Strategies to Cryopreserve Embryonic Axes of North American Oaks: Interactions of Moisture, Cryoprotectants and LN Exposure Rates in Quercus imbricaria

Jose Faria, Lisa Hill, Christina Walters, Tree Seed Laboratory, Federal University of Lavras, Brazil, USDA-ARS, National Center for Genetic Resources Preservation, United States

Quercus imbricaria is included in the red oak group (Lobatae) and is broadly distributed in the Midwestern US. The embryonic axes are about 1 mg dry mass and have 0.68 g H2O/g dw in the acorn. Their small size and considerable desiccation tolerance made them ideal to compare various approaches for cryopreservation. Drying over a stream of nitrogen gas for 120 min reduced axis water content to 0.15 g/g, with 55% survival following liquid nitrogen (LN) exposure. To test whether addition of cryoprotectants could improve survival, axes were bathed in Plant Vitrification Solution #2 (PVS2) for 5-60 min and tested for viability before and after LN exposure. Axes submitted to PVS2 treatments and exposed or not to LN attained survival of 100% (or close to) after two weeks of tissue culture. Differential scanning calorimetry (DSC) was used to infer water freezing and melting patterns. In axes dried without PVS2, water melted at about 178 J/g H2O and melting transitions were not observed in axes dried to less than 0.34 g/g when standard methods of 10oC/min scanning rates were used. Using similar DSC methods, the water melting signal was no longer apparent in axes soaked in PVS2 for 45 minutes. To evaluate water melting behavior during fast cooling and warming, axes were plunged into LN and scanned at 300oC/min, revealing broad endothermic events between -120 to -40oC in axes that received no cryoprotectant, which we interpreted as crystal growth during warming. The enthalpy for melting transitions was reduced to about 70 J/g H2O, but there was no effect on the water content at which the melting signal was not detected. From our collective results, we suggest that PVS2 alters the rate of lethal ice crystal formation during rapid cooling and warming allowing for greater survival of axes exposed to LN.