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ARS Home » Plains Area » Fort Collins, Colorado » Center for Agricultural Resources Research » Agricultural Genetic Resources Preservation Research » Research » Publications at this Location » Publication #403332

Research Project: Efficient and Effective Preservation and Management of Plant and Microbial Genetic Resource Collections

Location: Agricultural Genetic Resources Preservation Research

Title: Visualizing cryoprotectant permeation and location confined in plant cells and tissues

Author
item LEVINGER, N - Colorado State University
item SAMUELS, F - Colorado State University
item KRECKEL, H - Colorado State University
item PEARCE, KYLIE - Colorado State University
item Volk, Gayle

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/10/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: N/A

Technical Abstract: Cryopreservation of vegetatively propagated crop collections has become routine in many genebank and conservation programs. Although we know what 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.