Physiological responses of Eucalyptus in vitro axillary buds to cryopreparative desiccation and osmotic preculture: Effects of abscisic acid

The vegetative tissues of most angiosperm species, including Eucalyptus grandis, cannot withstand the excessive loss of tissue water required for successful cryopreservation without incurring extensive damage. Tolerance to water loss may be increased and the critical water content of the material depressed by the application of abscisic acid (ABA) or exposure to osmotic stress. Some plants, however, respond to such treatments by becoming increasingly resistant to water loss. This study demonstrated that the responses of E. grandis in vitro axillary buds tends towards the latter, since ABA pretreatment (5 mg/l ABA for 5 days) resulted in a significantly higher water content and higher survival in ABA-pretreated buds after 20 min drying over activated silica gel (at ambient conditions) than in non-treated buds. E. grandis buds also responded positively to a combination of osmotic preculture (0.4 M, 0.7 M and 1 M sucrose and glycerol), and 5 mg/l ABA, which resulted in the maintenance of viability for significantly longer periods at reduced water contents than sucrose and glycerol preculture alone. The results (including ultrastructural observations of the mersitem dome in treated axillary buds), therefore, demonstrated the intolerance of Eucalyptus in vitro axillary buds to excessive water loss, and explained the recalcitrant nature of the buds to cryopreparative regimes, since the lowering of water content is essential for reducing the formation of potentially lethal ice crystals.