Baskar Venkidasamy, Gangadhar Baniekal H, Park Se Won, Nile Shivraj Hariram
Department of Bioresources and Food Science, School of Life and Environmental Sciences, Konkuk University, Seoul, 143701, South Korea.
3 Biotech. 2016 Jun;6(1):88. doi: 10.1007/s13205-016-0402-1. Epub 2016 Mar 7.
The present study aims to investigate the numerous factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Chinese cabbage (Brassica rapa ssp. pekinensis). Factors affecting transformation efficiency, such as age of explants, Agrobacterium concentration, and effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were examined. The pre-cultured hypocotyls from young seedlings prior to exposure to Agrobacterium showed higher shoot regeneration. The plant transformation with the modest A. tumefaciens concentrations (0.8 OD) and the 3 days co-cultivation periods increased transformation efficiency. Plant growth hormones [1-naphthyl acetic acid (NAA) and 6-benzyl amino purine (BAP)] were essential for callus and shoot formation. Root formation was effective in half strength MS medium without supplementation of root-inducing hormones. To maintain selection pressure, plant subculture was carried out every 2 weeks with selective antibiotics. The putative transgenic plants were acclimatized in the greenhouse. Polymerase chain reaction was performed to confirm the integration of T-DNA into the genome of transgenic plants. A transformation efficiency of 15 % was obtained. This protocol allows effective transformation and indirect regeneration of Brassica rapa.
本研究旨在探究影响根癌农杆菌介导的大白菜（Brassica rapa ssp. pekinensis）遗传转化的诸多因素。考察了影响转化效率的因素，如外植体的年龄、农杆菌浓度、乙酰丁香酮的作用、农杆菌的预培养、感染及共培养时间。在接触农杆菌之前，来自幼苗的预培养下胚轴表现出更高的芽再生能力。使用适度的根癌农杆菌浓度（0.8 OD）和3天的共培养时间进行植物转化可提高转化效率。植物生长激素[1-萘乙酸（NAA）和6-苄基氨基嘌呤（BAP）]对愈伤组织和芽的形成至关重要。在不添加生根激素的1/2强度MS培养基中生根效果良好。为维持选择压力，每隔2周用选择性抗生素进行植物继代培养。将推定的转基因植株在温室中驯化。进行聚合酶链反应以确认T-DNA整合到转基因植物的基因组中。获得了15%的转化效率。该方案可实现白菜型油菜的有效转化和间接再生。
