Article

Review Male-sterility systems in pigeonpea and their role in enhancing yield

Plant Breeding (Impact Factor: 1.6). 01/2010;

ABSTRACT

Male-sterility has been successfully used for enhancing yield in a number of cereal and vegetable crops. In food legumes, this technology could never be used either due to non-availability of natural out-crossing system, or an efficient male-sterility system or both. Pigeonpea [Cajanus cajan (L.) Millsp.] is a partially cross-pollinated food legume and recent success in breeding a stable male-sterility system has allowed breeders to exploit hybrid vigour for increasing yields. The cytoplasmic-nuclear male-sterility (CMS)-based hybrids have recorded 28.4% yield superiority over local checks in farmersÕ fields. This paper besides summarizing the reports of all the genetic and CMS systems, also discusses the prospects of utilizing these male-sterility systems in commercial hybrid breeding programmes.

Review
Male-sterility systems in pigeonpea and their role in enhancing yield
K. B. S
AXENA
1,2
, R. S
ULTANA
1
, N. M
ALLIKARJUNA
1
, R. K. S
AXENA
1
, R. V. K
UMAR
1
, S. L. S
AWARGAONKAR
1
and
R. K. V
ARSHNEY
1,2
1
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, 502324, A.P., India;
2
Corresponding author: E-mail: k.saxena@cgiar.org and r.k.varshney@cgiar.org
With 1 figure and 4 tables
Received September 19, 2009/Accepted November 12, 2009
Communicated by J. Le
´
on
Abstract
Male-sterility has been successfully used for enhancing yield in a
number of cereal and vegetable crops. In food legumes, this technology
could never be used either due to non-availability of natural out-
crossing system, or an efficient male-sterility system or both. Pigeonpea
[Cajanus cajan (L.) Millsp.] is a partially cross-pollinated food legume
and recent success in breeding a stable male-sterility system has
allowed breeders to exploit hybrid vigour for increasing yields. The
cytoplasmic-nuclear male-sterility (CMS)-based hybrids have recorded
28.4% yield superiority over local checks in farmersÕ fields. This paper
besides summarizing the reports of all the genetic and CMS systems,
also discusses the prospects of utilizing these male-sterility systems in
commercial hybrid breeding programmes.
Key words: Cajanus cajan (L.) Millsp. pigeonpea genetic
male-sterility cytoplasmic-nuclear male-sterility micro-
sporogenesis natural out-crossing hybrid vigour
Male-sterility in plants is a phenomenon where the individuals
are unable to reproduce through natural means because of
their defective male-reproductive parts. Such plants reproduce
only when fertile pollen from other plants is placed on the
stigmatic surface of the male-sterile flowers through any
mechanical means such as deliberate manual efforts, wind or
insects. The phenomenon of male-sterility was recorded as
early as by Kolreuter 1763. Subsequently, its role in evolution
particularly that of dioecism was proposed by Darwin (1890).
Bateson et al. (1908) suggested that male-sterility in most cases
was controlled by recessive genes. Correns (1908) was the first
to demonstrate the role of some cytoplasmic (maternal) factors
in the expression of male-sterility. Subsequently, a large
number of reports have appeared on various aspects of male-
sterility in different crops. Such events can be brought about
either by nature or through human interventions. Like other
traits in living organisms, the male-sterility is also governed by
specific genetic factors which are generally recessive in nature.
Such genes are exposed during inbreeding and their mainte-
nance is affected by fertilization with the pollen that carries
counterpart dominant gene(s).
Male-sterility systems have played a great role in enhancing
productivity of many crops through exploitation of hybrid
vigour. In a plant system, the male-sterility is generally caused by
some specific bio-chemical events that hinder the normal
biological processes of pollen production. It is also observed
that all the male-sterility systems identified so far in different
crops could not be used in hybrid breeding programmes because
of non-availability of other complementary genetic systems
required for restoring their male-fertility. For effective utiliza-
tion of a male-sterility system in hybrid breeding, it is important
that the expression of both the male-sterility and its fertility
restoration are stable over years and locations. Such male-
sterility systems have been successfully exploited in breeding
high-yielding hybrid cultivars in sorghum [Sorghum vulgare (L.)
Monach], pearl millet [Pennisetum glaucum (L.) R. Br.], maize
[Zea mays (L.) spp. mays], sunflower [Helianthus annus (L.)],
castor [Ricinus communis (L.)] and more recently in rice [Oryza
sativa (L.)] and wheat [Triticum aestivum (L.)]. Besides these, in a
number of fruits and vegetable crops also, breeders have
successfully exploited hybrid vigour for enhancing their pro-
ductivity. Food legumes, on the contrary, could not take
advantage of this phenomenon because of their highly self-
pollinating nature that restricts large-scale seed production of
hybrids. Therefore, raising the productivity of food legumes has
been a long standing challenge for breeders.
Pigeonpea [Cajanus cajan (L.) Millsp.] is an important high
protein (20–22%)food legume of rainfedtropics andsub-tropics.
Globally, it is cultivated by small holding farmers on 4.9-m ha in
Asia, Africa and South America. It is primarily consumed as
decorticated dry splits, fresh or frozen peas. In spite of high
importance in nutrition of poor masses and dedicated efforts of
scientists, the productivity of pigeonpea in the last five decades
has remained lowat about700 kg/ha (http://faostat.fao.org/site/
339/default.aspx). Pigeonpea is unique among legumes as its
floral morphology allows both self as well as insect-aided cross-
pollination and their extents vary from one place to another.
