Ducrocq C, Sangwan R S, Sangwan-Norreel B S
Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules Verne, Amiens, France.
Plant Mol Biol. 1994 Sep;25(6):995-1009. doi: 10.1007/BF00014673.
This work describes a new method to obtain transgenic somatic embryos from Agrobacterium-infected immature zygotic embryos of Datura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene beta-glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency for Agrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.
本研究描述了一种从被农杆菌感染的紫花曼陀罗未成熟合子胚中获得转基因体细胞胚的新方法。与先前的转化方法相比，该方法具有诸多优势，如不存在愈伤组织阶段、平均转化率为76%以及再生频率高。实现最佳转化的关键步骤是胚阶段和短期预培养处理。使用标记基因β-葡萄糖醛酸酶和光学显微镜来鉴定有能力的胚性细胞，这些细胞在转化后经历胚胎发育的经典阶段。转基因以孟德尔方式传递给后代。通过直接体细胞胚胎发生再生的植株在细胞学和形态学上是一致的。我们还观察到：（1）合子胚转化不需要创伤或创伤诱导的分裂；（2）表皮细胞具有转化和再生的能力；（3）对农杆菌感染的能力具有发育阶段特异性。这种新方法应有助于开发常规转化难转化植物物种的新策略。
