College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, People's Republic of China.
Appl Biochem Biotechnol. 2013 Sep;171(2):543-52. doi: 10.1007/s12010-013-0391-z. Epub 2013 Jul 20.
Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l(-1) 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l(-1) α-naphthalene acetic acid and 6.0 mg l(-1) 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105-harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter-was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.
已制定出用于进行无融合生殖种百脉根（Eulaliopsis binata）再生和农杆菌介导转化的方案。最初，在添加有三种浓度 2,4-二氯苯氧乙酸（2,4-D）的 Murashige 和 Skoog（MS）基础培养基上孵育了四个百脉根基因型的种子。结果发现，在含有 3.0mg·l-1 2,4-D 的培养基上，36.2%的外植体发育出高度易碎的愈伤组织。基于愈伤组织诱导频率，选择内乡基因型进行再生和转化。在补充有 0.2mg·l-1α-萘乙酸和 6.0mg·l-1 6-糠基氨基嘌呤的 MS 基础培养基上培养的愈伤组织可发育出芽。随后，使用携带潮霉素磷酸转移酶（hpt）抗性基因和合成绿色荧光蛋白（GFP）基因的质粒 pCAMBIA1381 转化系统，将携带该质粒的根癌农杆菌（Agrobacterium tumefaciens）菌株 EHA105 转化到愈伤组织中。经过两轮潮霉素选择后获得了疑似转化的愈伤组织。通过 GFP 检测分析了疑似转基因愈伤组织中转基因的表达。通过聚合酶链反应和 Southern blot 分析对疑似转化芽进行分子鉴定，以确认 hpt 基因的存在和整合。
