绿色荧光蛋白基因转化柑橘胚性愈伤组织与高效再生

Transfer of green fluorescent protein gene into citrus embryogenic calluses and high efficient regeneration

采用根癌农杆菌介导法对13份柑橘胚性愈伤组织进行绿色荧光蛋白基因(gfp)的转化,短时间内获得了温州蜜柑、橘橙杂种GWZ等11份柑橘材料的82个转化细胞系,其中佛罗斯特脐橙和日辉橘最易转化,分别获得28个和25个转化细胞系。选取3份材料的不同转化细胞系进行gfp的PCR分析,均获得了预期的gfp片段。研究表明,GFP标记可以在转化早期快速检测并分离转化子,及时剔除逃逸体,获得纯合的转化系;高水平的gfp表达不影响转化细胞增殖、生长和分化。这些带有GFP标记的转化细胞系为柑橘的有关基础研究提供了良好的试材,目前正用于柑橘体细胞杂交和不同倍性愈伤组织的社会化控制现象与机理等研究。

Transformation of green fluorescent protein gene （gfp） into citrus embryogenic calluses of 13 cultivars mediated by Agrobacterium tumefaciens was conducted. Totally 82 transgenic lines from 11 cultivars were produced, among which 28 and 25 transgenic lines were obtained from Frost navel orange and Sunburst tangerine respectively. Further PCR analysis of 3 selected transformed cuhivars amplified the expected GFP fragment. This study proved that GFP as a vital marker could localize the transgene sites at an early stage, be helpful for discriminating escapes and chimeras, and physically separating in vitro putative transformants. High level of gfp expression did not affect transgenic cell division, somatic embryogenesis and further differentiation. These transgenic cell lines are valuable materials in citrus basic research and currently being used in studies on citrus somatic fusion and social control mechanism between calluses of different ploidy levels.