Genetic Transformation of Vigna Species: Current
Status and Future Perspectives
Darshna Chaudhary1, Manish Sainger1, Lingaraj Sahoo2 and Pawan K. Jaiwal1*
1

Advanced Centre for Biotechnology, M. D. University , Rohtak-124001, India,
Department of Biotechnology , Indian Institute of Technology , Guwahati, India* Corresponding author, e mail: jaiwalpawan@rediffmail.com

2

Abstract

Vigna species form an important component for vegetarian diet and sustainable agriculture.
During the last four-five decades their production is reducing or stagnating, which has resulted inescalation of their market price. To meet the demand of bourgeoning population, their yield is to beenhanced and stabilized in their traditional growing areas where they are exposed to numerous abiotic
and biotic stresses. Their improvement through conventional breeding is low or often not possible
due to the lack of sufficient and satisfactory level of genetic variability within the germplasm.
Biotechnological approaches are required to widen the gene pool beyond sexual boundaries. Much
progress has been made in in vitro regeneration of sexually mature plants, in majority of theserecalcitrant species, from seedling explants such as cotyledonary node and shoot tip via direct shootorganogenesis in V. mungo , V. radiata and V. unguiculata and considerable success in generating
plants from callus in V. angularis and from protoplast in V. aconitifolia . These protocols are used forgeneration of transgenics in major cultivated Vigna species. This paper provides an overview of invitro regeneration, state of the art, current status and critical analysis of the genetic transformation in
major Vigna species along with future directions for their improvement.

Introduction

The genus Vigna has 150 species, only sevenof them are cultivated, six Asian of subgenus Ceratotropis , V. radiata , V. mungo , V. angularis , V. aconitifolia and V. umbellata , and two African of subgenus Vigna , V. unguiculata and V. subterranea . These
species are grown for their protein rich edible seeds
and sprouts which are rich in minerals and vitamins.
The plants are also used as fodder. All Vigna species
fix atmospheric nitrogen and are of short duration,
therefore, they are grown as mixed/ intercrop or inrotation to improve nitrogen status of soil and to
break disease/pest cycles. The production of Vigna
species has not improved significantly due to theirprominent susceptibility to abiotic stresses such as
salinity and drought etc and to several fungal, bacterial or viral pathogens and insect pests (see Jaiwal
and Gulati, 1995, Sahoo and Jaiwal, 2008) The otheryield limiting factors are their indeterminate growthhabit, defective plant type, low harvest index, sensitivity to photoperiod and temperature, asynchronous
flowering and shattering pods and sprouting of seeds
in situ following rains. Genetic improvement ofthese species through conventional breeding has beenslow due the lack of desirable and satisfactory levelsof variability in germplasm. Genes conferring resistance to biotic and abiotic stresses are found in wildor related species (Tomooka et al ., 2004, Vaughan et
al ., 2004) which are sexually incompatible with thecultivated species. The only option left is to transfer
desirable genes from other sources for their quanti- 41 -tative and qualitative improvement through in vitro
regeneration and genetic transformation techniquesin more precise and faster ways as compared to traditional Mendelian methods. Besides crop improvement, these approaches also help in studying genefunction. The present review provides the current
status of genetic transformation undertaken for the
improvement of Vigna species and outlines the future
directions.
Genetic transformation of Vigna species
Like the large-seeded legumes, the genetictransformation of Vigna species have been difficultand challenging because of their recalcitrant nature toin vitro regeneration and genetic transformation (Sahoo and Jaiwal, 2008). During the last almost three
decades significant progress has been made to transform grain legumes including Vigna species (Gulati
and Jaiwal, 1995, Somers et al., 2003; Popelka et al .,2004; Eapen et al ., 2008, Sahoo and Jaiwal, 2008).
Gene transfer in these species requires a robust plantregeneration system from tissues amenable to routine
transformation method, efficient selection system forthe recovery of viable and fertile transgenic plantsfrom transformed explants at a reasonable frequency
and a time frame in culture to void somaclonal variation and possible sterility (Sahoo et al ., 2003).

