PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-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 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/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50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12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.

Transformation of PSAG12-ipt Gomphosis Gene into Potato

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(PSAG12-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 PSAG12 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.