Distant hybridization and alien gene introgression. In Biology and breeding of food legumes

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©CAB International 2011. Biology and Breeding of Food Legumes
(eds A. Pratap and J. Kumar) 81
6.1 Introduction
Chickpea (Cicer arietinum L.), lentil (Lens
culinaris Medik.), pigeon pea (Cajanus cajan
L. Millsp.), green gram (Vigna radiata L.
Wilczek), black gram (Vigna mungo L. Hepper),
common bean (Phaseolus vulgaris L.) and grass
pea (Lathyrus sativus L.) are among the impor-
tant food legume crops grown on 74 million
ha area with 64 million tons of global out-
put (FAO, 2010). These crops are an integral
part of subsistence agriculture with signifi-
cant contributions to dietary protein supply,
atmospheric nitrogen fixation and agricul-
tural sustainability (Ali and Kumar, 2009).
The average productivity of these crops is
846 kg/ha, which is dismally low compared
with their potential harvestable yield. This
is attributed to their cultivation on poor soils
under rainfed conditions by marginal farmers
with minimum care and, consequently, these
crops suffer severe yield losses not only due
to edaphic, abiotic and socio-economic fac-
tors but also to confounding effects of various
biotic stresses. Yield losses caused by various
fungal, bacterial and viral diseases are enor-
mous, besides parasitic weed menace at vari-
ous growth stages (Dita et al., 2006). Being rich
in protein, several insect pests also cause yield
losses to food legumes both under field condi-
tions and in storage (Clement et al., 1994, 1999).
Among abiotic stresses, drought, temperature
extremities and edaphic problems (salinity
and mineral toxicities) have great bearing on
their harvestable yield (Stoddard et al., 2006).
Since plant breeding in practice as an option
for crop improvement, efforts have been
made to search for genes imparting resistance
to these stresses within the cultivated species
and, to a limited extent, among their wild
relatives, but success has been limited to a
few diseases and insect pests, and is confined
to major gene(s) from the primary gene pool
in few food legume crops (Knott and Dvorak,
1976; Stalker, 1980; Prescott-Allen and
Prescott-Allen, 1986, 1988; Ladizinsky et al.,
1988; Hajjar and Hodgkin, 2007). To diversify
and broaden the genetic base of cultivated
germplasm, introgression of alien genes from
wild species needs to be persuaded vigor-
ously, not only to minimize the risk of stress
epidemics but also to make discernible yield
advances in these legume crops. Therefore,
pre-breeding efforts are urgently required
involving particularly those wild species that
carry useful alien genes for improving yield,
quality and stress resistance. In this chapter
we review the information on the present sta-
tus of wild gene pools, their evaluation, intro-
gression through distance hybridization and
future crossing potential, crossability barriers
and means to overcome them, strategies for
successful introgressions, and future pros-
pects in the selected legume crops.
6 Distant Hybridization and Alien Gene
Introgression
Shiv Kumar, Muhammad Imtiaz, Sanjeev Gupta and Aditya Pratap
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82 S. Kumar et al.
6.2 Wild Gene Pool: Present Status
Wild species are a rich reservoir of useful
alien genes that are no longer available within
the cultivated gene pool (Hawkes, 1977;
Doyle, 1988; Tanksley and McCouch, 1997).
Continuous efforts have been under way to
collect and conserve wild relatives of vari-
ous food legume crops in national and inter-
national gene banks (Plucknett et al., 1987;
FAO, 1996). Over the years, ICARDA has col-
lected and conserved, in its global germplasm
repository, 587 accessions representing 6 wild
Lens species from 26 countries, 270 accessions
of 12 wild Cicer species from ten countries
and 1555 accessions of 45 wild Lathyrus spe-
cies from 45 countries. Similarly, the ICRISAT
gene bank is reported to have 308 acces-
sions of 18 Cicer species from 19 countries,
555 accessions of 57 Cajanus species from 41
countries and 478 accessions of 47 Arachis
species from 7 countries in its wild gene pool
(Upadhyaya, personal communication). The
US Department of Agriculture, Agricultural
Research Service (USDA-ARS), Western
Regional Plant Introduction Station (WRPIS),
Pullman, Washington also has a collection of
4602 accessions of chickpea (Hannon et al.,
2001). In spite of being the largest collections,
these have major germplasm gaps at species
and genotype levels (Ferguson and Erskine,
2001), and a continuum in our efforts is very
much required to fill these gaps in wild gene
pools from the unrepresented areas of diver-
sity in the gene banks.
The gene pool concept of Harlan and
De Wet (1971) has been very helpful to plant
breeders for initiating a pre-breeding pro-
gramme for directed crop improvement.
Various species of major food legume crops
have been grouped into primary, secondary
and tertiary gene pools on the basis of crossa-
bility, cytogenetic, phylogenetic and molecu-
lar data (Table 6.1). The useful genes identified
in the primary gene pool are readily usable
for crop improvement. However, occurrence
of useful genes is much more frequent in the
secondary and tertiary gene pools of various
food legume crops (Kaiser et al., 1994; Collard
et al., 2001; Mallikarjuna et al., 2006; Tullu
et al., 2006). This requires the deployment of
much more effort and novel techniques for
integrating this invaluable resource of nature
into crop improvement programmes.
6.3 Evaluation of Wild Gene Pool
Sporadic efforts have been made in the past
to screen wild species of food legume crops
under field and controlled conditions in order
to identify useful alien genes for desired
traits. These efforts have resulted in identifi-
cation of valuable sources of resistance to key
diseases and insect pests in addition to use-
ful traits such as protein content, cytoplasmic
male sterility, fertility restoration and yield
attributes (Table 6.2).
Chickpea
Annual Cicer species have been evaluated
for reaction to ascochyta blight, fusarium
wilt, cyst nematode, leaf miner, seed beetle
and cold tolerance at ICARDA (International
Centre for Agricultural Research in the Dry
Areas), and a high level of resistance to each
stress has been identified (Table 6.2). Kumar
and Dua (2006) presented a list of possible
wild species as a source of useful alien genes
for chickpea improvement. Cicer judaicum is
reported to have resistance genes for asco-
chyta blight, fusarium wilt and botrytis grey
mould (van der Maesen and Pundir, 1984).
Greco and Di Vito (1993) reported valuable
sources of resistance to cyst nematode in Cicer
bijugum, Cicer pinnatifidum and Cicer reticula-
tum. Some wild accessions have shown resist-
ance to more than one stress (Singh et al., 1994;
Ahmad et al., 2005). For example, ILWC 7-1
of C. bijugum showed resistance to ascochyta
blight, fusarium wilt, leaf miner, cyst nema-
tode and cold, and ILWC 33/S-4 of C. pinnati-
fidum to ascochyta blight, fusarium wilt, seed
beetle and cyst nematode. Kaur et al. (1999)
reported significantly lower larval density of
helicoverpa pod borer on some of the acces-
sions of Cicer echinospermum, C. judaicum,
C. pinnatifidum and C. reticulatum. Recently,
150 accessions of wild chickpea have been
evaluated for resistance to helicoverpa pod
borer under field and greenhouse conditions
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Distant Hybridization and Alien Gene Introgression 83
Table 6.1. Different gene pools of selected legume crops
Crop Primary gene pool Secondary gene pool Tertiary gene pool References
Chickpea Cicer arietinum, C.
reticulatum C.
echinospermum,
C. bijugum,
C. pinnatifidum,
C. judaicum,
C. cuneatum,
C. chorassani-
cum,
C. yamashitae
Ladizinsky and
Adler, (1976a,
1976b); Ahmad
et al. (1988,
2005); van der
Maesen et al.
(2007)
Lentil Lens culinaris ssp.
culinaris,
L. culinaris ssp.
orientalis,
L. odemensis
L. ervoides,
L. nigricans
L. Lamottei,
L. tomentosus
Ladizinsky et al.
(1984);
Ladizinsky
(1999);
Muehlbauer and
McPhee (2005)
Pigeon pea Cajanus cajan,
C. cajanifolius
C. acutifolius, C. albicans,
C. confertiflorus,
C. lanceolatus,
C. latisepalous,
C. lineatus,
C. reticulatus,
C. scarabaeoides,
C. sericeus, C. trinervius
C. goensis,
C. heynei,
C. kerstingii,
C. mollis,
C. platycarpus,
C. rugosus,
C. volubilis and
other species
Smartt (1990);
Singh et al.
