Rachmawati Diah, Hosaka Takehiko, Inoue Eiichi, Anzai Hiroyuki
Gene Research Center, Ibaraki University, Ami, Ibaraki, Japan.
Biosci Biotechnol Biochem. 2004 Jun;68(6):1193-200. doi: 10.1271/bbb.68.1193.
The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now. Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar. In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified. Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for beta-glucuronidase (gus) and hygromycin resistance (hpt). Finally, the transformation efficiency of Rojolele variety using A. tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare. The seed fertility of transgenic Rojolele was more than 90%. The copy number of the transgene varied from one to three copies in the T(0) transgenic lines. Both the gus and the hpt genes were inherited and expressed in the progeny.
根癌农杆菌介导的转化系统被扩展到一种著名的爪哇稻品种Rojolele，该品种目前在印度尼西亚种植。此前，对于任何水稻品种，利用未成熟和成熟种子高效诱导愈伤组织均未成功。在本研究中，对碳源和培养基pH进行改良后，Rojolele成熟种子的愈伤组织在C培养基上呈现出紧密且结节状的外观。将成熟种子盾片来源的愈伤组织与携带质粒pAFT14的根癌农杆菌菌株EHA101或LBA4404共培养，该质粒包含β-葡萄糖醛酸酶（gus）基因和潮霉素抗性（hpt）基因。最终，使用根癌农杆菌菌株EHA101（pAFT14）的Rojolele品种的转化效率提高到约23%，与粳稻品种日本晴相似。转基因Rojolele的种子育性超过90%。在T(0)转基因株系中，转基因的拷贝数从1到3个不等。gus和hpt基因在后代中均能遗传并表达。
