| Indica rice(Oryza saliva L.) is a major cultivar in China. Its high efficiency transformation and large scale transgenic breeding has big strategic and practical significance to the national food security in the future. This thesis selectively used five typical and divergent indica varieties 93-11, MH63,307S, Bing-Long-Hong, and 893 as test materials and developed a widely applicable subculture medium, called WA, via medium components optimization. And then based on this optimized medium, a highly effecient indica rice genetic transformation system was set up. The major results obtained are as follow:1,Using MS basal medium supplemented with 2mg/l 2,4-D for callus induction, the well grown primary calli were induced from all five tested indica varieties, characterized in a yellowish color, compact texture, dry surface, and dense cytoplasm.2,A optimized medium widely applicable for different indica rice callus subculture (MS macronutrient components and N6 organic components,5×B5 micronutrient components,0.5g/l glutamine,2.8g/l proline), named WA medium, was developed via medium components adjustment. The adjusted components include the inorganic and organic nutrients and plant hormones based on the MS, N6 and B5 medium. Using this optimized subculture medium for subculture, the growth rate (GR) and the differentiation rate (DR) of primary calli derived from five indica varieties were significantly higher than those in MS, N6 or B5 medium. ANOVA analysis indicated that the differences of GR and DR detected between different subculture media and genotypes were significant (p＜0.01).3,Hygromycin resistance test showed that when 93-11 and 307S at a concentration of 50mg/l, MH 63 at 60mg/l, and Bing-Lang-Hong and 893 at 75mg/l hygromycin, their calli would stop growing. These data revealed that these hygromycin concentrations are the suitable concentration for callus selection of corresponding varieties after transformation.4,The hygromycin resistant calli were successfully obtained form all five indica varieties after primary callus subculture and post-transform selection on the WA medium. Subsequent GFP fluorescence signal detection and hph amplification confirmed that the percentage of positive transformants present in the varieties of 93-11, MH63,307S, Bing-Lan-Hong and 893 were 4.1%/2.7%,36.8%/25.0%,10.5%/8.5%,37.1%/34.3% and 43.8%/33.9%, respectively. The significant differences were still existent in the different genotypes as the data present above. In terms of growth status of calli, the developed resistant calli developed on the WA selection medium all showed the features of high vitality, compact texture and dense cytoplasm, indicating their strong embryogenesis.5,A high efficient indica rice transformation system was set up based on the optimized WA medium. Genetic transformation test indicated that the PCR detected positive ratio in the base of the resistant plantlents derived from five indica rice varieties were all above 70%, with a mean ratio up to 85%. The transformation efficiency calculated based on the number of inoculated calli was 2.7% for 93-11,20.2% for MH63,8.5% for 307S,24.0% for Bing-Lang-Hong, and 25.6% for 893, respectively.All of these results therefore verified that using the optimized WA medium to carry out callus subculture and subsequent genetic transformation is widely applicable for at least the tested five indica varieties.
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