However, most breeders in the past ignored this fact and handled
pigeonpea as a self-pollinated crop as far as its breeding
methodology was concerned. The International Crops Research
Institute for the Semi-Arid Tropics (ICRISAT), located at
Patancheru (India), focused pigeonpea research on hybrid
breeding by exploiting its partial (25–70%) natural out-crossing
(Saxena et al. 1990). To achieve success in this endeavour, it was
essential to breed a quality male-sterility system that would be
acceptable to commercial hybrid seed producers. In the past
35 years, a considerable research was undertaken by ICRISAT
on the breeding of various male-sterility systems in pigeonpea
(Saxena 2009). This paper, besides reviewing the origin and
nature of available male-sterility systems, discusses major
achievements in breeding high-yielding hybrids.
Plant Breeding 129, 125—134 (2010) doi:10.1111/j.1439-0523.2009.01752.x
2010 Blackwell Verlag GmbH
www3.interscience.wiley.com
Page 1
Genetic Male-Sterility (GMS) Systems in Pigeonpea
All the GMS systems reported so far in pigeonpea have
emerged from spontaneous mutations. This happens when a
male-fertility controlling dominant (Fr) nuclear gene mutates
to its recessive form under the influence of some natural
forces and with subsequent natural selfing of heterozygotes
(Frfr) the male-sterile genotypes (frfr) appear within the
population. Such genotypes, if not cross-pollinated by fertile
pollen, are eliminated from its parental population. In
comparison with highly self-pollinated crops, the elimination
of frfr genotypes is gradual in out-crossed species. Therefore,
such elimination processes depend on the rate of natural out-
crossing in a given population. In comparison with recessive
genes, the frequency of dominant male-sterile genes in nature
is very low (Kaul 1988). There are many instances where
progeny of some inter-specific and -generic crosses have also
produced male-sterile segregants. In genus Cajanus also, a
number of such wide crosses have produced male-sterile
segregants. These cases, however, were hardly pursued
further (Dundas 1990, Reddy et al. 1990) for use in breeding
programmes.
The male-sterile mutants have also been reported in some
mutagen-induced populations. In most cases, such mutants
could not be maintained either due to their tight association
with female-sterility or reproductive abnormalities such as
chromosome addition or deletion (Dundas 1990). If for
some reason, a chromosome with male-fertility (Fr) gene is
lost then male-sterility trait with frfr alleles will express but
such plants hardly reach their maturity due to poor vigour
and abnormal growth. In contrast, if the male-sterile mutant
gene is dominant then it is eliminated rapidly from the
population, particularly in a self-pollinated species. There-
fore, most spontaneous male-sterile mutants that have been
detected so far, are recessive. Relatively high occurrence of
non-allelic recessive male-sterility genes suggests that the
frequency of such natural mutations is quite high and their
deletion from the parental populations is rather slow.
According to Kaul (1988) the male-sterility in legumes that
is controlled by recessive genes was reported in broad bean
[Vicia faba (L.)], grass pea [Lathyrus sativus (L.)], groundnut
[Arachis hypogea (L.)], sunhemp [Crotalaria juncea (L.)],
soybean [Glycine max (L.) Merr.], pea [Pisum sativum (L.)]
white clover [Trifolium repens (L.)], common bean [Phaseolus
vulgaris (L.)], alfalfa [Medicago sativa (L.) spp. sativa] etc.;
while dominant genetic control of male-sterility was reported
in Trifolium repens.
Reports of genetic male-sterility systems in pigeonpea
Various GMS systems reported in pigeonpea are summarized
in Table 1. The first report on male-sterility in pigeonpea was
published by Deshmukh (1959). This spontaneous mutant
could not be maintained because of its tight linkage with
female-sterility. Reddy et al. (1977) made a deliberate search
for male-sterility in 7216 germplasm accessions sown at
ICRISAT in 1974. They selected 75 single plants which
remained green till the end of season and had a few pods,
suggesting absence of self-fertilization of flowers to affect
normal pod setting. These selections were female fertile and
had different types of anthers with variable fertility levels.
Among these, six plants with fully grown translucent anthers
and no pollen grains were selected for further studies and use
in hybrid breeding programmes.
Dundas et al. (1982) reported a male-sterile mutant within a
photo-insensitive pigeonpea breeding line. At about the same
time yet another genetic male-sterile spontaneous mutant was
selected in a breeding line B15B (Saxena et al. 1983). This
mutant was characterized by brown coloured arrow-head
shape anthers. Verulkar and Singh (1997) reported another
recessive male-sterile mutant in a population of cultivar ÔUPAS
120Õ. This mutant had translucent anthers, sparse podding and
delayed flowering.
Wanjari et al. (2000) recorded the first dominant gene in an
inter-specific progeny that controlled male-sterility in pigeon-
pea. Saxena and Kumar (2001) reported a genetic male-sterile
mutant that was selected from an inbred population of cultivar
ÔICPL 85010Õ. This mutant was characterized by small light
yellow anthers with no pollen grains. Venkateswarlu et al.
(1981) and Pandey et al. (1994) reported perhaps the similar
male-sterile gene that was linked to characteristic obcordate
leaves. In a segregating population of cross between obcordate
leaf genotype and cultivar ÔHY 3CÕ, a total of 13 obcordate
leaf type plants were found with 60–100% pollen sterility
(Venkateswarlu et al. 1981). The authors postulated a linkage
between male-sterility and obcordate leaf trait. They further
observed that all the male-sterile plants had modified keel that
exposed the flowers for out-crossing. Saxena et al. (1981)
reported the presence of partial male-sterile plants with sparse
pollen production in an F
2
population of cross MS 4A · QPL-1.
The pollen sterility in these plants ranged from 40% to 80%.
There was no intra-plant variation for pollen sterility. The pod
set on these plants varied in accordance with their pollen-
fertility. Gupta and Faris (1983) reported the identification of 11
male-sterile plants in a population of cross 0DT · ICPL 86.
