Development and characterization of Bt chickpea (Cicer arietinum L.) plants against its major pest, helicoverpa armigera (Hübner)
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Date
2013
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CCSHAU
Abstract
Genetic improvement of chickpea against the pod borer Helicoverpa armigera, either by traditional or molecular methods, is hampered due to limited genomic resources coupled with narrow genetic diversity in the elite gene pool. The present study reports the stable integration and inheritance of cry1Aa3 gene in chickpea cv. C-235 plants developed using a novel process of Agrobacterium-mediated genetic transformation without using tissue culture procedure (Patent No. 252590). Further, the transgenic chickpea lines of cvs. C-235 and HC-1 carrying cry1Ac gene have been developed and stability of integration and inheritance of cry1Ac gene has also been confirmed in this study. Transformation with cry1Ac gene following the non-selective/PCR detection system using direct plant PCR screening indicated the putative transgenic nature of plants and represented transformation frequency of 14.4% and 41% in cvs. C-235 and HC-1, respectively. The putative transgenic chickpea plants were analyzed adopting multiple evaluation strategies, such as PCR, ELISA and southern blotting, for selection of plants for further advancement. Quantitative assessment of Bt Cry toxins by ELISA in leaves of transgenic chickpea plants showed variation in expression of Cry1Aa3 toxin (91.0 to 154.0 ng g-1 FW) and Cry1Ac toxin (82.6 to 141.0 ng g-1 FW). Results obtained from Southern blotting using gene specific probe confirmed the single copy integration of the cry1Aa3 or cry1Ac gene into the chickpea genome which was further confirmed by real-time PCR assay for copy number detection using SYBR Green chemistry. The efficacy of transgenic chickpea plants expressing cry1Aa3 or cry1Ac gene against the target pest is required to judge the performance of the plants. The present study offers a suitable approach for development of chickpea plants with novel traits, with the possibility of developing marker free transgenic events, allows stacking of multiple genes and can be applicable across different genotypes/cultivars of chickpea, the recalcitrant grain legume crop.
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Keywords
Chickpea, Genetic transformation, Agrobacterium, Bt toxin, Real-time PCR