Genetic enhancement of pearl millet against Helicoverpa armigera expressing the entomotoxic Cocculus hirsutus trypsin inhibitor.

Expression of Cocculus hirsutus trypsin inhibitor (ChTI) gene in pearl millet via Agrobacterium-mediated transformation exhibited reduced damage and enhanced resistance by suppressing the growth and survival of Helicoverpa armigera. Cocculus hirsutus trypsin inhibitor (ChTI), a serine proteinase inhibitor, was isolated from the medicinal plant Cocculus hirsutus belonging to the family Menispermaceae. It is an 18 kDa thermotolerant monomeric protein with insecticidal activity against Helicoverpa armigera and other insect pests. To enhance pearl millet (Pennisetum glaucum L.) tolerance to Helicoverpa armigera (ear head worm), the ChTI gene encoding a protease inhibitor was introduced into pearl millet via genetic transformation. EHA105, a super virulent Agrobacterium tumefaciens strain carrying a binary vector driven by the CaMV35S promoter and terminated by the nos terminator (nosT), was used to develop transgenic plants. T1 events were screened by PCR using gene-specific primers, yielding a 383 bp amplicon. Quantitative expression of the ChTI gene by qRT-PCR analysis showed a 2-8-fold change in the T1 progeny. T1 plants expressing ChTI showed higher trypsin inhibitor activity than non-transgenic controls. Insect bioassay of transgenic plants against H. armigera showed a 60% mortality and 76.5% reduction in weight gain with less feeding damage compared to the non-transgenic control leaf. The transgenic pearl millet plants exhibited reduced damage and consequently lower ROS accumulation, as confirmed by DAB and NBT staining, thereby conferring enhanced tolerance to H. armigera.