Expression of Cocculus hirsutus trypsin inhibitor promotes endogenous defensive response against Helicoverpa armigera and enhanced levels of antioxidants

Abstract

Proteinase inhibitors are a group of defense related proteins, natural antagonists of proteinases, induced upon herbivory, play a defensive role against polyphagous insects and phytopathogens. Serine proteinase inhibitor isolated from Cocculus hirsutus (L.) Diels, Cocculus hirsutus trypsin inhibtor (ChTI) was found effective as antifungal, bactericidal and nematicidal agent. Tomato plants expressing ChTI have been developed by agro-infection with almost 27% transformation efficiency. Stable integration and expression of ChTI has been established by polymerase chain reaction (PCR), inhibitory assay and western blot assay. Transgenic plants showed increased fruit yield, antioxidants, phenolics, flavonoids and titratable acidity. Protein extracts of tomato plants inhibited Helicoverpa armigera (gut proteinases up to 40%. Transgenic plants MT2 and JT2 challenged with 2nd and 4thinstar H. armigera (Hubner) larvae, showed delayed larval growth with 100% mortality. The results put together suggest that ChTI is a potential candidate for developing transgenic plant with multiple biotic stress tolerance.   Key words: ChTI, Helicoverpa armigera, insect bioassay, transgenic tomato, trypsin inhibitory assay.