Enhancing Tolerance of Chickpea to Drought, Freezing, and Low

Temperature through Genetic Engineering with the codA and P5CSF-129A

Genes
Project ID: PS4

Project 1 April 2001 to 31 August 2004

Duration:

Principal Dr P.P. Saradhi

Investigators: Department of Environmental
Biology
School of Environmental Studies
New Delhi - 110 007, INDIA
E-mail: ppsaradhi@hotmail.com
saradhi@ndf.vsnl.net.in

Dr Kiran K. Sharma
Genetic Transformation
Laboratory
ICRISAT (International Crops
Research Institute for the Semi-
Arid Tropics)
Patancheru
Andhra Pradesh - 502 324,
INDIA
E-mail: k.sharma@cgiar.org

Dr Reto J. Strasser
Laboratory of Bioenergetics
University of Geneva
1254 Jussy, SWITZERLAND
E-mail:
reto.strasser@bioen.unige.ch

RATIONALE

Only about 13% of the total pulse crop yield is produced under irrigation. Pulses are
produced to a large extent in marginal agricultural areas with poor soil quality, since
their intrinsic characteristics allow production under harsh environmental conditions.
As a result, the crop plants have acquired primitive traits such as long duration,
indeterminate growth, bushy habit and flower drop, etc. rather than high yield. There
is also general agreement that the production of pulses is very much limited by
abiotic stress factors evoked by soil salinity, drought, and low temperature. A shift
from rainfed to irrigated cultivation is very unlikely, since pulses are not an important
cash crop and thus have a secondary status in the farming system.

Four different genes will be transferred into chickpea to increase the resistance to
salt, drought, and low temperature by metabolic pathway engineering.
 • P5CSF129A (mutant gamma-pyrrolidine-5-carboxylate synthetase) ->
glutamate to gamma-pyrrolidine-5-carboxylate -> proline
• mtID (mannitol-1-phosphate dehydrogenase) -> mannitol-1-phosphate
dehydrogenase to mannitol
• codA (cholin oxidase) -> choline to glycinebetaine
• annexin -> annexin protein

SUMMARY OF THE ACHIEVMENTS OF THE FIRST PROGRAM PHASE

adapted from the summary provided by the project partners

Being a good source of protein especially for vegetarians, chickpea (Cicer arietinum
L.) is grown as the most important pulse crop in India . Crops grown in over 90% of
arable land are exposed to abiotic stress. Chickpea is sensitive to drought, salinity
and frost/low temperature stresses, and the overall yield of chickpea is often
curtailed significantly due to its exposure to these stresses. Therefore, investigations
carried out in this project were aimed at enhancing the tolerance of chickpea to
drought, salinity and frost/low temperature stress by introducing the genes for
compatible solutes (the codA gene for glycinebetaine biosynthesis and the P5F129A
gene for proline biosynthesis), which are well established to impart abiotic stress
tolerance, through modern genetic manipulation technology. Both these genes could
be successfully introduced independently into chickpea through Agrobacterium
mediated transformation using half-embryonic axis with single cotyledon as explants,
following the protocols that were fine-tuned by us earlier. The putative transformants
selected after rigorous selection for over three months with regular (15 day)
subculture in the presence of 50 mg/l kanamycin were rooted in the presence of 50
mg/l kanamycin and/or 20 mM choline following a short treatment (3 sec) of the cut
end of the shoots with 100 mM IBA. A number of putative codA transgenic genotypes
could be successfully established in pots filled with garden soil and manure in the
ratio of 3:1. PCR analysis carried out independently with nptII and hpt primers
revealed that only five out of twenty six independent T0 genotypes well-established in
pots showed the presence of both nptII as well as hpt marker genes. Healthy
seedlings obtained from the seed of five independent T0 lines germinated in the
presence of 50 mg/l kanamycin and 20 mg/l hygromycin and were established in
pots. All the T1 plants established in the pots showed the presence of both nptII as
well as hpt marker genes suggesting the presence of the codA gene in them.
Western analysis confirmed the presence of the protein choline oxidase in all the
independent T0 and T1 lines that possessed npt II as well as hpt marker genes,
demonstrating that the prokaryotic codA gene had not only been integrated into the
genome of these transgenic lines, but could also be translated successfully. In the
subsequent season, seeds of nine independent codA transgenic genotypes (five of T2
generation and four of T1 generation were collected).
51 putative transgenic lines with P5CSF129A gene were advanced to T3 generation
and their confirmatory molecular analysis is currently underway. The seed material
was harvested from all 51 lines. PCR analysis showed over 75% of the selected
plants to be positive for nptII gene and showed transcription in RT-PCR analysis of
the mRNA. The P5CSF:129A positive plants showed the overproduction of proline in
the range of 2.0 to 5.0 fold in individual transgenic lines (10 lines tested) of
chickpea. The transgenic lines were advanced to T3 generations and will be used for
physiological, molecular and biochemical studies under glasshouse (for drought in
dry down experiments) during the second phase of this project.
Chlorophyll and fluorescence analysis is recognized as a useful tool for quick and
easy detection of the influence of many stress types on plants such as drought, heat,
cold etc.. Protocols were fine-tuned to determine abiotic stress tolerance, in
particular drought and salinity, using the Plant Efficiency Analyzer (PEA), a technical
set-up developed at the University of Geneva . These methods were adapted to
chickpea plants and experiments confirmed their utility in analyzing 9 chickpea
varieties under normal and stress conditions. Drought stress was the predominant
interest of the studies. It was shown that all 9 varieties suffered under drought
conditions, but recovered after rewatering. Depending on the variety, a recovery
between 65% and over 100 % in comparison to the unstressed control variety was
observed.

