Submitted to: Cryobiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/21/2005
Publication Date: 2/9/2006
Citation: Volk, G.M. and C. Walters. 2006. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection. Cryobiology 52:48-61. Interpretive Summary: Genetic resources of several species of plants that are propagated vegetatively are preserved at liquid nitrogen temperatures at the National Center for Genetic Resources Preservation. Cryoprotectant solutions are routinely used to prevent lethal damage in these plant tissues during the cryopreservation procedure; however, the mode of protection is unknown. We have developed methods to investigate the effect of these solutions on cell water behavior using differential scanning calorimetry and plant shoot tips (buds grown in vitro that contain a meristem and leaf primordia). We apply these methods to study the effects of plant vitrification solution 2 (PVS2), a common cryoprotectant solution that contains 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose. We show that exposure of plant shoot tips to this concentrated solution removes water that could freeze and limits ice crystal formation during the cooling process. Ethylene glycol and dimethyl sulfoxide replace water in the shoot tips and alter the biophysical properties of the water remaining in the cells. Garlic and mint shoot tips exposed to PVS2 for 30 minutes showed no signs of freezing or melting transitions upon cooling or warming. Glass transitions occurred at –115C. Our experiments suggest a mechanism of protection that involves some dehydration, but also a structuring of water molecules to inhibit ice formation but not necessarily induce glass formation. Our methods provide needed tools to study the thermodynamic effects of other solutions purported to have cryoprotectant activity. This information will allow us to develop cryoprotectant formulations that are highly protective and have low toxicity.
Technical Abstract: Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease the likelihood of lethal ice formation, but the extent of dehydration and the effect on water freezing properties is not known. We have examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips was comparable to dilute aqueous solutions. Exposure to PVS2 caused dramatic changes in the behavior of water in shoot tips: the enthalpy of melting transitions decreased to about 40 J.g H2O-1, the amount of water that did not freeze increased to about 0.7 g H2O.g dry mass-1, and a glass transition (Tg) at –115 degrees C was immediately apparent. Evaporative drying at room temperature was also slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that PVS2 components permeate into cells and replace some of the water. Within cells, these components limit ice crystallization that would otherwise occur in unprotected shoot tips. Protection by PVS2 components occurred at temperatures in which the solution had not yet formed a glass. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for different cryoprotection solutions in diverse shoot tip systems will identify the efficacy of cryopreservation protocols.