Analysis of defense mechanism against Agrobacterium-reinfection in plants regenerated from hairy roots.

携带pBI 121的发根农杆菌15834和根癌农杆菌LBA 4404感染烟草BY-2细胞，分别形成毛状根细胞和卡那霉素抗性（Km^R）细胞。然而，通过将pBI 121的T-DNA上的新霉素磷酸转移酶II（NPT II）基因导入毛状根细胞中，没有获得毛状根细胞的Km^R-PCR，尽管通过将Ri T-DNA导入Km^R克隆中获得了Km^R克隆的毛状根细胞。用野生型烟草植株、毛状根克隆再生植株和Km^R转基因烟草植株获得了类似的结果，这些转化实验表明，已经用Ri T-DNA转化的转基因植株不能再进行第二次农杆菌介导的T-DNA转化。转Km^R基因植株的毛状根克隆在不含卡那霉素的培养基中培养8个月后，仍能保持卡那霉素抗性和不依赖于植物生长素的生长特性。结果表明，两种T-DNA共存，T-DNA上的基因在植物基因组中正常表达，NPT Ⅱ基因直接导入烟草原生质体，从BY-2细胞中获得Km^R细胞，而从毛状根细胞中未获得Km^R细胞。农杆菌侵染的过程可分为四个阶段;（1）附着于植物细胞，（2）T-DNA转移到植物细胞，（3）T-DNA整合到植物基因组中，这些结果表明，携带Ri T-DNA的植物细胞的“免疫”反应可能发生在T-阶段，DNA整合到植物基因组中。

Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens LBA4404 harboring pBI121 infected tobacco BY-2 cells, forming hairy root cells and kanamycin-resistant (Km^R) cells, respectively. However, no Km^R-transformant of hairy root cells was obtained by introducing the neomycin phosphotransferase II (NPT II) gene on the T-DNA of pBI121 into the hairy root cells, although the hairy root cells of Km^R clones were obtained by introducing Ri T-DNA into Km^R clones. The similar results were obtained using the wild-type tobacco plant, the plants regenerated from hairy root clones and Km^R transgenic plants of Nicotiana plumbaginifolia.These transformation experiments suggest that transgenic plants which have been already transformed by Ri T-DNA cannot undergo a second Agrobacterium-mediated T-DNA transformation. Plants carrying Ri T-DNA,which is integrated into the host chromosome analogously with lambda phage DNA,are possibly immune to further infection with Agrobacteria.Both characteristics of the kanamycin resistance and the phytohormone-independent growth were stably maintained in the hairy root clones derived from the Km^R transgenic plants after culturing in the medium without kanamycin for 8 months. These results show that two types of T-DNA coexist and the genes on the T-DNAs are normally expressed in the plant genome.When NPT II gene was directly introduced into the tobacco protoplasts by electroporation, Km^R cells were obtained from BY-2 cells but not from the hairy root cells. The process of infection with Agrobacteria can be separated into the four stages ; (1) Attachment to the plant cells, (2) T-DNA transfer to the plant cells, (3)T-DNA integration into the plant genome, and (4) Gene expression from the integrated T-DNA.These results show that the "immune" response in the plant cells carrying Ri T-DNA may be occurred in the stage of T-DNA integration into the plant genome.