农杆菌介导的墨兰遗传转化

Agrobacterium-mediated transformation of Cymbidium sinensis

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200 μmol/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200 μmol/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。

Abstract:

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.