Male sterility
• When a plant fails to produce functional pollen, it becomes male sterile and the
phenomenon is male sterility. Male sterility in plants exhibits in diverse forms like
absence of normal anthers, reduction in anther size, varying degree of petaloidy,
abnormal meiosis followed by formation of empty, shrivelled microspores, normal
meiosis followed by abnormal development of microspores and normal meiosis but
failure of anthesis so that pollen is not shed.
• Based on its inheritance or origin, male sterility may be further divided into: (1) 'Nuclear
male sterility' (NMS), also called 'genic', 'genetic' or 'Mendelian', in which the male sterility
is governed solely by one or more nuclear genes; 'Cytoplasmic male sterility' (CMS) in
which male sterility comes about as a result of the combined action mitochondrial genes
with coupled nuclear genes in other words “maternally inherited deficiency in producing
viable pollen”.
Why male sterility
• Traditionally, the plant breeders are emasculating the anthers by hand to prevent self-
pollination for hybrid production and obviously the cost of such hybrid seeds are too high.
• On the other hand, use of male sterile lines enable the breeders to produce hybrid seeds
at low cost.
• Male-sterility plants provide crucial breeding tools to harness hybrid vigor, or heterosis,
in hybrid crops and also provide important materials to study stamen and pollen
development and cytoplasmic-nuclear genomic interactions.
• Hybrid vigor, or heterosis, refers to the phenomenon in which the progeny derived from
a cross of two inbred lines outperform the parent lines. For example, hybrid crops can
produce 15–50% higher yields than inbred varieties
Causes of Male Sterility
• Tapetum is the sporophytic tissue and innermost wall layer of the micro-sporangia in the
angiosperm plants. It plays an important role in the development of male gametophyte
(microspore). Tapetal degeneration is a programmed cell death (PCD) event with typical
cytological features of mitochondria and cytoskeleton degeneration, nuclear
condensation, oligonucleosomal cleavage of DNA, vacuole rupture, and endoplasmic
reticular swelling. Tapetal PCD at a specific developmental stage is crucial for pollen
fertility, and disruption of the timing of PCD, either early or delayed, results in pollen
abortion or male sterility
Result of Male sterility
• Male-sterility mutants can cause abnormal development of either the sporophytic or
gametophytic anther tissues. Most sporophytic male-sterility mutants affect primarily
tapeta and meiocytes (cells undergoing meiosis), leading to pollen abortion or pollenless
sterility. By contrast, gametophytic male-sterility mutants affect mainly the development
of microspores or pollen grains.
• The mitochondrial mutation which impairs the proper functioning of mitochondria,
leading to Cytoplasmic male sterility. Such mutated mitochondrial genomes would
obviously never have been selected for in natural conditions.
• Therefore, maternally inherited male sterility resulting from a specific (mitochondrial)
gene whose expression impairs the production of viable pollen without affecting the
plant.
Reason to choose CMS
• Nuclear male sterilities are generally recessive mutations which affect a huge number
of functions, the recessive inheritance of GMS made it difficult to fully utilize its
potential, because the resultant progeny is 50% sterile and 50% fertile. Hence the
breeders are more inclined towards CMS where 100% sterility is achieved.
Importance of CMS
• The ability to artificially suppress pollination and so prevent a plant’s self-fertilization,
thereby encouraging cross-pollination and higher-yielding seed through an effect
known as ‘hybrid vigor’.
• CMS, being extrachromosomal in inheritance, transmits its expression to the whole
progeny since the male gamete generally contributes a very small amount of
cytoplasm to the zygote.
• However, in many cases of CMS, parental genotype may totally or partially restore the
male fertility expression of the progeny of a CMS maternal seed line.
• The ability to genetically engineer such suppression of male fertility into elements in
the cytoplasm, rather than the nucleus — the result is transmission of desirable
characteristics through genes in the female line, and those genes cannot ‘escape’
uncontrollably via pollen.
Fertility Restoration
• CMS-based hybrid seed technology uses a three-line system, which requires three
different breeding lines: the CMS line, the maintainer line, and the restorer line (Figure
1a).
• The CMS line has male-sterile cytoplasm with a CMS-causing gene (hereafter termed
a CMS gene) and lacks a functional nuclear restorer of fertility (Rf, or restorer) gene or
genes (122), and is used as the female parent.
• The maintainer line has normal fertile cytoplasm but contains the same nuclear
genome as the CMS line, and thus serves as the male parent in crosses for the
propagation of the CMS line.
• The restorer line possesses a functional Rf gene or genes, and thus serves as the male
parent to cross with the CMS line to produce F1 hybrid seeds. In the F1 plants, the Rf
gene restores male fertility, and the combination of nuclear genomes from the CMS
line and the restorer line produces hybrid vigor.
