瓯柑愈伤组织的玻璃化法超低温保存研究

Studies of cryopreservation of callus of Citrus suavissima Hort.et Tanaka by vitrification

采用玻璃化法对瓯柑愈伤组织进行超低温保存研究.瓯柑愈伤组织在含5%二甲亚砜(DMSO)的MS培养基上预培养5d,室温下60%PVS2预处理20min,然后用100%PVS2于0℃处理40min,投入液氮(LN)保存,24h后在40℃水浴中迅速化冻,再用1.2mol/L蔗糖培养液洗涤3次.用氯化三苯四氮唑(TTC)法检测,其存活率可达85.62%.冻后的愈伤组织转移到MS+6-BA0.5mg/L继代培养基上,在黑暗中培养生长,存活率可达76.32%.分析讨论了瓯柑愈伤组织玻璃化法超低温保存中存在的一些问题及意义,并对其前景进行了展望.

A procedure was presented on cryopresevation of callus of Citrus suavissima Hort.et Tanaka by vitrification. Calli were precultured for 5 days in MS medium supplemented with 5 % DMSO. Then, they were loaded with 60 % PVS_2 for 20 min at room temperature and exposed to 100% PVS_2 at 0 ℃ for 40 min. Followed by changing the solution with fresh PVS_2, the calli were immered into LN directly and keep for 24 h. After rapid thawing in a water bath at 40 ℃ , the calli were washed with 1.2 mol/L sucrose solution three times and transferred onto MS medium supplemented with BA 0.5 mg/L . The cultures were kept in dark for one week pritor to exposure to the light. Survival rate of callus was 85.62 % by TTC examination and their regeneration rates reached to 76.32 %. The significance of cryopreservation of Citrus callus was summarized.