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超低温保存是一个复杂的过程,除了保存技术本身外,一些因素如材料的选择及细胞生理状态的调控等也直接影响着保存后细胞的存活与再生。本文以芒果子叶诱导的胚性培养物和愈伤组织细胞为试材,采用组织学观察、流式细胞分析和冷冻切片荧光观察等手段比较了其在超低温保存过程中的存活特性。结果表明胚性培养物细胞与愈伤组织细胞相比具有明显不同的细胞学特性,从而导致其在超低温保存过程中具有很强的耐脱水和低温能力,脱水处理30 min及超低温保存后存活率分别为96.7%和94.7%,而愈伤组织存活率则为0。利用二乙酸荧光素(FDA)荧光染色法和冷冻切片技术,观察到超低温保存后胚性培养物内存活细胞的分布。本研究结果证明试材细胞的生理状态对超低温保存的成功具有重要的影响,对未来如何更准确地评价超低温保存的效果和确定最佳的超低温保存程序也具有重要意义。
Cryopreservation is a complex process. In addition to the preservation of the technology itself, a number of factors, such as the choice of materials and the regulation of cellular physiological status, directly influence the survival and regeneration of the cells after preservation. In this paper, embryogenic cultures and callus cells induced by mango cotyledons were used as materials to compare their survival characteristics during cryopreservation with histological observation, flow cytometry analysis and frozen section fluorescence observation. The results showed that embryogenic cultures had significantly different cytological characteristics compared with callus cells, which resulted in their ability of dehydration and hypothermia during cryopreservation. After 30 min of dehydration and survival after cryopreservation 96.7% and 94.7% respectively, while the callus survival rate was 0. The distribution of viable cells in embryogenic cultures after cryopreservation was observed using fluorescein diacetate (FDA) fluorescence staining and cryosectioning. The results of this study demonstrate that the physiological state of the test cell has an important impact on the success of cryopreservation, and it is of great significance for the future to evaluate the effect of cryopreservation and to determine the optimal cryopreservation procedure.