Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP)

Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.