Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules Verne, 33 Rue Saint-Leu Ilot des Poulies, F-80039, Amiens Cédex, France.
Planta. 1992 Oct;188(3):439-56. doi: 10.1007/BF00192812.
The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.
植物中外源 DNA 的插入是通过根瘤农杆菌与宿主植物细胞之间的复杂相互作用来实现的。本文使用大肠杆菌的标记基因β-葡萄糖醛酸酶和细胞学方法，对根瘤农杆菌介导的转化的感受态细胞进行了特征描述，研究了转化的早期细胞事件，并鉴定了限制拟南芥转化的潜在宿主细胞障碍。在子叶和叶片外植体中，感受态细胞是正在去分化的叶肉细胞，这一过程是由创伤和/或植物激素诱导的。在植物激素预处理后，这些细胞位于外植体的切口表面或内部。在根外植体中，感受态细胞存在于去分化的中柱，只有在植物激素预处理后才会产生。无论其来源如何，感受态细胞都很小，等径，初生细胞壁薄，小而多泡，核突出，细胞质致密。在子叶和根外植体中，组织学计数和β-葡萄糖醛酸酶分析表明，预培养处理增加了推测的感受态细胞数量，这表明在没有植物激素处理的情况下，创伤后细胞的激活和细胞分裂不足以进行转化。将外植体暴露于根瘤农杆菌中 48 h 不会产生特征性的应激反应，也不会逐渐丧失活力或细胞死亡。然而，在感受态细胞中，宿主细胞壁多糖与细菌丝多糖之间的联系经常被观察到，这表明转化需要多糖-多糖接触。流式细胞荧光术和组织学分析表明，丰富的转化不仅需要细胞的激活（表现为核蛋白增加的早期状态），还需要细胞的增殖（在子叶组织中发生在许多倍性水平上）。在细菌感染前，用适当的植物激素处理可以使非感受态细胞成为感受态细胞：这有助于分析转化过程中的关键步骤，并有助于开发转化顽固植物的新策略。