With the aim to develop a quick test, preliminary experiments with detached leaves
were carried out (mimicking salt stress and drought conditions on filter paper and
with salt solutions). A first comparative analysis of putative transgenic chickpea lines
carrying the P5CSF129A gene revealed that the performance index (indication of
photosynthetic efficiency) of transgenic lines was significantly superior to that of wild
type.

***********************************************************************

Cicer arietinum L.
Contributors: F.J. Muehlbauer and Abebe Tullu<
Copyright © 1997. All rights Reserved. Quotation from this document should cite and
acknowledge the contributors.

1. Common Names
2. Scientific Names
3. Uses
4. Chemistry
1. Traditional Medicinal Uses
5. Origin
6. Botany
1. Taxonomy, Morphology and Floral Biology
2. Ecology
7. Crop Culture
1. Field Cultivation
2. Harvesting
8. Yields and Economics
9. Germplasm
10. Biotic Factors
11. References
12. Selected Experts

Common Names
Bengal gram (Indian), Chickpea (English), Garbanzo (Latin America),
Hommes, Hamaz (Arab world), Nohud, Lablabi (Turkey), Shimbra
(Ethiopia)

Scientific Names
Species: Cicer arietinum L.
Family: Leguminosae

Uses
Chickpea is grown in tropical, sub-tropical and temperate regions.
Kabuli type is grown in temperate regions while the desi type chickpea
is grown in the semi-arid tropics (Muehlbauer and Singh, 1987;
Malhotra et al., 1987). Chickpea is valued for its nutritive seeds with
high protein content, 25.3-28.9 %, after dehulling (Hulse, 1991).
Chickpea seeds are eaten fresh as green vegetables, parched, fried,
roasted, and boiled; as snack food, sweet and condiments; seeds are
ground and the flour can be used as soup, dhal, and to make bread;
prepared with pepper, salt and lemon it is served as a side dish
(Saxena, 1990). Dhal is the split chickpea without its seedcoat, dried
and cooked into a thick soup or ground into flour for snacks and
sweetmeats (Saxena, 1990; Hulse, 1991). "Sprouted seeds are eaten
as a vegetable or added to salads. Young plants and green pods are
eaten like spinach. A small proportion of canned chickpea is also used
in Turkey and Latin America, and to produce fermented food. Animal
feed is another use of chickpea in many developing countries. An
adhesive may also be prepared; although not water-resistant, it is
suitable for plywood. Gram husks, and green or dried stems and leaves
are used for stock feed; whole seeds may be milled directly for feed.
Leaves are said to yield an indigolike dye. Acid exudates from the
leaves can be applied medicinally or used as vinegar. In Chile, a
cooked chickpea-milk (4:1) mixture was good for feeding infants,
effectively controlling diarrhea. Chickpeas yield 21% starch suitable for
textile sizing, giving a light finish to silk, wool, and cotton cloth" (Duke,
1981).

