Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.
We report on generation of marker-free (&lsquo;clean DNA&rsquo;) 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&percnt;, 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.
Kumar
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Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi-284 003, India. sureshkumar3_in@yahoo.co.uk
Arul
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L
Talwar
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D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D015139:Blotting, Southern; D003433:Crosses, Genetic; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000378:metabolism; D007113:Immunity, Innate / Q000235:genetics*; D040582:Inheritance Patterns / Q000235:genetics; D007915:Lepidoptera / Q000502:physiology*; D016254:Mutagenesis, Insertional / Q000235:genetics; D012275:Oryza / Q000235:genetics* / Q000469:parasitology; D010572:Pest Control, Biological / Q000379:methods*; D010935:Plant Diseases / Q000235:genetics* / Q000276:immunology* / Q000469:parasitology; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D018744:DNA, Plant; D005819:Genetic Markers; D005966:Glucuronidase