Jordan M C
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Rd., Winnipeg, MB, R3T 2M9, Canada e-mail:
Plant Cell Rep. 2000 Nov;19(11):1069-1075. doi: 10.1007/s002990000246.
Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization.
通过粒子轰击进行小麦（Triticum aestivum L.）转化目前已在许多实验室中得以确立，但转化效率仍然普遍较低，且只有特定基因型才能获得最高效率。为了快速优化和改进小麦转化方案，需要一种能够早期检测转化细胞的非破坏性标记。我们评估了改造后的维多利亚多管水母绿色荧光蛋白（GFP）作为检测小麦转化细胞和组织标记的能力。用sGFPS65T基因构建体进行粒子轰击14天后，观察到发出绿色荧光的多细胞团簇，轰击后最早21天就能观察到表达gfp的芽（通常带有表达gfp的根）。这些芽可以从愈伤组织中分离出来并进一步培养，直到准备好转移到土壤中。仅基于gfp表达就可以筛选出转基因小麦植株，不过加入抗生素抗性作为选择标记可以提高效率。在一项比较轰击参数的实验中使用sgfpS65T作为标记基因，能够快速识别可作为优化目标的变量。