Chemistry
Chickpea seed has 38-59% carbohydrate, 3% fiber, 4.8-5.5% oil, 3%
ash, 0.2% calcium, and 0.3% phosphorus. Digestibility of protein varies
from 76-78% and its carbohydrate from 57-60%. (Hulse, 1991,
Huisman and van der poel, 1994). Raw whole seeds contain per 100 g:
357 calories, 4.5-15.69% moisture, 14.9-24.6 g protein, 0.8-6.4 % fat,
2.1-11.7 g fiber, 2-4.8 g ash, 140-440 mg Ca, 190-382 mg P, 5.0-23, 9
mg Fe, 0-225 µ g β-carotene equivalent, 0.21-1.1 mg thiamin, 0.12-
0.33 mg riboflavin, and 1.3-2.9 mg niacin (Duke, 1981; Huisman and
van der Poel, 1994). "Boiled and roasted seeds contain similar
amounts. Sprouting is said to increase the proportionate amounts of
ascorbic acid, niacin, available iron, choline, tocopherol, pantothenic
acid, biotin, pyridoxine, inositol, and vitamin K. Malic and oxalic acid
exudation from the leaves of the plant may soil and damage trousers
and shoes. Wild species often have similar glandular secretions" (Duke,
1981). The limiting amino acid concentrations are 0.52 for methionine,
1.45 for lysine and cystine, 0.71 for threonine and 0.16 for tryptophan
(Williams et al., 1994). The amino acid composition of seeds with
19.5% protein, 5.5% oil is (per 16 g N): 7.2 g lysine, 1.4 g methionine,
8.8 g arginine, 4.0 g glycine, 2.3 g histidine, 4.4 g isoleucine, 7.6 g
leucine, 6.6 g phenylalanine, 3.3 g tyrosine, 3.5 g threonine, 4.6 g
valine, 4.1 g alanine, 11.7 g aspartic acid, 16.0 g glutamic acid, 0.0 g
hydroxyproline, 4.3 g proline, and 5.2 g serine" (Duke, 1981; Huisman
and van der poel, 1994; and Williams et al., 1994). "Percent fatty acid
compositions are: 'Desi': oleic 52.1, linoleic 38.0, myristic 2.74, pactic
5.11, and steatic 2.05; 'Kabuli': oleic 50.3, linoleic 40.0, myristic 2.28,
palmitic 5.74, stearic 1.61, and arachidic 0.07%. The leaves contain 4-
8% protein" (Duke, 1981).

Traditional Medicinal Uses

Among the food legumes, chickpea is the most hypocholesteremic

agent; germinated chickpea was reported to be effective in controlling
cholesterol level in rats (Geervani, 19991). "Glandular secretion of the
leaves, stems, and pods consists of malic and oxalic acids, giving a
sour taste. In India these acids used to be harvested by spreading thin
muslin over the crop during the night. In the morning the soaked cloth
is wrung out, and the acids are collected in bottles. Medicinal
applications include use for aphrodisiac, bronchitis, catarrh, cutamenia,
cholera, constipation, diarrhea, dyspepsia, flatulence, snakebite,
sunstroke, and warts. Acids are supposed to lower the blood
cholesterol levels. Seeds are considered antibilious" (Duke, 1981).