Table 1: A summary of genetic male-sterility systems reported in pigeonpea
S. No. Authors Gene symbol Remarks
1 Deshmukh (1959) Male-sterility was associated with female-sterility
2 Reddy et al. (1977) Seven types of floral variants with varying degree
of male-sterility recorded
3 Reddy et al. (1978) ms
1
Translucent male-sterile anthers
4 Dundas et al. (1982) Photo-insensitive male-sterile mutant
5 Saxena et al. (1983) ms
2
Brown, arrow-head shape anthers; non-allelic to ms
1
6 Verulkar and Singh (1997) Single recessive gene control
7 Wanjari et al. (2000) Single dominant gene control
8 Saxena and Kumar (2001) ms
3
Under-developed anthers, non-allelic to ms
1
and ms
2
9 Venkateswarlu et al. (1981) Male-sterility linked to obcordate leaf type
10 Pandey et al. (1994) Male-sterility linked to obcordate leaf type
11 Saxena et al. (1981) Partial male-sterility with sparse pollen production
12 Gupta and Faris (1983) Recessive gene control
126 S
AXENA
,S
ULTANA
,M
ALLIKARJUNA
,S
AXENA
,K
UMAR
,S
AWARGAONKAR
and V
ARSHNEY
Page 2
Anthers of the male-sterile plants were small, white (later turned
brown) and non-dehiscent. They also reported another mutant
with non-dehiscent type of male-sterility where the pollen grains
were released only when the mature anthers were physically
ruptured. The pollen thus obtained was 70–80% sterile. These
mutants were not studied further.
Cytoplasmic-Nuclear Male-Sterility (CMS) Systems in
Pigeonpea
The other type of major reproductive abnormality leading to
male-sterility is caused together by specific nuclear and
cytoplasmic genetic factors. In most cases, the recessive
nuclear genes interact with specific genetic factors housed in
the cytoplasm of a cell and make an individualÕs anthers non-
functional leading to male-sterility. Such plants produce fertile
pollen when the recessive nuclear genes are replaced by their
dominant counterparts or the cytoplasmic male-sterility caus-
ing factors by fertility inducing genetic factors. Therefore, for
maintaining a male-sterile progeny and to produce fertile
hybrids, the individual pollen parents with different genetic
constitutions are required. Hence, the hybrid breeding tech-
nology based on this system involves three parents; a male-
sterile (A-) line, its maintainer (B-) line and a fertility restorer
(R-) line.
The CMS systems can arise either through spontaneous
mutation, intra-specific crosses, inter-specific crosses or inter-
generic crosses. The wide hybridization programmes such as
inter-specific and -generic crosses have been found to produce
a greater proportion (about 75%) of CMS systems (Kaul
1988). Scanning of literature on this subject shows that in the
dicots most CMS cases have arisen through inter-specific
crosses, while in monocots it is the inter-generic hybrids that
have yielded most CMS sources (Kaul 1988). As the expression
of CMS requires two different genetic systems, one each in
cytoplasm and nucleus, to come together in a single cell; the
frequency of spontaneously occurring mutants simultaneously
in both the entities (i.e., nucleus and cytoplasm) is quite low.
On the contrary in GMS system, only a single nuclear
mutation can lead to the development of male-sterility. Unlike
GMS controlling genes, the influence of environment (tem-
perature and/or photoperiod) on CMS controlling nuclear fr
and Fr genes is more prominent. This may lead to instability of
the expression of male-sterility and its fertility restoration.
Such unstable expressions are also sometimes influenced by the
genetic background of an individual.
Breeding of CMS lines in pigeonpea
Reddy and Faris (1981) made the first attempt to breed a
CMS line in pigeonpea using cytoplasm of a wild relative of
pigeonpea, then classified under a separate genus Atylosia.
To start this programme, they crossed a cultivated type (as
female) with pollen from two different wild relatives,
Atylosia sericea and Atylosia scarabaeoides. The fertile F
1
plants of these two crosses were used as male parent to
produce backcrosses with wild species as female parents. The
resultant BC
1
F
1
plants were male fertile while their BC
1
F
2
progeny segregated for male-sterility and fertility. The
maternally inherited male-sterility in these segregants was
found to be tightly linked with various floral abnormalities
such as petaloid anthers, free stamen or heterostyly. They
also reported that these segregants had different degrees of
female-sterility and, could never be stabilized as pure lines
therefore, could not be used in hybrid breeding programmes.
Ariyanayagam et al. (1993) attempted to develop CMS
through chemical and physical mutagens. A GMS line with
ms
2
gene, when treated with 0.025% sodium azide or
500 mg/kg of streptomycin sulphate, showed mutational
changes and expressed male-sterility that was maternally
inherited. This male-sterility was maintained only by het-
erozygote sibs that raised doubts about its nature and use in
hybrid breeding programme. The proportion of male-sterile
plants in these mutagenic progeny varied a lot and no good
male-sterile line could be derived Subsequently, a few CMS
systems were developed in pigeonpea and these are briefly
described below.
CMS with Cajanus sericeus (Benth.ex Bak.) van der Maesen
comb. nov. (A
1
) cytoplasm
Ariyanayagam et al. (1993) crossed C. sericeus with an
advanced breeding line of pigeonpea. The F
1
progeny of this
cross showed partial male-sterility but in F
2
generation a few
segregants expressed 100% pollen sterility. In the subsequent
backcross generations, for some reasons, these male-sterile
plants could not maintain their high levels of male-sterility. In
addition, it was also observed that some male-sterile plants
reverted back to male fertility when local environment,
particularly temperatures and photoperiods changed.
To stabilize the male-sterile trait, besides conventional
backcrossing, Ariyanayagam et al. (1995) also used multiple
cross genome transfer methodology. Both these approaches
yielded certain proportion of male-sterile segregants, but the
backcross derivatives were also found to be female-sterile and
failed to set any pod. The progeny derived from the genome
transfer scheme also produced a few male-sterile segregants
which were maintained by other pigeonpea inbred lines.
