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Male Sterility in Vegetables

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 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
432 OLERICULTURE–I : FUNDAMENTALS OF VEGETABLE PRODUCTION

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 (mt-
genome) 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.
MALE STERILITY IN VEGETABLES 433

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 (S-
cytoplasm) and recessive allele of restorer (maintainer allele; rf) is located in the nuclear genome.
434 OLERICULTURE–I : FUNDAMENTALS OF VEGETABLE PRODUCTION

TABLE 2
Genic male sterility or nuclear male sterility (GMS) in selected vegetable crops
Crop Salient features of gms
Tomato† 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.
Brinjal Monogenic recessive gene has been reported; monogenic recessive functional
sterility available in India and utilized for developing experimental crosses.
Pepper† More than 12 recessive genes have been reported; MS-12 (ms-509/ms-10) and ms-
3 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 (MS-
12 × 2025) hybrids. The ms-10 gene of MS-12 line is linked with taller plant
height, erect growth and dark purple anther.
Cauliflower Both recessive and dominant genes have been reported.
Cabbage 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.
Brussels sprout Recessive male sterile mutant has been reported but not utilized due to availability
and utilization of CMS system.
Broccoli Six recessive non-allelic genes have been reported; linkage of ms gene with bright
green hypocotyl.
Watermelon Recessive mutants have been reported; linkage of ms gene with delayed-green
(dg) seedling marker gene.
Muskmelon† 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).
Cucumber Monogenic recessive gene has been reported; limited scope of utilization because
of the availability and utilization of of gynoceous lines.
Summer squash Monogenic recessive gene has been reported; very limited scope of utilization
because of the availability of sex regulating mechanism using certain chemicals.
Onion Monogenic recessive genes are known but not utilized at commerial scale due to
availability and utilization of CMS system.
Carrot Recessive male sterile genes have been reported; not utilized at commercial scale
because of the availability of CMS lines as an alternative method.
Radish 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
MALE STERILITY IN VEGETABLES 435

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 Salient features of cms
Tomato Sterile cytoplasm has been derived from the distinct species through protoplast
fusion; restorer gene (Rf) is not available.
Pepper† 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.
Cole crops 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.
Onion† Two types of sterile cytoplasms, viz., S and T have been reported; S-cytoplasm is
most widely exploited.
Carrot† 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.
Radish† 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
436 OLERICULTURE–I : FUNDAMENTALS OF VEGETABLE PRODUCTION

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
MALE STERILITY IN VEGETABLES 437

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 Beet
FW 450 Beet, tomato
Gibberellic acid (GA3) Lettuce, onion, maize
Maleic hydrazide (MH) Cucurbits, onion, tomato
Naphthalene acetic acid (NAA) 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
438 OLERICULTURE–I : FUNDAMENTALS OF VEGETABLE PRODUCTION

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.
 MALE STERILITY IN VEGETABLES 439

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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