Han J-S, Kim C K, Park S H, Hirschi K D, Mok I- G
National Horticultural Research Institute, Rural Development Administration, Suwon, 441-440, South Korea.
Plant Cell Rep. 2005 Mar;23(10-11):692-8. doi: 10.1007/s00299-004-0874-z. Epub 2004 Oct 12.
We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.
我们描述了一种通过用携带二元载体pCAMBIA3301的根癌农杆菌AGL1菌株接种子叶外植体来生产转基因葫芦植株的方法，该二元载体包含抗草铵膦（bar）基因和β-D-葡萄糖醛酸酶（GUS）报告基因。当4日龄幼苗的子叶外植体在补充有0.1 - 0.001 mg/l L-α-（2-氨基乙氧基乙烯基）甘氨酸（AVG）的共培养基上与农杆菌共培养6 - 8天时，观察到最有效的细菌感染。在含有2 mg/l DL-草丁膦的选择培养基上培养2 - 3周后，推定转化的芽直接在子叶外植体的近端出现。这些芽在含有0.1 mg/l吲哚乙酸和1 mg/l DL-草丁膦的半强度MS培养基上培养3周后生根。获得转基因植株的频率为1.9%。通过组织化学GUS分析、聚合酶链反应和Southern印迹分析证实了转基因在T1代植株中的稳定整合和传递。T1代后代的遗传分离分析表明，转基因以孟德尔方式遗传。据我们所知，本研究首次展示了农杆菌介导的葫芦转化。
