Development of a Sri Lankan rice variety Bg 94-1 harbouring cry2A gene of Bacillus thuringiensis resistant to rice leaffolder [Cnaphalocrocis medinalis (Guenée)]

Abstract

The rice leaffolder (RLF) [Cnaphalocrocis medinalis (Guenée)] is a major lepidopteran insect pest of rice in Sri Lanka and the larvae cause significant yield losses annually. Insecticidal crystal protein gene, cry2A driven by the 35S cauliflower mosaic virus (CaMV) promoter with a double enhancer sequence was transformed into scutellum derived embryogenic calli of Bg 94-1 via Agrobacterium tumefaciens mediated transformation procedure. Transformation vector pCAMBIA 1305.1 containing the transgene construct CaMV 35S d-cry2A-NOS, hygromycin phosphotransferase and β-glucuronidase (GUS)-Plus was employed in this study. Transformed calli were subjected to 4 ‒ 5 rounds of hygromycin selection. Molecular analysis of the generated plants together with an insect bioassay using RLF larvae confirmed successful transformation and expression of this transgene in T0 generation of rice plants. A total of 19 putative transgenic plants could be confirmed from a total of 60 T0 generation plants examined. Insect feeding bioassay demonstrated that T0 transgenic rice plants are effective against RLF larvae, and showed approximately 89 % larval mortality after 3 days and 100 % mortality after 5 days of feeding. Further molecular analysis of T1 generation of the transgenic lines confirmed the stable integration of cry2A transgene into the Bg 94-1 genome. Findings of this study demonstrated that the cry2A gene of Bacillus thuringiensis (Bt) was successfully transformed into Bg 94-1 via Agrobacterium mediated transformation and have developed resistance against the RLF larvae. Thus, this study highlights the importance of developing transgenic rice, which can provide insect resistance against a major lepidopteran insect pest of rice in Sri Lanka.