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Chickpea is a highly nutritious grain legume crop, widely appreciated as a health food, especially in the Indian subcontinent. The major constraints on chickpea production are biotic (Helicoverpa, bruchid, aphid, ascochyta) and abiotic (drought, heat, salt, cold) stresses, which reduce the yield by up to 90%. Various strategies like conventional breeding, molecular breeding, and modern plant breeding have been used to overcome these problems. Conventionally, breeding programs aim at development of varieties that combine maximum number of traits through inter-specific hybridization, wide hybridization, and hybridization involving more than two parents. Breeding is difficult in this crop because of its self-pollinating nature and limited genetic variation. Recent advances in in vitro culture and gene technologies offer unique opportunities to realize the full potential of chickpea production. However, as of date, no transgenic chickpea variety has been approved for cultivation in the world. In this review, we provide an update on the development of genetically modified chickpea plants, including those resistant to Helicoverpa armigera, Callosobruchus maculatus, Aphis craccivora, as well as to drought and salt stress. The genes utilized for development of resistance against pod borer, bruchid, aphid, drought, and salt tolerance, namely, Bt, alpha amylase inhibitor, ASAL, P5CSF129A, and P5CS, respectively, are discussed.
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Vol.:(0123456789)
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Molecular Biotechnology
https://doi.org/10.1007/s12033-018-0096-1
REVIEW
An Update onGenetic Modification ofChickpea forIncreased Yield
andStress Tolerance
ManojKumar1,2· MohdAslamYusuf3· ManishaNigam4· ManojKumar2
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Chickpea is a highly nutritious grain legume crop, widely appreciated as a health food, especially in the Indian subcontinent.
The major constraints on chickpea production are biotic (Helicoverpa, bruchid, aphid, ascochyta) and abiotic (drought, heat,
salt, cold) stresses, which reduce the yield by up to 90%. Various strategies like conventional breeding, molecular breeding,
and modern plant breeding have been used to overcome these problems. Conventionally, breeding programs aim at devel-
opment of varieties that combine maximum number of traits through inter-specific hybridization, wide hybridization, and
hybridization involving more than two parents. Breeding is dicult in this crop because of its self-pollinating nature and
limited genetic variation. Recent advances in invitro culture and gene technologies oer unique opportunities to realize the
full potential of chickpea production. However, as of date, no transgenic chickpea variety has been approved for cultivation
in the world. In this review, we provide an update on the development of genetically modified chickpea plants, including
those resistant to Helicoverpa armigera, Callosobruchus maculatus, Aphis craccivora, as well as to drought and salt stress.
The genes utilized for development of resistance against pod borer, bruchid, aphid, drought, and salt tolerance, namely, Bt,
alpha amylase inhibitor, ASAL, P5CSF129A, and P5CS, respectively, are discussed.
Keywords Chickpea· Transgenics· Agrobacterium· Abiotic stress· Biotic stress
Abbreviations
ASAL Allium sativum leaf lectin
Bt Bacillus thuringiensis
CaMV35S Cauliflower Mosaic Virus 35S promoter
CSIRO Commonwealth Scientific and Industrial
Research Organisation
GM Genetically modified
ICRISAT International Crop Research Institute for
Semi-Arid Tropics
IARI Indian Agricultural Research Institute
IIPR Indian Institute of Pulses Research
MAS Marker-assisted selection
Mbps Mega base pairs
MT Million tons
mha Million hectares
P5CS Δ1-Pyrroline-5-carboxylate synthetase
Introduction
Chickpea (Cicer arietinum L.) is a winter legume crop
grown worldwide for human food and animal feed. It origi-
nated in southeastern Turkey and is now cultivated globally.
India contributes 75% to the total chickpea production in the
world. Seeds are the edible part of this plant with 17–22%
protein content [1]. They are eaten raw, roasted, as sprouts
or dal (decorticated split cotyledons), or in the form of flour.
* Manoj Kumar
manojyd1234@gmail.com
Manoj Kumar
manojbiochem16@gmail.com
Mohd Aslam Yusuf
aslambio@iul.ac.in
Manisha Nigam
m.nigam@hnbgu.ac.in
1 Department ofBiosciences, Integral University, Lucknow,
U.P.226026, India
2 Division ofPlant Microbe Interactions, CSIR-National
Botanical Research Institute, Rana Pratap Marg, Lucknow,
U.P.226001, India
3 Department ofBioengineering, Integral University, Lucknow,
U.P.226026, India
4 Department ofBiochemistry, Hemvati Nandan Bahuguna,
Garhwal University, Srinagar, Garhwal, Uttarakhand246174,
India
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There are two commercial classes of chickpea, namely, Desi
and Kabuli. The Desi type of chickpea has colored flowers
and seed coat, whereas the Kabuli type has white flowers and
seed coat. Morphologically, the height of chickpea plants
ranges from 12 to 28 inches and they have one- or two-
seeded pods. It is a self-pollinated crop with diploid chromo-
some (2n = 16), having a genome size of 738.09 Mbps [2].
Although chickpea is grown in over 50 countries, 90%
of the area under chickpea cultivation is in the developing
countries, with southern and South-East Asian countries
accounting for > 79% of the global production [3]. Moreo-
ver, the number of chickpea importing countries has been
consistently increasing, suggesting an increase in the global
demand. To meet these increased demands, the production
of chickpea needs to be increased, for which either the area
under cultivation has to be increased or crop improvement
strategies have to be employed to minimize the immense
losses in production caused by biotic and abiotic stresses.
