Department of Chemistry, Colorado State University, Fort Collins, CO, 80523, USA.
Department of Chemistry, Colorado State University, Fort Collins, CO, 80523, USA.
Cryobiology. 2024 Sep;116:104928. doi: 10.1016/j.cryobiol.2024.104928. Epub 2024 Jul 2.
The fundamental interactions between plant cells and cryoprotectants during vitrification are understudied in the field of plant cryopreservation. Within this area of research, real time cryoprotectant permeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice callus cells when in mixtures with other cryoprotectants. Specifically, we use coherent anti-Stokes Raman scattering (CARS) microscopy to observe the permeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibrational frequencies that correspond to C-D stretching modes of the cryoprotectant molecules. Additionally, we measure cell plasma membrane responses to PVS2 exposure using brightfield microscopy. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell plasma membrane responses observed and therefore permeation and cell plasma membrane response do not appear to be directly correlated. In addition, we observe that cryoprotectant permeation into plant cells occurs more quickly and more uniformly when cryoprotectants are in PVS2 solution than when they are in single component aqueous solutions.
在植物冷冻保存领域，植物细胞与冷冻保护剂之间的基本相互作用在很大程度上仍未得到研究。在该研究领域中，实时记录冷冻保护剂渗透进入植物细胞的情况则更为少见。在本研究中，我们监测了在与其他冷冻保护剂混合的情况下，个别冷冻保护剂进入水稻愈伤组织细胞的实时渗透情况。具体而言，我们使用相干反斯托克斯拉曼散射（CARS）显微镜，通过探测与冷冻保护剂分子的 C-D 伸缩模式相对应的振动频率，来观察 individually deuterated DMSO、ethylene glycol 和 glycerol 在植物玻璃化溶液 2（PVS2）中的渗透情况。此外，我们还使用明场显微镜测量细胞质膜对 PVS2 暴露的反应。我们得出结论，PVS2 成分渗透进入植物细胞的速度快于观察到的第一个细胞质膜反应，因此渗透和细胞质膜反应似乎没有直接关联。此外，我们观察到，当冷冻保护剂处于 PVS2 溶液中时，它们进入植物细胞的速度更快且更均匀，而当它们处于单一成分水溶液中时则不然。
