Hayashimoto A, Li Z, Murai N
Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, Louisiana 70803-1720.
Plant Physiol. 1990 Jul;93(3):857-63. doi: 10.1104/pp.93.3.857.
We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.
我们已经建立了一种高效的程序，用于水稻（Oryza sativa L.）品种日本晴和台北309的原生质体转化及可育转基因植株的再生。将原生质体与植物可表达的潮霉素抗性基因混合，并用25%（w/v）的聚乙二醇处理。在95微摩尔潮霉素B存在的情况下，对14日龄再生的原生质体进行严格的转化菌落筛选，持续12天。筛选后，每百万个处理过的台北309和日本晴原生质体分别获得450个和200个抗性菌落。对潮霉素抗性细胞系和再生植株的Southern杂交分析表明，转移的DNA有1至10个拷贝整合到了水稻基因组的1至4个位点。Southern DNA分析表明，导入的质粒DNA在整合之前可能通过分子间重组形成串联体。4株台北309和39株日本晴转基因水稻植株在温室中再生并生长至成熟。2株台北309和35株日本晴植株结出了有活力的种子。分析了台北309转基因植株的农艺性状以及自交转基因植株后代对潮霉素抗性性状的遗传情况。
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