(2006)
Mung bean Vigna radiata var.
radiata,
V. radiata var.
sublobata,
V. radiata var
setulosa
V. mungo var. mungo,
V. mungo var. var
silvestris, V. aconitifolia,
V. trilobata
V. angularis,
V dalzelliana,
V. glabrescens,
V. grandis,
V. umbellata,
V. vexillata
Smartt (1981,
1985); Dana
and Karmakar
(1990); Chandel
and Lester
(1991); Kumar
et al. (2004)
Urd bean V. mungo var.
mungo,
V. mungo var
sylvestris
Vigna radiata var. radiata,
V. radiata var. sublo-
bata, V. radiata var.
setulosa, V. aconitifolia,
V. trilobata
V. angularis,
V. dalzelliana,
V. glabrescens,
V. grandis,
V. umbellata,
V. vexillata
Dana and
Karmakar
(1990); Chandel
and Lester
(1991); Kumar
et al. (2004)
Common
bean
Phaseolus
vulgaris
P. coccineus, P. costari-
censis, P. polyanthus
P. acutifolius,
P. lunatus,
other
Phaseolus spp.
Debouck and
Smartt (1995);
Debouck (1999,
2000)
Grass pea Lathyrus sativus L. chrysanthus, L. cicera,
L. gorgoni, L. marmora-
tus, L. pseudocicera,
L. amphicarpus,
L. blepharicarpus,
L. chloranthus,
L. hierosolymitanus,
L. hirsutus,
Remaining
Lathyrus
species
Jackson and
Yunus, (1984);
Yunus and
Jackson (1991);
Kearney (1993);
Kearney and
Smartt (1995)
(Sharma, 2004). Potential accessions of C. retic-
ulatum that can provide genes for high yield
have also been reported by various workers
(Jaiswal and Singh, 1989; Singh and Ocampo,
1997; Singh et al., 2005).
Lentil
The Lens gene pool consists of many
wild relatives offering resistance to biotic
(Ahmad et al., 1997a, b) and abiotic stresses
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84 S. Kumar et al.
Table 6.2. Useful wild germplasm for introgression of alien genes in food legume crops
Crop Useful trait(s) Wild species Reference(s)
Chickpea Ascochyta blight
resistance
C. judaicum, C. montbretii,
C. pinnatifidum
van der Maesen and Pundir
(1984); Singh and Reddy
(1993)
Fusarium wilt
resistance
C. bijugum, C. judaicum,,
C. reticulatum
van der Maesen and Pundir
(1984); Kaiser et al.
(1994); Infantino et al.
(1996)
Botrytis grey mould
resistance
C. pinnatifidum, C. judaicum Singh et al. (1982); van der
Maesen and Pundir (1984)
Cyst nematode
resistance
C. bijugum, C. pinnatifidum,
C. reticulatum
Greco and Di Vito (1993); Di
Vito et al. (1996)
Phytophthora root rot
resistance
C. echinospermum,
C. bijugum, C. reticulatum,
and C. pinnatifidum
Knights et al. (2008)
Cold tolerance C. bijugum, C.
echinospermum and
C. reticulatum
Singh et al. (1990)
Helicoverpa pod borer
tolerance
C. bijugum, C.
echinospermum, C.
judaicum, C. pinnatifidum,
C. reticulatum, C. cuneatum
Kaur et al. (1999); Sharma
(2004)
Drought tolerance C. anatolicum,
C. microphyllum,
C. montbretii, C. oxydon
and C. songaricum
Toker et al. (2007)
Yield attributes C. reticulatum Jaiswal and Singh (1989);
Singh and Ocampo
(1997); Singh et al. (2005)
Lentil Anthracnose resistance Lens ervoides, L. lamottei,
L. nigricans
Tullu et al. (2006)
Ascochyta blight
resistance
L. ervoides, L. culinaris ssp.
orientalis, L. odemensis,
L. nigricans, L. montbretti
Bayaa et al. (1994)
Fusarium wilt
resistance
L. culinaris ssp. orientalis,
L. ervoides
Bayaa et al. (1995); Gupta
and Sharma (2006)
Powdery mildew
resistance
L. culinaris ssp. orientalis,
L. nigricans
Gupta and Sharma (2006)
Rust resistance L. culinaris ssp. orientalis,
L. ervoides, L. nigricans,
L. odemensis
Gupta and Sharma (2006)
Drought tolerance L. odemensis, L. ervoides,
L. nigricans
Hamdi and Erskine (1996)
Gupta and Sharma (2006)
Cold tolerance L. culinaris ssp. orientalis Hamdi et al. (1996)
Yield attributes L. culinaris ssp. orientalis Gupta and Sharma (2006)
Resistance to
orobanche
Lens ervoides, L. odemensis,
L. orientalis
Ferna’Ndez-Aparicio et al.
(2009)
Resistance to sitona
weevils
L. odemensis, L. ervoides,
L. nigricans, L. culinaris
ssp. orientalis
El-Bouhssini et al. (2008)
Grasspea Low ODAP content L. cicera Aletor et al. (1994); Siddique
et al. (1996); Hanbury
et al. (1999); Kumar et al.
(2010)
Continued
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Distant Hybridization and Alien Gene Introgression 85
Table 6.2. Continued.
Crop Useful trait(s) Wild species Reference(s)
Pigeon pea Cytoplasmic male
sterility
Cajanus cajanifolius,
C. sericeus, C. scarabae-
oides, C. acutifolius
Rathnaswamy et al.
(1999) Ariyanayagam et al.
(1993, 1995); Tikka et al.
(1997), Saxena and
Kumar (2003); Kalaimagal
et al. (2008)
High protein content Cajanus cajanifolius,
C. sericeus
Akinola et al. (1975); Dalvi
et al. (2008)
Sterility mosaic disease
resistance
C. sericeus, C. albicans Akinola et al. (1975); Singh
et al. (1993, 2005)
Phytophthora blight
resistance
C. sericeus, C. acutifolius,
C. platycarpus
Akinola et al. (1975);
Mallikarjuna and Saxena,
(2002)
Helicoverpa pod borer
resistance
C. scarabaeoides Verulkar et al. (1997)
Salinity tolerance C. albicans Subba Rao (1990)
Earliness C. platycarpus Saxena (2008)
Vigna MYMV resistance V. umbellata, V. trilobata,
V. mungo
Singh and Dikshit (2002);
Pandiyan et al. (2008)
Common
bean
Common blight
resistance
P. acutifolius Singh and Munoz (1999)
BGYMV resistance P. coccineus Osorno et al. (2003)
Resistance to root rot,
anthracnose and
angular leaf spot
P. coccineus Silbernagel and Hannan,
(1992); Mahuku et al.
(2003)
Heat tolerance P. acutifolius Federici et al. (1990)
Drought tolerance P. acutifolius Parsons and Howe (1984);
Markhart (1985)
Freezing tolerance P. angustissimus Balsubramanian et al. (2004)
Salt tolerance P. filiformis Bayuelo-Jimenez et al. (2002)
ODAP, β-N-oxalyl-L-α,β-diaminopropionic acid; MYMV, mung bean yellow mosaic virus; BGYMV, bean golden yellow
mosaic virus.
(Hamdi et al., 1996). A few attempts have been
made at ICARDA and advanced research
institutions to evaluate wild Lens taxa for
agro-morphological traits besides key biotic
and abiotic stresses (Erskine and Saxena,
1993; Bayaa et al., 1994, 1995; Hamdi and
Erskine, 1996; Hamdi et al., 1996; Ferguson
and Robertson, 1999; Tullu et al., 2006; see also
Table 6.2). The wild gene pool of lentil showed
drought tolerance in Lens odemensis and Lens
ervoides (Hamdi and Erskine, 1996; Gupta and
Sharma, 2006), and cold tolerance and earli-
ness in Lens culinaris ssp. orientalis (Hamdi
et al., 1996). Some of the wild accessions of
Lens showing combined resistance to asco-
chyta blight, fusarium wilt (ILWL 138) and
anthracnose disease (IG 72653, IG 72646, IG
72651) have also been identified (Bayaa et al.,
1995; Tullu et al., 2006). Gupta and Sharma
(2006) evaluated 70 accessions representing
four wild species/subspecies (L. culinaris ssp.
orientalis, L. odemensis, L. ervoides and Lens nig-
ricans) for yield attributes and biotic and abi-
otic stresses. This resulted in identification of
donors for resistance to powdery mildew in
L. c. ssp. orientalis (ILWL 200) and L. nigricans
(ILWL 37); rust and wilt resistance in all four
species; drought tolerance in L. nigricans; and
seeds per plant in L. c. ssp. orientalis (ILWL 90).
Some accessions of L. nigricans (ILWL 37) and
L. c. ssp. orientalis (ILWL 77) have multiple dis-
ease resistance and can be very useful sources
of alien resistance genes. El-Bouhssini et al.