Origin
van der Maesen (1972) believed that the species originated in the
southern Caucasus and northern Persia. However, Ladizinsky, (1975)
reported the center of origin to be southeastern Turkey. van der
Maesen (1987) recognized the southeastern part of Turkey adjoining
Syria as the possible center of origin of chickpea based on the
presence of the closely related annual species, C. reticulatum
Ladizinsky and C. echinospermum P.H. Davis. Wild C. reticulatum is
interfertile with the cultivated pulse and morphologically closely
resembles cultivated C. arietinum. It is regarded as the wild progenitor
of chickpea (Ladizinsky, 1975). "Botanical and archeological evidence
show that chickpeas were first domesticated in the Middle East and
were widely cultivated in India, Mediterranean area, the Middle East,
and Ethiopia since antiquity. Brought to the New World, it is now
important in Mexico, Argentina, Chile, Peru and the U.S. Also important
in Australia. Wild species are most abundant in Turkey, Iran,
Afghanistan, and Central Asia" (Duke, 1981).

Botany
Taxonomy, Morphology and Floral Biology

Cicer, which was classified under Vicieae Alef., was later reported to
belong to the monogeneric tribe, Cicereae (Kupitcha, 1977). The Genus
includes 9 annuals and 34 perennial herbs (van der Maesen, 1972; and
Muehlbauer, 1993). Crossability and fertility of hybrids in interspecific
crosses have been used as a basis to classify the annuals into 4
crossability groups. The first group includes the cultivated chickpea
(Cicer arietinum L.) (Ladizinsky et al., 1988) and C. reticulatum.
Chickpea plants can be described as "stems are branched, erect or
spreading, sometimes shrubby much branched, 0.2-1 m tall, glandular
pubescent, olive, dark green or bluish green in color. Root system is
robust, up to 2 m deep, but major portion up to 60 cm. Leaves
imparipinnate, glandular-pubescent with 3-8 pairs of leaflets and a top
leaflet (rachis ending in a leaflet); leaflets ovate to elliptic, 0.6-2.0 cm
long, 0.3-1.4 cm wide; margin serrate, apex acuminate to aristate,
base cuneate; stipules 2-5 toothed, stipules absent. Flowers solitary,
sometimes 2 per inflorescence, axillary; peduncles 0.6-3 cm long,
pedicels 0.5-1.3 cm long, bracts triangular or tripartite; calyx 7-10 mm
long; corolla white, pink, purplish (fading to blue), or blue, 0.8-1.2 cm
long. The staminal column is diadelphous (9-1) and the ovary is sessile,
inflated and pubescent" (Duke, 1981; Cubero, 1987; van der Maesen,
1987). Pod rhomboid ellipsoid, 1-2 with three seeds as a maximum,
and inflated, glandular-pubescent. Seed color cream, yellow, brown,
black, or green, rounded to angular, seedcoat smooth or wrinkled, or
tuberculate, laterally compressed with a median groove around two-
thirds of the seed, anterior beaked; germination cryptocotylar (Duke,
1981; Cubero, 1987 van der Maesen, 1987).

Ecology
Chickpea is a self-pollinated crop. Cross-pollination is rare; only 0-1 %
is reported (Singh, 1987; Smithson et al., 1985). Grown usually as a
rainfed cool-weather crop or as a dry climate crop in semi-arid regions.
Optimum conditions include 18-26°C day and 21-29°C night
temperatures and annual rainfall of 600-1000 mm (Duke, 1981;
Muehlbauer et al., 1982; Smithson et al., 1985). The Palouse region of
the states Washington and Idaho, appears to be well suited to chickpea
and can be characterized as having 18-25°C during the day and 5-10°C
during the night and a sufficiently long growing season (Muehlbauer et
al., 1982). California is very suited to the chickpea crop and it has
thrived in the coastal areas and in the Central Valley. Thrives on a
sunny site in a cool, dry climate on well-drained soils and grows on a
residual moisture in the post-rainy seasons of sub tropical winter or
spring of the northern hemisphere (Smithson et al., 1985). "Generally
grown on heavy black or red soils pH 5.5-8.6. Frost, hailstones, and
excessive rains damage the crop. Though sensitive to cold, some
cultivars can tolerate temperatures as low as -9.5°C in early stages or
under snow cover. Daily temperature fluctuations are desired with cold
nights with dewfall. Relative humidity of 21-41% is optimum for seed
setting. In virgin sandy soils or for the first planting in heavier soils,
inoculation is said to increase yield by 10-62%" (Duke, 1981). Although
spoken of as "day-neutral," chickpea is a quantitative long-day plant,
but flowers in every photoperiod (Smithson et al., 1985).

