Kojima M, Arai Y, Iwase N, Shirotori K, Shioiri H, Nozue M
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
Biosci Biotechnol Biochem. 2000 Apr;64(4):845-7. doi: 10.1271/bbb.64.845.
Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var. Shinano No. 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121). The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants). The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively. Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A. tumefaciens harboring a modified pBI121 for plasmid rescue. Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.
用针穿刺荞麦（甜荞品种信浓 1 号）幼苗的顶端分生组织，并用根癌农杆菌（LBA4404，pBI121）进行接种。接种后的幼苗生长至成熟，使其随机授粉以结籽（T1 代植株）。通过在含有遗传霉素（20 微克/毫升）的条件下萌发以及用 PCR 检测β-葡萄糖醛酸酶（GUS）基因来估计 T1 代植株的转化效率，结果表明分别有 36%和 70%的 T1 代植株发生了转化。从用携带经修饰的 pBI121 用于质粒拯救的根癌农杆菌转化的 T1 代植株中挑选出 4 株呈现突变形态的植株。对从这 4 株 T1 代植株拯救出的质粒进行 Southern 杂交分析表明，每个质粒都含有不同的荞麦基因组侧翼 DNA，这证明在这 4 株 T1 代植株中 T-DNA 整合到了基因组 DNA 的不同位点。