(2008) identified increased resistance to sitona
weevil in L. odemensis, followed by L. ervoides,
L. c. ssp. orientalis and L. nigricans.
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86 S. Kumar et al.
Grass pea
The wild gene pool is a rich reservoir of rare
alleles for grass pea improvement, which
have been evaluated sporadically to identify
zero/low ODAP (b-N-oxalyl-L-a,b-diami-
nopropionic acid) lines (Jackson and Yunus,
1984). A total of 1082 accessions belonging to
30 species were evaluated for 21 descriptors
and agronomic traits at ICARDA (Robertson
and Abd-El-Moneim, 1997). Assessment of
ODAP content in wild species of Lathyrus
indicated that in none of the species is it
absent (Aletor et al., 1994; Siddique et al., 1996;
Hanbury et al., 1999). On average, the ODAP
concentration in Lathyrus cicera was lowest,
followed by Lathyrus sativus and Lathyrus
ochrus (Aletor et al., 1994; Hanbury et al.,
1999). Evaluation of 142 accessions of L. cicera
at ICARDA showed a range of 0.073–0.513%
for ODAP content, which is much lower than
that in cultivated species (Kumar et al., 2010).
The accessions of L. cicera are also a good
source of earliness, orobanche tolerance and
cold tolerance (Robertson et al., 1996).
Pigeon pea
Evaluation of wild species of pigeon pea has
shown many desirable characteristics that
can be introgressed into cultivated species
to make them more adapted and produc-
tive. The species with useful traits are listed
in Table 6.2. These species have been reported
to carry genes for high protein content, salin-
ity tolerance, pod borer tolerance, sterility
mosaic resistance, wilt resistance, phytoph-
thora blight resistance and cytoplasmic male
sterility. Cajanus sericeus and Cajanus albicans
are rich in protein content, Cajanus reticula-
tus var. grandifolius is hardy and fire tolerant
(Akinola et al., 1975) and C. albicans is tolerant
to soil salinity (Subba Rao, 1988).
Vigna crops
A wild accession of Vigna radiata var. sublobata,
PLN 15, has been found to be the potential
donor for pods per plant and seeds per pod
(Reddy and Singh, 1990). Resistance to mung
bean yellow mosaic virus (MYMV) has been
reported in Vigna umbellata, Vigna trolibata
and Vigna mungo (Nagaraj et al., 1981; Singh
and Dikshit, 2002).
Common bean
Wild species of Phaseolus have been charac-
terized for biotic stresses. Wilkinson (1983)
reported Phaseolus coccineus as a poten-
tial source of high yield for common bean.
Resistance to angular leaf spot (Busogoro
et al., 1999), anthracnose (Hubbeling, 1957),
ascochyta blight (Schmit and Baudoin, 1992),
bean golden mosaic virus (BGMV) (CIAT,
1986; Beebe and Pastor-Corrales, 1991; Singh
et al.. 1997), bean yellow mosaic virus (BYMV)
(Baggett, 1956), common bean blight (CBB)
(Mohan, 1982; Schuster et al., 1983; Singh and
Munoz, 1999), root rot (Yerkes and Freytag,
1956; Azzam, 1957; Hassan et al.. 1971), white
mould (Abawi et al., 1978; Hunter et al., 1982)
and cold (Bannerot, 1979) are found in the
secondary gene pool. Some sources of resist-
ance have also been identified in the tertiary
gene pool. Resistance to ashy stem blight
(Macrophoma phaseolina) and fusarium wilt
(Fusarium oxysporum f. sp. phaseoli) (Miklas
et al., 1998b), BGMV (Miklas and Santiago,
1996), bruchids (Shade et al., 1987; Dobie et al.,
1990; CIAT, 1995, 1996), CBB (Coyne et al.,
1963; Schuster et al., 1983; Singh and Munoz,
1999), drought (Thomas et al., 1983; Parsons
and Howe 1984; Markhart, 1985; Federici et al.,
1990; Rosas et al., 1991), leafhopper (CIAT,
1995,1996) and rust (Miklas and Stavely, 1998)
are found in Phaseolus acutifolius.
6.4. Distant Hybridization
Crosses between species of the same or dif-
ferent genera have contributed immensely
to crop improvement, gene and genome
mapping, understanding of chromosome
behaviour and evolution in crops like rice,
wheat, maize, sugar cane, cotton, tomato,
etc. (Sharma, 1995). The ultimate goal of dis-
tant hybridization is to transfer useful genes
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Distant Hybridization and Alien Gene Introgression 87
from alien species into cultivated species, and
this has been very successful in a few crops
but not very encouraging for legume crops.
Stalker (1980) discussed the gaps between
hybridization and utilization, along with
approaches for the utilization of wild species
in food legumes. However, it is well recog-
nized that gene transfer through wide crosses
is a long and tedious process, due to lack of
homology between chromosomes of partici-
pating species in the cross and pre- and post-
zygotic crossability barriers between wild
and cultivated species. Utilizing the wild
gene pool in breeding programmes may also
be constrained by collection gaps in wild spe-
cies, with no information on genome relation-
ships, poor/limited screening of wild species,
linkage drag and genetic complexity of the
traits. Therefore, improvement through dis-
tant hybridization often takes longer in order
to recover genotypes associated with accepta-
ble agronomic background, and thus requires
a long-tem approach.
Crossability potential
The crossability of cultivars with wild species
is a prerequisite for alien gene introgression.
A large proportion of wild species are not
crossable with cultivated species, and con-
sequently of no use for crop improvement
through sexual manipulation. However, vari-
ability for crossability has been observed not
only among genotypes of cultivated species
but also among those of alien species in sev-
eral crops (Sirkka et al., 1993; Sharma, 1995).
Environmental factors can also influence
embryo development of interspecific hybrids,
and thereby the crossability potential (Percy,
1986; Sirkka et al., 1993; Tyagi and Chawla,
1999). Therefore, an understanding of the
extent of crossability is essential for successful
production of hybrids and their derivatives.
The early work on interspecific hybridiza-
tion in grain legumes has been reviewed by
Smartt (1979). Singh (1990) reviewed a wide
spectrum of hybridization work in the genus
Vigna, and Ocampo et al. (2000) in cool season
legume crops. During the past two decades
much information relating to possible gene
flow between legume crops and their wild
relatives, crossability barriers and methods
of overcoming them has been generated. This
has greatly enhanced the interest of breeders
in distant hybridization. This section sum-
marizes the crossability potential of different
food legume crops using various wild and
cultivated species.
Chickpea
Of the eight annual wild species, only Cicer
reticulatum and Cicer echinospermum have been
successfully crossed with chickpea (Ladizinsky
and Alder, 1976a; Ahmad et al., 1988, 2005;
Verma et al., 1990; Singh and Ocampo, 1993),
a technique regularly utilized in the ICARDA
chickpea breeding programme (Imtiaz, per-
sonal communication). Conventional crossing
has been successful in producing interspecific
hybrids between Cicer arietinum and C. reticu-
latum and between C. arietinum and C. echino-
spermum. Due to the presence of post-zygotic
barriers, abortion of the immature embryo
occurs for other interspecific crosses involv-
ing species from the tertiary gene pool such as
C. bijugum and C. judaicum (Ahmad et al., 1988;
Clarke et al., 2006). The availability of novel
tissue culture techniques and biotechnological
tools for circumventing crossing barriers has
brightened the prospects of transferring use-
ful traits from the tertiary gene pool (Shiela
et al., 1992; Mallikarjuna, 1999; Clarke et al.,
2006) and, as a result, hybrids were obtained
between C. pinnatifidum and C. bijugum
(Mallikarjuna, 1999).