Among the various abiotic stresses like drought, salinity,
cold, frost, and water logging, drought is a major cause of
damage and yield loss that can be as high as 40% of the
total production in world [4]. Biotic stresses include infes-
tations with insects (e.g., Helicoverpa armigera), bacteria
(Xanthomonas, causing bacterial blight; Burkholderia, caus-
ing bacterial leaf spot), fungus (Fusarium spp., Ascochyta
blight), viruses (alfa mosaic virus), and nematodes (Rotylen-
chulus reniformis causing dirty root). For utilization of bio-
technological approaches to genetically engineer transgenic
plants with improved resistance to stresses, knowledge of
the genetic makeup of plants is essential. Chickpea genome
was sequenced in a collaborative eort of scientists from
International Crops Research Institute for the Semi-Arid
Tropics (ICRISAT), India, and 20 other research organiza-
tions using 90 genotypes [2]. More than 28,000 genes and
several million genetic markers have been identified in the
chickpea genome. Low genetic variability among the cul-
tivars of chickpea has necessitated organized compilation,
documentation, and assessment of wild Cicer species for
use in chickpea variety enhancement programs [57]. Wide
hybridization and intragenic crosses to widen the genetic
make of cultivars are required. Moreover, biotechnological
approaches are needed for introgression of valuable traits
from the wild species of Cicer [6].
The first draft sequence of the Desi-type chickpea genome
was generated using next-generation sequencing platforms
[8]. These researchers assembled 520-Mb data, covering
70% of the predicted 740-Mb genome length, and > 80%
of the gene space. The genome analysis predicted 27,571
genes, and the expression analysis performed using 274mil-
lion RNA-Seq reads identified several tissue-specific and
stress-responsive genes. Gupta etal. assembled short-read
sequences of C. reticulatum into 416-Mb draft genome and
anchored 78% (327Mb) of the sequences to eight linkage
groups [9]. In this case, genome annotation predicted 25,680
protein-coding genes. Moreover, determination of molecular
diversity and population genetic structure was done using
15,096 genome-wide single nucleotide polymorphisms.
A comparison of gene sequences and nucleotide diversity
using 66 wild and domesticated chickpea accessions sug-
gested that the Desi-type chickpea was genetically closer to
the wild species than the Kabuli type.
Over the past few decades, transgenic technology has
emerged as an ecient tool to introduce foreign gene(s)
or to manipulate the expression of a self-gene in a plant
variety to increase its tolerance to stress. Models like super
chickpea, with several genes conferring tolerance to dierent
stresses stacked in a single variety, have been proposed [10].
Chickpea has been transformed for resistance against target
pests like H. armigera [1117], bruchid [18], and aphids
[19] as well as for conferring tolerance to abiotic stresses
like drought [20, 21] and salinity [22]. This review presents
an overview of the current status of genetic transformation
of chickpea aimed at improving its production and yield.
Nutritional Value
Chickpea is a rich source of essential nutrients like proteins,
carbohydrates, iron, folic acid, phosphorus, and dietary
fibers. The protein content varies significantly as a per-
centage of total dry seed mass before (17–22%) and after
(25.3–28.9%) dehulling. The seeds also have carbohydrate
(62%), fat (2.70–6.48%), dietary fiber (18–22%), minerals
(Cu, Fe, Zn, Mn, and Ca), vitamins (A, B1, B2, B3, B5, B6,
B12, C, D, E, K, and Biotin), and carotenoids [23]. Chick-
pea has higher levels of Mn, Zn, and P compared to those
present in other legumes [24]. The grain and flour of chick-
pea are characterized by high contents of monosaccharides,
disaccharides, and oligosaccharides. Dietary fiber includes
both soluble and insoluble fibers. The soluble fibers stabilize
blood sugar levels and help in the removal of bile (which
includes cholesterol) in the digestive tract by forming a gel-
like substance. The insoluble fibers increase stool movement
and prevent stomach disorder like constipation. The con-
sumption of chickpea routinely lowers health risks related to
cholesterol levels. For women, the uptake of chickpea lowers
the chances of breast cancer, provides protection from osteo-
porosis, and reduces post-menopausal problems. Generally,
legumes are reported to have low nutritive value because
of the presence of antinutritional factors, such as tannins,
phytic acid, trypsin inhibitor, and phenolics. Cooking has
been reported to improve the nutritive quality by destruction
or inactivation of the heat labile antinutritional factors [25,
26]. Germination also enhances the nutritive value of leg-
umes by inducing the formation of enzymes that eliminate or
reduce the antinutritional and indigestible factors in legumes
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[2729]. Another issue associated with chickpea consump-
tion is the presence of allergens that can cause IgE-mediated
hypersensitivity ranging from rhinitis to anaphylaxis [30].
Chickpea Consumption
India is the largest consumer of chickpea, where 90% of
chickpea consumed is of the Desi type. During 2015–2016,
India exported chickpea to Pakistan (35.60%), Algeria
(15.17%), Turkey (8.58%), Sri Lanka (8.07%), United Arab
Emirates (4.97%), and imported from Australia (74.40%),
Russia (16.49%), Tanzania (2.79%), Myanmar (0.92%), and
USA (0.74%) [31]. Nearly every part of chickpea, except
roots, leaves, and pods, is consumed in dierent forms.
Chickpea is added to numerous dishes, such as salads, soups,
and desserts, to impart taste. Sprouted chickpea, mixed with
other pulses, is an appetizer. Salted or sugared seeds are
consumed in roasted or boiled form. In developing countries,
chickpea is used as an animal feed. Husk, green/dried stem,
and leaves are used as stock feed.
