Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
Appl Biochem Biotechnol. 2013 Sep;171(2):450-68. doi: 10.1007/s12010-013-0359-z. Epub 2013 Jul 14.
An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 μM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).
建立了一种高效、可重复的茄子体内转化方法，以种子为外植体。将茄子种子感染携带 pCAMBIA 1301-bar 质粒的根癌农杆菌 EHA 105，并用 BASTA®筛选转化体。评估了影响体内种子转化的几个参数，如预培养时间、乙酰丁香酮浓度、表面活性剂、超声时间、真空压力和真空时间。通过 GUS 组织化学分析筛选疑似转化（T0）的茄子植株。在测试的不同组合和浓度中，当 18 小时预培养的茄子种子在含有 100 μM 乙酰丁香酮的农杆菌悬浮液中超声 20 分钟，真空过滤 3 分钟（真空度为 500 毫米汞柱）时，0.2% Silwett L-77 有利于农杆菌感染，并显示出最高的转化效率。在所评估的五个茄子品种中，Arka Samhitha 的转化效率最高，为 45.66%。通过 GUS 组织化学分析、聚合酶链反应和 Southern 杂交证实，该转基因已成功传递给后代植株（T1）。本研究中建立的体内方案将有利于将经济和营养上重要的基因转移到不同品种的茄子中，并且可以在更短的时间（约 27 天）内生产出转基因茄子植株。
