State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.