Production ofChickpea
Among the pulses, chickpea ranks as the third most important
crop with an annual global production of 10.1million tons
[32]. It is grown on stored soil moisture after the rainy season
in South Asia, eastern Africa, north-eastern Australia (subtrop-
ical regions), whereas it is grown in the rainy season in Canada
and Mediterranean climatic regions [33]. In Australia, Canada,
and the USA, the expanded chickpea production appears to
have positively influenced the yield worldwide. Globally, total
chickpea harvested area has been expanded from 9.63million
ha in 1980 to 12.65million ha in 2016. Global chickpea pro-
duction has also increased from 4.85million tons in 1980 to
12.09million tons in 2016. Countries that have emerged as
major chickpea producers are Ethiopia (Africa), Kazakhstan
and Yemen (West Asia), Uzbekistan and China (Central Asia),
Russia (Europe), Canada and USA (North America), and Aus-
tralia (Table2). In West Asia, Iran and Turkey are the domi-
nant chickpea producers with total productivity of 177,493 and
455,000 tons in 2016, respectively. In North America, chickpea
yield and production is highest in Mexico, followed by that in
Canada and USA. Argentina has become the highest producer
of chickpea in South America with a production 69,788 tons
in 2016 [34]. Chickpea is the largest pulse crop in Australia,
currently grown on > 0.67million ha, with a total production
of 874,593 tons in 2016 [34]. India produced approximately
17.56 MT pulses in 2016 and was the biggest chickpea pro-
ducer in the world with a production of approximately 7.8 MT
in 2016 (Fig.1). By the year 2050, the global pulse production
is expected to be 100 MT, from a cultivated area of 62mha,
with a yield per hectare of 1.6 tons [35] (Table1). Of the
total chickpea produced, about 75% is of the Desi type and
Fig. 1 Chickpea vs. total pulses
production in India 1980–2016
(Source: FAOSTAT, 2017)
Table 1 Development in pulses production, yield, and harvested
area throughout the world (Source: FAO world agriculture towards
2030/2050: revision 2012)
The figures show changing trend in the past and that expected in the
future
1961/63 2013/14 2050
Production (million tons) 44 73 100
Harvested area (million ha) 69 80.8 62
Yield (tons/ha) 0.6 0.9 1.6
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25% is of the Kabuli type. In India, Madhya Pradesh is the
highest chickpea producing state with a share of 41% of the
national production (Fig.2). The other major chickpea produc-
ing states are Rajasthan, Maharashtra, Andhra Pradesh, Telan-
gana, Uttar Pradesh, and Karnataka, which cover 95% the area
under chickpea cultivation [36]. In the past 3decades, the area
under chickpea cultivation in India has increased marginally to
8.3mha from a previous figure of 6.9mha, whereas the pro-
duction has increased by 40% (from 3.3 to 7.8MT). The global
growth rate of pulse production over the last decade has been
2.61%. The worldwide chickpea production has increased dur-
ing the past 3decades from 4.8MT (1980 average) to 12MT
(2017 average) because of the increase in grain yield from 504
to 956kg/ha during this period (Table2) [34]. The increase in
production in new regions, especially in Australia and North
America, has significantly contributed to the global increase
in chickpea production.
Constraints inProduction
The chickpea production is constrained by several factors
and the crop is susceptible to a range of abiotic (drought,
high temperature, salinity, cold and chilling, water logging,
Table 2 Worldwide chickpea production scenario in 1980 and 2016 (Source: FAOSTAT, 2017)
Region Country Area (ha) Yield (kg/ha) Production (tons)
1980 2016 1980 2016 1980 2016
Africa Algeria 42,100 20,617 376 955 15,839 19,707
Egypt 7297 611 1516 2153 11,069 1315
Ethiopia – 225,608 1968 – 444,146
Malawi 27,000 115,727 666 583 18,000 67,498
Morocco 65,500 88,030 680 500 44,580 44,062
Tunisia 78,000 6500 384 769 30,000 5000
United Republic of Tanzania 27,000 116,388 277 902 7500 104,980
West Asia Iran 130,600 433,356 645 409 84,300 177,493
Iraq 24,050 1441 540 4148 13,000 1159
Israel 3820 3580 1374 4148 5250 14,850
Jordan 2865 685 577 2953 1654 2023
Lebanon 2500 2937 1200 909 3000 2671
Kazakhstan – 4111 848 – 3487
Syrian Arab Republic 91,380 78,777 802 661 73,365 52,071
Turkey 240,000 351,687 1145 1293 275,000 455,000
Yemen – 17,981 3330 – 59,890
South Asia India 6,985,000 8,392,652 480 931 3,356,300 7,818,984
Bangladesh 128,000 8445 664 940 85,000 7940
Myanmar 70,194 363,870 542 1537 38,055 559,390
Pakistan 1,128,500 1,004,681 277 514 313,400 517,107
Nepal 50,000 9883 600 1104 30,000 10,914
Central Asia China 3044 4710 14,339
Uzbekistan – 2777 2320 – 6443
Europe Greece 16,315 5334 1024 1223 16,715 6527
Italy 13,745 13,940 1178 1601 16,200 22,328
Portugal 39,926 1782 368 743 14,710 1325
Russia – 357,945 893 – 319,908
Spain 90,100 33,157 673 800 60,700 26,552
North America Canada 59,800 1787 106,900
Mexico 134,659 66,316 1138 1833 153,248 121,567
USA – 70,879 1517 – 107,542
South America Argentina 5280 65,776 833 1061 4400 69,788
Chile 20,570 409 563 890 11,600 364
Australia – 677,444 1291 – 874,593
World (total) 9,631,007 12,650,078 504 956 4,854,451 12,092,950
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etc.) [37, 38] and biotic (diseases and pests) stresses [37].
