农杆菌介导的籼型水稻恢复系R752成熟胚转基因技术体系的优化

Optimization of Agrobacterium-mediated Transgenic Technology System for Indica Rice Restorer R752 by Using Mature Embryo

采用农杆菌介导法,对籼型晚稻恢复系R752成熟胚的遗传转化体系进行了优化。结果表明:在MSS培养基上的愈伤诱导率要高于在CCS培养基上的;碳源麦芽糖对提高胚性愈伤诱导率的效果优于蔗糖的;在4种诱导培养基(MSM、MSS、CCM、CCS)与2种分化培养基(MSB、MSK)的8种组合中,以MSM-MSB组合获得的绿苗分化率最高,达28.97%;不同激素对绿苗分化率的影响大小表现为NAA>KT>6-BA,分化培养基的最佳激素配比为2 mg/L 6-BA+0.5 mg/L NAA+1 mg/L KT;3种农杆菌菌株的侵染效率表现为EHA105>LBA4404>GV3101。最终建立了R752的高效转基因技术体系,其平均转化效率达到10.98%。

In this study, the Agrobacterium-mediated transgenic technology system for indica late rice restorer R752 by using mature embryo callus as recipient was optimized. The results showed that the MSS medium was better for callus induction from ma- ture embryo than the CCS medium. It was also found that maltose used as carbon source was better for callus induction than sucrose at the same concentration. The combination of callus induction medium MSM and differentiation medium MSB obtained the highest green plantlet production rate （28.97%）. The results of orthogonal experiment showed that the optimal concentration of 6-BA, NAA and KT in differentiation medium was 2.0 mg/L, 0.5 mg/L and 1.0 mg/L, respectively, and NAA had the greatest effect on green plantlet production rate, followed by KT and 6-BA in order. The infection efficiency of three tested Agrobacterium strains for primary calli induced from mature embryo revealed the following order： EHA105〉 LBALM04〉 GV3101. Using EHA105, the Bt gene was transformed into the mature embryo callus of R752 by the constructed high-efficient Agrobacterium-mediated transgenic technology system, and the average transformation efficiency was 10.98%.