| Rice is one of the most important grain crops in the world, which is widely cultivated and in great demand. Indica and japonica are the two main subspecies of rice. About 80% of world rice production is based on indica varieties. There is a big difference of tissue culture between indica and japonica rice. And there are much lower regeneration frequency for indica rice than for japonica rice. This is a barrier to the application of genetic engineer to rice breeding. Genetic transformation is based on high regeneration frequency. However, there is no efficient genetic transformation system for all the indica varieties. As indica rice is widely cultivated in China. Therefore, it is very important to improve the present technology for genetic transformation of indica rice. Mature embryo of five indica rice varieties (9311. Minghui63, Shuangkangmingzhan, Jiafuzhan and R527) was used as materials in the experiment. Effect of different medium and plant growth regulator types and concentrations on callus induction rate from mature embryo of five indica rice varieties was studied. The culture system of indica rice mature embryo was optimized, in order to establish a high frequency regeneration system. The physiological and biochemical characteristic of callus from mature embryo of five indica rice varieties was studied during induction. Effect of different antioxidants on callus browning from mature embryo of five indica rice varieties was studied, in order to provide a theoretical basis for callus browning problem during subculture. The main results were showed as follows:1. By using the mature embryos of 9311, Minghui63, Shuangkangmingzhan, Jiafuzhan and R527 as experimental materials, the optimum medium (MS) were determined. When using MS as basic medium, and adding 30 g/L sucrose,8g/L agar and 2 mg/L 2,4-D. adding different plant growth regulator types and concentrations in induction medium. The result showed that callus induction conditions of 9311. Minghui63, Shuangkangmingzhan, Jiafuzhan and R527 mature embryo were respectively 1mg/L KT,1 mg/L 6-BA,3 mg/L NAA; 1 mg/L KT,1 mg/L 6-BA.1.5 mg/L NAA; 0.5 mg/L KT.0.5 mg/L 6-BA.3 mg/L NAA; 1 mg/L KT,1 mg/L 6-BA.3 mg/L NAA; 0.5 mg/L 6-BA. In the appropriate conditions, callus induction rate of 9311, Minghui63. Shuangkangmingzhan. Jiafuzhan and R527 mature embryo were respectively 24.67%、28%、23.33%、27.33%、22.67%.2. The mature embryos of 9311. Minghui63. Shuangkangmingzhan, Jiafuzhan and R527 were selected, and they were inoculated on the appropriate induced medium respectively. Different inoculation time and different materials were used to study changes of the soluble protein contents. SOD. POD and CAT, MDA and PPO during the callus inducement. The correlation analysis between different indica rice callus induction rate and soluble protein content and changes of enzyme activity were studied during the callus inducement. The results showed that these had correlation with callus induction rate. Variation of soluble protein content and the induction rate was negative correlation; Variation of SOD activity and the induction rate was negative correlation; Variation of POD activity and the induction rate was positive correlation; Variation of CAT activity and the induction rate was significantly positive correlation; Variation of MDA and the induction rate was negative correlation; Variation of PPO activity and the induction rate was positive correlation.3. Effect of different antioxidants (PVP, VC and citric acid) on callus browning of different indica rice varieties (9311, Minghui63, Shuangkangmingzhan, Jiafuzhan and R527) were studied in subculture medium. The results showed that effect of different PVP, VC and citric acid concentrations on callus browning degree of 9311, Minghui63, Shuangkangmingzhan. Jiafuzhan and R527 were different. In the same antioxidant type and same concentration condition, responses of different antioxidants on different indica rice were different. Effect of 5 mg/L PVP on callus browning of different indica rice was better, and browning rate of different indica rice was 45%-65%. Inhibition of PVP on POD and PPO activities were better in callus from different indica rice. POD activity of callus from Shuangkangmingzhan, Minghui63 and R527 were inhibited at 5 mg/L PVP. POD activity of callus from 9311 and Jiafuzhan were inhibited at 15 mg/L PVP. PPO activity of callus from 9311, Minghui63, Jiafuzhan and R527 were inhibited at 5 mg/L PVP. POD activity of callus from Shuangkangmingzhan were inhibited at 15 mg/L PVP. The correlation analysis between different indica rice callus browning rate and POD and PPO activities were studied during the callus subculture. Variation of POD activity and the browning rate was significantly positive correlation. Variation of PPO activity and the browning rate was great significantly positive correlation. |
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