In India, chickpea is cultivated as a rain-fed crop (> 70%
area) on soil with low fertility and withholding capacity;
consequently, the crop often faces soil water stress at various
stages of growth. Among the various diseases that aict the
chickpea plants, Ascochyta blight, Fusarium wilt, root rot,
stem, and collar rot, and Botrytis gray mold are the major
ones. Similarly, insect pests, like gram pod borer (H. armig-
era), semi-looper (Autographa nigrisigna), cutworm (Agrotis
ipsilon), and bruchid (Callosobruchus chinensis) cause huge
losses to chickpea yield at various stages of crop growth
and during post-harvest storage. Soil salinity and alkalinity,
low and high temperature, terminal moisture stress, foggy
weather conditions, and lodging under high input environ-
ment are the major abiotic stresses that aect the yield [39].
We have detailed the various aspects of abiotic and biotic
stresses faced by chickpea in the following sections.
Abiotic Stress
Drought Stress
Drought means scarcity of water and is a usual feature in
semi-arid tropics. The plants acclimatize under drought
conditions through escape, avoidance, and tolerance mech-
anisms [4042]. Because of the heavy production losses
caused by drought, it remains the foremost and biggest chal-
lenge that needs to be addressed for increasing the chick-
pea production. Combating drought by traditional breeding
methods involves introgression of drought-tolerance charac-
ter to form tolerant varieties into the susceptible varieties to
form hybrids [for example, Pusa-1108, Pusa-2024, UPC 622,
RSG-44, and ICCV10 (Bharati)] with the drought-tolerance
trait.
Biotechnological intervention to improve chickpea for
drought-tolerance trait involves development of transgenic
chickpea tolerant to drought. Several studies have reported
the expression of drought-tolerance gene(s) driven by a
strong promoter in chickpea. The expression of P5CSF129A
under the CaMV35S promoter improved drought stress tol-
erance in chickpea by enhancing proline accumulation [20].
Similarly, the expression of AtDREB1A under the drought-
inducible Rd29A promoter influenced the mechanisms
underlying water uptake, stomatal response, transpiration
eciency, and rooting architecture in water-stressed chick-
pea plants [21] (Table3). Moreover, the whole-genome
sequencing of chickpea revealed that it contains 187 disease-
resistance gene homologs (RGHs), of which 153 RGHs are
anchored in pseudo-molecules. Compared to RGHs present
in the other genomes that have been sequenced (764 in Med-
icago truncatula, 506 in soybean, 406 in pigeon pea) [2],
153 RGHs in the chickpea genome are considerably less. It
is pertinent to mention here that plants use various types of
disease-resistance genes to sense the presence of pathogens
and induce defense response. The most abundant resistance
genes are those that encode proteins with nucleotide bind-
ing site (NBS) and leucine-rich repeat (LRR) domains [43,
44]. The NBS domain contains several conserved motifs that
are responsible for nucleotide binding and initiating a sig-
nal transduction cascade to activate the plant defenses [45].
The LRR region, on the other hand, is typically involved
in protein–protein interactions and pathogen recognition
specificity [4648]. The identification of disease-resistance
genes from whole-genome data on the basis of the pres-
ence of NBS and LRR regions provides insights into the
response of a crop to pathogens. Whole-genome sequenc-
ing has enabled genome-level investigations of the RGHs in
monocot and dicot species, such as Arabidopsis [49], rice
[50], Medicago [51], sunflower [52], sugarcane [53], and
chickpea [2]. Plant growth promoting rhizobacteria are also
being used to prevent drought stress-induced yield loss in
chickpea. The synergistic eect of Pseudomonas putida and
Bacillus amyloliquefaciens application has been reported to
ameliorate drought stress in chickpea [54].
Salinity
Chickpea is severely aected by soil salinity, which results
in huge yield losses. Reduction in germination, plant growth
(biomass), and seed size occurs in plants growing in saline
soil. The chickpea-growing areas like Australia and West
and Central Asia have saline soil. In India, some salinity-
tolerant lines have been identified, like Karnal channa
1 (CSG 8963) [55], which can be grown in saline soils
with electrical conductivity up to 6dS/m, and have been
released for commercial production [56]. Salinity causes
injury to plants by ion toxicity and osmotic stress leading
Fig. 2 State wise share to total production and area of chickpea in
India in 2015–2016 (Source: CommoditiesControl.com)
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Table 3 Chickpea transgenic plants developed for abiotic stress tolerance and biotic stress resistance by transgene integration
Traits Aected part of plant Potential genes Promoter used for gene expres-
sion
Chickpea cultivars Ecacy of potential genes References
Helicoverpa armigera Leaf, flower and pod Cry1A(c) CaMV35S ICCV-1 and ICCV-6 The expression level of
cry1A(c) gene was inhibitory
to the development of feeding
larvae of H. armigera
[6, 11]
Cry1Ac CaMV35S C235, BG256, Pusa 362, Pusa
372
> 80% larval mortality [7, 12]
Cry1Ac A-1 and ICCV-2 Transformed chickpea plants
expressing CrylAc protein
had high mortality (> 60%)
[8, 13]
Cry2Aa AtSSU Semsen and iccv 89314 98% larval mortality [9, 14]
Cry1X CaMV35S KAK-2 The chickpea plants harboring
the cry1X gene were resistant
to H. armigera and 49.6%
larval mortality was found
[50, 87]
Cry1Ab + Cry1Ac CaMV35S and Pcec P-362 Pyramided transgenic plants
with moderate expression
levels (15–20ng/mg of TSP)
showed high level of resist-
ance and protection against
pod borer larvae of H. armig-
era as compared to high level
expression of a single toxin
[52, 88]
Cry1Ab/Ac Soybean msg and rice actin1 DCP92-3 Expression of fused cry gene
under constitutive and pod-
specific promoter results in
increase of 77- and 110-fold,
respectively
[51, 62]
Cry1Aabc CaMV35S DCP92-3 Expression of a chimeric gene
encoding insecticidal crystal
protein Cry1Aabc of Bacillus
thuringiensis in Chickpea
(Cicer arietinum L.) confers
resistance to gram pod borer
[16, 15]
Cry1Ac Ubiquitin and rbcS ICCV89314 Conferred a high level of
protection against pod borer
infestation, where chloroplast
targeting system was found to
be more ecient in control-
ling this particular devastating
lepidopteran pest
[17, 16]
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Table 3 (continued)
Traits Aected part of plant Potential genes Promoter used for gene expres-
sion
Chickpea cultivars Ecacy of potential genes References
Bruchids resistance Seed Enzyme inhibitors: bean-amyl-
ase gene (α-AI1)
Semsen Larval growth reduction and
no emergence of adult Cal-
losobruchus spp.
