冰糖橙成年态节间茎段遗传转化体系的建立

Citrus genetic transformation using mature tissue as explant is important for the precocious evaluation of the genetically modified characteristics, which is especially indispensable for seedless cultivars like‘Bingtang’orange. In this study, a protocol was improved for regeneration and genetic transformation with mature tissues of‘Bingtang’orange. The internodal stem segments were sampled from adult plants grafted onPoncirus trifoliateand maintained in the greenhouse. The new improved disinfection could decrease the death rate from 27.2% to 0, and increase the survival rate from over 60% to approximately 80%. The highest regeneration frequency of adventitious buds (47.89%) were obtained in the co-culture medium and the shoot regeneration medium. For the transformation experiments, 30 mg·L-1of kanamycin and 500 mg·L-1of cefotaxime were added to the shoot regeneration medium. Three methods were tried to in vitro graft the adventitious buds onto citrange rootstock, and it was observed that the highest rate of success reached approximately 90% obtained by pressed "⊥"shaped method. The chit42 gene was transferred into‘Bingtang’ orange by methods mentioned above and transgenic plants were confirmed by PCR analysis, the transformation rate raised to 7.6%.