In vitro regeneration
A fast, reliable and efficient plant regeneration
system is essential for the development of a robust

genetic transformation procedure. However, grain
legumes in general and Vigna species in particular
are highly recalcitrant to in vitro regeneration. In
recent years significant progress has been made toregenerate them through proliferation of apical andaxillary meristems, de novo organogenesis and somatic embryogenesis (see Jaiwal and Singh, 2003).
Direct multiple shoot organogenesis from cotyledonary node and shoot tip of embryonal axis or seedlinghas been found more suitable for transformation as in
Vigna radiata (Jaiwal et al ., 2001; Sonia et al ., 2007),
V. mungo ( Saini et al ., 2003, Saini and Jaiwal, 2005)
and V. unguiculata (Chaudhary et al ., 2007).
Both these explants have pre-existing meristems which give rise to multiple shoots on induction
by cytokinins and are transformation competent. In
V. angularis , de novo organogenesis from epicotyl
has been used for developing transformants. Regeneration via somatic embryogenesis is preferred
for transformation in order to homogenously (nonchimeric) transformed plants as somatic embryos
develop from single cells. Although somatic embryogenesis has been obtained from cell suspension
cultures established either from primary leaf-derived
embryogenic calli in V. unguiculata (Kulothungan et
al ., 1995; Ramakrishnan et al ., 2005) or hypocotyl
derived calli in V. mungo (Saini et al ., 2004) andthrough embryogenic calli obtained from immature
cotyledons (Girija et al ., 2000) or primary leaf segments (Devi et al ., 2004) in V. radiata , it has beenvery difficult to convert somatic embryos into plantlets at a high frequency, therefore, organogenesis
pathways are to be followed. Further, in vitro plantregeneration in Vigna species is dependent on genotype, explant type, media composition and culture
conditions. These parameters are to be optimized forestablishing an optimal regeneration protocol.
Recent knowledge on in vitro regeneration
will soon allow introduction of genes that promotesomatic embryogenesis such as Wuschel (Zuo et al .,2002), Baby boom (Boutilier et al ., 2002) and esr-1(Bano and Chuo, 2002) into grain legumes to enhance their embryogenic response and improve theirregeneration potential.
Reporter and selectable marker genes
Screenable markers or reporter genes code for
products that detoxify selective agents or catalyze
specific reactions whose products are detectable.
The β-glucuronidase (uidA /gusA ) gene is widely
employed as reporter for transient and stable transformation studies in legumes including Vigna species (see Sahoo et al ., 2003). Localized gus activity- 42 -

in intact tissues can be visualized as blue-spots or-areas within the tissue. However, GUS assay is destructive in nature. The assay of green fluorescentprotein (GFP) gene of the jellyfish, a reporter geneis non-destructive and do not require any cofactor orexternal substrate. In plants, GFP accumulation does
not have any toxic effect. Use of GFP protein genehas been beneficial for screening of transformed
shoots at an early stage to avoid the chimeras. Noneof methods developed so far for Vigna species, except in V. angularis (Yamada et al ., 2001), exploredthis possibility.
Selectable marker genes are useful in selection
and maintenance of transformed cells/plants as they
allow the transformed cells to proliferate and growin the presence of a selective agent while the nontransformed cells either do not grow or multiply at aslow rate. The most commonly used selectable marker gene in legumes is neomycin phosphotransferase(nptII ) which imparts resistance to kanamycin or its
analogues, geneticin or paromomycin by inactivatingthem through phosphorylation. Kanamycin has beenused for the selection of transformants in V. radiata(Jaiwal et al ., 2001), V. mungo (Saini et al., 2003,
Saini and Jaiwal, 2005), V. unguiculata (Chaudhary
et al ., 2007) and V. angularis (Yamada et al ., 2001).
It neither impairs the normal shoot regeneration nor
shoot vigor but helps in the early identification ofgreen transformed shoots as the shoots emerging
from non-transformed cells are bleached (albino).
However, recovery of a large number of escapes orchimeric shoots in the presence of kanamycin is themain problem. Geneticin in conjunction with nptII
has been found to be more effective in selection oftransformed shoots in cowpea (Solleti et al ., 2008).
The hygromycin phosphotransferase (hpt ) genewhich confers resistance to hygromycin has also beenused in V. radiata (Mahalakshmi et al ., 2006) and V.
angularis (El-Shemy et al ., 2002) for the recovery oftransformants with minimum escapes. The efficiencyof transformation has been improved by replacing
antibiotic resistance genes with genes for herbicide
tolerance, as selectable marker. Bialaphos resistancegene (bar ) which acetylates and detoxify phosphinothricin has been efficiently used for selection oftransformants in V. radiata (Sonia et al ., 2007), V.
mungo (Muruganantham et al ., 2007), V. unguiculata(Popelka et al ., 2006) and V. angularis (Khalafalla
et al ., 2005). PPT selection is more efficient than
kanamycin. Delayed application of selective agent
after coculture was found to be more efficient for therecovery of transformants in V. unguiculata (Popelka
et al ., 2006). The concentration of selective agent (le-thal or sublethal), time of its application (i.e., more
precisely immediately after coculture or at a muchdelayed stage), and increments (gradual increasingor decreasing increments) need to be carefully assessed for a particular species and an explant (Sahoo
and Jaiwal, 2008). Judicious choice of selective
levels has remained as an important criterion for
the recovery of transformed cells at initial stage ofscreening. Studies on the use of positive selectable
markers such as phospho-mannose isomerase (pmi ),xylose isomerase (xyl A) etc and the elimination ofmarker genes that are considered environmentallysafe, are to be taken up in Vigna species (Eapen et
al ., 2008).
Gene transfer methods
The three methods, Agrobacterium- mediated, biolistics and electroporation have been used
for genetic transformation of Vigna species. Amongthem, A. tumefaciens mediated transformation is
the preferred method.