Lentil
Many successful attempts have been made to
develop interspecific hybrids, but still many
cross combinations are yet to be attempted
successfully. As far as the crossability sta-
tus of wild Lens taxa is concerned, L. c. ssp.
orientalis and L. odemensis are crossable with
cultivated lentil (Ladizinsky et al., 1984; Abbo
and Ladizinsky, 1991, 1994; Fratini et al., 2004;
Fratini and Ruiz, 2006; Muehlbauer et al., 2006),
although the fertility of hybrids depends on
the chromosome arrangement of the wild
parent (Ladizinsky, 1979; Ladizinsky et al.,
1984). Most accessions of L. c. ssp. orientalis
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88 S. Kumar et al.
cross readily with L. culinaris, and both are
genetically isolated from other species. Lens
nigricans and L. ervoides are not readily cross-
able with the cultivated lentil using conven-
tional crossing methods, due to hybrid embryo
breakdown (Abbo and Ladizinsky, 1991, 1994;
Gupta and Sharma, 2005). Crosses are pos-
sible between L. culinaris and the remaining
species, but they are characterized by a high
frequency of hybrid embryo abortion, albino
seedlings and chromosomal rearrangements
that result in hybrid sterility, if these seedlings
reach maturity (Abbo and Ladizinsky, 1991,
1994; Ladizinsky, 1993; Gupta and Sharma,
2005). Only four crosses have not resulted in
hybrids to date: L. c. ssp. orientalis × L. ervoides;
L. c. ssp. orientalis × L. nigricans (Ladizinsky
et al., 1984); L. c. ssp. tomentosus × L. lamottei
(Van Oss et al., 1997); and L. c. ssp. odemensis ×
L. ervoides (Ladizinsky et al., 1984), although
viable hybrids have been reported between
cultivated species and L. ervoides, L. odemensis
and L. nigricans with the use of GA3 (Ahmad
et al., 1995). Fratini et al. (2006) reported a high
correlation between crossing success and phe-
notypic similarity based on pollen morphol-
ogy and in vitro pollen length, together with
pistil and style length, indicating a good pre-
dictor of hybridization success between dif-
ferent species.
Grass pea
Interspecific hybridization has been success-
ful between L. sativus and two wild Lathyrus
species (L. cicera and L. amphicarpus) with
viable seeds (Davies, 1957, 1958; Khawaja,
1985; Yunus, 1990). Yunus (1990) crossed 11
wild species with L. sativus and found viable
seeds with L. cicera and L. amphicarpus only.
Other species formed pods but did not give
fully developed viable seeds (Yamamoto
et al., 1989; Yunus, 1990; Kearney, 1993).
Some other successful interspecific hybrids
reported in the genus Lathyrus were L. annuus
with L. hierosolymilanus (Yamamoto et al.,
1989; Hammett et al., 1994, 1996); L. articulatus
with L. clymenus and L. ochrus (Davies, 1958;
Trankovskij, 1962); L. cicera with L. blepharicar-
pus, L. gorgoni, L. marmoratus and L. pseudo-
cicera (Yamamoto et al., 1989; Kearney, 1993);
L. gorgoni with L. pseudocicera (Yamamoto et al.,
1989; Kearney, 1993); L. hirsutus with L. odora-
tus (Davies, 1958; Trankovskij, 1962; Khawaja,
1988; Yamamoto et al., 1989); L. marmoratus
with L. blepharicarpus (Yamamoto et al., 1989;
Kearney, 1993); L. odoratus with L. belinenesis
(Hemmett et al., 1994, 1996); L. rotundifolius
with L. tuberosus (Marsden-Jones, 1919); and
L. sylvestris with L. latifolius (Davies, 1957).
Pigeon pea
Hybridization studies have shown that C. cajan
can be successfully crossed with C. albicans,
C. cajanifolius, C. sericeus, C. scarabaeoides, and
C. lineatus (Reddy, 1981; Reddy and De, 1983;
Kumar et al., 1985; Pundir and Singh, 1985).
Reddy et al. (1981) reported that five species of
Cajanus (C. sericeus, C. scarabaeoides, C. albicans,
C. trinervius and C. cajanifolius) were crossable
with pigeon pea cultivars. However, C. crassus
var. crassus and C. platycarpus cannot be
crossed. With the help of in vitro embryo rescue
technique, a C. cajan × C. platycarpus cross has
also been successfully engineered (Dhanuj and
Gill, 1985; Kumar et al., 1985; Mallikarjuna and
Moss, 1995; Mallikarjuna et al. 2006; Saxena
et al., 1996). Shahi et al. (2006) attempted
crosses between C. cajan and C. platycarpus to
diversify the existing gene pool. Since the pol-
len of C. platycarpus failed to germinate on the
stigma of C. cajan, the former was used as the
female parent. However, hybrids of C. platy-
carpus with two cultivars of C. cajan var. Bahar
and Pant A3 survived through embryo cul-
ture. Mallikarjuna et al. (2006) were also able
successfully to cross C. platycarpus with culti-
vated pigeon pea by hormone-aided pollina-
tions, rescuing the hybrid embryos in vitro and
treating the hybrids with colchicines as these
were 100% sterile. Nevertheless, Cajanus scara-
baeoides has several undesirable characteristics
(Upadhyaya, 2006), but is cross-compatible
with cultivated pigeon pea and interspecific
gene transfer is possible through conventional
hybridization. C. acutifolius can also be success-
fully crossed with pigeon pea as a one-way
cross (Mallikarjuna and Saxena, 2005).
Vigna species
A number of studies undertaken on cross-
ability among different Vigna species have
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Distant Hybridization and Alien Gene Introgression 89
been reviewed by Dana and Karmakar (1990)
and Singh (1990). Most reports indicate that
V. radiata produced successful hybrids as
seed parent with V. mungo, V. umbellata and
V. angularis, although their reciprocal cross
hybrids were not viable. However, by using
sequential embryo rescue methods, the recip-
rocal hybrids between V. mungo and V. radiata
could be successfully produced (Gosal and
Bajaj, 1983a; Verma and Singh, 1986). V. mungo
was also successfully crossed with V. delzelli-
ana (Chavan et al., 1966), V. glabrescens (Dana,
1968; Krishnan and De, 1968) and V. trilobata
(Dana, 1966). In some cases, hybrid plants
could be obtained only through embryo rescue
technique, e.g. V. mungo × V. umbellata (Biswas
and Dana, 1975; Chen et al., 1983). Mung bean
× rice bean crosses were generated to incor-
porate MYMV resistance and other desir-
able traits into mung bean (Verma and Brar,
1996). However, genotypic differences were
observed in successful crosses. Furthermore,
four amphidiploids of mung bean (ML 267
and K 851) × rice bean (RBL 33 and RBL 140)
crosses were successfully produced and eval-
uated for different characters (Dar et al., 1991).
Singh et al. (2003) also produced successful
hybrids between V. radiata and V. umbellata,
and the hybrids possessed intermediate mor-
phology with MYMV resistance. Similarly,
Pal et al. (2005) were also successful in pro-
ducing interspecific crosses between V. mungo
and V. umbellata. Interspecific hybridizations
between cultivated cowpea (V. unguiculata
ssp. unguiculata and V. u. ssp. biflora) and wild
forms of cowpea (V. u. var. spontanea, V. u . ssp.
alba, V. u. ssp. stenophylla, V. u . ssp. pawekiae
and V. u. ssp. baoulensis) were attempted by
Kouadio et al. (2007), and the highest success
rate was obtained in crosses between cul-
tivated and annual inbred forms, although
hybridization between cultivated and wild
allogamous forms gave an intermediate rate
of success. The success rate was lower when
V. u. ssp. baoulensis was crossed with culti-
vated forms.
Crossability barriers
Crossability barriers developed during the
process of speciation frustrate breeders’
efforts in successful hybridization between
species of different gene pools. Reproductive
isolation, embryo or endosperm abortion,
hybrid sterility and limited levels of genetic
recombination are significant obstacles to
the greater use of wild germplasm. These
obstacles are in addition to those of undesir-
able linkages to non-agronomic traits once
gene flow has been achieved. These barriers
can prevent fertilization, reduce the number
of hybrid seeds, retard the normal develop-
ment of hybrid endosperm leading to embryo
death or can cause hybrid sterility. In nature,
there is selection bias towards strengthening
these barriers to avoid extinction of the spe-
cies by chaotic hybridization. In food leg-
ume crops several crossability barriers have
been reported, the most common being cross
incompatibility, embryo abortion at early
growth stage, inviability of F1 hybrids and
sterility of F1 hybrid and subsequent proge-
nies (Kumar et al., 2007). The pre-fertilization
cross incompatibility between parent species
arises when pollen grains do not germinate,
the pollen tube does not reach the ovary
or the male gametes do not fuse with the
female (Chowdhury and Chowdhury, 1983;
Shanmugam et al., 1983).
Chickpea
Both pre-zygotic and post-zygotic barriers
to interspecific hybridization in chickpea
have been reported (Croser et al., 2003). In
the case of pre-zygotic barriers, Mercy and
Kakar (1975) attempted to clarify incompat-
ibility barrier(s) present among Cicer genus.
