Rathore K S, Chowdhury V K, Hodges T K
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
Plant Mol Biol. 1993 Mar;21(5):871-84. doi: 10.1007/BF00027118.
We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2-4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was verified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T0 plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T0 plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.
我们已将bar基因与除草剂Basta结合使用，以筛选转化的水稻（Oryza sativa L. cv. Radon）原生质体，用于培育抗除草剂水稻植株。从由未成熟胚愈伤组织建立的可再生悬浮培养物中获得的原生质体，通过聚乙二醇（PEG）介导的DNA摄取进行转化。将原生质体来源的愈伤组织置于含有选择剂草丁膦（PPT，Basta的活性成分）的培养基上2 - 4周后，可筛选出转化的愈伤组织。对PPT具有抗性的愈伤组织能够再生植株。草丁膦乙酰转移酶（PAT）检测证实了bar基因在从PPT抗性愈伤组织获得的植株中的表达。唯一的例外是从同一个愈伤组织获得的两株植物，它们的基因组中整合了多个bar基因拷贝。通过Southern印迹分析验证了植株的转基因状态。在我们通过原生质体方法进行转化的系统中，逃逸植株极少。与报告基因gusA共转化的效率为30%。Radon的T0植株可自交结实。Southern分析证实bar和gusA基因均传递给了后代。这两个基因在T1和T2后代中均有表达。对T1后代植株的酶分析也显示出基因剂量反应，反映了它们的纯合和杂合状态。T0植株以及具有bar基因的后代植株的叶片对Basta处理具有抗性。因此，bar基因已被证明是用于水稻植株转化和培育抗除草剂植株的一种有用的选择和筛选标记。
