Al-Ramamneh E A, Sriskandarajah S, Serek M
Department of Agricultural Sciences, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, 1871, Frederiksberg C, Copenhagen, Denmark.
Plant Cell Rep. 2006 Nov;25(11):1219-25. doi: 10.1007/s00299-006-0190-x. Epub 2006 Jun 24.
A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9-12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l(-1) kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.
开发了一种根癌农杆菌介导的仙人指品种CB5遗传转化方案。从叶状枝外植体获得的愈伤组织，在新鲜的愈伤组织诱导培养基上继代培养9 - 12个月后，与根癌农杆菌LBA4404共培养。使用携带nptII基因（作为选择标记）和报告基因uidA的质粒构建体。通过在含有600 mg l(-1)卡那霉素的培养基上延长培养，获得了具有旺盛生长表型的转化仙人指愈伤组织。经过9个月的严格选择压力后，从最终培养基中去除卡那霉素，并在营养胁迫下培养转化的愈伤组织，导致形成了几个转基因不定芽。通过GUS染色（用于uidA基因）、ELISA分析和Southern印迹杂交（用于nptII基因）证实了转化。采用这种方法，实现了22.7%的转化效率。本研究中描述的总体结果表明，农杆菌介导的转化是这种仙人掌物种的一种有前景的方法。
