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WEL COME

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MAJOR ADVISOR:Dr. Y. Ravindrababu

Research Scientist,Center of excellance for research

on pulses,SDAU, S.K. Nagar

MINOR ADVISOR :Mrs.Sweta MishraAssistant Professor,

Dept. of Biotechnology,C.P.C.A,

SDAU, S.K. Nagar

BIOTECHNOLOGICAL INTERVENTIONS FOR CHICKPEA IMPROVEMENT

Seminar Presentation on

SPEAKER:KANAK SAXENA

Reg. No:- 04-01034-2012

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Introduction – Area, Production, Productivity & Distribution

Biotechnological Approach, It’s Need In Chickpea

Source Of Resistance Major Production Constraints Biotechnological Approaches To

Overcome Various Impediments Conclusions Future Thrust

CONTENTS

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• Chickpea (Cicer arietinum) is a self pollinated crop of family Leguminaceae grown in semi-arid regions of the world

• Evidence shows that chickpea was first cultivated in Turkey, as early as 7000BC• It is believed to have been domesticated from Cicer reticulatum, a close wild

relative• India is the Largest producer & importer. It ranks third among food legumes in

production (FAO, 2012)• In 2009-10 chickpea has marked significant increase in area (8.45mha) highest in

last 10 yrs (AICRP on chickpea,kanpur)• Similarly, the crop production (7.35mt) also surpassed last 50 yr record with

increased productivity (858 kg/ha) ever recorded in the history of india.(AICRP on chickpea,kanpur)

INTRODUCTION

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International Crops Research Institute for the Semi-AridTropics (ICRISAT), India

20,267

National Bureau of Plant Genetics Resource (NBPGR), India 14,704

International Centre for Agricultural Research inDryland Areas (ICARDA), Syrian Arab Republic

13,462

Australian Temperate Field Crops Collection, Australia 8,655

Western Regional Plant Introduction Station, USDA-ARS, Washington State University, USA

6,763

National Plant Gene Bank of Iran, Iran 5,700

Plant Genetic Resources Institute, Pakistan 2,146N.I. Vavilov All-Russian Scientific Research Instituteof Plant Industry, Russian Federation

2,091

Plant Genetic Resources Department, Turkey 2,076

Institute of Biodiversity Conservation, Ethiopia 1,173

CHICKPEA GERMPLASM HELD AT GLOBAL RESEARCH INSTITUTES

Upadhyaya et al.,2012ICRISAT, Hyderabad

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GENERAL INFORMATION Of CHICKPEA.

FEATURES SPECIFICATIONS IN CHICKPEA

Common name Gram, Chana

Botanical genera Cicer

Cultivated species arietinum

Related wild species

Cicer reticulatum

Chromosome number

Diploid (Desi - 2n= 16) (Kabuli – 2n =18)

Genome size 740 Mb

cultivated chickpeas

DESI (small seeds, angular shape, and dark coloured seeds with a high percentage of fibre)KABULI (large seeds, owl-head shape, cream coloured seeds with a low percentage of fibre).

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Madhya pradesh, RajasthanBihar,Uttar Pradesh,Andhra pradeshGujarat, Maharashtra

Chickpea producing states in IndiaGlobal production of Chickpea

Gaur,et al.,(2010)

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Chickpea Area tones (Million ha)

Production (Million tones)

Productivity (kg/ha)

World 10.33 7.9 767

India 7.33 5.8 808

Gujarat 0.24 0.21 977

Area, Production and Productivity (2011-12)

FAO (2011)

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Annual species of chickpea and their importance

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Source Species Somatic

chromosome number

Traits

Cicer arietinum 16Ascochyta blight,

Fusarium wilt, Drought, Cold, Leaf minor

Cicer cuneatum 16 Leaf minor, Ascochyta blight

Cicer judaicum 16 Leaf minor, Cold

Cicer pinnatifidum 16 Leaf minor, Cold

Cicer echinospermum 16 Leaf minor, Cold

Cicer reticulatum 16 Fusarium, Cold , Blight

Cicer bijugum 16 Blight, Cold, Cyst nematode

New Delhi Chopra (2000)

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• It also contains nutritionally important minerals (calcium, potassium,

phosphorus, iron, zinc and magnesium). • Limited in the sulfur containing amino acids methionine (1.3–1.6%) and

cysteine (2.5–3.0%). • Free from anti nutritional factors.• Chickpea seeds are considered as Hypocholestric Agent (Because of its

superior fiber over many cereals and consumption of even smaller amounts of chickpea improves insulin secretion and controls blood sugar levels)