Agrobacterium -mediated transformation
Agrobacterium Ti plasmid-based vector isthe best system for plant transformation due to its
simplicity, high frequency of precise transfer andintegration of transgenes with single copy insertion,
low incidence of transgene silencing and ability totransfer long stretches of DNA (Veluthambi et al .,2003).
Vigna radiata
Pal et al . (1991) were the first to recover primary transformants on kanamycin selection fromcotyledons of V. radiata inoculated with A. tumefaciens . The transgenic nature of transformants was
confirmed by dot blot and MUG assays. However,no evidence was presented for stabilization andinheritance of transgenes. Phogat et al . (1999) regenerated transgenic calli on kanamycin (100 mg/l)
selection medium from primary leaf explants cocultured with A. tumefaciens . The transformed calli
were found resistant up to 750 mg/l of kanamycin,
exhibited β-glucuronidase activity and showed integration of nptII gene by Southern analysis. However, plants could not be recovered from transgenic
calli. Jaiwal et al . (2001) successfully recovered
fertile transgenic plants from agro-inoculated cotyledonary node explants on kanamycin selection medium at a frequency of 0.9 %. Molecular analysis
of primary transformants revealed integration andexpression of transgenes but no data for inheritanceof transgenes to progeny was presented. Mahalak-shmi et al . (2006) developed transgenic mungbean
plants from primary leaf explant on hygromycin selection medium at a frequency of 2 %. Integrationof marker gene in T0 and its inheritance in T1 wasshown. Sonia et al . (2007) generated transgenic
plants carrying Phaseolus vulgaris α -amylase inhibitor I gene, on PPT selection at a frequency of 1.5%.

Vigna mungo
Karthikeyan et al . (1996) obtained transformed calli of V. mungo by cocultivating segments of primary leaves with A. tumefaciens . The
stable integration of nptII in transgenic calli was
confirmed by Southern hybridization and the expression was demonstrated by neomycin phosphotransferase assay. However, transgenic calli could
not be regenerated into plants. Saini et al . (2003)
developed fertile transgenic plants from cotyledonary node explants (without cotylendons) inoculatedwith A. tumefaciens . The transgenic nature wasconfirmed by stable GUS activity, integration ofuidA gene by Southern analysis and inheritanceof transgene to progeny in Mendelian fashion.
Mechanical wounding of explants prior to inoculation with Agrobacterium , time lag in regeneration
due to removal of cotyledons from explants and asecond round of selection on kanamycin at rooting
stage were found to be critical for transformation.
However, the efficiency of transformation of meristematic cells in the axil of cotyledonary node waslow (1%) which may be attributed to the presenceof limited number of regenerable cells whose capacity for regeneration were short lived-in explantand inefficient T-DNA delivery to regenerable cells(Saini and Jaiwal, 2005). An Agrobacterium -mediated transformation of shoot apex explants wasdeveloped to overcome these limitations. A significant improvement in transformation efficiency
from an average of 1% to 6.5% was obtained. Evidence for stable integration of transgenes and theirinheritance to progeny were presented. Shoot apex
explants are preferred because of their high regeneration potential with minimal tissue culture manipulations in a less genotype dependent fashion.
Saini and Jaiwal (2007) optimized the conditions
for enhanced transformation of cotyledonary nodeand generated stable transgenic plants at a frequency of 4.3%. Murugananthan et al . (2007) developed
herbicide (Basta®) tolerant V. mungo plants using
cotyledonary node and shoot tip explants (fromseedlings germinated in vitro from immature seeds)
and A. tumefaciens harboring a binary vector car-- 43 -

