Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties
Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties
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DOI：
10.1007/bf00195194
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发表时间：
1996-08-01
期刊：
影响因子：
4.3
通讯作者：
Hodges, TK
中科院分区：
文献类型：
--
作者：
Aldemita, RR;Hodges, TK
水稻遗传转化的研究在粳稻品种中已经证实了由根癌农杆菌介导的，并扩展到包括更多的籼稻品种。未成熟胚接种A.根癌农杆菌At 656（pChTL 56）或LBA 4404（pTOK 233）。实验条件最初开发的菌株At 656处理的未成熟胚，基于瞬时和稳定的β-葡糖醛酸苷酶（GUS）的活动。然而，在G418（pCNL 56含有npt II基因）上选择后的植物再生没有发生。使用相同的基本协议。但是用LBA 4404（pTOK 233）处理水稻的未成熟胚，导致有效地（约27%）产生了粳稻品种Radon的转基因植物，并且对籼稻品种IR 72和TCS 10产生了可接受的效率（1-5%）。转化基于对潮霉素的抗性（pTOK 233含有hpt基因）、GUS活性的存在（来自gusA基因）、用于检测整合的gusA基因的Southern印迹以及GUS活性以孟德尔3：1分离比传递给后代。Southern印迹表明在大多数转化体中整合了两到三个拷贝的基因。粳稻和籼稻品种的转基因植株都是自花可育的，在这方面与种子生长的植株相当。促进根癌农杆菌转化水稻的关键因素似乎是使用胚作为外植体，使用潮霉素作为选择剂（其不干扰水稻再生），在pTOK 233的二元载体上存在某些vir基因的额外拷贝，以及在胚与农杆菌共培养期间维持高浓度的乙酰丁香酮用于诱导vir基因。
Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pChTL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable beta-glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol. but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1-5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the explant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.