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

摘要

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) crylAc 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 crylAc and nptII to 1.12% and elimination of escapes. Southern analysis of T-0 and T, plants confirmed co-integration and segregation of the T-DNA bearing crylAc and nptII genes into the genomic DNA of transgenic chickpea plants. The CrylAc protein accumulated maximum from 14.5 to 23.5 ng mg(-1) extractable protein in To and T, transgenic chickpea plants. About 80-85% To 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 CrylAc 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 crylAc gene in chickpea has shown effective resistance in transgenic plants to the major pod borer insects. (c) 2005 Elsevier Ireland Ltd. All rights reserved.