They found the evidence of a low molecular
weight inhibitory substance, possibly a pro-
tein present in the stylar and stigmatic tissues,
inhibiting the germination and tube growth
of the pollen. One of the reasons reported for
the failure of interspecific crosses is the pres-
ence of localized sticky stigmatic secretion at
the time pollen needs to be placed directly on
the most receptive part of the stigma (Croser
et al., 2003). However, Ahmed et al. (1988) and
Ahmed and Slinkard (2004) demonstrated a
post-zygotic barrier(s) to crossing incompat-
ibility rather than a pre-zygotic. They used
seven of the eight wild annual Cicer species,
belonging to the secondary and tertiary gene
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90 S. Kumar et al.
pools in reciprocal crosses with cultivated
chickpea, and confirmed that the zygote
was formed in all interspecific crosses. The
embryos showed continued and retarded
growth at different rates in various crosses
but eventually aborted at an early pro- embryo
stage in all crosses, except for C. arietinum ×
C. echinospermum. There is thus clear evidence
confirming post-zygotic barriers in interspe-
cific hybridization; however, further research
is required to establish the exact causes of
endosperm breakdown leading to embryo
abortion, which might now be more feasible
with the availability of new tools.
Lentil
Strong crossability barriers exist among Lens
species that limit the utilization of the wild
gene pool for lentil improvement. In some
crosses, such as L. culinaris × L. tomentosus,
the problem of chromosome pairing was
observed between the participating genomes
(Ladizinsky, 1979). In some L. culinaris ×
L. culinaris ssp. orientalis crosses, the hybrid
embryo ceased growing but the endosperm
shows no sign of disintegration (Ladizinsky,
1993). In contrast, Abbo and Ladizinsky (1991)
observed that the endosperm was either
abnormal or lacking in L. culinaris × L. c. ssp.
orientalis crosses. Hybrids showed varying
degrees of fertility, usually due to chromo-
some translocations and subsequent prob-
lems with chromosome pairing at meiosis,
in Lens culinaris × L. nigricans (Goshen et al.,
1982; Ladizinsky et al., 1984). Fertility is often
very low, with little viable pollen produced in
anthers, and varies depending on the acces-
sion in L. culinaris × L. c. ssp. orientalis crosses
from 2% to 69% (Ladizinsky et al., 1984). These
problems can occur in the F1 and also persist
in later generations, causing partial or com-
plete sterility. Albino seedlings can also occur
in the F1 generation and thus prevent hybridi-
zation success (Ladizinsky and Abbo, 1993).
Another common problem is that hybrid
embryos cease to grow about 7–14 days after
pollination due to endosperm degeneration,
and thus need rescuing in order to obtain via-
ble hybrids (Ladizinsky et al., 1985; Ahmad
et al., 1995). Hence, L. culinaris × L. ervoides or
L. culinaris × L. nigricans crosses need embryo
rescue techniques in order to develop mature
hybrid plants (Cohen et al., 1984; Abbo and
Ladizinsky, 1991).
Vigna crops
In Vigna crops a slow rate of pollen growth, in
addition to abnormalities in stigmatic and sty-
lar regions, could be one of the major causes
for low percentage of pod set in V. radiata × V.
umbellata and V. mungo × V. umbellata crosses
(Thiyagu et al., 2008). However, the ploidy
level and style length difference may not be
major barriers in the case of Vigna species, as
the long-styled female parent V. radiata could
be successfully crossed with the short-styled
male parent V. trilobata. Crosses between dip-
loid × tetraploid (V. radiata × V. glabrescens)
(Krishnan and De, 1968; Chen et al., 1989)
and tetraploid × diploid (V. glabrescens ×
V. umbellata) were also successful. In many
studies crossability was genotype depend-
ent (Rashid et al., 1988). It was observed that
strong pre-fertilization barriers were present
in the cross between V. radiata and V. umbel-
lata, and growth and lethality of interspecific
hybrid seedlings were influenced by the gen-
otypes of both parental species (Kumar et al.,
2007). Male sterility in F1 plants and subse-
quent generations in interspecific crosses of
Vigna could be attributed to meiotic irregu-
larities: for example, unequal separation of
tetrads and female sterility to degeneration
of megaspores during megasporogenesis
(Pandiyan et al., 2008). One fertile pod with
two hybrid seeds was obtained when V.
angularis was used as a male parent; conse-
quently, a partly fertile interspecific hybrid
was obtained. Among the post-fertilization
barriers, production of shrivelled hybrid
seed with reduced or no germination (hybrid
inviability), development of dwarf and non-
vigorous plants and death of F1 plants at criti-
cal stages of development (hybrid lethality)
are the most common crossability barriers
(Biswas and Dana, 1975). These barriers were
of varying degrees in most of the interspecific
crosses (Dana, 1964; Al-Yasiri and Coyne,
1966; Biswas and Dana, 1976; Chowdhury
and Chowdhury, 1977; Machado et al., 1982;
Chen et al., 1983; Gopinathan et al., 1986).
Sidhu (2003) produced interspecific hybrids
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Distant Hybridization and Alien Gene Introgression 91
of V. radiata with V. mungo and V. trilobata.
Although the crosses between V. radiata and
V. trilobata were successful, the seeds pro-
duced between V. mungo and V. trilobata had
very poor germination and the germinated
seedlings did not survive. Cytological analy-
sis revealed irregular chromosome behaviour
at diakinesis/metaphase I. In some of the
interspecific crosses of Vigna, hybrid sterility
has been observed to be of segregational type
and was due mainly to interchange, inversion
and possibly the duplication-deficiency type
of structural heterozogosities in the F1 indi-
viduals (Dana, 1964; Biswas and Dana, 1975;
Karmakar and Dana, 1987).
Strategy to overcoming
crossability barriers
With better understanding of the processes
involved in pollen germination, pollen tube
growth and fertilization, the opportunities
to manipulate these processes toward the
development of viable and fertile interspe-
cific hybrids have improved considerably.
Various measures to crossability barriers
were reviewed by various workers (Sharma
and Satija, 1996; Singh and Munoz, 1999), and
are summarized in Table 6.3.
Embryo rescue protocols
The advent of in vitro techniques such as
embryo and ovule culture, coupled with in vivo
hormonal treatments, has greatly increased
the scope of distant hybridization in food leg-
ume crops where post-fertilization barriers
(zygotic abortion mechanisms) are common
(Gupta and Sharma, 2005; Clarke et al., 2006;
Fratini and Ruiz, 2006; Mallikarjuna et al.,
2006). In wide crosses where few embryos are
produced, the efficiency of recovering viable
hybrid plants may also be enhanced by callus
induction from the embryo and subsequent
regeneration of plantlets. These procedures
are also directed towards obtaining more effi-
cient survival of embryos in situations where
very immature embryos are to be cultured.
Wide crosses that do not produce viable seeds
could also be obtained through embryo cal-
lus production and subsequent regeneration
and rooting of the callus. The possibility of
increasing crossability also exists by predis-
posing crop embryos to alien endosperm and
then using plants raised from those embryos
to cross with the alien species. Hybridization
of cultivated lentil with L. ervoides and L. nig-
ricans results in pod development that is
arrested within 10–16 days after pollination
and finally yields shrivelled, non-viable seeds
(Ladizinsky et al., 1985), but can be rescued by
a two-step in vitro method of embryo–ovule
rescue to obtain successful distant hybrids
(Cohen et al., 1984). However, Ahmad et al.
(1995) and Gupta and Sharma (2005) could
not produce hybrids using the same tech-
nique. Fratini and Ruiz (2006) developed a
protocol in which hybrid ovules were rescued
18 days after pollination. Fiala (2006) also
obtained L. culinaris × L. ervoides hybrids using
the Cohen et al. (1984) protocol. In addition,
one viable L. culinaris ssp. culinaris × L. lamot-
tei hybrid was also produced in this study. In
chickpea, Clarke et al. (2006) suggested that
the appropriate time to rescue C. arietinum ×
C. bijugum hybrids is the early globular stage
of embryogenesis (2–7 days). In contrast,
C. arietinum × C. pinnatifidum hybrids abort
later (15–20 days) at the heart-shaped or tor-
pedo stages, and are easier to rescue in vitro.
Genotype also plays a significant role in the
ability of immature selfed ovules to germinate
in vitro. Thus the development of appropri-
ate and efficient in vitro protocols for rescu-
ing immature hybrid embryos is a necessity
for these legume crops to secure alien gene
resources available for their improvement.
Chromosome doubling
Colchicine-induced allopolyploids have been
raised from most of the semi-fertile and com-
pletely seed-sterile F1 hybrids in Vigna hav-
ing high pollen fertility and seed set (Dana,
1966; Pande et al., 1990), and some of these
allopolyploids were used as a bridge species
in wide crosses. In pigeon pea, Mallikarjuna
and Moss (1995) attempted chromosome
doubling of diploid F1 hybrids of Cajanus
platycarpus × C. cajan to obtain tetraploid F1
hybrids. Selfing in successive generations had
given rise to mature seeds with introgression
of a resistance gene to phytophthora blight
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92 S. Kumar et al.
