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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections

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Title: Cryoprotectants and their components induce plasmolytic responses in sweet potato suspension cells

Author
item Volk, Gayle
item Caspersen, Ann

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2017
Publication Date: 8/3/2017
Citation: Volk, G.M., Caspersen, A.M. 2017. Cryoprotectants and their components induce plasmolytic responses in sweet potato suspension cells. In Vitro Cellular and Developmental Biology - Plants. 53:363-371.

Interpretive Summary: Field-maintained genebank collections of clonally propagated crops are vulnerable to biotic and abiotic threats. Therefore, it is important that genebanks identify strategies to secure these field collections. Cryopreservation has emerged as a cost-effective strategy to conserve clonal collections for the long term. Specific genetic combinations, or cultivars, of clonally propagated crops can be cryopreserved using 1 mm vegetative shoot tips as propagules. Shoot tips are excised and treated with cryoprotectants to ensure survival after liquid nitrogen exposure. Highly concentrated vitrification solutions are cryoprotectants that remove water and prevent ice crystallization within cells after plunge-cooling into liquid nitrogen. We determined the membrane permeability of two vitrification solutions, PVS2 and PVS3, and their components, using sweet potato suspension cells as a model system. We found that ethylene glycol and DMSO were membrane permeable. In contrast, sucrose and glycerol were membrane impermeable. Because PVS2 includes both permeable (DMSO and ethylene glycol) and impermeable components (glycerol and sucrose) and PVS3 includes only membrane impermeable components (glycerol and sucrose), it is likely that the two vitrification solutions differ in their functions as cryoprotectants.

Technical Abstract: Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane-permeable or membrane-impermeable. In this work, the osmotic responses of sweet potato (Ipomoea batatas (L.) Lam.) suspension cell cultures were observed after treatment with Plant Vitrification Solution 2 (PVS2; 15% DMSO, 15% ethylene glycol, 30% glycerol, 0.4 M sucrose), Plant Vitrification Solution 3 (PVS3; 50% glycerol, 50% sucrose), and their components at 25oC and 0oC. At either 25oC or 0oC, cells plasmolyzed after exposure to PVS2, PVS3 and their components, as a result of being placed into a concentrated solution. Cells then deplasmolyzed when the plasma membrane was permeable to the solute, as water re-entered to maintain the chemical potential. Cell suspension cells deplasmolyzed in the presence of 15% DMSO or 15% ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25oC than at 0oC. According to our observations, glycerol and sucrose were membrane impermeable. Thus, PVS2 includes both permeable (DMSO and ethylene glycol) and impermeable components (glycerol and sucrose) and PVS3 includes only membrane impermeable components (glycerol and sucrose), making it likely that the two vitrification solutions differ in their functions as cryoprotectants.