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Journal of Applied Genetics 51(3), 2010, pp. 243-257

Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance

S. Kumar, L. Arul, D. Talwar


Abstract: We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

Key words: biolistic transformation, Bacillus thuringiensis, Bt transgenic rice, gene-expression-cassette, marker-free transgenic, Oryza sativa, Scirpophaga incertulas, yellow stem borer.

Correspondence: S. Kumar, Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi-284 003, India; e-mail: sureshkumar3_in@yahoo.co.uk

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