Table 6.3. Methods of overcoming crossability barriers in food legumes
Method Cross combination Reference(s)
Reciprocal crosses Vigna radiata × V. mungo Verma and Singh (1986), Ravi et al.
(1987)
Phaseolus vulgaris × P.
coccineus
Rabakoarihanta et al. (1979)
P. vulgaris × P. lunatus Leonard et al. (1987)
Growth regulators V. radiata × V. umbellata Gupta et al. (2002)
V. mungo × V. umbellata Chen et al. (1978)
Embryo rescue V. radiata × V. unguiculata Tyagi and Chawla (1999)
V. mungo × V. radiate Gosal and Bajaj (1983a,b)
V. radiata × V. trilobata Sharma and Satija (1996)
V. radiata × V. radiata var.
sublobata
Sharma and Satija (1996)
V. marina × V. luteola Palmer et al. (2002)
V. glabrescens × V. radiata Chen et al. (1990)
V. vexillata × V. unguiculata Gomathinayagam et al. (1998)
V. unguiculata × V. mungo Shrivastava and Chawla (1993)
Cajanus cajan × C. cajanifolius Singh et al. (1993)
C. cajan × C. platycarpus Singh et al. (1993), Shahi et al. (2006)
C. cajan × Rhynchosia aurea Singh et al. (1993)
C. platycarpus × C. cajan Shahi et al. (2006), Mallikarjuna and
Moss (1995), Mallikarjuna et al. (2006)
C. cajan × C. scarabaeoides –
C. cajan × C. acutifolius –
P. vulgaris × P. lunatus Kobuyama et al. (1991)
P. vulgaris × P. acutifolius Harlan and de Wet (1971)
P. vulgaris × P. acutifolius Cabral and Crocomo (1989), Andrade-
Aguilar and Jackson (1988)
Lens culinaris × L. orientalis Ladizinsky et al. (1985), Ahmad et al.
(1995)
L. culinaris × L. odemensis Goshen et al. (1982), Fratini and Ruiz
(2006)
L. culinaris × L. tomentosus Ladizinsky and Abbo (1993)
L. culinaris × L. ervoides Cohen et al. (1984), Ahmad et al. (1995),
Fiala (2006), Fratini and Ruiz (2006)
L. culinaris × L. lamottei Fiala (2006)
L. culinaris × L. nigricans Cohen et al. (1984), Fratini and Ruiz
(2006)
L. orientalis × L. odemensis Ladizinsky et al. (1985), Goshen et al.
(1982)
L. orientalis × L. tomentosus Ladizinsky and Abbo (1993), van Oss
et al. (1997)
Cicer arietinum × C. reticulatum Ladizinsky and Adler (1976a, b)
C. arietinum × C.
echinospermum
Pundir and Mengesha (1995)
C. arietinum × C. pinnatifidum Mallikarjuna (1999)
C. arietinum × C.bijugum Clarke et al. (2006)
Chromosome doubling
using colchicine
V. radiata × V. mungo Pande et al. (1990)
V. radiata × V. trilobata Dana (1966)
Use of bridge species (V. mungo × V. radiata) × V.
angularis
Gupta et al. (2002)
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Distant Hybridization and Alien Gene Introgression 93
disease from C. platycarpus. In cases where
cultivated species cannot tolerate a large por-
tion of alien chromosome, irradiation tech-
niques have been successfully used. Among
food legumes, irradiation techniques have
been successful in recovering fertile plants
in F1 and subsequent generations in interspe-
cific crosses in Vigna. Pandiyan et al. (2008)
reported increased pod set in interspecific
V. radiata × V. umbellata crosses developed
from gamma ray- irradiated parental lines.
Reciprocal crossing
Reciprocal differences in wide crosses are also
very common, and can be due to chromo-
somal imbalance in the endosperm, the role of
the sperm nucleus in differential endosperm
development or the alteration of endosperm
development by pollen through the effects of
antipodal cells, which are assumed to supply
nutrients during early endosperm develop-
ment (Beaudry, 1951). If disharmony between
the genome of one species and cytoplasm
of the other is a cause of a fertilization bar-
rier, reciprocal crosses can be successful in
recovery of hybrids. For example, while a
V. mungo × V. radiata cross was unsuccessful,
its reciprocal cross, V. radiata × V. mungo, pro-
duced successful hybrids (Verma and Singh,
1986; Ravi et al., 1987). Interspecific hybridi-
zation between V. nakashimae and V. angularis
was successful in both directions and viable
seeds were produced, while V. riukinensis pro-
duced successful hybrids when used as male
parent only with V. angularis and V. umbellata
(Siriwardhane et al., 1991). In general, using
a female parent with higher chromosome
number is more successful than the reciprocal
method.
Use of bridge species
When useful genes are available in secondary
and tertiary gene pools and direct hybridi-
zation between cultivated and wild species
does not result in fertile hybrids, involve-
ment of a third species as a bridge species
has often been used for introgression of alien
genes. For example, attempts at hybridizing
Lens culinaris with L. lamottei and L. nigricans
have not yielded fertile hybrids. This offers
the possibility of transferring the genes for
resistance to ascochyta blight and anthracnose
to L. culinaris by using L. ervoides as a bridge
species, with the embryo rescue technique as
a means of broadening the resistance gene
base in the cultivated species (Ye et al., 2002;
Tullu et al., 2006). Transfer of bruchid resist-
ance from wild Vigna species is difficult due
to cross incompatibility. By using the bridge
species V. nakashimae, the bruchid resistance
of V. umbellata is transferred to azuki bean
(Tomooka et al., 1992, 2000). However, bridge
crosses will work only under the condition
where species A hybridizes with species B but
not with species C, and species B and C form a
viable hybrid. Based on the close relationship
reported in perennial Cicer anatolicum, C. retic-
ulatum and C. echinospermum, the bridge-
crossing approach deserves further attention.
Growth hormones
In wide crosses, if the hybrid seeds die when
their embryos are too small to be cultured,
post-pollination application of growth regu-
lators such as gibberellic acid, naphthalene
acetic acid, kinetin or 2, 4-D (dimethylamine),
singly or as in combination, may be help-
ful in maintaining the developing seeds by
facilitating division of the hybrid zygote and
endosperm. Mallikarjuna (1999) observed that
the only way to obtain interspecific hybrid in
chickpea is by the application of growth regu-
lators to pollinated pistils, to prevent initial
pod abscission and to save the aborting hybrid
embryos by embryo rescue techniques. Some
interspecific crosses have been successful in
Phaseolus (Stalker, 1980), Cajanus (Singh et al.,
1993) and Cicer (Shiela et al., 1992) by appli-
cation of growth regulators after pollination.
This suggests that further breakthroughs in
wide crossing may be possible through the
exploitation of growth regulators followed by
embryo rescue. In vivo hormonal treatments
have also greatly helped in recovery of inter-
specific hybrids in Vigna. A true-breeding
Vigna mungo × V. radiata derivative was recip-
rocally crossed with V. angularis, and the pol-
linated pistils were treated with after GA3 24
and 78 h of pollination.
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94 S. Kumar et al.
Backcrossing
In wide crosses, plants in initial generations
are generally of inferior nature with poor
expression of desired traits. This requires
advancing the cross populations up to F8/F9
generations for recovery of desired types. In
many cases the crosses are abandoned mid-
way due various reasons, in spite of reports
that useful recombinants could be recovered
in later generations (F10–F12) of an interspecific
cross (Singh and Dikshit, 2002). Therefore,
delayed segregation often causes problems in
identification and utilization of useful recom-
binants in interspecific crosses. This problem
can be overcome through backcrossing F1
hybrids with cultivated species in early gener-
ations. Mallikarjuna et al. (2006) introgressed
the Cajanus platycarpus genome into cultivated
pigeon pea by backcrossing embryo-rescued F1
hybrids with cultivated pigeon pea followed
by in vitro culture of aborting embryos of BC1
progeny. Similarly, one or more backcrosses to
the recurrent parent are often required in com-
mon bean to restore fertility of hybrids when
crossed with Phaseolus acutifolius and P. parvi-
folius. Using P. acutifolius as female parent of
the initial F1 cross, and/or first backcrossing
P. vulgaris × P. acutifolius hybrid on to P. acuti-
folius, is often more difficult than using P. vul-
garis as the female parent of the initial cross
and backcrossing the interspecies hybrid on
to P. vulgaris (Mejia-Jimenez et al., 1994). The
choice of parents (Parker and Michaels, 1986;
Federici and Waines, 1988; Mejia-Jimenez et al.,
1994) and use of the congruity backcross (i.e.
backcrossing alternately to each species) over
recurrent backcrossing (Haghighi and Ascher,
1988; Mejia-Jimenez et al., 1994) facilitate inter-
specific crosses of common and tepary beans,
in addition to recovery of fertility and more
hybrid progenies.