Saxena et al. (1996) carried forward the selections of Ari-
yanayagam et al. (1995) through additional hybridization and
selection of male-sterile plants. This led to the development of
male-sterile lines such as CMS 85010A, CMS 88034A and
CMS 13091A (K. B. Saxena, Unpublished data). From these
populations, Saxena et al. (2006) selected male-sterile lines that
revert back to full male fertility under low temperature and
shorter days (Table 2) and again to full male-sterility under
high temperature and longer days.
CMS with Cajanus scarabaeoides (L.) Thou. var. pedunculatus
(Reynolds and Pedley) van der Maesen comb. nov. (A
2
)
cytoplasm
In an attempt to develop a stable CMS line, Ariyanayagam
et al. (1993) crossed C. scarabaeoides as female parent with a
pigeonpea line ICPL 85030. The F
1
plants were partial male-
sterile. In the backcross progeny, some promising male-sterile
plants were identified but no stable CMS line could be bred.
Tikka et al. (1997) reported the development of a CMS line by
crossing a cultivated type with its wild relative C. scarabaeoides
as a female parent. The resultant F
1
plant was partial male-
sterile and in F
2
a number of male-sterile segregants were
recovered. Subsequently, a perfect male-sterile maintainer line
ICPL 288 was also identified. The fertility restoration of this
male-sterile line was also found among fertile F
2
segregants.
This male-sterile source was used in developing experimental
hybrids in Gujarat state of India.
Male-sterility systems in pigeonpea 127
Page 3
Saxena and Kumar (2003) also crossed C. scarabaeoides as a
female parent with four pigeonpea cultivars. Among F
1
s, a
progeny derived from cross C. scarabaeoides · ICPL 88039
was completely male-sterile. To stabilize this source of male-
sterility, backcrosses were made with ICPL 88039 as recurrent
parent and all the plants in BC
1
F
1
through BC
6
F
1
generations
were male-sterile. They also reported eight fertility restorers
and six male-sterility maintainers. This allowed breeding of
genetically diverse hybrids for different cropping systems.
Saxena (2008) reported that fertility restoration in hybrids
involving this CMS was not perfect and a large variation (50–
95%) was observed for pollen fertility. This variation could be
due to differential inter-genomic or cytoplasmic–genomic
interactions. Abdalla and Hermsen (1972) opined that poly-
morphism, arising due to differential genes, can also yield
inconsistent expressions of both male-sterility and fertility
restoration.
CMS with Cajanus volubilis (Blanco) Blanco (A
3
) cytoplasm
Wanjari et al. (1999) selected a number of male-sterile segre-
gants with maternal inheritance from a cross involving
C. volubilis and a cultivated type. These selections, however,
could not be used in any hybrid breeding programme due to
lack of fertility restoring genotypes.
CMS with Cajanus cajanifolius (Hains) van der Maesen comb.
nov. (A
4
) cytoplasm
Rathnaswamy et al. (1999) unsuccessfully attempted to breed
a CMS line by crossing C. cajanifolius as male parent with a
GMS line as female parent. All the progeny from this cross
were male fertile. Saxena et al. (2005) crossed ICPW 29, an
accession of C. cajanifolius, a wild relative of pigeonpea, as
female parent with pigeonpea line ICPL 28. According to De
(1974), C. cajanifolius resembles with cultivated types in most
morphological traits and differs by only a solitary gene. The
inter-specific F
1
hybrid plants grown in 2001 expressed
variable extents of pollen sterility and one plant with 60%
pollen sterility was backcrossed to ICPL 28. This was followed
by six backcrosses to substitute the nuclear genome of wild
species with that of the cultivated type. This substitution led to
enhanced male-sterility that was fully maintained by its
recurrent pigeonpea parent. This male-sterile source is the
best among those identified so far and it was designated as
ICPA 2039. It was found to be highly stable male-sterile line
across environments and years (Dalvi et al. 2008a, Saxena
2008) and never showed any morphological deformity. To
develop diverse pigeonpea hybrids this male-sterile source has
now been transferred into a number of genetic backgrounds.
CMS with Cajanus cajan (L.) Millsp. (A
5
) cytoplasm
Rathnaswamy et al. (1999) crossed a GMS line with Cajanus
acutifolius (F.V. Muell.) van der Maesen comb. nov. as male
parent and all the F
1
plants were male fertile. Mallikarjuna and
Saxena (2002) crossed C. acutifolius as a female parent with
pigeonpea accession ICP 1140 with only 1.5% pod set. The use
of gibberellic acid (at 50 mg/l) in backcrosses enhanced the
pod set to 6% but the seeds, thus obtained, were under
developed and failed to germinate. To overcome this problem,
the developing embryos were rescued and successfully cultured
in artificial media (Mallikarjuna and Moss 1995). Encouraged
with the success of embryo rescue technology, Malikarjuna
and Saxena (2005) again crossed six pigeonpea cultivars as
female parent with two accessions (ICPW 15613, ICPW 15605)
of C. acutifolius. The F
1
s involving pigeonpea lines ICPL
85010, ICPL 85030 and ICPL 88014 produced a few male-
steriles with some plants exhibiting up to 100% pollen sterility.
The anthers of these male-sterile plants were shrunken and
pale yellow in colour. Such male-steriles maintained their
sterility when crossed to their respective wild relative acces-
sions. Most of the cultivated accessions when crossed to these
male-steriles restored the male fertility of the plants. An
exception to this was HPL 24, where F
1
progeny produced
both male-sterile and fertile plants. This suggests the presence
of both fr and Fr genes in its nuclear genome. Further
backcrossing with this line and selection for pollen sterility
helped in stabilizing the male-sterility (K. B. Saxena, Unpub-
lished data). Interestingly, HPL 24 was bred from a cross
involving C. sericeus, another wild species (Saxena 2008), and
this suggested that besides C. acutifolius the fr genes may also
be present in C. sericeus.
