农杆菌介导的墨兰遗传转化  

Agrobacterium-mediated transformation of Cymbidium sinensis

作　　者：谢利[1] 王芬[1] 曾瑞珍[1] 郭和蓉[1] 周玉亮[1] 张志胜[1] 

机构地区：[1]华南农业大学广东省植物分子育种重点实验室,广东广州510642

出　　处：《生物工程学报》2015年第4期542-551,共10页Chinese Journal of Biotechnology

基　　金：广东省农业科技攻关项目(No.2007A020300009-3);广东省教育部产学研项目(No.2012B091100480)资助~~

摘　　要：转基因育种是快速定向改良兰花育种目标性状的有效方法,但迄今未见有关墨兰转基因育种的研究报道。试验以‘企剑白墨’墨兰Cymbidium sinensis cv.‘Qijianbaimo’的根状茎为受体材料,研究了影响农杆菌介导墨兰遗传转化效率的因素,以建立有实用价值的墨兰遗传转化技术体系。结果表明,受体的预培养时间、乙酰丁香酮的添加方式及浓度、农杆菌工程菌液浓度(OD600)、侵染时间和共培养时间均对‘企剑白墨’根状茎的GUS瞬时表达率有显著影响。以预培养39 d的根状茎尖为材料,在添加200μmol/L乙酰丁香酮,OD600为0.9的工程菌液中侵染35 min后,转入添加200μmol/L乙酰丁香酮的共培养基中培养7 d时,‘企剑白墨’根状茎的GUS瞬时表达率最高,为11.67%。采用上述条件对‘企剑白墨’墨兰进行遗传转化,经PCR鉴定和GUS染色检测,从400株再生植株中获得了3株转基因植株,转化率为0.75%。研究表明通过农杆菌介导法对墨兰进行遗传改良是可行的。Genetic transformation is an effective method to improve breeding objective traits of orchids. However, there is little information about genetic transformation of Cymbidium sinensis. Rhizomes from shoot-tip culture of C. sinensis cv. ‘Qijianbaimo＇ were used to establish a practical transformation protocol of C. sinensis. Pre-culture time, concentration and treating methods of acetosyringone, concentration of infection bacteria fluid（OD600）, infection time, and co-culture time had significant effects on β-glucuronidase（GUS） transient expression rate of C. sinensis cv. ‘Qijianbaimo＇ rhizome. The GUS transient expression rate of rhizome was the highest（11.67%） when rhizomes pre-cultured for 39 d were soaked in bacterium suspension（OD600=0.9） supplemented with 200 μmol/L acetosyringone for 35 min, followed by culturing on co-culture medium supplemented with 200 μmol/L acetosyringone for 7 d. Under this transformation conditions, 3 transgenic plantlets, confirmed by GUS histochemical assay and PCR, were obtained from 400 regenerated plantlets, and the genetic transformation rate was 0.75%. This proved that it was feasible to create new cultivars by the use of Agrobacterium-mediated genetic transformation in C. sinense.

关 键 词：墨兰 农杆菌介导 遗传转化 根状茎 

分 类 号：S682.31[农业科学—观赏园艺]