Towill L E, Mazur P
The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.
Plant Physiol. 1976 Feb;57(2):290-6. doi: 10.1104/pp.57.2.290.
Haplopappus gracilis and Acer saccharum tissue culture cells are extremely sensitive to freezing injury, and exhibit a decrease in survival from 98% at -1 C to 4% at -3 C (Haplopappus) and 92% at -3 C to 13% at -5 C (Acer) when suspended in distilled H(2)O, seeded at -1 C, and then cooled by 0.1 C/minute. Similar results are obtained when cells are suspended in growth medium. The extent of shrinkage of cells during freezing can be duplicated by exposure of the cells to plasmolyzing solutions of nonpenetrating substances (Delta T(f) = 1.86 phivm). Solutions of sucrose and glycerol that produce extensive plasmolysis cause a decrease in survival within 3 to 5 minutes at room temperature, and the higher the molality to which the cell is exposed the greater the injury. Also, the rate of rehydration of the plasmolyzed cell and of the frozen cell affects its survival, with the slower rate being more beneficial. The close correlation between the decrease in survival at subzero temperatures and the decrease in survival when cells are placed in solutions having osmolalities, which could produce the same extent of shrinkage as these killing temperatures, suggests that this shrinkage is related to freezing injury in tissue culture cells.
细叶单冠菊和糖槭的组织培养细胞对冻害极为敏感，当悬浮于蒸馏水中、在-1℃接种并以0.1℃/分钟的速度冷却时，细叶单冠菊的存活率从-1℃时的98%降至-3℃时的4%，糖槭的存活率从-3℃时的92%降至-5℃时的13%。当细胞悬浮于生长培养基中时也会得到类似结果。细胞在冷冻过程中的收缩程度可通过将细胞暴露于非穿透性物质的质壁分离溶液（ΔT(f)=1.86φvm）来重现。能引起广泛质壁分离的蔗糖和甘油溶液在室温下3至5分钟内会导致存活率下降，细胞暴露于其中的重量摩尔浓度越高，损伤越大。此外，质壁分离细胞和冷冻细胞的复水速率会影响其存活率，速率越慢越有利。零下温度下存活率的降低与细胞置于具有与这些致死温度相同收缩程度的渗透压溶液中时存活率的降低之间的密切相关性表明，这种收缩与组织培养细胞的冻害有关。