Crop Culture
Field Cultivation

Chickpeas are propagated from seeds. "Seed is broadcast or (more

often) drilled in rows 25-60 cm apart, spaced at 10 cm between seeds,
at a depth of 2-12 cm with soil well pressed down. Soil is worked into a
rough tilth, clods broken and field-leveled. Seed is sown in spring (late
March-mid April in Turkey, United States; February-March-April around
the Mediterranean) when the ground has warmed or when the rains
recede (mid-September to November, rarely later in India and Pakistan;
September-January or April, Ethiopia) depending on the region"
(Smithson et al., 1985). Seeding rates vary from 25-40 kg/ha to 80-120
kg/ha, depending on the area and seed type (Smithson et al., 1985).
Chickpea may be cultivated as a sole crop, or mixed with barley,
lathyrus (grasspea), linseed, mustard, peas, corn, coffee, safflower,
potato, sweet potato, sorghum, or wheat. In rotation it often follows
wheat, barley, rice, or tef (van der Maesen, 1972). "In India, chickpeas
are also grown as a catch crop in sugarcane fields and often as a
second crop after rice. Although usually considered a dry-land crop,
chickpeas develop well on rice lands.
In most areas, chickpeas are intercultivated once about 3-4 weeks after
sowing; thereafter, the crop develops enough shade to smother weeds.
In other areas light weedings are recommended. On poor soils, manure
or compost is beneficial. Seed inoculation improves yield only for crops
grown for the first time or after rice, where Rhizobium populations are
naturally low or absent. Irrigation at 45 and 75 days after planting is
useful (Duke, 1981). Fertilizers or manure have often failed to increase
yields substantially because of fixation of P by soils and the
accumulation of nutrients in the upper layer of the soil which are often
dry (Smithson et al., 1985).

Harvesting

Chickpeas mature in 3-7 months and the leaves turn brown/yellow

during maturity. For dry seeds, the plants are harvested at maturity or
slightly earlier by cutting them close to the ground or uprooting. The
plants are stacked in the field for a few days to dry and later the crop is
threshed by trampling or beating with wooden flails. The chaff is
separated from the grain by winnowing. Tall cultivars are suitable for
mechanized harvesting in which case combines can be used.
Chickpeas are usually stored in bags, but are more subject to insect
damage than when stored in bulk. Proper cleaning, drying, and
aeration are necessary to control seed beetles. A thin coating with
vegetable oil can reduce storage damage. Sometimes baskets, made
from twisted rice straw, are used as storage containers.

Yields and Economics

Greater and more stable yields are the major goals of plant breeding
programs. Chickpea yields usually average 400-600 kg/ha, but can
surpass 2,000 kg/ha, and in experiments have attained 5,200 kg/ha.
Yields from irrigated crops are 20-28% higher than yields from rainfed
crops. Two types of chickpea are recognized, desi (colored, small
seeded, angular and fibrous) and kabuli (beige, large seeded, rams-
head shaped with lower fiber content) types (Malhotra et al., 1987). In
a 3-cultivar trial in India, dry matter yields ranged from 9,400 to
12,000 kg/ha. In India, chickpea or gram ranks 5th among grain crops,
and is the most important pulse crop (Smithson et al., 1985). In India
and Pakistan, chickpeas are consumed locally, and about 56% of the
crop is retained by growers (Duke, 1981). In United States and Europe,
chickpeas are marketed dried, canned, or in various vegetable
mixtures. In Europe, mashed chickpeas from the Mediterranean are
sold canned. Mashed chickpea mixed with oils and spices (hummus) is
a popular hors d'oeuvre in the Mediterranean Middle East (Duke,
1981). In 1975 to 1994, on the average, Asia produced 5-6,000,000
MT, yields ranging from 570-766 kg/ha, led by India, which produced 4-
5,000,000 MT, ranging from 500-900 kg/ha; Africa produced 250-
364,000 MT, with yields ranging from 600-660 kg/ha. North and Central
America produced 180-260,000 MT, while averaging 1,600 kg/ha.
Europe produced 50-118,000 MT, while averaging 750 kg/ha (FAO,
1976, 1994). The major chickpea growing countries are India, Pakistan,
and Turkey in Asia, Ethiopia in Africa, California and Washington state
in the U.S., Mexico and Australia (FAO, 1994). Chickpea production
increased from 1980 to 1990 by about a million tons (at 1.8 %
annually), and there was a 5.6 % increase in yield over the decade
(Oram, P.A. and M. Agcaoili, 1994). Further increases in yield could be
attained from the use of germplasm/wild relatives, for identification of
new genes, and from new combinations of favorable genes already
existing (Muehlbauer et al., 1988).

