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Enhancing Tolerance of Chickpea to Drought, Freezing, and Low Temperature through Genetic Engineering with the codA and P5CSF-129A Genes Project ID: PS4
Project Duration:
1 April 2001 to 31 August 2004
Principal Investigators:
Dr P.P. SaradhiDepartment of Environmental BiologySchool of Environmental StudiesNew Delhi - 110 007, INDIAE-mail: [email protected]@ndf.vsnl.net.in
Dr Kiran K. SharmaGenetic Transformation LaboratoryICRISAT (International Crops Research Institute for the Semi-Arid Tropics)PatancheruAndhra Pradesh - 502 324, INDIA
E-mail: [email protected]
Dr Reto J. StrasserLaboratory of BioenergeticsUniversity of Geneva1254 Jussy, SWITZERLANDE-mail: [email protected]
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 PHASEadapted 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.
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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 g -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.
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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
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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.
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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.
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