[10, 18]
Stem and leaf α-Amylase inhibitor gene
isolated from the Phaseolus
vulgaris
Phytohemagglutinin promoter K850 Bioassay results revealed a
significant reduction in the
survival rate of bruchid
weevil C. Maculatus reared
on transgenic chickpea seeds.
All the transgenic plants
exhibited a segregation ratio
of 3:1
[43, 72]
Aphid resistance Leaf Allium sativum leaf agglutinin
(ASAL)
CaMV35S and rolC ICCV 89314 ASAL: decreased survival of
aphids by only 11–42%
[11, 19]
Sulfur amino acid enrichment Seed Sunflower seed albumin (SSA):
serine acetyltransferase (SAT)
CaMV 35S Semsen and amethyst SSA: high Met in normal soil
conditions, while high Met
and Cys in high nitrogen to
low sulfur conditions
[53, 89]
Drought tolerance Root P5CSF129A CaMV 35S C 235 The overexpression of
P5CSF129A gene resulted in
negative eects of drought
stress in chickpea by enhanc-
ing Proline accumulation
[12, 20]
AtDREB1A Rd29A C235 The implicit influence
of rd29A::DREB1A on
mechanisms underlying water
uptake, stomatal response,
transpiration eciency, and
rooting architecture in water-
stressed plants
[13, 21]
Salt tolerance Root Vigna Δ1-pyrroline-5-
carboxylate synthetase (P5CS)
CaMV 35S Annigeri Transgenics lines were grown
to maturity and set normal
viable seeds under continuous
salinity stress (250mM NaCl)
without any reduction in plant
yield in terms of seed mass
[14, 22]
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to dehydration. This adversely aects plant process, such
as photosynthesis, metabolism, and growth. The response
of plants to dehydration and salinity stress includes percep-
tion and transduction of stress signals via signaling cascades
and activation of stress-responsive genes. The development
of salt-tolerant transgenic chickpea is one of the ways to
ameliorate salinity stress. A salt-tolerance gene, Vigna
Δ1-pyrroline-5-carboxylate synthetase (P5CS), was suc-
cessfully transformed into chickpea plant, under the control
of CaMV35S promoter; the transgenic plants could grow to
maturity and could set normal viable seeds under continuous
salinity stress (250mM NaCl) without any reduction in plant
yield in terms of seed mass [22].
Biotic Stress
A number of biotic stresses reduce the chickpea yield enor-
mously. The major biotic constraints are insect pests and
diseases. To overcome these problems, development of
resistance in chickpea has been attempted using dierent
strategies, as detailed below.
Insect Resistance
Helicoverpa armigera Helicoverpa armigera is a major
insect pest that causes loss of yield in chickpea as well as
in many other crops. Spraying of chemical insecticide is the
method used by farmers for its control [57, 58]. However,
extensive chemical spraying leads to environmental pollu-
tion and human health-related issues, besides development
of resistance to the insecticides in insects upon regular use
[59, 60]. The development of Bt-transgenic chickpea using
Agrobacterium-mediated transformation provided a new
strategy for generating insect-resistant chickpea [16, 61,
62]. Agrobacterium tumefaciens mediated genetic transfor-
mation of chickpea using Bt-cry1Ac gene driven by a con-
stitutive promoter, and a green tissue-specific promoter was
done in chickpea [63]. The Bt gene(s) produces δ-endotoxin
protein, which forms pores in the mid-gut of insects and is
specifically toxic to lepidopterans. 13 GM crops generated
using this strategy are currently undergoing trials under con-
fined field conditions. Besides, GM rice, brinjal, chickpea,
mustard, and cotton have cleared the approval of regulatory
bodies. In India, Bt-cotton, which was the first GM crop to
get approval, is cultivated in south, central, and north zones
of the country. The GM cotton varieties released harbor cry
1Ac and cry1Ac and cry2Ab genes for resistance against cot-
ton bollworm and have been hugely successful. In a joint
program, Assam Agricultural University, India, and Com-
monwealth Scientific and Industrial Research Organisation
(CSIRO), Australia, have developed marker-free transgenic
chickpea plants expressing Bt genes (Cry2Aa) that provide
resistance to gram pod borer, H. armigera, infestation. This
technology is under field trials for agronomic performance.