Biotic Factors
The main fungi that affect chickpea are Fusarium oxysporum
Schlechtend.:Fr. f. sp. ciceris (Padwick) Matuo & K. Sato, causing the
plant to wilt and Ascochyta blight caused by Ascochyta rabiei.
Ascochyta blight is the most serious disease in North India, Pakistan,
the U.S. and the Middle East (sometimes causing l00% losses)
(Smithson et al., 1985). Blight causes brown spots on leaves, stems,
pods and seeds (Kaiser, 1992). Other fungi known to attack chickpea
include leaf spot (Alternaria sp.), Ascochyta pisi, rust (Uromyces
ciceris-arientini), gray mould (Botrytis cinera), powdery mildew
(Leviellula taurica), Pythium debar-yanum, P. ultimum, dry root rot
(Rhizoctonia bataticola), R. solani, foot rot (Sclerotium rolfsii),
Sclerotinia sclerotiorum, wilt (Verticillium albo-atrum). Some of these
fungi may become of economic importance. Viruses isolated from
chickpea include alfalfa mosaic, pea enation mosaic, pea leaf roll, pea
streak, bean yellow mosaic, and cucumber mosaic (Duke, 1981; Kaiser,
1988; Smithson et al., 1985; van Emden et al., 1994). Pod borer
(Helicoverpa armigera), the most important pest, and feeds on leaves
and developing seeds (Smithson et al., 1985). Cutworms (Agrotis sp.),
lesser armyworms (Spodoptera exigua) and leaf minor. Groundnut
aphid (Aphis craccivora), pea aphid (Acyrthsosiphon pisum), cowpea
bean seed beetle (Callosobruchus maculatus), and Adzuki bean seed
beetle (C. chinensis) are also important. "Many storage insects
specifically Bruchid sp. are a serious pest of stored chickpea.
Chickpeas stored as dhal harbor fewer bruchids. Callosobruchus
chinensis lowers seed viability. For control of bruchids, dusting with
BHC, DDT, derris, lindane, or pyrethrum or fumigation with methyl
bromide, have been recommended" (Duke, 1981). In general,
estimates of yield losses by individual pests, diseases or weeds range
from 5-10 % in temperate regions and 50-100 % in tropical regions
(van Emden, 1988).
Among the abiotic factors, drought stands to be the number one
problem in major chickpea growing regions because the crop is grown
on residual moisture and the crop is eventually exposed to terminal
drought (Johansen et al., 1994). In west Asia and North African
countries, low temperature causing freezing injury or death or delayed
onset of podding reduces yield tremendously (Singh, 1987). Heat and
salinity problems are relatively important following drought and cold
stresses (Singh et al., 1994).

Germplasm
Chickpea germplasm is maintained at two International centers
(ICRISAT in India and ICARDA in Syria) and at National centers including
the Vavilov institute in Russia, the USDA-ARS Regional Plant
Introduction Station at Pullman in the U.S. and other gene banks.
Tremendous variation for economically important traits has been
documented and improved cultivars have been developed and
released. Variation for Flower and seed color and size, growth duration,
yield, and biomass, disease resistance, quality traits (cooking time,
amino acid content, flatulence and digestibility) are recorded. 'Kabuli'
type chickpeas (Mediterranean and Middle Eastern origin) generally
have the largest seeds, and grow well under irrigation. Desi chickpeas
(Indian distribution) have smaller seeds, and yield better in Indian
subcontinent, Ethiopia and often elsewhere. Hybrids between Kabuli
and Desi have produced strains with medium-size seeds and fair yields.
The bulk of chickpeas grown in developing countries are from
unselected land races. Germplasm with resistance to major diseases
has been identified and genes for important diseases have been
named (Muehlbauer and Singh, 1987).

