Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium.
EMBO J. 1984 Aug;3(8):1681-9. doi: 10.1002/j.1460-2075.1984.tb02032.x.
Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km and Cm). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.
嵌合基因由胭脂碱合成酶启动子和编码序列组成，可指定对卡那霉素、氯霉素或氨甲喋呤的抗性，通过重组（通过嵌合基因构建体和 pGV3850 中存在的同源 pBR322 序列）插入非致癌 Ti 质粒载体 pGV3850 中。这些共整合子在根癌农杆菌中被用于通过共培养感染烟草的单个原生质体。在适当抗生素的存在下，抗性特性允许在组织培养中选择转化的愈伤组织。此外，由于用于原生质体转化的 Ti 质粒是非致癌的，因此可以从抗性愈伤组织中再生出表型正常和可育的植物。我们已经表明，这些完全分化的植物组织表现出抗性特性（Km 和 Cm）的功能性表达。携带嵌合基因的所有植物均正常发育、开花和结籽。对其中几个抗性特性的遗传分析表明，它们是孟德尔遗传的。这些结果是模型实验，证明可以使用非致癌 Ti 质粒衍生物系统地将感兴趣的基因转移到植物基因组中；并且转化的植物能够正常生长和分化，从而为研究基因表达和植物细胞发育提供了自然环境。
