Production Of Rice Lines Resistant To Bacterial Blight And Stem Borer Via Genetic Engineering

Rice is important grain crop, rice bacterial blight and stem borer are the main disease and pest in rice production. Breeding resistant rice variety is the most effective strategy in controlling bacterial blight and stem borer. In this research factors that influence transformation efficiency mediated by Agrobacterium of rice varieties that are mainly planted in Shandong province named 11301 and Yujing 6 were discussed. On the basic of these researches, we optimized the parameter that involved in transformation mediated by AKrobucterium and established an excellent system for the transformation of rice calli. Also factors that influence regeneration efficiency of rice calli were discussed and we created an optimal regeneration system for these two varieties. As the result, we successful ly transformed the blight resistance gene Xa21 gene and stem borer resistance gene crylA (b) gene into rice immature embryos and rice calli and got transgenic rice plants.Using Agrobacterium strains EHA105(with pCXK1301) and LBA4404 (with pCDAbLMS) to transform rice immature embryos and calli and according to the transient transformation efficiency showed by Gus straining, we analysed factors that influence transformation efficiency. As the result, we found that 1130 1 had higher transformation efficiency than Yujing 6: that immature embryos had higher transformation efficiency than ceil I i ; that when concentrat ion of Agrobacterium was ODç±¾u=0. 5 and 100 M As was added into cocultural media, immature embryos and calli got their highest transformation efficiency.About the differentiation and regeneration of transformed resistant rice calli, hormone determined their direction of differentiation. When KTNAA is 50, calli of 11301 and Yujing 6 reached their highest differentiation efficiency. Drying treatment and high infiltration treatment can obviously enhanced differentiation efficiency by 2 to 3 times. High concentration of agar in differentiation media can also enhance the differentiation efficiency of transformed calli. Adding Met into root bear ing media can make root of rice regenerated rough and strong. Under the best conditions, differentiation efficiency of calli of Yujing6 can reach 40% and H301 36%.Through transformation mediated by Agrobacterium and subsequent select ion and regeneration, we got 22 Xa21 transgenic rice plants and 10 Bt traiisgenic plants. All transonic rice plants grew and bred nomally in fields. ITK analysis and Southern Blot analysis demonstrate that Xa21 gene and Bt gent1 had entered and integrated into genome of transgenic rice plants. Primary exam mat ion (Gus staining and PCR analysis) of T1 progeny of transgenic plants showed that Xa21 and Bt genes can inherited into Tl progency and they showed segregation of 3:1, which is corresponding to Memlelian hereditary principle. Resistance examination showed that transgenic plants of T1 progency showed obvious blight resistance and stem borer resistance.Ideal rice variety should be multiresistant. This object can be realized by transforming many a gene into the same receptor material. Yao fang yin et al. used Xa21 transgenic rice plants(with Gus marker gene) and stably inherited pure rice lines with Bt gene(with Bar marker gene) to hybridize and got a lot of F1 progency plants. Primary examination confiraed that rice plants with both Xa21 gene and Bt gene had been got. This will show its significant value to both research and application of rice transgenic engineering.