Carbohydrate 48.2–67.6%Protein 12.4–31.5%Starch 41–50%Fat and lipids 6 – 8 %Fiber 10–20%

Nutritional Composition Of Chickpea

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The major production constraints of chickpea (Cicer arietinum L.) and their solutions

Acharjee et al., (2013)Plant Science

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Production losses in chickpea due to stresses Country Mean

Prod. (tone)

Estimated production losses (tone)

Abiotic stress Biotic stress

Drought/Heat

Cold Salinity AB V Hel. Total

Bangladesh 69 27 0 10 0 7 14 75

China 210 126 0 17 - - 42 185

India 4329 2026 269 569 318 501 925 4759

Myanmar 110 55 0 11 0 0 22 88

Nepal 17 7 3 0 0 1 4 20

Pakistan 455 182 68 114 91 91 114 706

Middle East 916 532 133 85 218 57 145 1170

Africa 267 160 5 40 19 22 44 290

America 171 72 23 39 0 30 43 207

Europe 79 47 4 5 10 3 8 77

Australia 83 42 13 8 4 8 21 138

World 6700 3276 518 898 660 720 1382 7715AB – Ascochyta blight, V- Virus, Hel- Helicoverpa pod borer

13ICRISAT, Hyderabad Kumar et al., (1996)

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Sources of resistance to Biotic and Abiotic stresses in chickpea identified at ICARDA and

ICRISAT. Biotic Stress Source of resistance

Ascochyta blight ILC 72, 200, 3279, 3856, 5902,6090 ; ICC 1069

Botrytis grey mold ICC 1084, 1102, 3540, 4065

Fusarium wilt ILC 54, 240, 256, 336, 487, WR 315 ; ICC 11550, 12467

Pod borer ICC 506, 6663, 10619, 10667; ICCV 7

Abiotic Stress Source of resistance

Cold ILC 8262, 482 M

Drought ICC 4958

Syria Saxena., (1993)

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Why we need biotechnological approach ?

Cross incompatibility

lack of genetic variation

Narrow genetic base

Biotic and abiotic stresses

Limitation Embryo Rescue and Other tissue culture techniques

Transgenic development

Molecular breeding

Identification of new genes.

Strategies for improvement

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Biotechnological Approaches For Chickpea Improvement

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• The main constraint to the transfer of desired traits into cultivated chickpea from wild Cicer relatives is the presence of post-zygotic barriers which result in abortion of the immature embryo

• Optical microscopy shows that most C. bijugum hybrids aborted at very early stages of embryo development 4–8 days after pollination, compared to C. Pinnatifidum hybrids which reached the heart-shaped stage from 10 to 12 days after pollination.

• These observations suggest that the appropriate time to rescue chickpea C. bijugum hybrids is at the early globular stage of embryogenesis (2–7 days old).

• In contrast hybrids between chickpea C. pinnatifidum abort later (up to 15–20 days old) at the heart shaped or torpedo stages.

• Rescue of hybrid embryos in vitro and regeneration of hybrid plantlets could allow chickpea breeders to transfer desirable traits from wild relatives of chickpea.

(ICRISAT) Hyderabad Clarke,et al., 2005

Embryo rescue and plant regeneration in vitro of selfed chickpea (Cicer arietinum L.) and its wild annual relatives

Barriers to interspecific hybridisation in Cicer in situ. Pre-zygotic: (a) pollen tubes of C. bijugum (wild) grow down the style to fertilise ovules of C. arietinum (chickpea) within 24 h. Post-zygotic: (b) a very immature 7 day old hybrid globular embryo and elongated suspensor cells (c) Selfed ovules had an embryo sac containing endosperm, spherical suspensor cells, globular embryo (d) heart-shaped embryo . Clarke,et al., 2005

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Regeneration of plantlets from rescued chickpea ovules.(a) Dissected pod showing ovules (b) dissected ovule showing stage of development of an 8 day old from the ovule coat and endosperm; (c) placing embryo in direct contact with the filter paper bridge and medium in a fresh culture tube. (d) growing embryo erupts through the wall of the ovule at 3 weeks; (e) hypocotyl and/or roots develop at 6–12 weeks;(f) shoots are micropropagated on solid MS medium, Clarke,et al., 2005

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The most important pest of chickpea widely distributed throughout world.

