Generation of insect resistant marker-free transgenic rice with a novel cry2AX1 gene

Abstract

A synthetic cry2AX1 gene, consisting sequence of cry2Aa and cry2Ac genes, driven by green tissue specific rbcS promoter was used to develop insect resistant marker-free transgenic rice events. The gene of interest cry2AX1 and the selectable marker gene hpt were present in two independent T-DNAs of the Agrobacterium strain, C58C1. Seventy seven hygromycin and GUS positive T0 transgenic plants were recovered. Among them, 48 were found to be co-transformed with cry2AX1. The Cry2AX1 protein expression level in T0 transgenic events ranged from 0.027 to 0.110 μg/g of fresh leaf tissue. The toxicity level of Cry2AX1 protein was assessed through in vitro bioassay against target insects. The larval mortality ranged from 46.66 to 66.67 per cent against rice leaffolder (C. medinalis) while it ranged from 60 to 100 per cent against rice yellow stem borer (S. incertulas) in T0 transgenic plants. A marker free transgenic event, which expressed 0.043 μg/g of Cry2AX1 in fresh leaf tissue exhibited 45 and 55 per cent larval mortality against rice leaffolder and rice yellow stem borer, respectively in T2 generation