两种遗传标记筛选物在马铃薯Desiree遗传转化应用中的研究

以马铃薯茎段为外植体,采用农杆菌介导法,将带有Bar和hpt基因的植物表达质粒载体转入马铃薯栽培品种Desiree,并对遗传转化过程中的预培养时间、抑菌素浓度、筛选物抗除草剂Basta和潮霉素的浓度等因素进行了研究.结果表明:预培养时间2 d转化效率最高;头孢霉素浓度为300 mg/L时,能够有效的抑制农杆菌的生长;以除草剂Basta为筛选物时,适宜的筛选压为0.6 mg/L,用潮霉素(Hyg)作为筛选物时,以15 mg/L为最合适;获得的转基因植株经PCR分析,证实Bar基因己经整合到马铃薯基因组DNA中,从而建立了以除草剂Basta或以潮霉素为遗传标记筛选物的马铃薯栽培品种Desiree的遗传转化体系,为利用基因工程进一步改良马铃薯品质奠定了基础.

Studies on Application of Two Genetic Marking Substances in Agrobacterium-Mediated Transformation of Potato

In In this paper,genetic transformation of potato cultivar Desiree was conducted utilizing the Agrobacterium tumefaciens strain EHA105 that contains the binary vector pYLTAC7 with the phosphinothricin resistance(Bar) and hygromycin phosphotransferase(hpt) genes.The time of pre-cultivation,concentrations of Cefotaxime,Hygromycin and Herbicide Basta were studied.Our results suggested that: The optimal pre-cultivation time was 2d;Cefotaxime(Cefo),at the concentration of 300mg/L,was able to effectively suppress the growth of Agrobacterium after co-cultivation;The optimal concentrations for Basta and Hygromycin were,respectively,0.6 and 15mg/L and The presence of the Bar gene in the genomic DNA of transgenic plants was verified by PCR amplification.A stem transformation and regeneration system was devised for potato Desiree using Basta or Hygromycin as a selective marker.