Liau C-H, You S-J, Prasad V, Hsiao H-H, Lu J-C, Yang N-S, Chan M-T
Institute of BioAgricultural Sciences, Academia Sinica, 11529 Taiwan, Republic of China.
Plant Cell Rep. 2003 Jun;21(10):993-8. doi: 10.1007/s00299-003-0614-9. Epub 2003 Apr 3.
The present protocol was aimed at establishing a routine transformation procedure via Agrobacterium tumefaciens for an important Oncidium orchid cultivar. An expression vector containing hptII and gusA genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the Oncidium orchid genome by A. tumefaciens. Protocorm-like bodies (PLBs) derived from protocorms, were the target explants for transformation. The transformation was performed through two stages of cocultivation, the first stage occurring on antibiotic-free medium for 3 days, and the subsequent stage on medium containing 100 mg/l timentin for 1 month. Among 1,000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on 5 mg/l hygromycin selection medium. A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines that were positive for beta-glucuronidase were randomly selected and confirmed by Southern, northern and western blot analyses. These results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants. The present transformation method reported is suitable for improving the Oncidium orchid through genetic engineering.
本实验方案旨在建立一种通过根癌农杆菌对一种重要的文心兰品种进行常规转化的方法。一个含有由花椰菜花叶病毒（CaMV）35S启动子驱动的hptII和gusA基因的表达载体通过根癌农杆菌成功导入文心兰基因组。源自原球茎的类原球茎体（PLBs）是转化的目标外植体。转化通过两个共培养阶段进行，第一阶段在无抗生素培养基上进行3天，随后阶段在含有100 mg/l替门汀的培养基上进行1个月。在1000个接种的PLBs中，有108个推定转化的PLBs在5 mg/l潮霉素选择培养基上增殖。共获得了28株独立的转基因兰花植株，从中随机选择了6个β-葡萄糖醛酸酶呈阳性的转基因株系，并通过Southern、Northern和Western印迹分析进行了确认。这些结果表明外源DNA已成功整合到兰花基因组中，并在这些兰花植株中进行了转录和翻译表达。所报道的目前这种转化方法适用于通过基因工程改良文心兰。