Cause losses in chickpea yields up to 90-100 %

Due to presence of highly acidic exudates from the trichomes of chickpeas. Several chickpea genotypes have low-to-moderate level of resistance against pod borers but these levels are unstable because of the variable insect population and climatic conditions.

Pod borer (Helicoverpa armigera)

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Plant material C 235, BG 256, Pusa 362 and Pusa 372 (IARI,Delhi)cotyledonary nodes (CNs)

Transformation method

Agrobacterium tumefaciens strains LBA 4404

plasmid p35SGUS-INT promoter CaMV35SReporter gene bacterial (uidA) gene for b-glucuronidase (GUS)Gene of interest Bacillus thuringiensis (Bt) cry1Ac gene and kanamycin

resistance (nptII)

National Botanical Research Institute, Lucknow Sanyal, et al.,2005

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera

• 12-15 explants are cocultivated with agrobacterium culture for 48 hr. and then explant is incubated in fresh culture medium for 30 days.

• Putative transformed shoots are subcultured in selective medium for 15 days and plants are allowed to regenerate in glasshouse.

• Such plants are transformed transgenic plant and after selfing they were allowed to produce seeds.

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Different stages of in vitro regeneration of the T0 transgenic chickpea.

Sanyal, et al.,2005 National Botanical Research Institute, Lucknow

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Molecular and protein expression analysis of Transgenic plants

Quantitative determination of expressed Cry1Ac protein in different T1 chickpea plants.

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• The most important pests of stored grain legume seeds are bruchid beetles.

• High iinfestation rate because of the high fertility and short generation times of bruchid beetles.

• Many insecticidal proteins and molecules of plant origin such as lectins, α-amylase inhibitor and protease inhibitor can retard insect growth and development when ingested (Ussuf et al 2001).

• The α-amylase inhibitor markedly suppressed the α-amylase activity in the larval midgut of the weevils. Since α-amylase inhibitor is easily inactivated by cooking, introducing this gene into host plants can be regarded as a safe strategy.

Bruchid beetles (Coleoptera: Chrysomelidae: Bruchinae)

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• α-Amylases, the target of αAI-1, are key enzymes for starch digestion and have been shown to be vital for bruchid development.

• Chickpea seeds expressing αAI-1 in their cotyledons is highly resistant to the bruchid species C. chinensis, C. maculatus, C. analis.

Agrobacterium-mediated transformation of chickpea with α-amylase inhibitor gene for insect resistance

Entomology Research Institute,Chennai Ignacimuthu,et al.,(2006)

Plant material C. arietinum L. cv. ICCV 89314 (ICRISAT),embryonic axis (explant)

Transformation method

Agrobacterium tumefaciens strains LBA 4404

plasmid pCAMBIA35SASAL

promoter seed-specific promoter, phytohemagglutinin (PHA) CaMV35S promoter.

Reporter gene gusA geneGene of interest α-amylase inhibitor gene (αAl1) isolated from the seeds of

Phaseolus vulgaris L. (common bean)

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Caulogenic response of embryonic axis explants of chickpea. (A) Shoot initiation from embryonic axis explant. (B) Shoot bud formation from embryonic axis. (C) Multiple shoot formation after subculturing on fresh medium. (D) Shoot elongation on GA3 medium. (E) Root initiation from elongated shoot. (F) Earthen pot with well developed plant

Entomology Research Institute ,Chennai Ignacimuthu, (2006) Ignacimuthu,et al.,(2006)

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PCR amplifi cation of GUS gene from transformed chickpea embryonic axis explants resulting from infection with Agrobacterium LBA 4404 strain. Lane 1, Marker lambda DNA/HindIII digest; lanes 2-3, negative control (undigested DNA from plants); lanes 4-5, positive control (plasmid DNA); lanes 6-7, negative control (digested DNA from plants); lanes 8-15, transformed chickpea plants.

Entomology Research Institute ,Chennai Ignacimuthu,(2006) Ignacimuthu,et al.,(2006)

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Southern hybridization of HindIII digested DNA from transgenic chickpea plants using α-amylase gene probe.

Western blot analysis confirmed the presence of α-amylase inhibitor protein.

Molecular Analysis

Entomology Research Institute ,Chennai

Transgenic plants produced fertile seeds & Segregation of α-amylase inhibitor gene to next generation demonstrated that inheritance occurred in Mendelian ratio of 3:1.

Bioassay study proved that the transgenic seeds showed increased resistance to the bruchid weevil than the control seeds.

