一种马铃薯高效无标记转基因技术的建立

用农杆菌介导法转化马铃薯栽培品种紫花白的叶盘,通过1/4 MS培养基预培养、热激处理、低pH、高糖培养基共培养,之后利用PCR直接检测转化体,结果表明遗传转化效率可达5.1%,建立了马铃薯无标记转基因技术.该技术受基因型的限制小,用于其它3个不同的栽培品种东北白、晋薯7号和早大白,遗传转化效率亦达到了4.1%~8.3%.利用这项无标记转基因技术,在载体构建时就剔除了标记基因,遗传转化后直接分化培养,不必对转化细胞进行抗性筛选,缩短了遗传转化周期,省去了费时费力的标记基因剔除步骤,亦为重复转化聚合多个优良基因提供了便利.

Development of Potato High Efficiency Marker-free Transgenic Technology

The production of marker-free transgenic plants is a critical advance for both plant biotechnology research and commercial development.Several methods have been reported to create marker-free transgenic plants,for example co-transformation,transposable,site-specific recombination,or intrachromosomal recombination.Not only these methods are time-consuming and inefficient,but they are also employed on the assumption that isolation of transmants without a selective marker gene is not feasible.In this paper,the genetic transformation efficiency of potato mediated by Agrobacteria tumefaciens was clearly improved through nutrient limitation pre-culture,heat shock and co-culture on high-glucose medium and a technology have been developed to directly generate marker-free transgenic plants,which permits the removal marker gene of binary vector and direct selection of transformed cells or shoots after PCR analysis.Using this technology,commercially available varieties showed a percentage of PCR-positive shoots of 5.1% for cultivar Zihuabai,4.1% for cultivar Dongbeibai,5.7% for cultivar Jinshu 7,and 8.3% for cultivar Zaodabai.Because this technology does not require time-consuming marker gene removal through genetic segregation or site-specific DNA-deletion systems,it may provide a reliable and efficient tool for both generation of marker-free transgenic plants and re-transformation to stack multiple interest genes in potato for comercial use.