The The. Divalent Insect-resistant Genes Cry1c * / Cry2a * Super Rice In Cold Regions Songjing Genetic Transformation

Songjing9, a super rice variety in cold regions, was widely planted in the first temperature-accumulated area in Heilongjiang province, which was damaged severely by stripped stem borer (SSB). This study engaged to establish a tissue culture condition for Songjing9, optimize the Agrobacterium-mediated transformation condition and transfer the insect-resistant genes, cry1C* and cry2A*, into Songjing9 genome by infecting Songjing9 calli with Agrobacterium mixtures. The transgenic rice plants containing low copy number of foreign genes, expressing Bt at a relatively high level, exhibiting normal agronomic traits, were selected by Basta screening, PCR, Southern blot and insect bioassay at T₀ generation, and by RT-PCR, Bt protein quantitative detection and agronomic traits investigation at T1 generation. The main results of this study included:(1) a medium with N6B5 as basic nutrients and 2mg/L2,4-D, 0.2mg/L6-BA added was established, which was suitable for the inducion and growth of the calli of the super japonica rice, Songjing9;(2) a highly efficient transformation system fitting super rice varieties with indica inheritance in cold regions was developed, and the super japonica rice, Songjing9, was transformed with a mixture of two Agrobacterium strains containing cry1C* and cry2A* expression vector respectively, and the transformation efficiency and regeneration ratio were 12.74% and 46.71% respectively;(3) at T₀ generation, a system for evaluating the insect-resistant ability among different transgenic plants, with TT51 and wild-type Songjing9 as control, was developed, and the transgenic plants harboring cry1C*, cry2A* and both cry1C* and cry2A* conferring high resistances to insect were obtained;(4) at T1 generation, the cry1C* line T1C-3 and the cry2A* line T2A-2 showed the highest expression of cry1C* and cry2A*, the cry1C* and cry2A* co-transformed line T1C+2A showed a relatively higher expression of cry1C* and cry2A*, the transcription and translation of cry1C* and cry2A* were the highest in leaf tissue, and nearly at the same or slightly different level at stem and panicle tissue; SSB showed selective feeding character on the leaves of different transgenic lines, when the type of Bt genes were the same, SSB selected the transgenic leaves containing low contents of Bt protein, and when the Bt type were different and both expressed at a high level, SSB fed on the leaves of cry2A* plants firstly; seed-setting rate of some transgenic rice was significantly lower than that of control plants, and the agronomic traits of three lines, T1C-3, T2A-2 and T1C+2A, expressing Bt at a high level, were similar to wild type Songjing9.