Ignacimuthu, (2006) Ignacimuthu,et al.,(2006)

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Major diseases in chickpea Fusarium wilt (Fusarium oxysporum f. sp. ciceris) Ascochyta blight (Ascochyta rabiei)

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Resistant varieties

(JG 11, JG 130, JGK 1, JGK 2,WR-215)

Resistant varieties (PBG 1, BG 267)

Fusarium wilt Fusarium oxysporum f. sp ciceris

Ascochyta blight (Ascochyta rabiei)

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In this study,two mapping populations, ‘C 214’ x ‘WR 315’ and ‘C 214’ x ‘ILC 3279’ were developed for mapping FW resistance and AB resistance respectively.

‘C 214’ is a well-adapted FW and AB susceptible desi variety. ‘WR 315’ is a desi variety resistant to race 1, race 2, race 3, race 4 and race 5 of

FW. ‘ILC 3279’ is AB resistant kabuli variety. After screening 371 SSR markers on parental lines and genotyping the mapping

populations with polymorphic markers, two new genetic maps

(C 214 x WR 315) and (C 214 x I LC 3279) were developed. Analysis of genotyping data collected on mapping population for resistance to FW

in field conditions two major and novel QTLs are identified and for resistance to seedling resistance and adult plant resistance for AB under controlled and field conditions.

(ICRISAT), Hyderabad Sabbavarapu,et al.,(2013)

Molecular Mapping Of QTL For Resistance To Fusarium Wilt (Race 1) And Ascochyta Blight In Chickpea (Cicer Arietinum L.)

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QTLs for FW resistance QTLs for resistance to AB

QTL analysis for FW and AB resistance based on (C 214 X WR 315) and(C214 x ILC3279) population of chickpea

(ICRISAT), Hyderabad Sabbavarapu ,(2013)Sabbavarapu,et al.(2013)

Two novel QTLs for resistance to FW and four QTL for resistance (both SR and APR) to AB.

The QTLs identified and markers linked with the promising QTLs are useful resource for genomics assisted breeding for resistance to FW and AB.

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• Drought and salinity are two of the most important abiotic stresses that alter plant water status and severely limit plant growth and development.

• Drought causes a considerable (~50-60%) annual yield losses.

• Due to continued depletion of ground water level and demand for irrigation has led to the salinization of arable lands. Therefore, it is imperative to develop sustainable cultivars tolerant to drought and salinity.

Drought and Salinity

34 ICRISAT, Hyderabad (BMC Genomics )

The present study was undertaken to generate a comprehensive resource of drought and salinity responsive ESTs in chickpea with following specific objectives:

(i) To generate drought-responsive ESTs from water-stressed root tissues of both drought-tolerant and drought-sensitive genotypes.(UG-1)

(ii) To generate salinity responsive ESTs from root tissues of NaCl treated plants of salinity-tolerant and salinity-sensitive genotypes.(UG-2)

(iii) To identify unigenes of chickpea based on ESTs generated in this study as well as public domain ESTs. (UG-3 & UG-4 )

(iv) To functionally annotate the identified chickpea unigenes(v) To identify correlated expression between genes.(vi) To discover SSRs for developing potential markers.

Varshney,et al., (2009)

A comprehensive resource of drought and salinity responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.)

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• The relative effects of drought and salinity on the growth pattern were observed the growth of the drought-tolerant genotype (ICC 4958) was observed to be better compared to drought-sensitive genotype (ICC 1882) in all the cases of drought stress implications.

• Similarly, the salinity-sensitive genotype (ICCV 2) exhibited a relatively more stunted growth pattern than salinity-tolerant genotype (JG 11) when these genotypes were exposed to salinity stress.

• Root tissues from both drought and saline stressed plants were harvested for total RNA extraction and subsequent cDNA library construction.

• A total 18,435 high quality ESTs were obtained.

• The average length of these high quality ESTs was 569 bp.

• All EST sequences were deposited in the dbEST division of GenBank (GR390696-GR410171 and GR420430-GR421115).

ICRISAT, Hyderabad (BMC Genomics) Varshney,et al., (2009)

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Sequence annotation Analysis of chickpea unigenes with related legume and plant ESTs

ICRISAT, Hyderabad (BMC Genomics) Varshney, (2009)

Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding.

Varshney,et al., (2009)

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Morphological and reproductive damage due to cold – A: Growth pattern under warm and cold conditions; B: Anthocyanin accumulation in stressed plants (arrow); C: Anthocyanin accumulation in leaves (arrow); D: Burning of leaf tips (arrow); E: Floral dimorphism; F: Normal flower setting pod (arrow); G and H: Floral abortion (arrows); I: Pod abortion (arrow) J: Normal pod set.

