Visualizing cryoprotectant permeation and location confined in plant cells and tissues

Authors: LEVINGER, NANCY - Colorado State University; SAMUELS, FIONNA - Colorado State University; KRECKEL, HEIDI - Colorado State University; PEARCE, KYLIE - Colorado State University; Volk, Gayle

Submitted to: Acta horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2023
Publication Date: 2/27/2025
Citation: Levinger, N.E., Samuels, F., Kreckel, H.D., Pearce, K.C., Volk, G.M. 2025. Visualizing cryoprotectant permeation and location confined in plant cells and tissues. Acta horticulturae. 1421:47-54. https://doi.org/10.17660/ActaHortic.2025.1421.7.
DOI: https://doi.org/10.17660/ActaHortic.2025.1421.7

Interpretive Summary: The USDA-ARS National Plant Germplasm System maintains more than 30,000 accessions as plants in the field, greenhouse, screenhouse or tissue culture. These collections of plants are often referred to as the clonal collections because they are propagated using vegetative techniques, such as grafting, rather than by seeds. Cryopreservation technologies are used at the USDA-ARS National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado to secure clonal collections in liquid nitrogen. Many collections are backed up as shoot tips using vitrification cryopreservation methods. These methods rely on concentrated solutions of cryoprotectants containing dimethyl sulfoxide, ethylene glycol, glycerol and sucrose to remove freezable water, increase solute concentrations and protect membranes and proteins. This manuscript summarizes recent discoveries about cryoprotectant localization within plant cells using Coherent anti-Stokes Raman scattering (CARS) microscopy. Cryoprotectants are found to be membrane permeable and non-uniform in their tissue and subcellular localization.

Technical Abstract: Cryopreservation of vegetatively propagated crop collections has become routine in many genebank and conservation programs. Although we know which cryopreservation agents (CPAs) work for some samples, commonly used solutions, such as PVS2 and PVS3 are not universally cryoprotective and the how these solutions protect cells and tissues from freezing damage remains elusive. Permeation by CPAs into plant cells and shoot tips has been inferred by their responses, such as plasmolysis (shrinking) and deplasmolysis (swelling), observed in brightfield microscopy studies. However, these studies do not demonstrate when CPAs enter plant materials nor where the CPAs ultimately reside in living plant cells and tissue. We use coherent anti-Stokes Raman microscopy experiments to visualize CPAs as they permeate into living plant cells allowing us to measure exactly when and where the CPA goes in the plant material. This non-invasive imaging technique detects CPA location based on the unique molecular vibrational signatures, making it possible to determine the precise time and location of the CPAs as they interact with living rice callus cells and mint shoot tips. These studies demonstrate that cryoprotectant permeation occurs on substantially faster time scales compared to cellular responses, e.g., plasmolysis and deplasmolysis, which have traditionally been interpreted as due to CPA permeation. Furthermore, these studies reveal that CPA molecules permeate virtually all cells, even those displaying no response in brightfield microscopy.