Fernández-Piñán Sandra, López Jennifer, Armendariz Iker, Boher Pau, Figueras Mercè, Serra Olga
Department of Biology, Universitat de Girona.
Department of Biology, Universitat de Girona;
J Vis Exp. 2019 Mar 29(145). doi: 10.3791/59119.
Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant's genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed in roots.
农杆菌属是获得转基因植物最广泛使用的方法之一，因为它有能力将自身的T-DNA转移并整合到植物基因组中。在此，我们展示了两种用于对马铃薯（Solanum tuberosum）植株进行基因改造的转化系统。在根癌农杆菌转化中，叶片被感染，转化细胞被筛选出来，然后使用植物激素在18周内再生出一株完整的新转化植株。在发根农杆菌转化中，通过用针注射细菌来感染茎，使用红色荧光标记检测新出现的转化毛状根，并去除未转化的根。在5至6周内，得到的植株是一个野生型地上部分与完全发育的转化毛状根的复合体。为了增加生物量，可以切除转化的毛状根并使其自我繁殖。我们应用了两种农杆菌介导的转化方法来获得由木栓质生物合成基因启动子驱动表达GUS报告基因的根。文中提供了GUS染色步骤，可实现启动子诱导的细胞定位。在这两种方法中，转化的马铃薯根在栓质化的内皮层和外皮层中均显示出GUS染色，此外，在发根农杆菌转化的根中，在侧根的发生部位也检测到了GUS活性。这些结果表明，发根农杆菌可以成为研究根中表达基因的一种快速替代工具。