CMS with Cajanus lineatus (W. & A.) van der Maesen comb.
nov. (A
6
) cytoplasm
In 2002 rainy season, a naturally out-crossed partial male-
sterile plant was observed in an open-pollinated population of
C. lineatus (K. B. Saxena, Unpublished data) and the
morphology of this plant was very different from rest of the
population. The vegetative cuttings of this plant were raised in
a glasshouse and out of five cuttings planted only two survived
and the plants were found to be male-sterile. These were
Table 2: Changes in the propor-
tions of male-sterile (S) and male-
fertile (F) plants in the environ-
ment-sensitive lines selected from
ICPA 85010
Observation date
Progeny number
82 83 90 86 149
SFSFSFSFSF
4 September 22 1 21 0 23 0 21 5 20 0
22 September 18 5 6 15 4 19 0 26 11 9
17 October 4 19 1 20 2 21 0 26 2 18
14 November 4 19 2 19 2 21 0 26 2 18
16 February 22 1 21 0 23 0 19 5 20 0
14 March 22 1 21 0 23 0 19 4 19 0
Pods plant
-1
in converted
fertile plants
76–201 97–188 147–260 87–191 126–209
S, sterile; F, fertile.
128 S
AXENA
,S
ULTANA
,M
ALLIKARJUNA
,S
AXENA
,K
UMAR
,S
AWARGAONKAR
and V
ARSHNEY
Page 4
crossed with pigeonpea line ICPL 99044 and produced normal
pod set. The F
1
plants grown in 2004 season were partial male-
sterile. Back-crosses (BC
1
F
1
) were made with ICPL 99044 and
out of 20 plants grown five were partial male-sterile. In BC
4
F
1
generation, 167 plants were examined for pollen viability and it
ranged from 92% to 100%. The plants showing 100% male-
sterility were crossed with four pigeonpea lines in 2008 season.
At present this CMS source is in BC
5
F
1
stage with perfect
male-sterility maintenance system available.
CMS with Cajanus platycarpus (Benth.) van der Maesen comb.
nov. (A
7
) cytoplasm
Cajanus platycarpus, a wild species in the tertiary gene pool of
pigeonpea, is cross incompatible with cultivated types and,
therefore, hormone-aided pollinations coupled with embryo
rescue techniques were employed to obtain viable F
1
and
BC
1
F
1
progeny (Mallikarjuna et al. 2006). In BC
2
F
1
genera-
tion, a progeny (BC
2
-E) with low pollen fertility was selected.
Within this progeny two plants with 100% pollen sterility were
selected and crossed with a set of pigeonpea cultivars. The
examination of their F
1
s showed that the hybrid involving
cultivar ÔICPL 85010Õ maintained complete male-sterility,
whereas cultivars ÔICPL 88014Õ and ÔICP 14444Õ restored male
fertility. The detailed studies on this new CMS source are in
progress.
Blockages in Microsporogenesis of Male-Sterile
Genotypes
The differentiation of meristematic tissues into pollen mother
cell (PMC) is brought about by a series of bio-chemical events
led by inductive photoperiod and thermal changes. This is
followed by various development changes to produce pollen
grains. In the determination of male-sterility in crop plants, the
anther wall and in particular the tapetum, plays an important
role of producing and transporting critical enzymes, hormones
and nutrients that are essential for the growth of PMCs and
any abnormality in the anther wall development leads to the
production of defective pollen grains. Vasil (1967) deliberated
that during the process of meiosis any abnormality in the
supply of nutrients generally leads to aberrant outputs such as
large and more number of PMCs. Abnormal vacuolization or
fusion of cells into multi-nuclear syncytia, or degeneration of
the tapetal layer leads to the abnormal development and
separation of PMCs. The normal development of PMCs in
general is arrested either premeiotic, during meiosis or in
postmeiotic stages of growth.
Reddy et al. (1978) reported identification of the first GMS
system in pigeonpea. The cytological studies on the fertile and
sterile siblings showed that the microsporogenesis in the two
genotypes was similar up to tetrad formation stage. The
differences between the two emerged when the tetrads in the
male-sterile plants failed to be released and leading to
degeneration of tetrads through vacuolation. The tapetum
continued to persist even when the tetrads degenerated. On the
contrary, in the fertile plants, tapetum began to degenerate
during the formation of tetrad and disappeared during male
gametophyte development. In case of male-sterility, the callose
is synthesized because of the presence of high concentrations of
cellular calcium (Worrall et al. 1992). Ketti et al. (1994)
conducted further studies on the persistence of callose and
tapetum in the ms
1
type of male-sterility and reported the
accumulation of callose and persistent tapetum during post-
meiotic stages. They further deliberated that a gradual
reduction in the concentration of polysaccharides and RNA
proteins in the tetrads were responsible for disorientation of
cytoplasm leading to malnutrition and poor tetrad growth.
Saxena et al. (1983) reported male-sterility, where the
anthers were brown and shrivelled. Dundas et al. (1981)
revealed that the degeneration of microspores occurred at
the tetrad stage through rupturing of nuclear membrane and
collapse of the outer wall resulting.
Dundas et al. (1982) while reporting a new source of GMS
observed that in the male-sterile plants the PMCs count was
almost double than their fertile counterparts. The abnormal
enlargement of PMCs and their number was associated with
the failure of adjacent PMC walls to separate. The breakdown
of microsporogensis of this male-sterile occurred at prophase I.
The delayed and incomplete anther wall development
appeared to be responsible for PMC degeneration. Similar
observations were also reported by Murthi and Weaver (1974)
in cotton. Frankel and Galum (1977) suggested that early
breakdown of microsporogenesis was associated with varying
degrees of impairment of fertility of female gametes. Contrary
to this theory, the female fertility in this male-sterile line was
normal.
