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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。
Transgenic breeding is an effective method to rapidly improve the target traits of orchid breeding, but so far there is no report on transgenic breeding of Cymbidium. In this study, the rhizomes of ’Cymbidium sinensis cv.’Qijianbaimo’ from ’Jianjian White Ink’ were used to study the factors that affect the Agrobacterium-mediated genetic transformation efficiency of Cymbidium to establish practical Cymbidium genetic transformation Technology System. The results showed that GUS transient expression of ’Jianjian White’ rhizome was pre-cultured, the way and concentration of acetosyringone added, the concentration of Agrobacterium tumefacien, OD600, infection time and co-culture time Rate has a significant impact. The rhizome tips pre-cultured for 39 days were used as the materials. After the cells were infected with 200 μmol / L acetosyringone and OD600 of 0.9 for 35 min, they were transferred into the co-culture medium supplemented with 200 μmol / L acetosyringone When cultured for 7 days, the GUS transient expression rate of ’Jian Jian Bai Mo’ rhizome was the highest, which was 11.67%. Using the above conditions, ’Sijian White Ink’ was cultivated in Cymbidium fortunei. Three transgenic plants were obtained from 400 regenerated plants with the transformation rate of 0.75% by PCR and GUS staining. Studies have shown that Agrobacterium-mediated genetic modification of Cymbidium is feasible.