6.5. Successful Examples of Alien
Gene Introgression in Food Legumes
Successful examples of alien gene introgres-
sions in food legumes are limited to a few, for
various reasons (Table 6.4). Genes for disease
and insect resistance, male sterility and fertility
restoration, and yield attributes have been
transferred into cultivated species of various
legume crops. For example, successful intro-
gression of drought tolerance from Cicer reticu-
latum (Hajjar and Hodgkin, 2007), yield genes
from C. reticulatum (Singh et al., 2005) and tol-
erance to ascochyta blight, cyst nematode and
leaf miner have been documented. In lentil,
some progress has been made in introgres-
sion of alien genes for resistance to ascochyta
blight, anthracnose and cold in cultivated
lentil (Hamdi et al., 1996; Ye et al., 2002; Fiala,
2006). Successful examples of using crossable
wild species in pigeon pea breeding include
development of a highly cleistogamous line
(Saxena et al., 1992); genetic dwarfs (Saxena and
Sharma, 1995); phytophthora blight resistance
(Reddy et al., 1996; Mallikarjuna and Saxena,
Table 6.4. Successful examples of introgression in food legumes
Crop Wild relatives Character Reference(s)
Chickpea Cicer reticulatum Cyst nematode Di Vito et al. (1996)
C. reticulatum Yield Jaiswal and Singh (1989),
Singh et al. (2005)
C. reticulatum Cold tolerance Singh et al. (1995)
Lentil Lens orientalis Cold tolerance Hamdi et al. (1996)
Agronomic traits Abbo et al. (1992); ICARDA
(1995)
Lens ervoides Anthracnose resistance Fiala (2006); Tullu et al.
(2006)
Pigeon pea Cajanus sericeus Male sterility Ariyanayagam et al. (1995)
C. scarabaeoides Male sterility Tikka et al. (1997)
Mung bean Vigna mungo YMV resistance, plant
type traits
Singh and Dikshit (2002)
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Distant Hybridization and Alien Gene Introgression 95
2002); high-protein lines (Saxena et al., 2002);
cytoplasmic male sterile (CMS) lines (Saxena
et al., 2006); cyst nematode resistance (Saxena
et al., 1990); salinity resistance (Subba Rao et al.,
1990); and helicoverpa tolerance (Reed and
Lateef, 1990). Some successful examples of
alien gene introgression in food legume crops
are described below.
Yield genes
The notion that wild relatives are a prospec-
tive source of genes for biotic stress tolerance
only has been dismantled with convincing evi-
dence of introgression of yield QTLs from the
wild progenitors in some crops, including oats
(Frey et al., 1983), rice (Xiao et al., 1996) and
tomato (Tanksley et al., 1996; Fulton et al., 2000).
The possibilities of introgression of desirable
alien genes from wild to cultivated chickpea
have been explored (Jaiswal and Singh, 1989;
Verma et al., 1990; Singh et al., 2005). Studies
have shown that, besides disease resistance
and drought tolerance, wild Cicer species have
genes for desirable yield components such as
high number of fruiting branches and pods
per plants (Singh et al., 1994). In chickpea,
alien genes for productivity have been trans-
ferred from Cicer echinospermum, C. reticulatum
(Singh and Ocampo, 1997) and C. reticulatum
(Singh et al., 2005). Singh and Ocampo (1997)
transferred some genes from C. echinospermum
and C. reticulatum into cultivated chickpea and
observed up to 39% increase in seed yield fol-
lowing the pedigree method. Singh et al. (2005)
also reported introgression of yield genes and
disease resistance genes from C. reticulatum
to cultivated variety L550, with interspecific
derivatives showing 6–17% yield advantage.
A cross between Pusa 256 and C. reticulatum
was made and their F1 was again crossed with
the wilt-resistant variety Pusa 362. Further
selection concluded with the development of
Pusa 1103, which is a high-yielding early vari-
ety with resistance to wilt, root rot and stunt
virus and tolerance to drought and heat (Hajjar
and Hodgkin, 2007; Kumar et al., 2010). Singh
and Dikshit (2002) introgressed yield genes in
mung bean from urd bean with 15–60% yield
advantage. The derivatives from mung bean ×
urd bean crosses exhibit many other desirable
features such as lodging resistance, synchrony
in podding and non-shattering (Reddy and
Singh, 1990).
Disease resistance
In chickpea, introgression of resistance to cyst
nematode from Cicer reticulatum has been
reported, with promising lines under evalua-
tion at ICARDA (Di Vito et al., 1996; Ocampo
et al., 2000). Recently, resistance to anthrac-
nose found in Lens ervoides germplasm has
been exploited in Canada by introgressing
resistance genes into cultivated backgrounds
(Fiala, 2006; Tullu et al., 2006). This successful
use of L. ervoides holds promise as a source
of genes for resistance to other diseases, and
possibly for plant habit, biomass production
and other important agronomic and market-
ing traits. Further exploitation of L. ervoides
and the other wild Lens species is war-
ranted. Derivatives from mung bean × urd
bean crosses exhibit a higher level of MYMV
resistance (Gill et al., 1983). A few mung
bean × ricebean and mung bean × Vigna radi-
ata var. sublobata crosses having a high degree
of resistance to MYMV were also recovered
(Verma and Brar, 1996). Three mung bean
cultivars, HUM 1, Pant Moong 4 and IPM99-
125, and one urd bean cultivar, Mash 1008
(Sandhu et al., 2005) have been developed
from mung bean × urd bean crosses. These
cultivars have improved plant types, in addi-
tion to higher MYMV resistance and synchro-
nous maturity. In common bean, successful
introgressions of alien genes imparting CBB
(Freytag et al., 1982; Park and Dhanvantari,
1987; Miklas et al., 1994a, b), fusarium root
rot (Wallace and Wilkinson, 1965) and white
mould (Abawi et al., 1978; Dickson et al.,
1982; Lyons et al., 1987; Miklas et al., 1998a)
from Phaseolus coccineus have been reported.
In contrast, resistance to halo blight from the
common bean was incorporated into P. coc-
cineus (Ockendon et al., 1982). A high level
of resistance to CBB was transferred from
tepary to common bean (Coyne et al., 1963;
McElroy, 1985; Scott and Michaels, 1992;
Singh and Munoz, 1999).
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96 S. Kumar et al.
Insect pest resistance
The major production constraint of food
legumes is susceptibility to bruchids
(Callosobruchus chinensis L.) that eat seeds in
storage. One accession of wild mung bean
(Vigna radiata var. sublobata) exhibited com-
plete resistance to azuki bean weevils and
cowpea weevils (Fujii et al., 1989), which
has successfully been used in breeding pro-
grammes (Tomooka et al., 1992). Vigna mungo
var. silvestris) is also reported to be immune to
bruchids (Fujii et al., 1989; Dongre et al., 1996).
Recently, rice bean (V. umbellata) has been iden-
tified as being of use because many accessions
show complete resistance to bruchids and it is
a cultivated species. Efforts are in progress at
AVRDC to utilize V. r. var. sublobata for resist-
ance to bruchids. Similarly, sources of resist-
ance to leaf miner were used successfully in
a chickpea breeding programme at ICARDA
to develop promising breeding lines with leaf
miner resistance for North Africa and West
Asia (Singh and Weigand, 1996).
Male sterility and fertility restoration
Several wild relatives were used in hybridi-
zation with Cajanus cajan, and male sterile
plants were isolated from the segregating
populations. Ariyanayagam et al. (1995)
crossed C. sericeus with C. cajan and isolated
male sterile plants from the BC3F1 population.
Tikka et al. (1997) developed a CMS line using
C. scarabaeoides cytoplasm. Male sterile plants
were also isolated from an interspecific cross
of C. cajanifolius with C. volubilis. Saxena and
Kumar (2003) developed a CMS sterile line,
cms 88039A, using C. scarabaeoides (ICPW 89)
and an early-maturing line of C. cajan (ICPL
88039). Similarly, two CMS lines, CORG
990052A and CORG 990047A, were devel-
oped by interspecific hybridization of C. cajan
and C. scarabaeoides (Kalaimagal et al., 2008).
Experimental hybrids based on cytoplasmic
male sterility derived from C. scarabaeoides
and C. sericeus in pigeon pea are currently
being evaluated in multi-environment tri-
als. One recently released hybrid, GTH 1, has
male sterile cytoplasm from C. scarabaeoides.