In the GMS reported by Saxena et al. (1983), the develop-
ment of sporogeneous tissues and young PMCs was similar in
the male-fertile and sterile plants. In the male-sterile plants,
however, PMC degeneration occurred at young tetrad stage
with the rupturing of nuclear membrane and collapse of outer
cell walls. The vacuoles developed in the tapetal cells
metaphase I and by tetrad stage the entire cell gets vacuolated.
In this case, the precocious degeneration of tapetum ending its
role as a nutrient source for PMCs (Echlin 1971) could be
responsible for tetrad breakdown. Similar results were also
reported by Kaul and Singh (1966) in Hordeum vulgare;
Overman and Warmke (1972) in Sorghum vulgare and Reddy
and Reddi (1974) in Pennisetum typhoides.
In all the three GMS systems, the blockages in the
microsporogenesis occured at different stages of development
which also determined their anther morphology. Studies of
Saxena et al. (1983) and Saxena and Kumar (2001) showed
that if an individual plant carries two male-sterility inducing
genes, then the one which expresses first and hinders the
normal process of microsporogenesis, determines the pheno-
type of the anthers and the other genes become redundant as
far as their expression is concerned. Cytological examination
of sparse pollen producing flowers revealed that their tetrad
formation was normal but soon after this, only a portion of
microscopores collapsed. Further, the locules of anthers within
individual flowers varied in the proportions of microspore
degeneration (Saxena et al. 1981). The cause of this partial
breakdown of microsporogenesis could not be ascertained.
Ariyanayagam et al. (1995) working with C. sericeus-derived
CMS lines, reported that meiosis in the male-sterile plants
proceeded normally until the release of microspores and this
was followed by vacuolation and degeneration of protoplasm.
Cytological investigations with C. acutifolius-derived CMS
showed that the process of meiosis in the male-sterile plants
proceeded normally till the onset of tetrad stage (Malikarjuna
and Saxena 2005), but their further growth was arrested and
the tetrads remained inside the tapetum layer. This resulted in
the loss of cell contents and collapse of the process of
microsporogenesis.
Male-sterility systems in pigeonpea 129
Page 5
A detailed study by Malikarjuna and Kalpana (2004)
identified two different kinds of male-sterile plants in a cross
involving a cultivated pigeonpea as female parent and
C. acutifolius as male parent. These two male-sterile variants
had different anther morphology. In type I, the anthers were
shrivelled with brown colour, while in type II male-steriles, the
plants had pale white shrivelled anthers. These variants also
differed in their microsporogenesis. The PMCs of type I male-
sterile plants remained in prophase stage and subsequent
processes of meiosis were arrested. The PMCs enlarged
normally and once nucleus grew, further cell division did not
take place. In these plants, persistence of tapetum was also
observed. In type II plants, the anthers were translucent and
microsporogenesis continued up to tetrad stage but the tetrads
failed to separate and produce pollen grains. This was followed
by collapse of anther development process, a sort of postme-
iotic arrest of microspore development. Dalvi et al. (2008b)
studied cytogenetics of A
4
CMS and reported an early
breakdown of tapetum. In these plants, the anthers were
under-developed and the male-sterility expressed at tetrad
stage, where the tetrad wall failed to degenerate and resulted in
the degeneration of its contents.
It can be concluded that GMS in pigeonpea occurs due to
two primary reasons. The first process is characterized by the
development of brown and shrivelled anthers followed by
premeiotic breakdown of PMCs. In the other process, the
anthers are pale white or translucent accompanied by post-
meiotic breakdown of PMCs.
It is now well understood that the CMS trait is expressed
due to impairment of pollen formation processes that result
from interaction of the nuclear and the mitochondrial
genomes. Pollen maturation requires great amounts of energy
(Zhao et al. 2000). It is well known that there is many fold
increase in the number of mitochondria in the tapetal tissue
and PMCs during pollen development. In sugarbeet and
wheat, low temperatures cause CMS like microspore distur-
bances as microspores and tapetum cells are more sensitive
than the female reproductive organs and oxidative processes
are responsible for this development (Kuranouchi et al. 2000).
It is also believed that the mitochondria have a major role to
play in the expression of CMS trait. In pigeonpea, there is only
one report (Sivaramakrishnan et al. 2002) that deals with the
assessment of mitochondrial genome of the CMS plants.
Inheritance of Male-Sterility and Fertility Restoration
Systems
With one exception, all the reported sources of GMS are
controlled by a single recessive gene pair. Reddy et al. (1978)
designated the GMS gene as ms
1
, while Saxena et al. (1983)
reported a non-allelic relationship between ms
1
and ms
2
genes.
They also reported that during microsporogenesis the ms
2
is
expressed at an earlier stage than that of ms
1
gene. The male-
sterility reported within ICPL 85010 population (Saxena and
Kumar 2001), was also controlled by a single recessive gene
(ms
3
) and it was also non-allelic to ms
1
and ms
2
genes. They
further reported that all the three male-sterility genes were
independent and when present within a plant system, expressed
independently at different stages of microsporogenesis. The
first to express is ms
2
, followed by ms
3,
and finally ms
1
gene.
The translucent type of GMS reported by Verulkar and Singh
(1997) was also controlled by a single recessive gene but its
allelic relationship with ms
1
which also has translucent anthers
was not studied. Saxena et al. (1981) reported single recessive
genetic control of sparse pollen production in pigeonpea.
Among various CMS sources reported, the genetics has been
reported for only A
4
type of CMS. Dalvi et al. (2008a) studied
genetics of fertility restoration in five crosses. Of these, in three
crosses a single dominant gene, while in one cross two
dominant genes with duplicate gene action restored the
fertility. In the fifth cross also two dominant genes with
complimentary action governed the fertility. Further investi-
gation into the origin of fertility restoring lines showed that
these Fr genes were randomly distributed in the germplasm.
