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encoding enzymes required for the biosynthesis of
osmoprotectants or late embryogenesis proteins, detox-
ification enzymes and modification of membrane lipids
(Ishizaki-Nishizawa et al., 1996; McKersie et al., 1996;
Xu et al., 1996; Hayashi et al., 1997; Bhatnagar-Mathur
et al., 2008; Reddy et al., 2012; Rodziewicz et al., 2014).
Osmotic adjustment (osmotic regulation) is one of the
useful mechanisms for improving abiotic stress toler-
ance, especially if osmoregulatory genes can be triggered
in response to drought, salinity and high temperature
(Reddy et al., 2012). Sharmila et al. (2009) showed that
H 2 O 2 produced by a prokaryotic osmoregulatory choline
oxidase gene ( codA ) as a by-product during synthesis of
glycine-betaine is responsible for building a stronger
antioxidant system in chloroplasts of transgenic
chickpea plants. Similarly, at ICRISAT, the P5CSF129A
gene encoding the mutagenized D1-pyrroline-5-
carboxylate synthetase (P5CS) for the overproduction
of proline was introduced in chickpea. In several of
these transgenic events the accumulation of proline in
leaves increased significantly when the plants were
exposed to water stress, along with a decrease in free
radicals as measured by a decrease in the MDA levels, a
lipid peroxidation product (Reddy et al., 2012).
To date, genetic transformation has been reported in
all the major legume crops such as Vigna spp., C. arieti-
num , C. cajan , Phaseolus spp., Lupinus spp., Vicia spp., P.
sativum , soybean, groundnut, pigeon pea and chickpea
(Sharma & Lavanya, 2002; Reddy et al., 2012).
TILLING populations have been developed for several
legumes. For example, in the model legumes Medicago
(12,000 M2 plants; Rogers et al., 2009) and Lotus
(4904 M2 lines; Perry et al., 2009) mutant populations
were developed for use in reverse genetics. In the case
of crop legumes, over 3000 M3 lines were developed in
common bean and evaluated with root nodulation tests
by Porch et al. (2009). In peanut a TILLING population
of 10,000 lines has been established, and a subset of this
population investigated for allergenicity (Tadege et al.,
2009). In chickpea, a TILLING population of ~3500 lines
has been developed and is being used to identify candi-
date genes for drought tolerance (M. Thudi, personal
communication). The use of NGS technologies for
TILLING may increase the application of TILLING in
crop legumes (Kudapa et al., 2013).
EcoTILLING is a variant of TILLING, except that its
objective is to discover naturally occurring polymor-
phisms as opposed to experimentally induced mutations
(Kudapa et al., 2013). Single nucleotide polymorphisms
(SNPs), small insertions and deletions, and variations in
microsatellite repeat number can be efficiently detected
using the EcoTILLING technique (Kudapa et al., 2013).
For example, in legumes this method has been used to
develop molecular markers for cyst nematode candidate
resistance genes in soybean (Liu et al., 2012). In mung-
bean it has been proven to be a valuable method for
detecting polymorphisms in a collection that was previ-
ously shown to have limited diversity (Barkley & Wang,
2008).
1.7.9 targeting induced local lesions in
genomes (tILLING)
Recently, Kudapa et al. (2013) in their review reported
that validation of genes through genetic transformation,
RNAi or virus-induced gene silencing (VIGS) is a
time-consuming process in legumes, mainly due to lack of
efficient transformation systems in legumes. This situation
has promoted the application of TILLING to study gene
function. In TILLING, candidate genes are screened across
a mutant population (with point mutations), and line(s)
with the mutation for the target gene are identified
(McCallum et al., 2000). If the identified line exhibits the
expected phenotype for the candidate gene, the function
of the candidate gene is supported. The TILLING approach
could be preferred over RNAi for irreversibly inhibiting or
eliminating the target genes in commercial crop plants
since it avoids genetic transformation and increases sta-
bility of the phenotype (Barkley & Wang, 2008).
1.8 Conclusions and future prospects
Food legumes are affected by abiotic stresses like salinity,
water stress (drought and waterlogging), extreme tem-
peratures (heat and cold) and nutrient deficiency, which
ultimately lead to huge economic losses globally. Like
other plant species, the breeding process in food legumes
consists of four stages: (i) creating variations with
hybridizations and induced mutations; (ii) selection in
early generations; (iii) evaluation of selected lines; and
(iv) release of varieties (Toker & Mutlu, 2011).
The biotechnological approaches of resistance
breeding have provided several improved varieties of
food legumes with tolerance to abiotic stresses. There is
no substitute for these approaches, and they will con-
tinue to be the mainstay in the future. However, efforts
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