Thermal Analysis of the Plant Encapsulation-Dehydration Cryopreservation Protocol Using Silica Gel as the Desiccant

The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination. This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16°C for 5 h decreased bead water content from ca.75 to 28% fresh weight (3.8 to 0.4 g.g^-1 dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g.g^-1 dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. T_g mid-point temperatures ranged from -78 to -51°C (cooling) and from -88 to -54°C (warming) [at cooling and warming rates of 10 and 5°C min^-1, respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16°C). This reduces bead moisture content to a critical point (ca. 0.4 g.g^-1 dry weight) at which stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.