Direct evidence that cryoprotectant mixtures facilitate individual component permeation into living plant cells

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.