rying bar and uidA genes. Transgenes were shown to
be stably integrated and transmitted to progeny in Mendelian fashion. Immature cotyledonary node explants
produced a high frequency of transformed plants (7.6%)
than shoot tip explants (2.6%). Bhomkar et al . (2008)engineered salt stress tolerance in V. mungo by introducing glyoxalase I (gly I) gene under a novel constitutive
Cestrum Yellow Leaf Curling Virus (CmYLCV) into regenerable cells present at cotyledonary node of embryonic axis using A. tumefaciens . The T1 plants expressing
glyoxalase I activity survived and set seeds under NaCl
stress (100 mM).

Vigna angularis
Stable transformants of V. angularis were developed using A. tumefaciens -mediated gene transfer toepicotyl explants and subsequent selection on kanamycin (Yamada et al ., 2001) or hygromycin (El-Shemy etal ., 2002) or PPT (Khalafalla et al ., 2005) containing
medium. The transformation system based on Agroinfection of epicotyl explants was also used to transfer
Phaseolus vulgaris α-amylase inhibitor and D6 fatty aciddesaturase genes into V. angularis to impart resistanceagainst bruchids (Ishimoto et al ., 1996, Yamada et al ,2005) and to produce poly unsaturated fatty acids (Chenet al ., 2005) respectively.
Vigna unguiculata
Garcia et al . (1986) were the first to demonstratesusceptibility of cowpea to A. tumefaciens by inducing the formation of crown gall tumors (that produce
opines) at the wound site. Primary leaf discs of cowpea
plant were inoculated with A. tumefaciens strain C58C1
harboring a non-oncogenic Ti plasmid that contained
two copies of a chimeric kanamycin resistant gene. By
culturing the leaf discs on selective medium, kanamycin
resistant calli were obtained. However, whole plants
could not be regenerated from the transformed callus
tissue. Southern blot hybridisation revealed the integration of kanamycin resistant gene into the plant genome.
Muthukumar et al . (1996) obtained transformed shootson hygromycin selection medium from the mature
deembryonated cotyledons inoculated and cocultured
with A. tumefaciens carrying pUCD 2340 that contained hpt gene. The shoots were rooted on B5 basal
medium containing hygromycin and 17 rooted shoots(plantlets) were established in soil where only four ofthem survived and set seeds. Southern blot hybridization
confirmed the integration of hpt gene in the genome of
only one transgenic plant whose seeds failed to germinate. Hence transfer of transgene into progeny could not
be confirmed. Sahoo et al . (2000) reported recovery ofchimeric transgenic plants of cowpea under kanamycin- 44 -