References
• Cubero, J.I. 1987. Morphology of chickpea. p. 35-66. In: M.C. Saxena and K.B.
Singh (eds.), The Chickpea. CAB. International, Wallingford, Oxon, OX10 8DE,
UK.
• Duke, J.A. 1981. Handbook of legumes of world economic importance. Plenum
Press, New York. p. 52-57.
• Food and Agriculture Organization of the United Nations. 1976 Production
Yearbook. Rome, Italy.
• Food and Agriculture Organization of the United Nations. 1994. Production
Yearbook. Rome, Italy.
• Geervani, P. 1991. Utilization of chickpea in India and scope for novel and
alternative uses. p. 47-54. In: Uses of Tropical Grain Legumes: Proceedings of
 Consultants' Meeting, 27-30 March, 1989. ICRISAT Center, Patancheru, Andhra
Pradesh, India.
• Hawtin, G.C., K.B. Singh and M.C. Saxena. 1980. Some recent development in
the understanding and improvement of Cicer and Lens. p. 613-623. In: R.J.
Summerfield and A.H. Bunting (eds.), Advances in Legume Science. Proceedings
of the International Legume Conference, Kew, 31 July-4 August 1978, held under
the auspices of the Royal Botanic Garden, Kew, the Missouri Botanical Garden,
and the University of Reading, UK.
• Huisman, J. and A.F.B. van der Poel. 1994.Aspects of the nutritional quality and
use of cool season food legumes in animal feed. p. 53-76. In: F.J. Muehlbauer and
W.J. Kaiser (eds.) Expanding the Production and Use of Cool Season Food
Legumes. Kluwer Academic Publishers. Dordrecht, The Netherlands.
• Hulse, J.H. 1991. Nature, composition and utilization of grain legumes. p. 11-27.
In: Uses of tropical Legumes: Proceedings of a Consultants' Meeting, 27-30
March 1989, ICRISAT Center. ICRISAT, Patancheru, A.P. 502 324, India.
• Johansen, C., B. Baldev, J.B. Brouwer, W. Erskine, W.A. Jermyn, L. Li-Juan, B.A.
Malik, A. Ahad Miah and S.N. Silim. Biotic and abiotic stresses constraining
productivity of cool season food legumes in Asia, Africa and Oceania. p. 175-194.
In: F.J. Muehlbauer and W.J. Kaiser (eds.) Expanding the Production and Use of
Cool Season Food Legumes. Kluwer Academic Publishers. Dordrecht, The
Netherlands.
• Kaiser, W.J., W. Schaad, G.I. Mink and R.O. Hampton. 1988. Workshop: Seed
pathogens of food legumes. p.515-517. In: R.J. Summerfield (ed.), World Crops:
Cool Season Food Legumes. Kluwer Academic Publishers, Dordrecht, The
Netherlands.
• Kaiser, W.J. 1992. Epidemiology of Ascochyta rabiei. P. 117-134. In: K.B. Singh
and M.C. Saxena (eds.), Disease Resistance Breeding in Chickpea. ICARDA,
Aleppo, Syria.
• Kay, D. 1979. Food legumes. Tropical Development and Research Institute (TPI).
TPI Crop and Product Digest No. 3. p.48-71. Tropical Products Institute, London,
UK.
• Ladizinsky, G. 1975. A new Cicer from Turkey. Notes of the Royal Botanic
Garden Edinburgh 34: 201-202.
• Ladizinsky, G., B. Pickersgill and K. Yamamoto. 1988. Exploitation of wild
relatives of the food legumes. p.967-78 In: R.J. Summerfield (ed.), World Crops:
Cool Season Food Legumes. Kluwer Academic Publishers, Dordrecht The
Netherlands.
• Malhotra, R.S., R.P.S. Pundir and A.E. Slinkard. 1987. Genetic resources of
chickpea. p.67-81. In: M.C. Saxena and K.B. Singh (ed.), The Chickpea. C.A.B.
International Cambrian News Ltd, Aberystwyth, UK.