COLD STRESS

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• Low temperature injury is one of the most significant causes of crop damage worldwide.

• Cold acclimatization processes improve the freezing tolerance of plants.To identify genes of potential importance for acclimatzation to the cold and to elucidate the pathways that regulate this process, global transcriptome expression of the chickpea was analyzed using the cDNA-AFLP technique.

• Researchers are able to discover genes on the basis of their polymorphism or differential expression patterns.

• cDNA-amplified length polymorphism (cDNA-AFLP) is a sensitive, reproducible and efficient technology for the discovery and identification of genes.

• The main advantages of the cDNA-AFLP method 1.cDNA-AFLP does not require any prior knowledge of gene sequences.

2. It allows the detection of rarely expressed genes. • These features make cDNA-AFLP an ideal system for genome-wide

expression analysis.

IRAN Dinari,et al.,2013

Identification of Upregulated Genes under Cold Stress in Cold-Tolerant Chickpea Using the cDNA-AFLP Approach

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• In this study, the cDNA-AFLP technique was employed to isolate transcripts during low-temperature stress in the chickpea and demonstrate that the expression of several differentially expressed genes increased during cold acclimatization using real-time RT-PCR

• They generated 4800 transcript-derived fragments (TDFs) but considered only those cDNA fragments that seemed to be up-regulated during cold acclimatization. Fifty-four fragments were then cloned and sequenced.

• Comparative analysis show that 77 percent of TDFs belonged to known genes related to putative function i.e metabolism (31 percent), transport (10 percent), signal transduction pathways (15 percent) and transcription factors (21 percent).

• Approximately 15% of the up-regulated genes in investigation were involved in signal transduction. The mitogen-activated protein kinase (Jk649803) was up-regulated during cold acclimatization.

Dinari,et al.,2013 IRAN

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The cDNA-AFLP is a powerful technique for investigating the expression pattern of chickpea genes under low-temperature stress and to identify those genes that could increase the cold tolerance of the chickpea plant. Dinari,et al. 2013

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• In order to increase the concentration of the nutritionally essential sulphur amino acids in seed protein, a transgene encoding a methionine rich protein, sunflower seed albumin (SSA), was transferred to chickpea

(Cicer arietinum L).• In the current work, the methionine content of chickpea seeds has been

increased by adding a seed-expressed transgene encoding SSA, containing 16% methionine residues. The accumulation of SSA in chickpeas appeared to stimulate sulphur assimilation in the seeds.

• Seeds of non-transgenic chickpea, cultivar Semsen and transgenic chickpea line 45S were planted in greenhouse and different mineral nutrient solution was applied to each of four groups of chickpea The four treatments are referred to as:

high N, high S (HN-HS), high N, low S (HN-LS), low N, high S (LN-HS) and low N, low S (LN-LS).

Australia (Journal of Experimental Botany) Chiaiese,et al ., 2004

Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added,transgenic sink for organic sulphur

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Effects of sulphur and nitrogen nutrition and SSA genotype on the total methionine (%)

Introduction of a seed-expressed (SSA) transgene for a methionine rich protein into chickpea led to increased accumulation of sulphur amino acids in matureseeds of the transgenic lines.Methionine concentrations is high in the seeds of chickpea genotypes grown in the LN-HS nutrient that had a high sulphur-to-nitrogen ratio.

Chiaiese,et al 2004 Chiaiese,et al ., 2004

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Rescue of hybrid embryos in vitro and regeneration of hybrid plantlets could allow chickpea breeders to transfer desirable traits from wild relatives of chickpea.

The stable transgenic chickpea plants expressing cry1Ac gene have shown effective protection against damage by lepidopteran insect.

QTLs identified and markers linked with the promising QTLs are useful resource for genomics assisted breeding. For ex. resistance to FW and AB.

Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding.

Transgenic chickpea seeds that accumulated SSA gene contained more methionine.

CONCLUSION

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Various work has been done in generating transgenic chickpeas to tackle various stresses under changing climatic conditions. The future of chickpea production depends on the availability of cultivars that are resistant to both biotic and abiotic stresses.

The use of insect-resistant chickpeas will result in the decreased use of chemical pesticides but may not provide the desired yield advantage. So we have to improve chickpea production not only in terms of quantity, but also in nutritional quality.

This approach also help farmers adapt to climate change, in the process generating social, economic and environmental benefits for resource‐poor farmers.

FUTURE THRUST

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