The Study On Transformation Of Herbicide-resistant Gene And Multiple Insect-resistant Genes In PTGMS Line Of Rice

The harms of insects and weeds were an important reason for reduction of rice output, which caused the annual loss of global rice production reach to several decade billions in US dollar. In order to improve the insect resistance of hybrid rice, facilitate the direct seeding,hybrid seed production and weed control, avoid the worry about security for antibiotics selection marker,the researches on construction of new plant expression vector including herbicide and insect resistant genes and transformation of photoperiod-sensitive and /or thermo-sensitive genic male sterile (PTGMS) line in rice were carried through. Successful breeding of new PTGMS line with herbicide resistance and insect resistance, will be expected to play important roles in the herbicide and insect resistance hybrid rice breeding, the direct seeding and weed control, and the mechanization of hybrid rice seed production.A new plant expression vector was constructed, which included herbicide resistant gene Epsps and three insect resistant genes (potato proteinase inhibitor gene Pin II, Bacillus thuringiensis endotoxin gene Bt cryI(A) and Galanthus nivalis agglutinin gene GNA). In which, Epsps gene was not only used as a selection marker in transformation, but also endowed rice with the characteristic of herbicide resistance to facilitate the weed control, direct seeding and mechanization of hybrid rice seed production.Four genes linked together in T-DNA boundary were delivered into embryogenic calli of PTGMS line 7001S via particle bombardment and Agrobacterium-mediated transformation methods. By particle bombardment method, 167 regenerated plantlets were gained from 1160 calli, and the transformation frequency was 17.4%. Only 6 plantlets were regenerated form 270 calli by Agrobacterium-mediated transformation method, the transformation frequency was 2.2%. The integration of Epsps and insect -resistant genes in rice genome have been approved by PCR and Southern blot, the copy number of exogenous genes ranged from 1 to 2. Excluding the transgenic plants in which only 1 to 3 genes were detected, there were 81 transgenic plants with four genes. For the individuals contained 1 to 3 genes only, the gene defect would be take place in course of particle bombardment transformation. These transgenic plants with four genes were treated by herbicide glyphosate and performed different resistance to glyphosate, which would be resulted from the expression of herbicide-resistant gene in different degrees between individuals. After glyphosate treatment, 53 transgenic plants with good glyphosate resistance and normal seed setting were selected out.