Marker-free transgenic Bt rice engineered for resistance to yellow stem borer and leaffolder

A chimeric cry2AX1 gene, consisting of sequences from cry2Aa and cry2Ab genes, with insecticidal activity was employed in development of marker-free insect-resistant transgenic Bt rice. The genes of interest, cry2AX1 and hptII (selectable marker gene) were on two different T-DNAs of Agrobacterium strain, C58C1. A total of 130 hygromycin-resistant T₀ transgenic rice events which were generated using two different cry2AX1 gene constructs showed positive for gusA gene. Among them, fifty events were identified as positive for cry2AX1, exhibiting a co-transformation frequency of 38.46 per cent. The expression of Cry2AX1 protein in T₀ transgenic rice events varied between 0.023 and 0.133 μgg⁻¹ of fresh leaf tissue. The larval mortality in T₀ transgenic plants ranged from 20 to 95 per cent against the rice yellow stem borer (Scirpophaga incertulas) and 33.33 to 100 per cent against the rice leaffolder (Cnaphalocrocis medinalis). In T₁ generation, two marker-free transgenic rice events were identified by segregation analysis. The T₂ progeny plants derived from the marker-free homozygous transgenic events, expressing 0.117 μgg⁻¹ of Cry2AX1 in fresh tissues, caused 100 per cent and 83 per cent larval mortality in rice yellow stem borer and rice leaffolder, respectively. However, in whole plant insect bioassay experiment, the larval mortality of both the target insects ranged between 10 and 50 per cent.