6.6 Future Strategy for Alien Gene
Introgression
Advanced backcross-QTL strategy
Since the mid-1990s, convincing evidence at
both morphological and molecular levels has
accumulated for the utility of wild progeni-
tors and related species as donors of produc-
tivity alleles. Productivity-enhancing genes/
QTLs (quantitative-trait loci) have been intro-
gressed in oats from Avena sterilis (Frey et al.,
1983), in tomato from Lycopersicon pimpinelli-
folium and L. parviflorum (Tanksley et al., 1996;
Fulton et al., 2000), in rice from Oryza rufipogon
(Xiao et al., 1996) and in chickpea from Cicer
reticulatum (Singh et al., 2005). Novel breeding
strategies such as AB-QTL (advanced back-
cross-QTL) have been deployed to exploit the
worth of the progenitor and related species
as this helps minimize the negative effect of
linkage drag associated with alien gene intro-
gression (Tanksley and Nelson, 1996). The
related species of mung bean, such as Vigna
umbellata and V. angularis, have compara-
tively higher productivity and their relation-
ship with mung bean offers an opportunity
for the introgression of some productiv-
ity alleles using AB-QTL strategy. Another
related species, V. mungo, and the wild pro-
genitor of mung bean, V. radiata var. sublobata,
may also contribute some productivity alleles
to the elite mung bean lines using the same
approach.
Looking for genes based
on molecular maps
The traditional approach in utilizing exotic
germplasm is to screen the phenotype of
entries from a gene bank for a clearly defined
character and to use them in a crossing pro-
gramme in order to introduce the genes into
cultivated germplasm. Although effective for
qualitative traits, only a small proportion of
the genetic variation has been exploited for
crop improvement as a result of this strategy
(Tanksley and McCouch, 1997). Availability
of genetic linkage maps based on molecular
markers has opened up new opportunities in
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Distant Hybridization and Alien Gene Introgression 97
the utilization of hitherto unexploitable exotic
germplasm. This requires a paradigm shift
from selecting potential parents on the basis of
phenotype to evaluating them directly for the
presence of useful genes, through the integra-
tion of molecular tools. A gene-based approach
to screening exotic germplasm has already
been successfully used in rice and tomato for
improving yield levels (Tanksley et al., 1996;
Xiao et al., 1996). Recently, good progress has
been made in generating genomic resources
for food legume crops that will be very useful
in genetic mapping and QTL analysis in these
crops (Varshney et al.2009). With the use of
DNA profiles, the genetic uniqueness of each
accession in a gene bank can be determined
and quantified. Molecular marker technol-
ogy allows a targeted approach to the selec-
tion and introgression of valuable genes from
a range of genetic resources while retaining
the integrity of valuable genetic background
through forward and background selection.
Recombination DNA technology
Transgenic approaches provide new options
for broadening the genetic base in those cases
where current options are lacking in their effi-
cacy or existence. Plant genetic transformation
techniques such as Agrobacterium-mediated
transformation and direct gene delivery sys-
tem (biolistics) allow the precise transfer of
genes from any organism into either plant
nuclear or chloroplast genomes. Many iso-
lated plant genes are now being transferred
between sexually incompatible plant spe-
cies. In chickpea and pigeon pea, helicoverpa
pod borer is a major insect pest for which no
genetic solution exists. This requires devel-
opment of transgenics having Cry genes
from the soil bacterium Bacillus thuringiensis
to combat the menace of helicoverpa pod
borer. The recent report of a Bt. chickpea is
an encouraging step towards improvement
of food legumes for difficult traits such as
pod borer resistance (Acharjee et al., 2010).
Similar is the case for botrytis grey mould
in chickpea, where efforts are under way to
construct a resistance against this disease. For
gene introgression purposes, difficult species
falling in tertiary and quaternary gene pools
may turn out to be important sources of alien
genes. For example, identification and clon-
ing useful genes from Phaseolus filiformis,
P. angustissimus and P. lunana and successful
regeneration and transformation of common
bean may facilitate gene introgression in the
future.
Protoplast technology
Somatic hybridization using protoplast fusion
has potential to overcome pre- and post-zy-
gotic barriers to interspecific hybridization
(Powers et al., 1976; Davey et al., 2005). It is
possible to regenerate plants from a number
of legume species, including Pisum (Ochatt
et al., 2000), Trifolium (Gresshoff, 1980), Lotus
(Ahuja et al., 1983) and Melilotus (Luo and Jia,
1998), and asymmetric protoplast fusion has
been used for Medicago improvement (Tian
and Rose, 1999; Yuko et al., 2006). However,
only a few reports of successful regeneration
of plantlets are available in legumes (Li et al.,
1995). Initially, protoplast-derived tissues in
rice bean were obtained although no shoot
regeneration could be obtained. Shoot regen-
eration from protoplasts of Vigna sublobata
has more recently been reported by Bhadra
et al. (1994), with the maximum protoplast
yield being obtained from 5-day-old seed-
lings. There are no reports at the time of writ-
ing of successful growth or regeneration of
protoplasts from Lens species. Rozwadowski
et al. (1990) cultured protoplasts from lentil
epicotyl tissue, and around 6% of protoplasts
developed into cell colonies.
Doubled haploids
Doubled haploid breeding is an important
approach in many crop species, including
wheat, barley, rice, maize and canola, to fix the
hybrid immediately. Implementation of dou-
bled haploids increases selection efficiency
and allows new varieties to be bred up to 5
years faster than with conventional breeding
methods alone. Haploids may be produced
from either immature pollen cells, immature
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98 S. Kumar et al.
egg cells or following asymmetric chromo-
some elimination after interspecific hybridi-
zation. Several attempts have been made to
develop anther and microspore culture sys-
tems for chickpea (Huda et al., 2001; Vessal
et al., 2002; Croser et al., 2006), common bean
(Peters et al., 1977; Munoz-Florez and Baudoin
1994a, b), field pea (Croser et al., 2006) and
pigeon pea (Pratap et al., 2009). In chickpea,
cultivars responsive to isolated microspore
cultures have been identified and the induc-
tion of sporophytic development achieved in
uninucleate microspores via the application
of heat stress (32.5°C) pre- treatment to the
buds (Croser et al., 2006). Due to difficulty in
derivation of green haploid regenerants these
species have been defined as recalcitrant to
androgenesis, although some progress has
been made towards standardizing callus
induction media and culture conditions in
some of these crops. However, the produc-
tion of a successful double haploid system
in chickpea has been reported (Grewal et al.,
2009). A review of the literature on doubled
haploid production in Fabaceae (Croser et al.,
2006) indicated that none of these approaches
had been successful in producing haploid
plants in food legumes, but the early stages
of isolated microspore division have been
observed.
6.7 Prospects
Productivity of food legume crops is affected
by various biotic and abiotic stresses. There is
thus an urgent need to widen the cultivated
gene pool of these crops by incorporating
genes for economically important traits from
diverse sources. Wild species have proved to
be an important reservoir of useful genes, and
offer great potential for the incorporation of
such genes into commercial cultivars. Many
of the useful alien genes are expected to be
different from those of the cultivated species,
and are thus useful in broadening the base of
resistance to various stresses. Recently, OTLs
(oligogenic traits) that have been identified
for yield traits in wild species of pulse crops
may enhance agronomic and market values
of cultivated varieties. The molecular marker
technique can also be used for authentication
of interspecific hybrids (Yamini et al., 2001).
There is a need to identify high-crossability
genes in food legumes, as has been identified
in wheat cultivars such as Chinese Spring
(Luo et al., 1993; Sharma, 1995). Identification
of such genes in food legumes can bring non-
crossable species within the ambit of alien
gene transfer technology. There are major
gaps in germplasm collections of wild spe-
cies and their evaluation in food legumes
that need to be filled, in order to progress
further inroads in alien gene introgression.
Continuing advances in wide-crossing tech-
niques, such as embryo culture and develop-
ment of novel crossing strategies, are creating
greater accessibility in wild gene pools of
many crops. The success rate of gene trans-
fer in such wide crosses can be increased by
knowledge of chromosome pairing mecha-
nisms and their genetic control. The modern
tools of molecular biology, such as mono-
clonal antibodies and in situ hybridization
using various DNA probes, may soon make
it possible to study the switching on and off
of various genes in diverse tissues of the fer-
tilized ovule, and control over the levels and
movements of both exogenous and endog-
enous growth substances within the develop-
ing seed. It is likely that continuing advances
in structural genomics and genetic engineer-
ing will result in new strategies for alien gene
introgression.
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