超低温保存诱发百子莲胚性愈伤组织延迟性细胞死亡特征

Characteristics of Cryopreservation-induced Delayed-Onset Cell Death in Embryonic Callus of Agapanthus praecox ssp.orientalis

为了探究超低温保存诱发延迟性细胞死亡(cryopreservation-induced delayed-onset cell death, CIDOCD)对冻后细胞活性的影响,本研究以百子莲(Agapanthus praecox ssp. orientalis)胚性愈伤组织为试验材料,通过TTC染色法、伊文思蓝法定性定量检测超低温保存冻后恢复培养阶段细胞活性;采用TUNEL检测分析PCD变化规律;实时荧光定量PCR检测PCD相关基因表达。结果发现恢复培养6 h较0 h细胞存活率下降约8.29%,至24 h活性逐渐回升,30 h后细胞活性再次降低(较24 h降低约10.43%)。同时检测到明显的TUNEL阳性荧光变化,PCD发生比例在恢复培养0~36 h后期增多(30 h较24 h增加11.79%左右)。表明百子莲胚性愈伤组织超低温保存冻后发生CIDOCD,影响恢复培养实际冻存效率,6 h和30 h是活性下降关键时间点。Caspase、DAD1等PCD相关基因的差异性表达也证明了延迟性PCD在恢复培养阶段的发生。本研究为进一步完善超低温保存诱发PCD的分子网络及优化冻后恢复培养基以提高冻存效果提供参考。

Inordertoinvestigatetheeffectsofcryopreservation-induceddelayed-onsetcelldeath(CIDOCD)on cell activity after cryopreservation,the embryonic callus of Agapanthus praecox ssp.orientalis was used as experimen-tal material.TTC and Evans Blue staining were used to detect the cell viability in the recovery stage after cryop-reservation.TUNEL detection was used to analyze the change of PCD.Differential expression of PCD related genes was detected by quantitative real-time PCR.The results showed that the survival rate after recovery de-creased by 8.29%at 6 h compared with 0 h.And it increased gradually at 24 h and decreased again at 30 h(about 10.43%lower than that at 24 h).Obvious TUNEL positive fluorescence was detected simultaneously,and the PCD percentage increased at the later stage of 0~36 h(30 h increased by 11.79%compared with 24 h).These results in-dicated that CIDOCD occurred after cryopreservation,which affected the actual cryopreservation efficiency in re-covery stage.6 h and 30 h were the key time points of activity decline.The differential expression of PCD related genes such as caspase and DAD1 also proved the occurrence of delayed PCD in recovery stage.This study pro-vides reference for further improving molecular network of PCD induced by cryopreservation and optimizing recovery medium to improve cryopreservation effect.