encountered in intercrossing these species. Sometimes the plants can be easily crossed but the hybrid formed is sterile. In other cases, the hybrid resulting from such crosses get aborted at embryonic stages. In the worst situations, there are some morphological reproductive barriers which do not even allow the crossing and fertilization to occur easily. Various strategies such as bridge crossing, somatic hybridization, induced polyploidy, embryo rescue and use of plant growth regulators can be employed to overcome these problems and accomplish successful interspecific hybridization in widely related species. When two species cannot be intercrossed to produce a fertile hybrid, a third species which can be easily crossed with both the parental types can be used to produce a bridge cross. R.C Buckner and his colleagues have used this method to cross Italian ryegrass (Lolium multiflorum) with tall fescue (Festuca arundinacea) using meadow fescue (Fescue pratensis) as the bridging species (Aquaah,2007).

Somatic hybridization is a technique that can be used to bypass the mating barriers between widely related species. The individual somatic cell protoplasts from both the species are fused using polyethylene glycol (PEG) or electric fusion and a somatic cell hybrid is formed. This somatic hybrid is later regenerated into a full hybrid plant. Solanum brevidens and S. phureja have been used in somatic hybridization technique to introduce disease resistance for Potato leaf roll virus and Potato virus X into the S. tuberosum species. The nuclear genome can be rendered inactive by treating with Iodoacetate to get the desired hybrids (Chahal and Gosal, 2002). Embryo rescue can be employed in situations when there are risks of embyo abortion or loss of viability of the hybrid seeds. In research regarding the production of hybrids between Cicer arietinum and C. bijugum (wild variety), it was demonstrated that the hybrid produced from the cross did not yield viable seeds. However, if the embryos are cultured on the suitable media they can be regenerated into plantlets and then transferred to soil (Clarke et al., 2006). Plant growth regulators such as auxins and gibberlins can be used to save the weak hybrid plants from the crosses that do not survive under natural conditions. Induced polyploidy is helpful to partially or completely overcome the sterility in wide intercross hybrids (Fehr et al., 1980).

Asexual Reproduction

Asexual reproduction in plants is of two types: Vegetative propagation and apomixis. Some plants naturally reproduce vegetatively through tubers (e.g. potato), rhizomes (e.g. ginger), bulbs (e.g. onion) etc. Others are artificially propagated vegetatively using various methods like grafting, layering, budding etc.. Vegetative reproduction is extremely common in perennial plants, especially in grasses and aquatic plants. However, there are very few species that rely solely on vegetative reproduction. Mostly the species that reproduce vegetatively also reproduce sexually through seed (e.g. Trifolium repens)(Burdon,1980).

Apomixis is the process by which plants reproduce asexually through seed (Nogler, 1984). Nogler (1984) divided apomixis into three main groups according to the origin and development of the maternal embryos: apospory, diplospory and adventitious embryony. In aposporous species, embryo sacs are formed from the nucellar cells while in diplospory, the megaspore develops from the reproductive tissue but meiosis fails partially. In adventitious embryony, embryo develops directly from a somatic cell of the megagametophyte. Apomixis may be facultative or obligate. It is said to be facultative when some progeny from a plant may result from a normal meiosis and/or normal fertilization in addition to the apomictic progeny. Apomixis is obligate when all the progeny are maternal and there is no chance of developing a progeny from sexual reproduction in that plant. A major potential application is “hybrid crops that clone themselves” (Carman et al., 1985). Because the hybrids formed by wide hybridization may be sterile, they can only be asexually propagated and apomixis will facilitate their asexual propagation through seed. Other advantages of apomixis include uniformity of plants and virus free propagation because viruses are usually not transmitted through seeds. However, apomictic breeding has not realized its potential because there are very few economically important apomictic crops. Apomixis is less widespread than vegetative reproduction, although it has been reported from at least 30 families of flowering plants (Grant, 1981), and it is especially common in grasses.  

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