The CMS line is propagated by crossing with the maintainer
line; the maintainer and restorer lines can produce seeds
by self-pollination. The CMS line is crossed with the
restorer line to produce male-fertile hybrids.
1. Production of Male Sterility Plants
• Extensive progress has been made in cell and tissue specific gene expressing and
discovery of novel genes which can potentially inflict tissue ablation through genetic
engineering led to the successful production of male sterile plants partially or
completely. The basic concept adapted in the strategy is to target the expression of
gene encoding a cytotoxic driven under the control of an anther specific promoter.
• Tapetal cells are targeted and make them dysfunction or breakdown of the key
molecules inside the tapetal cells leading to non-functional pollen grain, which result
in male sterile lines.
2. According to the studies following reports have been implicated in the production of
male-sterile plants:
a) Tapetum specific expression of cytotoxic degradation enzyme using barnase RNase
system.
b) Premature dissolution of microsporocyte callose wall leading to male sterility.
c) Inhibition of particular enzyme or protein production by antisense technology.
d) Induction of mitochondrial dysfunction by transgenic expression of unedited RNA.
• In one of the earliest transgenic works on induction of male sterility, synthetic gene RNA-
T1 from the fungus Aspergillus oryzae have been employed by Quacus (1998). In another
attempt premature dissolution of the callose wall result in male sterility was observed in
transgenic tobacco.
• Before meiosis, (in angiosperms) microspores synthesise callose deposition continues
through the meiosis and microspore is surrounded by callose. The callose wall is
breakdown after meiotic process. For example, male sterility in petunia has been
attributed to the early appearance of active callose and β-1.3-glucanase in the anther
locule.
• As a consequence, this led to premature dissolution of callose wall surrounding the
microspore cells. Transgenic tobacco expressing modified glucanase from tapetum
specific promoters led to premature dissolution of callose after prophase I of meiotic
stage. Transgenic tobacco exhibit reduced male sterility, ranging from complete to partial
male sterility.
• This demonstrates that premature callose degradation is sufficient to cause male sterility
and suggest that callose is indispensable for the formation of normal microspore cell wall.
The callose is not exactly part of the cell wall but it participates in the formation of physical
barrier against pathogenic attack and also prevents all cohesion and fusion.
TA 29 Barnase Male Sterility:
➢ One of the most effective and classic case of male sterility induction was carried out
by the complete destruction of RNA molecule in tapetal cells. It was demonstrated
that chimaeric RNase and barnase gene driven under the control of tapetum specific
promoter TA 29.
➢ Where it can induce male sterility in tobacco and oil seed rape. The tissue specific TA
29 gene is highly regulated and specifically expressed in tapetum cells of anther.
Expression of TA 29 barnase fused gene consequently acts as cytotoxic and selectively
destroys the tapetal cell layer by complete elimination of RNA molecule.
➢ Tapetal cell is a nutritive layer which surrounds the pollen sac in the anther pollen
development requiring nutrition obtained from tapetal cells. Therefore, destruction
of tapetal cells cut off food supply, arrest pollen development or non-functional pollen
will be formed to produce sterile plants.
➢ The destruction of tapetum by RNase and RNase T1 gene derived from bacterium
Bacillus amyloliquefaciens and fungus Aspergillus oryzae, respectively. The same TA
29 RNase gene combination induced sterility was also produced in maize and other
vegetable species.
TA 29 Barstar Male Fertility:
➢ Expression of a novel gene known as barstar derived from the same bacterium Bacillus
amyloliquefaciens lead to the production of normal male fertile plant without
interfering its pollen development. Bacillus amyloliquefaciens expressing barnase
gene has corresponding inhibitor protein called barstar. Barstar is produced
intracellularly and protect bacteria from the harmful role played by barnase by
forming stable complex with the barnase in the cytoplasm.
➢ The gene 1.5 kilobase barstar was fused to tapetum specific TA 29 gene upstream
fragment that comprises all regulatory segments targeted for tapetum specific
expression. Mariani et al., 1992 introduced TA 29 barstar into oil seed rape by
employing Agrobacterium Ti plasmid and biolophase selectable marker gene. Barstar
expressing transformants were male fertile, produced normal anther with well-
developed anther tapetal cells.
Fertility Restoration by Barnase-Barstar System:
➢ Once male sterility is induced, plants are then can be exploited for introduction of
hybrid seed production and plants are maintained as male sterile lines. Restoration of
fertility has been accomplished by crossing male sterile plants with male fertile plants
expressing barstar gene.
➢ To determine whether TA 29 barstar gene expression could inhibit barnase activity in
tapetal cells and led to male fertile restoration, Mariami selected TA 29 barstar gene
containing male fertile plants and crosses with male sterile parental plants expressing
TA 29 baranse single copy germlines.
