Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay:: A novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Post-storage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.