Agrobacterium mediated transformation of sugarcane for borer resistance using Cry 1Aa3 gene and one-step regeneration of transgenic plants

Sugarcane borers are the major biotic pest in sugarcane that accounts nearly 25-30% yield loses. Most of the present sugarcane varieties are not resistant to borers. Development of borer resistance in sugarcane through transgenic technology could be the best approach. Cry series of genes are known for development of insect pest resistance in plants in particular for the most damaging order 'Lepidoptera'. The sugarcane (Saccharum hybrids) genotype CoC671 was transformed by Agrobacterium mediated method. Cry 1Aa3 gene driven by CaMV35S promoter and Ocs terminator with selectable marker nptII driven by Nos promoter and terminator harboring in binary vector pBinAR was transformed into Agrobacterium tumefaciens strain EHA 105 was used in sugarcane transformation. Young leaf roll discs were infected and regenerated plants were obtained in one step after infection with minimal callus phase. Three to four shoots per explant were developed after 7-10 days incubation. Regenerated shoots were allowed to grow further. Two months old putative transgenic plants were taken for molecular characterization. Out of 65 plants tested by PCR, 24 plants were positive for Cry1Aa3 gene. This represents nearly 28% of the transformation efficiency. All these plants were analyzed by Southern blot hybridization for integration of the inserted gene. Four plants showed single copy number and another two showed more than two integrations. All those 24 plants that are positive by PCR were subjected to ELISA test for expression of foreign protein. It was observed that four transgenic plants showed around ten-fold high level expression and another two plants showed with moderate levels, 3-5 fold expression. © Society for Sugar Research & Promotion 2009.