Cryopreservation of Desiccation-Sensitive Axes of Camellia sinensis in Relation to Dehydration, Freezing Rate and the Thermal Properties of Tissue Water

Abstract

The inter-relationships between water content, rate of freezing to −196 °C, thermal properties of water, and survival were studied in excised embryonic axes of tea seeds. Three freezing rates were used: 10°C min −1 , about 200°C min −1 and rapid freezing (attained by plunging samples into nitrogen slush). Differential scanning calorimetry yielded three categories of melting endotherms: those without endothermic peaks (no freezable water), those with a broad endothermic peak with onset temperature varying with water content, and those with an additional sharp peak at about −2°C. When axes were cooled slowly, the sharp peak was present at high water contents (> 1.8 g H 2 O g −1 dw) and was diminished as axes were dried. The sharp peak was not apparent when axes were subjected to rapid freezing. Axes at moisture contents and subjected to freezing rates such that the sharp peak was present in the melting endotherms did not survive in tissue culture. Axes rapidly dried to water contents between 1.1 and 1.6 g H 2 O g −1 dry mass prior to rapid freezing showed 100 % survival in tissue culture. Ultrastructural studies of freeze fractured replicas showed considerable freezing damage at intermediate freezing rates, but good preservation of subcellular detail under rapid freezing conditions. It is suggested that the sharp peak observed on melting endotherms represents the melting of pure water arising from ice crystals formed during freezing. If axes with a sufficiently high water content are cooled at a freezing rate that prevents the formation of large ice crystals, damage is minimized and survival enhanced. If axes are dried to water content close to the level of non-freezable water, the additional stress of freezing is deleterious.