Development of diamondback moth resistant transgenic cabbage and cauliflower by stacking Cry1B and Cry1CBt genes

Abstract

Cabbage and cauliflower are extensively cultivated Cole vegetables all around the world. These are highly prone to the most notorious insect pest Plutella xylostella (diamondback moth), causing huge yield losses to the growers. Conventional breeding for host plant resistance against this pest is limited due to lack of resistance source in the available germplasm. The chemical control measures are cumbersome, costly, health hazardous and environment unfriendly. Therefore, using quaternary gene pool for incorporation of resistance genes from unrelated sources such as bacterial derived insecticidal Bt toxins through genetic engineering is a viable option. Genetic manipulation of plants through Agrobacterium-mediated transformation is limited by a multitude of factors resulting in poor transformation efficiency. Hence, optimization of plant transformation method suitable for a particular crop is highly essential. Here in we report the optimization of plant transformation protocol for cabbage and cauliflower by the manipulation of various key parameters, including the type of explants, Agrobacterium strains, bacterial cell density, age of the explant, preculture period, media formulations etc. The binary vector pPIPRA560 containing stacked Bt genes (Cry1B/Cry1C) and npt II/Basta as selectable marker was chosen for plant transformation. As a proof of concept, initially the effectiveness of the Bt genes against diamondback moth was validated in transgenic Arabidopsis. Insect bioassay revealed 100% mortality within 48 h of feeding. Based on the exciting results, the same construct was used for transforming Indian popular cultivars of cabbage (‘Golden Acre’) and cauliflower (‘Pusa Meghna’ and ‘Pusa Snowball K1’). The presence of the Bt genes in the transgenic lines was confirmed by PCR and expression through RT-qPCR. Insect bioassay revealed 100% mortality in some of the plants tested so far.