➢ Both barnase and barstar gene co-expressed specifically in the tapetal cells of anther
led to the formation of barstar-barnase stable complex. In the F1 progeny ratio of
fertile to sterile would be 2:1 ratio. All F1 progeny from the crosses that were
expressing both chimeric genes were male fertile led to the conclusion that TA 29
barstar gene functioned as a dominant suppressor of TA 29 barnase gene activity (Fig.
21.2)
• Plants which are restored to fertility are in distinguish from those of wild-type plants
develop normally and exhibited normal anther dehiscent process. They have well
developed tapetal cell layers and produce vast amount of functional pollen grain. Thus,
effectiveness of chimeric TA 29 barnase and TA 29 barstar genes system in the male-
sterility induction and male fertility restoration may permit the breeding of genetic
engineering hybrid plant.
• Transgenic lines exhibiting the barstar gene have also been developed in Indian Mustard,
Brassica juncea, to develop a complete male sterility and restoration system for heterosis
in this crop. Transgenic mustard lines containing a modified barstar were also raised using
modified sequence of the barstar gene.
• Another example of obtaining parental male sterility in transgenic tobacco by blocking the
synthesis of flavanols and starch in pollen grain has been reported by Mutsuda (1996).
Reduction in levels of flavanols and sporopollenin of the pollen wall has been demon-
strated to be associated with abnormal microspores and abnormal growth of pollen tube
led to reduced fertility.
• Pollen cells have plenty of actin, which is an essential protein in cell function, blocking the
production of actin by antisense actin gene expression leading to male sterile plants in
crop plants.
• Phenyl ammonia lyase, key enzyme (PAL) in phenyl propanoid pathway, responsible for
the synthesis of flavanoids. Introduction of sense or antisense PAL cDNA of sweet potato
under the control of tapetum specific rice promoter into tobacco generated transgenic
plants. This exhibited reduced pollen fertility. The pollen fertility of these plants droped
from 8% to 6%.
• Reduction of PAL activity in anther was correlated with the number of fertile pollen grain
at flowering stage. These results demonstrated that manipulation of phenyl propanoid
pathway by transgene provides a powerful tool for a alternative of phenyl propanoid in
pollen and lead to reduced fertility.
• Molecular control of male fertility has been studied in Brassica. The gene, BcP1, essential
for production of functional pollen grain has been manipulated by their expression using
antisense approach. The specific down regulation of BcP1 further demonstrated the
importance of this gene during pollen development.
• Transgenic Arabidopsis plants in which the BcP1 gene expression is blocked either in
tapetum or in developing microspore using Lat52 promoter show arrest in pollen
development leading to pollen abortion and exhibit male sterile phenotype.
• Apart from antisense approach or production of degradative enzymes for flavanoid
biosynthesis, deploying unedited gene targeted to tapetal mitochondria induces male
sterility. The expression of transgene designed to contain the unedited atp 9
mitochondrial gene (u atp 9) fused to the yeast COXTV led to the production of male sterile
plants. In contrast expression of edited transgene in the same plant led to the production
of male fertile plants with normal pollen development.
• RNA editing in plant mitochondria is a post-transcription process improves protein
synthesis of mitochondrial proteins which are basically derived from edited mRNA.
However, several exceptions have been recorded. Expression of unedited gene led to the
production of non-functional proteins and produce male sterile plants.
• The modification of mitochondria was correlated with presence of translated product of
unedited atp 9 and a significant decrease in oxygen consumption in non-photosynthetic
tissue. The high reduction of CO2 consumption in root meristems of male sterile plants
confirms improvement of mitochondrial function.
• Due to the drop in respiration, transgenic male sterile plants are unable to maintain the
high energy level (ATP) required for anther development and micro-sporogenesis. These
factors suggested that RNA editing is indispensable in the production of functional
proteins.
• Carbohydrate was shown to play a critical role in anther and pollen development. Ac-
cording to studies, different male sterile lines were shown to be characterised by altered
carbohydrate metabolism. Once carbohydrates are produced in source region, they are
transported to sink region or inactive sink tissues by unloading pathway.
• An extracellular invertase bound to cell wall hydrolyses to transport sucrose. The
importance of extracellular invertase to assimilate partitioning and source sink regulation
by supplying carbohydrate to sink tissues via the apoplast has been suggested. The specific
interference with phloem unloading at metabolic signalling during pollen formation will
be a potential valuable approach to induce male sterility in various crop plants.
• Goet (2001) reported cloning of a gene encoding an extracellular invertase isoenzyme
from tobacco that shows a specific spatial and temporal expression in anthers. Transgenic
tobacco plants transformed with an antisense construct of extracellular invertase under
its own promoter are blocked in early pollen development, thus, causing male sterility.
Several male sterile genes expressed in transgenic plants for the production of male sterile
plants has been summarised in the Table 21.1
