Department of Crop and Soil Sciences, Washington State Univ., 99164-6420, Pullman, WA, USA.
Plant Cell Rep. 1993 Sep;12(11):612-6. doi: 10.1007/BF00232809.
Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.
已从几个实验室的原生质体中再生出可育的小麦植株。本研究的目的是开发一种利用原生质体作为靶细胞的转化系统。原生质体是从花药衍生愈伤组织的细胞悬浮液中分离出来的。使用携带 Basta 抗性（BAR）基因的 pBARGUS 或 pBAS 通过电穿孔将原生质体转化。在 3 个独立的实验中，共从电穿孔转化的原生质体中产生了 2761 个愈伤组织。在含有 10mg/l 草丁膦（PPT）的选择性培养基上，有 6 个愈伤组织存活下来，PPT 是一种完全抑制未转化小麦愈伤组织生长的浓度。5 个 PPT 抗性愈伤组织表现出草丁膦乙酰转移酶（PAT）活性，而第 6 个可能是突变体。转化的小麦愈伤组织能耐受高达 2560mg/l 的 PPT 浓度。Southern blot 分析证实了 BAR 基因已整合到小麦基因组中。整合的 DNA 序列可能已部分甲基化，并至少串联重复一次。这些结果表明，通过原生质体直接基因转移的电穿孔介导法可产生稳定转化的小麦愈伤组织。
