Transgenic Chickpea Research Articles

Estimation of Outcrossing Rate in Chickpea (Cicer Arietinum L.) Sown in Autumn

Gene flow via outcrossing from transgenic plants to relatives will be one of the most important concerns to grow of the transgenic chickpea (Cicer arietinum L.) in European Union (EU). This report is therefore focused on spontaneous outcrossing rate in chickpea. A total of 39 kabuli type mutants with white flower and one desi type with pink flower were grown to estimate spontaneous outcrossing rate. Outcrossing rate ranged from 0.0 to 1.25% in mutant materials. Since labelling threshold for transgenic contamination in food and feed in European Union (EU) is 0.9%, outcrossing rate of 1.25% is higher than threshold of 0.9% in EU, and this result suggests that cultivation of transgenic chickpea will be under high risk to be contaminated chickpeas in neighbourhood fields.

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

Efficient procedure for Agrobacterium-mediated transformation of excised cotyledonary nodes of recalcitrant grain legume chickpea was optimized to result in the production of stable transgenic plants expressing the 2.2 kb native, truncated Bacillus thuringiensis (Bt) cry1Ac gene driven by the CaMV35S promoter and nptII gene for kanamycin selection. The factors evaluated were the preparation of excised cotyledonary nodes, their pre-conditioning, Agrobacterium density, acetosyringone concentration, sonication, vacuum treatment, co-cultivation period in presence of dithiothreitol and l-cysteine followed by kanamycin selection regime. Combination of these physical and physiological conditions during agroinoculation, co-cultivation and selection on kanamycin were found to be critical determinants resulting into increased competence for regeneration, co-transformation frequency of cry1Ac and nptII to 1.12% and elimination of escapes. Southern analysis of T 0 and T 1 plants confirmed co-integration and segregation of the T-DNA bearing cry1Ac and nptII genes into the genomic DNA of transgenic chickpea plants. The Cry1Ac protein accumulated maximum from 14.5 to 23.5 ng mg −1 extractable protein in T 0 and T 1 transgenic chickpea plants. About 80–85% T 0 plants were fertile and majority of them showed Mendelian segregation of the nptII gene in 3:1 ratio to their progenies. Insect feeding bioassay on transformed chickpea plants (T 0 and T 1) with larvae of pod borer insect Helicoverpa armigera showed high levels of toxicity to insects and protection of transgenic plants. Transformed chickpea plants expressing Cry1Ac protein above 10 ng mg −1 soluble protein showed 80–85% protection and high mortality (>80%) of insects while plants expressing between 5 and 10 ng mg −1 resulted into early pupation, significant loss in weight (45–55%) and moderate mortality of insects. Expression of truncated native Bt cry1Ac gene in chickpea has shown effective resistance in transgenic plants to the major pod borer insects.

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea.

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea ( Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 microM thidiazuron (TDZ) and 2 mM lysine and 2 m M threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 micro M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l(-1 )PPT. Increasing the concentrations of PPT to 15 mg l(-1) reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.

In Vitro Regeneration from Internodal Explants and Somaclonal Variation in Chickpea (Cicer arietinum L)

Protocols have been developed for the in vitro regeneration of plants from calli derived from internode explants of chickpea (Cicer arietinum L) cv Pusa-372. Callusing was induced on both B5 and MS media supplemented with different combinations and concentrations of auxins and cytokinins, but shoot regeneration was achieved only in B5 medium supplemented with 4.0 mg l−1 IAA and 0.5 mg l−1 BAP after serial subculture of callus on media with increasing concentration of IAA and constant concentration of BAP. Rooting could not be achieved in in vitro regenerated shoots on any one of the media tried. Complete plantlets were, therefore, developed through grafting of the in vitro regenerated shoot on established root stock. The grafting methodology was found to be highly efficient and reproducible. The somaclones developed produced viable seeds which showed variability in terms of seed colour and seed weight. Thus, the protocols developed in this study remove one important bottleneck in the development of transgenic chickpea.

Expression of cryIA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae

Two strains of chickpea (Cicer arietinum L.) ICCV-1 and ICCV-6, were used for transgenic plant generation. Embryo axis of mature seed devoid of the root meristem and the shoot apex was used as experimental material. The explants were cultured in medium containing MS macro salts, 4 × MS micro salts, B5 vitamins, 3.0 mg l−1 BAP, 0.004 mg l−1 NAA, 30 mg l−1 sucrose and cultured at 26 °C in dark, 24 h prior to bombardment. Gene delivery to the explants was carried out using a Bio-Rad Biolistic 1000/He particle gun. A chimaeric, truncated bacterial cryIA(c) gene construct was developed for plant expression with the CaMV35S promoter, nos terminator, an initiatory kozak sequence and a translational enhancer (STAR-P) sequence of tobacco mosaic virus. This cryIA(c) gene was cotransferred with a plasmid containing nptII gene as the selection marker. Transgenic kanamycin resistant chickpea plants were obtained through multiple shoot formation and repeated selection of the bombarded explants. Molecular analyses of the transformants revealed the presence of the transferred functional cryIA(c) gene in plant. Insect feeding assay indicated that the expression level of the cryIA(c) gene was inhibitory to the development of the feeding larvae of Heliothis armigera Hubner, the chickpea pod-borer