selection using shoot apices and A. tumefaciens but did
not provide evidence of stable transformation. Popelka
et al . (2006) were the first to demonstrate the stabletransmission and expression of two cointegrated genes,
bar and uidA in the progeny of transgenic plants with afrequency of 0.001 to 0.003. They recovered transgenic
plants by inoculating the longitudinally bisected embryonic axes with cotyledon attached without shoot androot apices with hyper virulent Agro strain AGLI harboring a binary vector that contained a selectable marker(bar ) and a reporter gene (uidA ). This protocol required
5-8 months from explant preparation to harvested T1
seeds.
Chaudhary et al . (2007) improved the efficiencyof transformation from 0.1 (Popelka et al ., 2006) to
1.9%. Their transformation protocol is based on the direct shoot regeneration from the cotyledonary node explants without both the cotyledons. Removal of both thecotyledons not only reduces the size of the explants but
also delayed the shoot regeneration response which was
found to be effective for the selection of transformation
events. Mechanical wounding of the explants at nodal
regions prior to Agro infection and a second round of
selection at rooting stage were found to be critical in V.
unguiculata as in other Vigna species (Saini et al ., 2003,
Saini and Jaiwal, 2005, Sonia et al ., 2007).
Solleti et al . (2008) used Agrobacterium -mediated transformation for the introduction of the bean (Phaseolus vulgaris ) α-amylase inhibitor-1 (α AI-1) gene into
cowpea to confer resistance to bruchids (Callosobruchussps)- a storage pest. They enhanced the recovery oftransgenic plants to an average of 1.67% by the use ofadditional copies of vir genes in resident pSB1 vectorin Agro strain LBA4404, thiol compounds during cocultivation and a geneticin based selection.
Raji et al . (2008) demonstrated the compatibilityof in vitro plant regeneration protocol from embryonicaxes with Agrobacterium -mediated transfomation using
nptII as a selectable marker gene and paromomycin as aselective agent. PCR, uniform expression of uidA geneand Southern hybridization analysis of primary transformants revealed the integration of nptII gene into thecowpea genome. However, transmission of transgene toprogeny has not been demonstrated.
Nevertheless, these transformation systems arelaborious, time consuming, likely to be genotype- dependent and present low frequency of germline transformation (Ivo et al ., 2008).
Direct DNA transfer methods
Particle bombardment
Particle bombardment which relies upon the acceleration of DNA-coated particles into target cells ismuch preferred option for gene delivery to recalcitrant
grain legumes, Vigna species in particular, circumventing the host specificity as well as genotype dependenceby Agrobacterium . To date, only a few reports are available on transformation of Vigna species using particle
gun method. Kononowicz et al . (1993, 1997) demonstrated the possibility of transforming and obtaining
cowpea plants with introduced genes. However, stable
integration and expression of transgenes in progeny ofcowpea was not shown. Bhargava and Smigocki (1994)
observed transient expression of gus gene in the germinating embryos of V. aconitifolia , V. mungo and V. radiata , 18 to 24 h after particle bombardment. The raisedplantlets were rooted on kanamycin selection medium
but their transgenic nature was not confirmed. Ikea etal . (2003) optimized the conditions for delivery of plasmid that contained uidA and bar genes into embryonicaxes explants of cowpea using biolistic PDS-100/ He
particle delivery system. Putative transformed α wererecovered from the bombarded explants on bialophas(3 mg/l) selection medium. Molecular analysis of T1, T2
and T3 plants showed the transfer of genes into a small
proportion of these progenies and there was no evidence
for stable integration. Ivo et al . (2008) introduced themutant ahas gene (coding for acetolactate synthetase)and gus gene into the shoot apical meristem of cowpea,
which was fully exposed by removing primordial leaves,
excised from mature seeds. The putative transformed
shoot were selected on shoot regeneration medium containing 200µ-M imazapyr, a herbicide molecule capableof systemically translocating and concentrating in apicalmeristematic region of the plant. This selection coupledwith a multiple shooting allowed for recovery of transgenic cowpea lines at a frequency of germ line transformation. The L2 and L3 layers of the meristem should
receive the DNA coated particles as these layers dividein the anticlinal and periclinal plans to form the shoots.
Southern analysis of T0 plants confirmed the presence ofboth ahas and gus genes and their transmission to T1 andT2 progenies in Mendelian fashion.
Electroporation mediated transformation
Owing to the difficulties in regenerating Vigna
species from protoplast and callus cultures, electroporation of intact tissues and organs has been attemptedwith promising results. Penza et al . (1991) showed thatseed-derived embryos of V. unguiculata could take up
and transiently express a chimeric gene after passiveor electroporation-mediated DNA transfer. Akella andLurquin (1993) demonstrated that seedlings generated
from cowpea embryos after imbibition in the presenceof plasmid DNA harbouring gus gene, expressed the gus
transgene quite efficiently in a variety of tissues includ-ing apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and cationic liposome increased both the proportion of embryo
derived seedlings expressing the chimeric gene and thelevel of gene expression. However, they did not attempt
to grow the seedlings into transformed plants.