• Muehlbauer, F.J. and K.B. Singh. 1987. Genetics of chickpea. p. 99-125. In: M.C.
Saxena and K.B. Singh (eds.), The Chickpea. CAB. International, Wallingford,
Oxon, OX10 8DE, UK.
• Muehlbauer, F.J., 1996. Advances in the production of cool season legumes.
American Journal of Alternative Agriculture 11: 71-76.
• Muehlbauer, F.J., R.J. Redden, A.M. Nassib, L.D. Robertson and J.B. Smithson.
1988. Population improvement in pulse crops: an assessment of methods and
techniques. p.943-966. In: R.J. Summerfield (ed.), World Crops: Cool Season
Food Legumes. Kluwer Academic Publishers, Dordrecht, The Netherlands.
• Oram, P.A. and M. Agacoili. 1994. Current status and future trends in supply and
demandof cool season food legumes. p. 3-49. In: F.J. Muehlbauer and W.J. Kaiser
(eds.), Expanding the Production and Use of Cool Season Food Legumes. Kluwer
Academic Publishers. Dordrecht, The Netherlands.
• Robertson, L.D., K.B. Singh, W. Erskine and Ali M. Abd El Moneim. 1996.
Useful genetic diversity in germplasm collections of food and forage legumes
from West Asia and North Africa. Germplasm Resources and Crop Evolution 43:
447-460.
• Simon, C.J. and R.M. Richard, 1995. Development and use of core subsets of cool
season food legume germpasm collection. HortScience 30: 907.
• Singh, K.B. 1987. Chickpea breeding. p.127-162. In: M.C. Saxena and K.B.
Singh (eds.), The Chickpea. CAB International, UK.
• Singh, K.B., R.S. Malhotra, M.H. Halila, E.J. Knights and M.M. Verma. Current
status and future strategy in breeding chickpea for resistance to biotic and abiotic
stresses. p. 572-591. In: F.J. Muehlbauer and W.J. Kaiser (eds.), Expanding the
Production and Use of Cool Season Food Legumes. Kluwer Academic Publishers.
Dordrecht, The Netherlands.
• Smithson, J.B., J.A. Thompson and R.J. Summerfield. 1985. Chickpea (Cicer
arietinum L.). p. 312-390. In: R.J. Summerfield and E.H. Roberts (eds.), Grain
Legume Crops. Collins, London, UK.
• Van der Maesen, L.J.G., 1972. A monograph of the genus with special reference
to the chickpea (Cicer arietinum L.), Its ecology and cultivation. Commun. Agric.
University, Wageningen, Dordrecht, The Netherlands.
• Van der Maesen, L.J.G. 1972. Cicer L. a monograph of the genus, with special
reference to the chickpea (Cicer arietinum L.), its ecology and cultivation.
Mededlingen landbouwhogeschool (Communication Agricultural University)
Wageningen 72-10. 342 p.
• Van der Maesen, L.J.G. 1987. Cicer L. Origin, history and taxonomy of chickpea.
p.11-34. In: M.C. Saxena and K.B. Singh (ed.), The Chickpea. C.A.b.
International Cambrian News Ltd, Aberystwyth, UK.
• Van Emden, H.F., S.L. Ball and M.R. Rao. 1988. Pest disease and weed problems
in pea lentil and faba bean and chickpea. p. 519-534. In: R.J. Summerfield (ed.),
World Crops: Cool Season Food Legumes. ISBN 90-247-3641-2. Kluwer
Academic Publishers, Dordrecht, The Netherlands.
• Williams, P.C., R.S. Bhatty, S.S. Deshpande, L.A. Hussein and G.P. Savage. 1994.
Improving nutritional quality of cool season food legumes. p. 113-129. In: F.J.
Muehlbauer and W.J. Kaiser (eds.), Expanding the Production and Use of Cool
Season Food Legumes. Kluwer Academic Publishers, Dordrecht, The
Netherlands.