Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu 630003, India.
Plant Sci. 2011 Sep;181(3):258-68. doi: 10.1016/j.plantsci.2011.05.011. Epub 2011 May 27.
A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)).
已开发出一种简单有效的农杆菌介导转化方案，可用于转化具有农业应用价值、对非生物胁迫敏感的籼稻品种 ADT43。从 4 天大的水稻幼苗的叶片基部取材，在添加了 2.5mg/L 2,4-D 和 1.0mg/L 盐酸硫胺素的 LS 培养基上，最容易诱导愈伤组织的形成。将水稻愈伤组织浸入农杆菌悬浮液（菌株 EHA105，OD600=0.8）中，在黑暗条件下于 25±2°C 的 LS30-AsPC 培养基上共培养 2 天。基于 GUS 表达分析，发现用 100μM 乙酰丁香酮共培养 10min 是递送 gus 基因的最佳条件。愈伤组织对农杆菌感染非常敏感，我们发现共培养 30%LS、10g/L PVP、10%椰子水和 250mg/L 替莫西林可以将坏死反应的水平最小化，从而将最终的转化效率提高到 9.33%。对转基因植物的分子和遗传分析表明，外源基因在这些植物的后代（T1）中整合、表达和遗传。转基因植物（T0 和 T1）中转基因的拷贝数从 1 到 2 不等。
