Conventional two-step cooling (Day & Brand 2005) will remain the method of choice for cryopreservation, together with high throughput methods of viability assessment, e.g. flow cytometry (Fleck et al. 2006). Alternative approaches such as vitrification using encapsulation dehydration (Harding et al. 2004) and the use of novel cryoprotectants to alleviate oxidative stress related injuries (Day et al. 2000) will be investigated and developed further for organisms recalcitrant to conventional methodologies. Where robust methods can be developed, with good levels of post-thaw viability, strains may be maintained only in a frozen state. This will greatly reduce the amount of staff-time and consumables needed for strain maintenance. Cryopreservation research into the mechanisms of freeze-induced cell injury will continue using physical methods such as cryomicroscopy (Fleck et al. 2006) and Differential Scanning Calorimitry as well as biochemical approaches focussed on free-radical injuries and oxidative stress (Fleck et al. 2000; 2003). Some of these approaches have potential for environmentally orientated research (Johnstone et al. 2006) and it is planned to expand their application during Oceans 2025. In particular, research relating to stress adaptive mechanisms in polar microalgae will be investigated with reference to their avoidance of intracellular ice formation.

There is evidence that suboptimal preservation methods may influence phenotype or productivity of metabolites (Ryan et al. 2001) and this phenomena could also occur in algae (Müller et al. submitted). Studies on the effects of optimal and suboptimal cryopreservation protocols on phenotypic and genotypic stability will be undertaken. Growth rates, metabolic status, chemotaxonomic markers (e.g. pigments) and a variety of molecular biology approaches (including AFLP) will be used as stability indices for cryopreserved and cultured strains. Viability, cellular and morphological characteristics will be tested immediately after preservation and yearly thereafter. Organisms studied for genotypic stability will include the recently-sequenced alga Chlamydomonas reinhardii CC 1690.   

link to CCAP page

References

Day JG & Brand JJ (2005) Cryopreservation methods for maintaining cultures. In: Algal Culturing Techniques. Andersen RA (ed) Academic Press, New York. p 165-87.

Day JG, Fleck RA & Benson EE (2000) Cryopreservation-recalcitrance in microalgae: novel approaches to identify and avoid cryo-injury. J appl Phycol 12, 369-77.

Day JG, Achilles-Day U, Brown S & Warren A (2006) Cultivation of algae and protozoa. In: Manual of Environmental Microbiology. Hurst CJ, Knudsen GR, McInerney MJ, Stezenbach LD & Walter MV (Eds). ASM Press, WashingtonD.C. (in press)

Day JG, Lukavský J, Friedl T, Brand J, Campbell CN, Lorenz M & Elster J (2004) Pringsheim's living legacy: CAUP, CCALA, CCAP, SAG & UTEX. Nova Hedwigia 79, 27-37

Fleck RA, Benson EE, Bremner DH & Day JG (2000) Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: A new approach for developing protistan cryopreservation strategies. Free Rad Res 32, 157-70.

Fleck RA, Benson EE, Bremner DH & Day JG (2003) Studies of antioxidant protection in freeze-tolerant and freeze-sensitive microalgae: Applications in Cryopreservation protocol development. CryoLetters 24, 213-28.

Fleck RA, Pickup RW, Day JG& Benson EE (2006) The use of flow-cytometry and cryomicroscopy to characterise cryopreservation-induced injuries in Euglena gracilis. Cryobiology (in press).

Harding K, Day JG, Lorenz M, Timmermann H, Friedl T, Bremner DH & Benson EE (2004) Introducing the concept and application of vitrification for the cryo-conservation of algae “A mini review”. Nova Hedwigia 79, 207-26.

Hoef-Emden K & Melkonian M (2003) Revision of the genus Cryptomonas (Cryptophyceae): a combination of molecular phylogeny and morphology provides insights into a long-hidden dimorphism. Protist 154, 371-409.

Johnstone C, Day JG, Staines  H & Benson EE (2006) An in vitro oxidative stress test for determining pollutant tolerance in algae.Ecological Indicators. (in press)

Müller J, FriedlT, Day JG, Harding K, Hepperle D &  Lorenz M (submitted) Assessing genetic stability of microalgae after cryopreservation using amplified fragment length polymorphism (AFLP).

Ryan MJ, Jeffries P, Bridge PD & Smith D (2001) Developing cryopreservation protocols to secure fungal gene function. CryoLetters 22, 115-24.