Genetic transformation of Brassica napus with the antifungal chitinase gene
2013
Khan, I. (The University of Agriculture, Peshawar (Pakistan). Inst. of Biotechnology and Genetic Engineering) | Khan, M.S. (The University of Agriculture, Peshawar (Pakistan). Inst. of Biotechnology and Genetic Engineering) | Ilyas, M. (The University of Agriculture, Peshawar (Pakistan). Inst. of Biotechnology and Genetic Engineering) | Rajab, H. | Shah, S.H. | Jalal, A.
Brassica napus is one of the most important sources of vegetable oil in the world. Efficient and reproducible protocols were developed for Agrobacterium-mediated transformation of B. napus in the current study. Synthetic chitinase (NIC) gene encoding antimicrobial protein in plasmid pEKH was transformed to Abasyn-95 cultivar of B. napus to produce transgenic plants. The role of a number of factors such as different ratios of growth regulators, various concentrations of growth hormones and chemicals, which can directly or indirectly influence the process of transformation, was evaluated. Hypocotyls and cotyledons from in vitro grown seedlings were used as explants for transformation. Culturing of the explants on solidified MS plates supplemented with different hormone ratios and concentrations showed that the media supplemented with 2 mg L-1 2, 4-dichlorophenoxyacetic acid (2, 4-D), 0.5 mg L-1 benzylaminopurine (BAP) and 0.1 mg L-1 naphthaleneacetic acid (NAA) is the best combination among all tested conditions for callus induction. Maximum shooting was observed on MS medium containing 0.5 mg L-1 BAP and 2 mg L-1 2, 4-D. The transgenic nature of the calli and regenerated shoots was confirmed by PCR analysis via NIC gene specific primers using their isolated genomic DNA as template.
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