| In this research, three varieties of Ind ica rice and two sterile lines were chosen as the materials. The factors affecting regeneration were studied systematically and steady regeneration system was established. A plant expression vector used for the transformation of Ind ica rice by Agrobacterium tumefaciens was constructed. Factors affecting transformation by Agrobacterium tumefaciens were studied through orthogonal experiment and transformation was system established. The fusion gene of waxy and GUS was transferred into Ind ica rice, and the transgenic calli was detected. The results were summarized as follows:1. The construction of the expression vector of anti-sense waxy geneA plant expression vector cassette and a plant expression vector p13WG3.1 were constructed in this experiment. It contained a GUS gene, which was regulated by the upstream regulatory sequence of waxy gene, and a marker gene Hypromycin phosphotransferase. Both the genes were used in pre-experiment. The anti-sense waxy-GUS fusion gene in the plant expression vector, which was regulated by the upstream regulatory sequence of waxy gene was the material of target gene transformation.2 The establishment of Ind ica rice regeneration systema. Influence of 2, 4-D concentration on callus inductionInfluence of l~5mg/L 2, 4-D on callus induction was studied and all varieties had the highest frequency in the NB media with 2mg/L 2, 4-D. The frequency of Yue xiang zhan were 79. 17%, Yue feng zhan 62. 50%, Pei ai 64S 12. 50%, GD-1S 70. 00%,and Yue taiB 95. 83%.b. Influence of genotype on callus inductionCallus induction frequency of five genotypes showed significant difference in the same media. The highest was Yue tai B95.83%, and the lowest was Pei ai 64S 12.50%.c. Influence of inoculating waysEmbryos were inoculated in two ways: putting level or vertically onto the media.The results showed that putting embryos level onto the media was better than putting vertically, and the difference of frequency was 60% at the most.d. Influence of medium composition on callus induction of Pei ai 64SInfluence of media type, organic nutrition, organic nitrogen, PGR and trace element were studied to induce callus from Pei ai 64s,a sterile line of which callus inductionfrequency was relatively low.The results showed the best media was MB + 2mg/L 2, 4-D + 0. 5mg/L BA + 30g/L maltose, The frequency was 56. 48%.e. Factors influencing shoot regenerationFrequency of differentiation can be increased greatly through two-step differentiation, pre-differentiation treatment, dehydration treatment and regulating osmotic pressure. The frequency was all up to 80%, but there was difference among them.f. Seedling regeneration under low 2, 4-D concentration and lightCallus induced under low 2, 4-D concentration and light can differentiate directly after transferred onto differentiation media. The differentiation was over 80%.3 The establishment of Agrobacterium-mediated transformation systema. The selection pressThe suitable concentration of Hygromycin was 20mg/L;Hygromycin of 10mg/L was used for mature embryos of four genotypes; In the period of seed germination, 150 mg/L Hygromycin was suitable for four genotypes.b. Definition of selection pressureCef was used as antibiotic to restrain the propagation of Agrobacterium at the concentration of 0-600mg/L. And the result showed 500mg/L was suitable.c. Influence of genotype on transformationThere was difference of GUS expression rate among the factor genotypes, that were Yue xiang zhan 17. 65%, Yue feng zhan 25. 88%, Pei ai 64S 12. 76%, GD-1S 49. 36%.d. Optimization of factors influencing transformation transferredThe influence of other factors on transformation were studied by orthogonal experiment and the best condition was MB co-cultured media, 300umol/L As, 10g/L Glu, motionless infection, no As in callus pre-culture period, pH5.8, pre-cultured 3days and the infection of 3min.e. The detection of GUS gene...
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