青天葵组培物超低温保存研究

Tissue Cultures Cryopreservation of Nervilia fordii

目的用超低温方法保存青天葵组培物。方法采用单因子比较法、均匀设计法考察生长周龄、冷冻保护剂、预培养时间、降温方式、化冻方式等因素对青天葵组培物超低温保存后细胞生活力的影响。结果青天葵组培物较佳的超低温保存程序为:5周龄的组培物,在4℃进行低温锻炼12 d,以12.5%DMSO+0.25%LH为冷冻保护剂,在4℃静置处理30 m in,然后在-18℃,停留1 h后投入液氮中保存,材料取出后,在20℃化冻,无菌洗涤后,再接种,组培物能够存活并继续生长。结论超低温保存方式可以成功保存青天葵组培物种质资源。

Objective To preserve the tissue cultures of Nervilia fordii using cryopreservation method. Methods Suitable factors in cryopreservation （ - 196℃ ） were studied by monofactorial and uniform design. Results The experiments showed that five - weeked tissue cultures were the best material for cryopreservation. Tissue culures treated for 12days at 4℃ were the best cryotolerant. The optimal cyroprectants was 12.5% DMSO ＋ 0.25% LH. The best freezing procedure was to reduce the temperature from 0℃ to - 18℃ ,and stay at that temperature for lh, then drop them in liquid nitrogen（ - 196℃ ） to be preserved. 20℃ was good for melting the frozen material. Unfrozen tissue cultures survived when they were recultured . Conclusion Tissue cultures of Nervil iafordii can be preserved by cryopreservation method .