In 2009, Sungro Seeds Ltd., Delhi, licensed the Cry2Aa
technology developed at the Assam Agricultural University
and the ASAL technology developed at the Bose Institute
(https ://iscb.epfl.ch/files /conte nt/sites /iscb/files /share d/
Techn ology %20Tra nsfer /ASAL_Cry_color 1_updat ed.pdf).
Moreover, Maharashtra Hybrid Seeds Company Limited
(Mahyco), India, has also oered to purchase the license of
these technologies. Recently, Das etal. reported the devel-
opment and characterization of transgenic chickpea lines
with a domain-shued chimeric gene, cry1Aabc, for pro-
tection against H. armigera [15].
Bruchids The susceptibility of chickpea to bruchids leads
to extensive damage of the stored crop and results in post-
harvest losses [64]. These bruchids are also known as azuki
bean weevil (C. chinensis). Chemical treatment of seeds
is the method used to prevent bruchid infestation; how-
ever, it is not recommended because seeds are consumed.
New approaches, such as molecular breeding and genetic
engineering, may be utilized for the safety of stored seeds
without any chemical application [65]. Insecticidal proteins
of plant origin, such as lectins, α-amylase inhibitors, and
protease inhibitors, are toxic to these pests and retard their
growth and molting, when ingested [66, 67]. The α-amylase
inhibitor (αAI) gene from common bean (Phaseolus vul-
garis) when expressed in pea plants conferred resistance to
infestation by bruchid weevil, Callosobruchus maculatus
[68, 69]. The αAI suppressed the activity of α-amylase in
the larval mid-gut of the weevils [70, 71]. The αAI1 gene
was cloned under a strong seed-specific promoter, phytohe-
magglutinin (PHA), which regulates the expression of the
gene in chickpea seeds. In a similar study, the alpha-amylase
inhibitor gene isolated from P. vulgaris was introduced into
chickpea by Agrobacterium-mediated plant transformation
method [72]. Although the eciency of transformation was
low, the transformed plants showed considerable resistance
to storage pests. The toxicity eects of alpha amylase inhibi-
tor produced by P. vulgaris have been studied [73, 74] and
no serious side eects have been reported. In a comparative
study by Lee etal., it was demonstrated that αAI transgenic
peas were not more allergenic than beans or non-transgenic
peas in mice [75]. Moreover, in view of the fact that α-AI
is simply inactivated upon cooking, introducing α-AI gene
into host plants can be regarded as a safe approach.
Aphids Carbohydrate-binding lectins present in leaves have
insecticidal activity against various insects of the orders
Coleoptera, Diptera, Lepidoptera, and Homoptera. ASAL,
a mannose-binding homodimeric protein functions as an
antagonist to numerous sucking pests in the homopteran
group, including cowpea aphid (Aphis craccivora). A num-
ber of chickpea cultivars are more susceptible to aphid infes-
Molecular Biotechnology
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tation than others; for example, cultivars having a low den-
sity of trichomes are more sensitive than others. Chemical
treatments may be necessary for the control of aphids; how-
ever, they have developed resistance to a number of insec-
ticides widely used in India. The expression of ASAL gene
from garlic resulted in enhanced tolerance to sucking pests
[19]. This ASAL technology has been eectively verified
in various plants, like mustard, tobacco, and rice [7678].
Transgenic chickpea plants’ resistant to the sucking pests
has been developed by transferring the ASAL gene. White
fly tolerance has been implicated as an essential trait in
controlling plant diseases. The ASAL technology has been
perfected for tissue-specific expression of the ASAL gene
using site-specific promoters in transgenic chickpea [19].
Recently, Tma12 protein was identified from an edible fern,
Tectaria macrodonta (Fee) C. Chr., and was found to be
insecticidal to whitefly (Bemisia tabaci). Transgenic cotton
lines that expressed Tma12 at 0.01% of the total soluble
leaf protein were found to be resistant to whitefly infestation
in contained field trials, with no detectable yield penalty
[79]. Thus, Tma12 could be a potential candidate for engi-
neering whitefly resistance in chickpea.
Diseases
Fusarium oxysporum Chickpea production is severely
aected by the disease condition caused by Fusarium
oxysporum. Fusarium wilt leads to permanent drooping of
plants and this pathogen is transmitted very quickly in the
field. Being a soil-borne fungus, the remedy for this con-
dition is sanitation or plantation of wilt-resistant varieties.
Various chickpea varieties of the Desi type, like DCP 92-3
(water lodging tolerant and wilt resistant), BGD 103 (bold
seeds, resistant to Fusarium wilt), BGD 72 (high yielding
and large seeded), IPC 97-67 (SCS-3), and of the Kabuli
type, like IPCK 2002-29 or SHUBHRA (moderately resist-
ant to wilt), IPCK 2004-29 or Ujjawal (moderately resist-
ant to Fusarium wilt), MNK 1 (Fusarium wilt resistant),
and BG 1105 (medium early, moderately resistant to dry
root rot, wilt, and with excellent cooking quality), have
been released in India through breeding [80]. WR315 is a
well-known Fusarium wilt-resistant chickpea cultivar [81].
Further breeding to improve for more yield parameters can
be beneficial for production as well as for disease resist-
ance. Recently, scientists from IIPR and ICRISAT, India,
have developed a molecular marker-assisted backcross-
ing (MABC) variety using a Desi chickpea cultivar, Vijay,
as donor, to introgress resistance to Fusarium wilt race 2
(Foc2) in another elite Desi cultivar, Pusa 256 [82].
