An Efficient Agrobacterium-mediated Genetic Transformation Using cry1F gene in Castor (Ricinus communis L.) for protection Against lepidopteran Insects

Abstract

Castor (Ricinus communis L.) is an essential non-edible, pharmaceutical, and industrial oilseed as well as vulnerable to foliage feeders like Spodoptera litura  which resulted in a loss of production. This report focuses on the development of  an optimized protocol for the transformation of castor shoot apices by  Agrobacterium tumefaciens strain LBA4404 containing plasmid construct pBIN1F  harboring neomycin phosphotransferase (nptII), as selectable marker gene and  Bacillus thuringiensis var. aizawai (Bt) cry1F gene controlled by cauliflower mosaic  virus 35S promoter. Several parameters like O.D., concentration of kanamycin and  acetosyringone were optimized and produced a significant difference in the  transformation efficiency. Co-cultivation time and seedling age were factors, with overall transformation efficiency of 2.0% in 15 days-old seedlings and co-cultivated  for 3 days. The surviving and actively developing shoot apices were validated for  gene integration by molecular analysis after being preliminarily screened on  kanamycin. Furthermore, PCR, qRT-PCR and insect bioassay were used to confirm  the putative primary transformants. When bioassayed against newly hatched  Spodoptera litura hatchlings, these putative transgenics with cry1F gene caused  significant (≤93%) insect mortality. Cry1F gene expressing transgenic plants had  adequate defence against Spodoptera litura when exposed to castor.