Articles
Visualizing cryoprotectant permeation and location confined in plant cells and tissues
Article number
1421_7
Pages
47 – 54
Language
English
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 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 bright-field 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 scattering 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 peppermint 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 bright-field microscopy.
Although we know which cryopreservation agents (CPAs) work for some samples, commonly used solutions, such as PVS2 and PVS3 are not universally cryoprotective and 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 bright-field 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 scattering 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 peppermint 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 bright-field microscopy.
Authors
N.E. Levinger, F.M.D. Samuels, H.D. Kreckel, K.C. Pearce, G.M. Volk
Keywords
cryoprotectant imaging, cryoprotectant permeation, coherent anti-Stokes Raman scattering microscopy, rice callus cells, peppermint shoot tips
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