Preparing Plant Vitrification Solution 2
Katheryn Chen, Department of Soil and Crop Sciences, Colorado State University, 307 University Ave., Fort Collins, Colorado 80523.
Remi Bonnart, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521<EMAIL_ADDRESS>Outline
- Background
- Recipe
- Step-by-step instructions
- Variations
- References
- Additional information
- Acknowledgments
The PVS2 recipe is available to download here.
1. Background
Successful preservation of living plant tissue in liquid nitrogen (-196 oC) depends upon the ability to prevent damage to the cells. Ice crystallization that occurs during the freezing process, particularly the intracellular formation of large ice crystals, poses an acutely lethal threat. While a phase change from liquid to solid necessarily occurs during a transfer to extreme low temperatures, vitrification—the process by which liquids solidify into metastable glass—can preserve cellular integrity.
Vitrification techniques use cryoprotectants that osmotically desiccate the tissue without severely damaging it. Glycerol-based cryoprotectants, such as Plant Vitrification Solution 2 (PVS2; Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993) benefit from glycerol’s high viscosity and ability to be supercooled below -70 oC.
PVS2 was developed by Dr. Akira Sakai in the 1980s to cryopreserve 1 mm apical meristems from plants grown in tissue culture. Dr. Sakai outlines his personal experience developing PVS2 in ‘Development of PVS-Based Vitrification and Encapsulation–Vitrification Protocols’ (Sakai et al., 2008). He first tested PVS2 on nucellar cells of navel oranges, which resulted an 80% survival after rapid rewarming (Sakai et al., 1990).
The development of PVS2 as a cryoprotectant, coupled with an osmoprotection treatment (loading solution) to prepare the tissues for dehydration, was a significant in the field of clonal plant cryopreservation. This critical technology has greatly expanded the utility of meristematic cryopreservation; since its development, it has been used and adapted to a wide-ranging, diverse taxa including a variety of tropical plants (Sakai et al., 2008).
2. Recipe
PVS2 Cryoprotectant Recipe (0.4 M sucrose + ½ MS): 250 mL
- Glycerol (30% w/v) = 75.0 g
- Ethylene glycol (15% w/v) = 37.5 g or 33.7 mL
- Sucrose (0.4 M) = 34.23 g
- Dimethyl sulfoxide (15% w/v) = 37.5 g or 34.1 mL
- Murashige & Skoog basal medium with vitamins (PhytoTech Labs M519) = 0.55 g
- Bring to volume with distilled water
- Adjust pH to 5.8 with potassium hydroxide
- Filter sterilize using syringe filter or Stericup units (0.45 microns or smaller)
3. Step-by-step instructions
This section describes to how make PVS2 at the USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. This formulation has