Effect of Environment on Male-Sterility Systems
Ariyanayagam et al. (1995) while attempting to breed a CMS
system at ICRISAT (17N) using multiple genome transfer
approach identified a progeny where the male-sterile segre-
gants, expressed 100% pollen sterility from of March to June
but it started producing small quantities of pollen grains in
July. They attributed it to the reduction in mean temperature
and rise in humidity. Saxena (2009) reported the results of a
detailed study at ICRISAT with CMS lines derived from
C. sericeus under a selfing cage (to avoid the entry of pollen-
carrying insects) and reported that the process of conversion of
male-sterile plants to male fertility started at the end of
September and continued up to middle of November. Also, it
was observed that there was a genetic variation for this trait
among and within progeny. Such converted male-fertile plants
reverted back to male-sterility in the month of February
(Table 2). These observations suggested that shortening of
daylengths and reduction in temperatures induced male
fertility, while high temperatures and longer days maintained
male-sterility. The detailed studies on this subject are in
progress.
Molecular Characterization of Male-Sterile Lines
Although the CMS-based hybrid pigeonpea technology is now
ready for use with high yield potential, their genetic and
molecular basis is yet to be investigated. Recent advances in
pigeonpea genomics as a part of International Pigeonpea
Genomics Initiative (Varshney et al. 2009) offer a good scope
to characterize CMS (A-) lines along with their maintainers
(B-) and restorer (R-) lines at molecular level. The identification
of molecular markers associated with fertility restoring gene(s)
is also a potential research areas under consideration.
Recently, a number of DNA marker systems such as
restriction fragment length polymorphism (RFLP), randomly
amplified polymorphic DNA (RAPD), diversity arrays tech-
nology (DArT) and simple sequence repeats (SSRs) have been
used for pigeonpea genetic diversity analysis (Nadimpalli et al.
1993, Ratanparkhe et al. 1995, Burns et al. 2001, Yang et al.
2006, Odeny et al. 2009, Saxena et al. 2009a,b) but only two
reports are available on the characterization of A-, B- and R-
lines. Sivaramakrishnan et al. (2002) compared RFLP patterns
using maize mitochondrial DNA-specific probes in three
pigeonpea CMS progeny with A
2
cytoplasm and two GMS
lines. The results confirmed the maternal inheritance of male-
sterility. Similarly, RAPD markers (Souframanien et al. 2003)
were used for the identification of pigeonpea CMS lines
derived from two crosses (C. scarabaeoides · C. cajan and
C. sericeus · C. cajan). They also reported adequate
polymorphism to differentiate among A-, B- and R-lines with
130 S
AXENA
,S
ULTANA
,M
ALLIKARJUNA
,S
AXENA
,K
UMAR
,S
AWARGAONKAR
and V
ARSHNEY
Page 6
certain random primers. Recently, SSR genotyping of 37 A-,
38 B- and 84 R-lines was conducted at ICRISAT with 148 SSR
markers (R. K. Saxena, Unpublished data). The main aim of
this work was to obtain finger printing data of promising A-,
B- and R-lines and to select parents for developing mapping
populations for tagging the fertility restoration (Fr) genes in
pigeonpea (Varshney et al. 2009). This study has provided a
total of 41 markers polymorphic across 159 lines used with an
average of 3.1 alleles and polymorphism information content
(PIC) value of 0.41 per marker. As evident by PIC values,
among B-lines (0.39) a relatively higher genetic dissimilarity
was observed when compared with A- (0.34) and R- (0.37)
lines. Based on these results, eight mapping populations
including four F
2
and four back-cross (BC
1
F
1
) populations
are being developed. These populations will be used for
genotyping with SSR and DArT markers. The genotyping data
along with phenotyping data are likely to identify the markers
associated with Fr genes in pigeonpea.
As mentioned earlier, CMS is caused by impaired micro-
sporogenesis, which results in anthers with abortive pollen.
CMS is maternally inherited and expressed through interac-
tions of nuclear genes with cytoplasmic determinants assigned
to the mitochondrial genome (Budar and Berthome 2007).
Generally, CMS is accompanied by changes in the pattern of
plasmid-like DNAs, in expression of several genes and in the
mitochondrial genome structure (Zubko 2004). With an
objective of identification and expression of such genes,
mitochondrial genomes have been investigated in detail in
several species such as pepper (Kim and Kim 2006), sugar beet
(Satoh et al. 2004), rice (Wang et al. 2006), Brassica napus
(Carlsson et al. 2007) and maize (Allen et al. 2007), However,
it is still unknown which regions of the mitochondrial genome
interact with nuclear genes to cause CMS in pigeonpea. In this
direction, some efforts have been initiated at ICRISAT, in
collaboration with J. Craig Venter Institute (JCVI), USA, to
sequence the mitochondrial genomes of ICPA 2039 (A-line),
ICPB 2039 (B-line), ICPH 2433 (hybrid) and ICPW 29 (wild
species) by using 454/FLX sequencing technology. This study
should shed some light on the changes in the pattern and
expression of mitochondrial genes that leads to CMS in
pigeonpea.
Utilization of Male-Sterility Systems in Pigeonpea
Breeding
Population improvement
Broader genetic base and high recombination frequency are
the two key parameters that help in breeding for the desired
end products. In crops like pigeonpea, the extent of recombi-
nation is rather limited due to predominant selfing. This
adversely affects the selection efficiency. The use of double,
three-way, or composite crosses helps in broadening the
genetic base of segregating populations to some extent but
the inherent genetic linkages restrict the desired level of gene
reshuffling. To overcome this challenge in the self-pollinated
crops, Suneson (1956) and Jensen (1970, 1978) proposed
mating schemes among selected genotypes using cumbersome
hand pollination procedures. Also at ICRISAT, a dual
population breeding scheme using a recessive trait (obtuse
leaf) was implemented (Green et al. 1981) in pigeonpea. To
facilitate the genetic recombination in pigeonpea, Byth et al.
