Bechtold N, Jaudeau B, Jolivet S, Maba B, Vezon D, Voisin R, Pelletier G
Unité de Génétique et d'Amélioration des Plantes, INRA, 78026 Versailles Cedex, France.
Genetics. 2000 Aug;155(4):1875-87. doi: 10.1093/genetics/155.4.1875.
In planta transformation methods are now commonly used to transform Arabidopsis thaliana by Agrobacterium tumefaciens. The origin of transformants obtained by these methods has been studied by inoculating different floral stages and examining gametophytic expression of an introduced beta-glucuronidase marker gene encoding GUS. We observed that transformation can still occur after treating flowers where embryo sacs have reached the stage of the third division. No GUS expression was observed in embryo sacs or pollen of plants infiltrated with an Agrobacterium strain bearing a GUS gene under the control of a gametophyte-specific promoter. To identify the genetic target we used an insertion mutant in which a gene essential for male gametophytic development has been disrupted by a T-DNA bearing a Basta resistance gene (B(R)). In this mutant the B(R) marker is transferred to the progeny only by the female gametes. This mutant was retransformed with a hygromycin resistance marker and doubly resistant plants were selected. The study of 193 progeny of these transformants revealed 25 plants in which the two resistance markers were linked in coupling and only one plant where they were linked in repulsion. These results point to the chromosome set of the female gametophyte as the main target for the T-DNA.
目前,植物体内转化方法常用于通过根癌农杆菌转化拟南芥。通过接种不同花期的植株并检测导入的编码β-葡萄糖醛酸酶(GUS)的标记基因的配子体表达,对这些方法获得的转化体的起源进行了研究。我们观察到,在处理胚囊已达到第三次分裂阶段的花朵后,转化仍可发生。在用携带受配子体特异性启动子控制的GUS基因的农杆菌菌株浸润的植物的胚囊或花粉中未观察到GUS表达。为了确定遗传靶点,我们使用了一个插入突变体,其中一个对雄配子体发育至关重要的基因已被携带巴斯塔抗性基因(B(R))的T-DNA破坏。在这个突变体中,B(R)标记仅通过雌配子传递给后代。用潮霉素抗性标记对这个突变体进行再次转化,并筛选出双抗性植株。对这些转化体的193个后代的研究发现,有25株植物中两个抗性标记呈顺式连锁,只有1株植物中它们呈反式连锁。这些结果表明雌配子体的染色体组是T-DNA的主要靶点。