Ascochyta Blight Ascochyta blight is the most severe dis-
ease in almost every chickpea-growing country. A total
yield loss can occur as a result of Ascochyta infestation
and it can also reduce the quality of chickpea produced.
The causal fungus of this blight is Ascochyta rabiei. This
fungus is known to infect the plant parts that are above the
ground. The blight forms gray patches on the leaves, stems,
and pods, which change into lesions of brown color. These
brown to dark brown dots are the actual fruiting bodies of
fungus, called pycnidia, which get arranged in concentric
rings as the disease progresses. The blight can be managed
using an integrated approach of pest management involving
steps like the use of resistant seed varieties, crop rotation,
seed treatments, and application of fungicides.
Eorts have been made to understand the nature of path-
ogen infection and presence of genetic variability within
chickpea cultivar to fight blight. Resistance to Ascochyta
blight has been found in chickpea cultivars as well as in
wild accessions. Breeding programs focus on identifying and
transferring these genes to generate new hybrids. Molecular
tools are necessary to aid conventional breeding approaches
for introgression of genes into chickpea genotypes. Dier-
ent molecular markers associated with major QTLs for
resistance to blight are linked for marker-assisted selection
(MAS). Gil etal. used transgressive inheritance to select
new allelic combinations for developing new chickpea mate-
rials that were resistant to blight and had large seeds, as well
[83]. They observed significant dierences in the expres-
sion of defense pathway-related genes among the dierent
chickpea cultivars upon infection with A. rabiei. Recently,
using whole-genome re-sequencing of 69 chickpea geno-
types, “functional makers” were developed for MAS and
genomic selection for resistance to Ascochyta blight [84].
Garg etal. reported the identification of QTLs for resist-
ance to Ascochyta blight and Fusarium wilt in a recombinant
inbred population of chickpea [85].
Transgenic Approach forChickpea
Production
Transgenic chickpea is developed either by gene gun [86]
or Agrobacterium-mediated method [1114, 62, 87, 88].
Chickpea has mainly been transformed for expression of Bt
genes. Das etal. recently reported the generation of trans-
genic chickpea plants harboring codon-optimized synthetic
cry1Aabc gene [15]. Based on extensive molecular analy-
ses and insect bioassays, they selected three independent
chickpea events with single locus integration in the genome,
which showed high expression of the transgene and high
insect mortality (up to 100%) based on detached leaf bio-
assay at T4 generation. Because Bt toxins are harmless to
human beings, animals, and a broad range of non-target
pests, they are suited for incorporation into Integrated Pest
Management (IPM) programs [89]. To date, no transgenic
chickpea has been approved for cultivation in the world.
Molecular Biotechnology
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However, the Government of India allowed the field trials of
six transgenic crops, namely, rice, cotton, maize (corn), mus-
tard, brinjal, and chickpea, in 2015 [90]. Dierent transgenes
from various sources that have been transformed into chick-
pea for development of abiotic and biotic stress resistance
are listed in Table3.
Marker-Assisted Selection vs. Transgenesis
Improved crop productivity is a highly desirable trait in the
hybrids. Breeders were able to develop new improved varie-
ties using the methods of selection and hybridization; how-
ever, such methods are time consuming and can be hastened
by the use of molecular markers. Marker-assisted breeding
is based on the transfer a specific allele at the target locus in
a donor line to a recipient line while selecting against donor
introgression across the rest of the genome [91]. MAS is
used when the trait of interest is present within the gene
pool of the crop. Molecular breeding has reduced the time
for development of new varieties from 25 to 7–10years.
In contrast, genetic transformation or transgenesis helps to
make use of important genes that were earlier not amenable
to transfer. Transgenesis is an eective means for improving
crop genetic makeup for deriving favorable traits. Genetic
engineering techniques permit the simultaneous use of
several desirable genes in a single event, thus, allowing
the introduction of novel genes/traits into elite cultivars.
However, transgenic plants are associated with serious con-
cerns about biosafety and environment, whereas in the case
of MAS, biosafety and intellectual property rights are not
the major issues; the narrow gene pool of the species, is
instead, a bigger limitation. Breeding by transgenesis has
several advantages over breeding by MAB. Transgenesis
is a direct means for introducing a gene or genotype to a
genome in order to produce a target trait [92]. Modern breed-
ing approaches like marker-assisted recurrent selection and
genomic selection are being deployed for enhancing drought
tolerance in chickpea. Marker-assisted backcross breeding
(MABB) was undertaken by crossing chickpea cultivars
Pusa 362 (recurrent parent) and ICC 4958 (donor parent)
to introgress QTL-hotspots for root and yield traits under
water-stressed condition [93]. The introgression lines with
superior root traits and higher yield could be released for
planting in drought prone environments [93]. Using MAS
approaches together with the use of appropriate genetic
diversity, databases, analytical tools, well-characterized
drought stress scenarios, and weather and soil data, new
varieties with improved drought resistance corresponding to
grower preferences can be introduced in target regions [94].
For the identification and evaluation of chickpea wilt-resist-
ant lines against F. ox ys por um f. sp. ciceris (Schlechtends),
Ahmad etal. tested the germplasm in the field for selec-
tion of wilt-resistant lines and also identified PCR-based
molecular markers to use MAS for selection of the desir-
able cultivars [95].
