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

Authors: PEARCE, K - Colorado State University; SAMUELS, F - Colorado State University; Volk, Gayle; LEVINGER, N - Colorado State University

Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2024
Publication Date: 7/2/2024
Citation: Pearce, K.C., Samuels, F., Volk, G.M., Levinger, N.E. 2024. Direct evidence that cryoprotectant mixtures facilitate individual component permeation into living plant cells. Cryobiology. 116. Article e104928. https://doi.org/10.1016/j.cryobiol.2024.104928.
DOI: https://doi.org/10.1016/j.cryobiol.2024.104928

Interpretive Summary: Collections of clonally propagated plant genetic resources of fruits, nuts, and some vegetables are vulnerable when kept in field, greenhouse, or screenhouse conditions. When methods are available, these collections can be secured in liquid nitrogen as either dormant buds or shoot tips. For successful shoot tip cryopreservation, the cryoprotectant solution Plant Vitrification Solution 2 (PVS2) containing dimethyl sufoxide, ethylene glycol, glycerol, and sucrose is applied as part of a detailed procedure. The timeframe and extent of cryoprotectant permeation into plant cells is not known. This research monitors the real time permeation of individual cryoprotectants into rice suspension cells when they are in mixtures of solutions with other cryoprotectants. We determined 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. We also conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell membrane responses observed and therefore permeation and cell membrane response do not appear to be directly correlated.

Technical Abstract: 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 ermeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice suspension cells when in mixtures with other cryoprotectants. Specifically, we used coherent anti-Stokes Raman scattering (CARS) microscopy to observe the ermeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibration frequencies that correspond to C-D stretching modes. Additionally, we used brightfield microscopy to observe cell membrane responses to PVS2 exposure. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell membrane responses observed and therefore permeation and cell 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.