K. Datta, A. Vasquez, G.S. Khushi and S.K. Datta

Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, P.O. Box 3127, 1271 Makati City, Philippines

     The New Plant Type (NPT) rice has been developed with the target to increase the yield potential of rice by 20-25% (Khush 1995). However, the yield potential of the NPT rice is limited by poor grain fifing and susceptibility to insect pest and diseases (Khush and Peng 1996). Genetic engineering can be used to complement the plant breeding efforts aiming to stabilize yield and increasing economic value by incorporating disease and insect pest resistance to NP’!’ rice. The introduction of selectable marker gene into NPT lines though genetic engineering was reported before (Alam et a!. 1996). In this paper we report introduction of Bt gene [crylA(b)] which confers the resistance to stem borer directly into a NPT rice line to achieve a high level of stem borer resistance.

    Stem borer causes serious damage in NPT rice. Bacillus thuringiensis, the common soil bacterium produces crystals containing specific insecticidal proteins, which kill lepidopteran insects by binding to and creating pores in the midgut membranes. Earlier we reported the transgenic rice with Bt gene conferring resistance to stem borer (Alam et a!. 1998; Datta et al. 1998; Tu eta!. 1998;). The use of transgenic NPT rice plants expressing Bt proteins could be effective approach for controlling lepidopteran insect infestation on NPT rice.

      A truncated chimeric crylA(b) gene driven by the constitutive 35S promoter from CaMV, two tissue specific promoters, pith tissue and PEP-carboxylase (PEPC) gene for green tissue from maize were successfully introduced into the NPT (1R65600-42-5-2) line. A total of around 800 putative transgenic Bt rice plants were produced by microprojectile bombardment. Nine to twelve days old immature embryos were used as initial explant. We observed different integration pattern of Bt gene with variable copy number shown in Southern analysis (Fig. 1). More than 20 independently transformed plants were confirmed for integration of the crylA(b) gene. About 190 plants showed the exact size banding pattern (1.8 kb). The putative transgenic plants appeared to be healthy in the CL4 transgenic greenhouse. However, due to grain filling problems only 20% of the plants turned out to be fertile and normal.

    The transgene driven by different promoters showed a wide range of expression of Bt protein in western blot analysis with polyclonal antibody. Insect bioassay by a stem cutting method showed the mortality rate of neonate larva of yellow stem borer (Scirpophaga incertulas) up to 100% in some selected lines. Molecular analysis of the transgenic plants were positively correlated with the bioassay data. Selected transgenic lines are being grown for the next generation to obtain homozygosity. Present study will help the protection of NPT rice against the insect attack.