(1981) proposed the use of GMS genotypes and natural out-
crossing. Faris (1985) developed six recurrent selection popu-
lations at ICRISAT using GMS lines and natural out-crossing
for enhancing yield but the gains were discouraging. The
success of such population breeding schemes in practical
pigeonpea breeding, however, is yet to be established.
Hybrid breeding
GMS-based hybrids
Presence of partial natural out-crossing and development of a
stable male-sterility system offered a unique opportunity to
exploit hybrid vigour in pigeonpea. Efforts in this direction
were initiated at ICRISAT by using the male-sterility system
discovered by Reddy et al. (1978). The experimental hybrids
demonstrated significant heterosis over control cultivar. Out of
182 GMS hybrids tested at ICRISAT, 59 demonstrated more
than 60% yield advantage over the best control. From this
programme the worldÕs first pigeonpea hybrid ICPH 8 was
released for cultivation in India (Saxena et al. 1992). To the
best of our knowledge this is the first ever commercial hybrid
in any food legume. In farmers Õ fields, this hybrid recorded 31–
40% yield advantage over the best control. This was followed
by the release of five more pigeonpea hybrids. Which exhibited
high levels of standard heterosis (Table 3). But, none of these
could reach farmersÕ fields at commercial level and the main
hindrance was the large-scale seed production of female
parent. As the male-sterility is controlled by a pair of recessive
gene (msms) and it can only be maintained by crossing it to
heterozygous (Msms) plants. The progeny of this cross
(msms · Msms) will segregate in to 50% male-fertile (Msms)
and 50% male-sterile (msms) plants. Therefore, identification
of the fertile segregants within female population was primary
requirement of large-scale seed production and it was not
found commercially viable.
CMS-based hybrids
Soon after achieving the long awaited breakthrough of
developing a stable CMS line, a number of experimental
hybrids were synthesized and tested. In these hybrids, 25–
110% standard heterosis was recorded in station trials. Among
these, two hybrids ICPH 2671 and ICPH 2740 were found
promising. In multi-location trials, conducted for 4 years
ICPH 2740 recorded 35.8% superiority over the control
(Fig. 1). During 2009, the best performing hybrid ICPH
Table 3: GMS-based pigeonpea hybrids released in India
Character ICPH 8 PPH 4 CoH 1 CoH 2 AKPH 4104 AKPH 2022
Year released 1991 1994 1994 1997 1997 1998
Adaptability Central Zone Punjab Tamil Nadu Tamil Nadu Central Zone Maharashtra
Plant type Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate
Days to maturity 125 137 117 125 135 190
Yield superiority
over check (%)
30–41 14 19–22 35 64 25–35
Male-sterility systems in pigeonpea 131
Page 7
2671, was evaluated in 1248 on-farm trials in four Indian states
(Table 4). In these trials ICPH 2671, on average, recorded
28.4% yield advantage over local control. Under intercropping
conditions this hybrid was outstanding and when grown in
combination with soybean (1 : 5 ratio) and produced the
highest yield of 3300 kg/ha was recorded in a large (1.1 ha)
field. It appears that the CMS-based hybrids hold a great
promise for enhancing the stagnant productivity of pigeonpea.
Summary and Outlook
Pigeonpea is known to have tremendous genetic variability but
breeders have not been able to exploit it for the genetic
enhancement of yield. In the last 50 years, more than 100 pure
line pigeonpea varieties have been released in India alone
(Singh et al. 2005), but the national productivity has remained
stagnant around at low levels. A number of genetic studies
conducted on this subject in pigeonpea revealed that a
considerable extent of non-additive genetic variance is also
present in the crop which was never utilized in breeding high-
yielding cultivars (Saxena and Sharma 1990) and invariably
additive genetic variances were utilized for the enhancement of
yield and other related traits. Successful breeding of a CMS
system has given a golden opportunity to breeders to utilize
non-additive genetic variation for increasing pigeonpea pro-
ductivity.
The Herculean breeding efforts at ICRISAT, spanning over
30 years, culminated with the selection of an excellent CMS
system. Breeders have now developed technologies for large-
scale seed multiplication of hybrids and their parents. The
presence of high level of realized heterosis in farmersÕ fields has
opened the way for commercialization hybrid pigeonpea
technology. It is expected that the development of a large
number of hybrid combinations will allow selecting specifically
adapted hybrids with even high levels of hybrid vigour. These
developments have demonstrated that now a long-awaited
quantum jump in pigeonpea yields is possible.
Acknowledgements
Authors express their sincere thanks to Mr Gopinath Shinde for typing
and putting the manuscript into shape.
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134 S
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    [Show abstract] [Hide abstract] ABSTRACT: With 2 figures and 6 tables Male sterility is described for the first time in lupin crop species Lupinus angustifolius L. and Lupinus luteus L. and is also characterized in the Andean lupin, Lupinus mutabilis Sweet. In L. angustifolius and L. luteus, male-sterile plants were identified in artificially induced mutation populations, while in L. mutabilis, both naturally occurring and induced male-sterile plants were selected. For L. angustifolius artificially induced sterility, the segregation ratios in F1, F2 and backcrosses showed a single-gene recessive inheritance and was concluded to be of a nuclear rather than cytoplasmic form. In L. luteus, male-sterile plants were recovered from an M3 mutation population derived from cv. ‘Wodjil’, and several were consistent with that of single recessive gene, most likely nuclear. A naturally occurring sterility in L. mutabilis was concluded to be cytoplasmic with identification of restorer and maintainer genotypes. The trait in L. mutabilis has greatly increased the rate of F1 seed set with zero selfing. Male sterility could be useful for increasing crossing efficiency in breeding programmes, for exploiting heterosis and for interspecific hybridization.
    Full-text · Article · Jul 2011 · Plant Breeding