Direct observation of common cryoprotectant permeation into rice callus by CARS microscopy

Authors: SAMUELS, F - Colorado State University; PEARCE, K - Colorado State University; SODERLUND, S - Colorado State University; STICH, D - University Of Colorado; Bonnart, Remi; Volk, Gayle; LEVINGER, N - Colorado State University

Submitted to: Cell Reports Physical Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2023
Publication Date: 7/19/2023
Citation: Samuels, F., Pearce, K.C., Soderlund, S., Stich, D.G., Bonnart, R.M., Volk, G.M., Levinger, N.E. 2023. Direct observation of common cryoprotectant permeation into rice callus by CARS microscopy. Cell Reports Physical Science. 4(7). Article e101469. https://doi.org/10.1016/j.xcrp.2023.101469.
DOI: https://doi.org/10.1016/j.xcrp.2023.101469

Interpretive Summary: Plant genebanks use cryopreservation technologies to back-up collections of plant materials that are not conserved as seeds. Shoot tip cryopreservation methods use cryoprotectant solutions with glycerol, dimethyl sulfoxide, ethylene glycol, and sucrose to prepare samples to be placed into liquid nitrogen. The permeability of cryoprotectant solution components is not well-understood. This manuscript describes the localization of cryoprotectant components within individual rice cells. It demonstrates that all the components enter within the cells on different timescales. This information helps explain the mechanism by which cryoprotectants facilitate successful cryopreservation of plant materials.

Technical Abstract: Cryoprotectants are vital to ensuring that undifferentiated plant cells and organized shoot tips can survive liquid nitrogen exposure during cryopreservation and subsequent warming. The fundamental interaction between cryoprotectants and cells and shoot tips is an understudied area and the exact mechanism for protection remains elusive. By coupling coherent anti-Stokes Raman scattering (CARS) microscopy with brightfield microscopy, we determine how quickly the widely used cryoprotectants dimethyl sulfoxide, ethylene glycol, and glycerol permeate into living plant cells and how this permeation correlates with cellular responses. Some cellular responses observed with brightfield microscopy, such as plasmolysis and deplasmolysis, occur in response to cryoprotectant permeation. The observational results from the combination of brightfield and coherent anti-Stokes Raman microscopy reported here show that cellular response and cryoprotectant permeation occur on different timescales and that cryoprotectant molecules permeate virtually all cells, even those displaying no response in brightfield microscopy.