Conclusions andFuture Prospects
With the advent of new techniques in biotechnology, pro-
ductivity of nutritionally valuable crops is increasing. In
economically important food crops, like chickpea, it is very
essential to use the tools of genome editing for improve-
ment of desirable traits alongside conventional breeding
approaches. In countries like Pakistan, India, and Australia,
most of the land under chickpea cultivation is rain-fed, and
therefore, incorporation of drought-tolerance trait would be
a boon. The institutes like ICRISAT, Indian Agricultural
Research Institute (IARI), and Indian Institute of Pulses
Research (IIPR) in India are routinely developing drought-
tolerant varieties of chickpea through breeding. The toler-
ance trait present in the wild varieties of chickpea is utilized
by breeders to develop drought-resistant varieties by intro-
gression of drought-responsive genes into the susceptible
varieties. These hybrid varieties perform well for the desired
trait but over a period of time, these genes are lost from the
population and have to be brought again through breeding
or by maintaining the selection pressure. Thus, the quality
of such materials has to be checked frequently. Other demer-
its related to the traditional method are the linkage drag,
which comes with the desired genes during introgression. In
contrast, transgenic approaches are relatively fast and hold
great promise for pyramiding of several candidate genes for
conferring multiple-desired traits in transgenic plants.
Acknowledgements The study was supported by the New Initiative (as
a Cross Flow Technology project) “Root Biology and its Correlation
to Sustainable Plant Development and Soil Fertility” from Council of
Scientific and Industrial Research (CSIR), and project titled “Char-
acterization of gene(s) responsible for tyloses formation in chickpea
during Fusarium oxysporum infection” from Science & Engineering
Research Board (SERB), New Delhi, India. The manuscript communi-
cation number assigned to this manuscript by the Dean, R&D, Integral
University, Lucknow, is IU/R&D/2018-MCN000228.
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... In spite of such prolonged practices, the chickpea transformation frequency was highly unpredictable and as a result globally no transgenic varieties have been commercially approved for cultivation (Kumar et al. 2018). In such a situation, the development of reliable and reproducible transformation method to overcome the existing difficulties is still imperative. ...
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... The development of transgenic chickpea lines showing resistance to H. armigera is considered as one of the best approaches to counter yield loss (Asharani 2009). The genes utilized for development of resistance against pod borer, bruchid, aphid, drought and salt tolerance, namely, Bt, alpha amylase inhibitor, ASAL, P5CSF129A and P5CS, respectively, are discussed by several researchers (Das et al. 2017;Kumar et al. 2018;Aggarwal et al. 2018;Bhowmik et al. 2019). Till date, confined field trials of transgenic chickpea for insect-resistant trait are reported; however, the efforts require constant support to bring forth viable transgenics, addressing all regulatory issues. ...
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Cicer reticulatum L. is the wild progenitor of the fourth most important legume crop chickpea (C. arietinum L.). We assembled short-read sequences into 416 Mb draft genome of C. reticula-tum and anchored 78% (327 Mb) of this assembly to eight linkage groups. Genome annotation predicted 25,680 protein-coding genes covering more than 90% of predicted gene space. The genome assembly shared a substantial synteny and conservation of gene orders with the ge-nome of the model legume Medicago truncatula. Resistance gene homologs of wild and domesticated chickpeas showed high sequence homology and conserved synteny. Comparison of gene sequences and nucleotide diversity using 66 wild and domesticated chickpea accessions suggested that the desi type chickpea was genetically closer to the wild species than the kabuli type. Comparative analyses predicted gene flow between the wild and the cultivated species during domestication. Molecular diversity and population genetic structure determination using 15,096 genome-wide single nucleotide polymorphisms revealed an admixed domestication pattern among cultivated (desi and kabuli) and wild chickpea accessions belonging to three population groups reflecting significant influence of parentage or geographical origin for their cultivar-specific population classification. The assembly and the polymorphic sequence resources presented here would facilitate the study of chickpea do-mestication and targeted use of wild Cicer germplasms for agronomic trait improvement in chickpea.
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Chickpea is a valued crop and provides nutritious food for an expanding world population and will become increasingly important with climate change. Production ranks third after beans with a mean annual production of over 10 million tons with most of the production centered in India. Land area devoted to chickpea has increased in recent years and now stands at an estimated 13.5 million hectares. Production per unit area has slowly but steadily increased since 1961 at about 6 kg/ha per annum. Over 1.3 million tons of chickpea enter world markets annually to supplement the needs of countries unable to meet demand through domestic production. India, Australia, and Mexico are leading exporters. Chickpea is comprised of Desi and Kabuli types. The Desi type is characterized by relatively small angular seeds with various coloring and sometimes spotted. The Kabuli type is characterized by larger seed sizes that are smoother and generally light colored. Dal is a major use for chickpea in South Asia while hummus is widely popular in many parts of the world. Research efforts at ICRISAT, ICARDA, and national programs have slowly but steadily increased yield potential of chickpea germplasm.
Article
Appearance and size of chickpea (Cicer arietinum L.) seeds are key factors for the market in the Mediterranean Basin driven by consumer preferences. Hence, kabuli large seeds are sold on the market at higher price than the desi seeds. In this crop, Ascochyta blight (caused by Ascochyta rabiei (Pass.) Lab.) is a serious disease causing major losses in yield. Thus, developing large-seeded kabuli cultivars resistant to blight would be of great importance to farmers. In this study, the use of transgressive inheritance to select new allelic combinations for seed size was applied to develop new chickpea materials with large seeds and resistance to blight. Crosses between five different advanced lines of kabuli chickpea genotypes with medium-large seed size and resistant to blight were performed. As a results of the selections carried out during 10 successive years, 11F5:9 lines resistant to blight and with large seed size were selected to be released as future varieties. The markers SCY17590 and CaETR were employed to confirm blight resistance of the material developed.