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

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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 borerScirpophaga incertulas (Lepidoptera: Pyralidae). The transgenicindica rice harbours a translational fusion of 2 differentBacillus thuringiensis (Bt) genes, namelycry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an eliteindica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the markerhpt 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.

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  1. National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, 110 012, New Delhi, India

    S. Kumar, L. Arul & D. Talwar

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  1. S. Kumar
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Correspondence to S. Kumar.

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Kumar, S., Arul, L. & Talwar, D. Generation of marker-free Bt transgenicindica rice and evaluation of its yellow stem borer resistance. J Appl Genet 51, 243–257 (2010). https://doi.org/10.1007/BF03208854

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