Ishikawa Masaya, Suzuki Mitsuteru, Nakamura Toshihide, Kishimoto Tadashi, Robertson Albert J, Gusta Lawrence V
Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan 305-8602.
Ann Bot. 2006 Mar;97(3):453-9. doi: 10.1093/aob/mcj049. Epub 2006 Jan 3.
Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival.
Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments.
Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant.
The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.
冷冻保存是一种保存植物细胞培养物及其遗传完整性的实用方法。长期以来，人们一直认为植物细胞培养物的冷冻保存最好在迟滞期末期或指数生长期早期的细胞中进行。在这些阶段，细胞体积小且无液泡。这一观点基于使用传统慢速预冻方案的研究以及通过荧光素二乙酸酯染色或2,3,5-三苯基氯化四氮唑测定法确定的存活率。在此重新审视这一经典问题，以确定使用最新开发的方案和用于确定存活率的再生测定法冷冻保存无芒雀麦悬浮培养物的最佳生长阶段。
使用三种方案对不同生长阶段的细胞进行冷冻保存：慢速预冻、快速预冻和玻璃化。还确定了细胞渗透压、含水量以及对冷冻、热和盐胁迫耐受性的阶段依赖性趋势。在所有情况下，通过处理后细胞的再生来测定存活率。
与玻璃化方法相比，慢速预冻和快速预冻方案导致更高的细胞存活率。对于所有使用的方案，使用指数生长期后期或稳定期早期的细胞可获得最佳再生，而迟滞期末期或指数生长期早期的细胞存活率最低。指数生长期后期的细胞具有高含水量和高渗透压的特征，并且对冷冻、热和盐胁迫的耐受性最强，而指数生长期早期的细胞含水量低、渗透压低，耐受性最差。
结果与利用迟滞期末期或指数生长期早期的细胞进行冷冻保存的经典概念相反。冷冻保存的最佳生长阶段可能取决于所冷冻保存的物种或细胞培养物，并且每种细胞培养物都需要重新研究。冷冻保存后的阶段依赖性存活率与无芒雀麦细胞中对非生物胁迫的耐受性水平成比例相关。
