Ramesh Sunita A, Kaiser Brent N, Franks Tricia, Collins Graham, Sedgley Margaret
School of Agriculture and Wine, Waite Campus, University of Adelaide, Glen Osmond, SA, 5064, Australia.
Plant Cell Rep. 2006 Aug;25(8):821-8. doi: 10.1007/s00299-006-0139-0. Epub 2006 Mar 14.
A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.
针对甜杏仁品种“Ne Plus Ultra”的叶片外植体，开发了一种用于农杆菌介导转化的方案，该方案采用卡那霉素或甘露糖筛选。再生芽在含有15 μM卡那霉素的培养基上进行筛选（阴性筛选），而在阳性筛选策略中，芽在添加了15 g/l蔗糖的2.5 g/l甘露糖培养基上进行筛选。基于对来自独立株系的单个假定转化芽进行PCR分析，相对于所测试的初始叶片外植体数量，卡那霉素/nptII筛选的转化效率为5.6%，甘露糖/pmi筛选的转化效率为6.8%。对六个随机选择的PCR阳性芽进行Southern杂交分析，证实每个芽中都存在nptII转基因，并且通过PCR鉴定出含有pmi转基因的五个随机选择的株系与pmi DNA探针呈现阳性杂交。本研究中使用的阳性（甘露糖/pmi）和阴性（卡那霉素）筛选方案极大地提高了杏仁的转化效率，这通过PCR和Southern杂交得到了证实。本研究还表明，在杏仁中，甘露糖/pmi筛选方案是合适的，并且与卡那霉素/nptII筛选方案相比，能够产生更高的转化效率。
