Peña L, Cervera M, Juárez J, Navarro A, Pina J A, Navarro L
Departamento Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, E-46113-Moncada, Valencia, Spain E-mail:
Plant Cell Rep. 1997 Sep;16(11):731-737. doi: 10.1007/s002990050311.
We have previously developed procedures for the efficient production of sweet orange (Citrus sinensis L. Osbeck) and Carrizo citrange (C. sinensis L. Osbeck×Poncirus trifoliata L. Raf.) transgenic plants using an Agrobacterium tumefaciens-mediated transformation and shoot tip grafting in vitro regeneration system. We now report on the optimization of the cocultivation, regeneration and selection conditions for efficient and reliable production of transgenic lime (C. aurantifolia Swing.) plants. Improved transformation frequencies were obtained by cocultivating the explants with Agrobacterium on feeder plates. Optimum regeneration of transgenic shoots was obtained by exposing the explants to darkness for 2 weeks and by using kanamycin at 100 mg/l as selective agent. Attempts to use geneticin as selection antibiotic were not successful. Shoot tip grafting of regenerated shoots on Troyer citrange seedlings resulted in 100% successful production of transgenic plants. The presence and expression of the transferred genes in the regenerated plants was verified by β-glucuronidase histochemical and fluorimetric assays, neomycin phosphotransferase ELISA assays, PCR and Southern analyses.
我们之前已经开发出了利用根癌农杆菌介导的转化以及茎尖嫁接体外再生系统高效生产甜橙（Citrus sinensis L. Osbeck）和卡里佐枳橙（C. sinensis L. Osbeck×Poncirus trifoliata L. Raf.）转基因植株的方法。我们现在报告关于高效且可靠地生产转基因酸橙（C. aurantifolia Swing.）植株的共培养、再生和选择条件的优化。通过在饲养平板上使外植体与农杆菌共培养获得了更高的转化频率。通过将外植体置于黑暗中2周并使用100 mg/l的卡那霉素作为选择剂，获得了转基因芽的最佳再生效果。尝试使用遗传霉素作为选择抗生素未成功。将再生芽茎尖嫁接到特罗耶枳橙幼苗上，转基因植株的生产成功率达100%。通过β-葡萄糖醛酸酶组织化学和荧光测定、新霉素磷酸转移酶ELISA测定、PCR和Southern分析验证了再生植株中转移基因的存在和表达。
