---

作 者 ：吴旺泽1，彭晓莉2*，刘小平1

期 刊 ：西北植物学报 2012年 5期 页码：895~901

关键词：PSAG12-ipt；遗传转化；马铃薯；细胞分裂素(CTK)；根癌农杆菌；

Keywords:PSAG12-ipt, genetic transformation, potato, cytokinin (CTK), Agrobacterium tumefaciens,

摘 要 ：以根癌农杆菌介导法将P_SAG12-ipt嵌合基因导入马铃薯栽培品种，对影响马铃薯遗传转化的多种因素进行系统研究。结果表明：马铃薯茎段分化效率高于叶片，马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25 mg/L+NAA 0.25 mg/L+2,4-D 0.25 mg/L，添加1% Na₂SO₃能有效防止褐化；茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50 mg/L和75 mg/L；外植体预培养2 d，OD₆₀₀为0.2~0.5的农杆菌浓度侵染8 min、共培养3 d后进行选择培养能有效地提高植株再生能力。用P_SAG12和ipt双重PCR检测再生植株，阳性转化率为65.8％。Southern blotting结果表明，转基因植株多以单拷贝形式整合进马铃薯基因组中。

Abstract:A population of transgenic plants was produced by the transformation of internodal explants of potato using an Agrobacterium tumefaciens LBA4404-based vector containing a gomphosis gene (P_SAG12-ipt).The results showed that intermodal explants were more effective for transformation than leaf explants.The regeneration strategy utilised a three-step protocol,a 2 days pre-culture on the MS medium containing 6-benzylaminopurine (6-BA) 0.25 mg/L,α-naphythy lacetic acid (NAA) 0.25 mg/L,2,4-dichlorophenacetic acid (2,4-D) 0.25 mg/L，and supplemented 1% Na₂SO₃,followed the explants of pre-culture were incubated 8 min in Agrobacterium tumefaciens LBA4404 suspension (OD₆₀₀ 0.2~0.5),last,the explants were co-cultured 3 days on basal medium without supplemented any phytohormones.After 3 days co-cultivation,the explants of internode and leaf were transferred to basal medium supplemented phytohormones,1% Na₂SO₃,200 mg/L cefotaxime and 75 mg/L kanamycin until to regenerated plants.Transgenic plants were identified utilizing P_SAG12 and ipt gene dual primer by PCR.The positive transformation rate was 65.8%.Southern blotting analysis identification showed that most ipt gene were induced into potato genome only one copy.

---