Chowira et al . (1995) attempted electroporation of axillary nodal meristem of mature plants of pea,
lentil, soybean and cowpea and produced the T1 transgenic plants from the seeds developed on the chimericbranch grew out from the elctroporated nodal meristem.
Although T2 soybean and lentil plants were obtained,no transgenic T2 cowpea plants were reported. Electroporation of whole plant tissue might results in unstable
pattern of inheritence of the introduced trait. Subsequentattempts in electroporation of axillary buds of pea witha construct containing coat protein gene of Pea Enation
Mosaic Virus (PEMV) lead to the generation of transformed plants resistant to PEMV (Chowira et al . 1996).
Adensoye et al . (2008) used electroporation
method for introduction of an insecticidal cry1Ab andan antibiotic resistance nptII gene into the nodal buds ofcowpea. T1 seeds derived from electroporated branches
were selected on a medium containing geneticin. Stable
integration of cry1Ab gene into cowpea genome at T1
was confirmed by PCR and Southern blot analysis. T1
plant of cowpea also showed resistance to Maruca vitrata larvae and protection of cowpea from damage.
Other novel transformation technologies
Although Vigna species are susceptible toAgrobacterium , difficulties in regenerating whole plants
especially from callus, cell suspension and protoplastnecessitated the development of alternative transformation methods that essentially avoid tissue culture
and regeneration procedures. Further the regeneration
steps are time consuming, labour intensive and required
skilled personnel and are associated with somaclonalvariations or morphological abnormalities exhibited by
the regenerated plants and reduced fertility. There are
only a few species for which transformation system (likeflower dip, vaccum infilteration of Agrobacterium etc.)
avoiding tissues based regeneration system, has beendeveloped which includes the modal plants Arabidopsis
thaliana , Medicago truncatula and Lotus japonica andsome legumes like soybean, peanut and pigeonpea. A
non-tissue culture based approach should be followed
for generating transgenics in Vigna species.
Improvement in transformation efficiency
The various approaches, such as mechanicalwounding of explants prior to Agrobacterium inoculation, use of supervirulent plasmids carrying extra cop- 45 -ies of virulence genes, presence of acetosyringone
in bacterial inoculation and cocultivation medium,inclusion of thiol compounds ( L-cysteine, DTT etc.)
in cocultivation medium, lower pH (lower than 5.86.0) of cocultivation medium, low temperature (<25° C) during infection and coculture, concentrationof bacteria and duration of cocultivation have beenused to improve transformation efficiency in Vigna
species.
Conclusions and future directions
Vigna species are cheap and rich source ofprotein for vegetarian diet and are important forsustainable agriculture. Their improvement throughconventional breeding has hampered due to limited
gene pool and sexual-incompatibility with wild andrelated species, the reservoir of desirable genes.
Conventional breeding is to be supplemented bybiotechnological means to transfer desirable genes
from sources across the sexual boundaries. The recalcitrant nature of Vigna species has so far hindered
the successful application of transformation technology. However, success has been achieved to regenerate them from seedling explants via adventitious
or direct shoot organogenesis. These protocols areused for Agrobacterium -mediated transformation in
all the cultivated Vigna species with low to moderate frequency in more or less genotype independent
manner. Regeneration from callus, cell suspension
and protoplast cultures except V. aconitifolia and V.
angularis still remain problematic. Transgenics withintegrated marker or reporter genes mostly up to T0
or T1 level have been developed. The stability andexpression of transgene(s) in subsequent progenies
have not been investigated. Agronomically desired
genes in some cases have been introduced but thesetransgenics are so far confined to the research laboratories only. Transformation through bombardmentof DNA-coated particle to regenerable tissue such as
seedling meritems, electroporation of intact plants
and in planta transformation are to be explored.
Improvement in transformation frequency in Vigna
species is required. A high throughput transformation system is not only essential for introduction ofdesirable genes for their better yield and nutritional
quality but also required to explore gene functions
through reverse genetic techniques. The new tools of
genomics, proteomics and metabolomics would allow better understanding of vital processes of thesefood legumes for their improvement. In addition, forthe sake of end user acceptance, the research should
focus on (1) development of methods avoiding antibiotic or herbicide resistance genes as selectable- 46 -

marker and (2) selecting genes for the desirable
traits for the transfer and (3)strategies for the seed
distribution system, where the end user in the developing countries is benefitted and not only industries
in developed countries. Further, there is a need of asustained coordinated research with long term funding and involvement of private sector for generationof transgenics in these tropical food legumes so as
to meet the daily protein requirement of millions ofpeople of the developing countries.
Acknowledgement
Financial support from DBT, New Delhi to the laboratory of PKJ for the work on genetic improvementof legumes is gratefully acknowledged.
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