title: Encapsulation dehydration colligative cryoprotective strategies and amplified fragment length polymorphism markers to verify the identity and genetic stability of euglenoids following cryopreservation.
abstract: An encapsulation/dehydration procedure was developed for Euglena gracilis Klebs as a 'model alga' to examine various cryoprotective regimes combined with controlled rate cooling to cryopreserve other Euglenoid taxa. Cryoprotective variables were optimised to enable reproducible growth following a combination of alginate encapsulation, sucrose osmotic dehydration, air desiccation, methanol treatment, cooling to -40 degrees C and plunging into liquid nitrogen (LN). Amplified Fragment Length Polymorphism (AFLP) analysis was adapted to: (i) verify algal identity by discriminating between different Euglenoids and (ii) examine the genetic stability of algal cultures prior to various stages of cryoprotective treatments and following exposure to LN. AFLPs were highly reproducible (> 99%) as reliable diagnostic markers, where a single DNA fragment change accounted for -0.4% of the detectable variation in an AFLP pattern. AFLP changes were detected in cryoprotective treatments following LN exposure. Successive stages of the dehydration and desiccation treatments did not accumulate AFLP changes indicating these are random events.
authors.affiliation: Plant Conservation Group, University of Abertay Dundee.
authors.forename: Keith
authors.initials: K
authors.lastname: Harding
authors.forename: Julia
authors.initials: J
authors.lastname: Miller
authors.forename: Hella
authors.initials: H
authors.lastname: Timmermann
authors.forename: Maike
authors.initials: M
authors.lastname: Lorenz
authors.forename: John G
authors.initials: JG
authors.lastname: Day
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedl
mesh_terms: D000464:Alginates / Q000737:chemistry; D054458:Amplified Fragment Length Polymorphism Analysis; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002470:Cell Survival; D015925:Cryopreservation; D003890:Desiccation; D016822:Euglenida; D014644:Genetic Variation; D005838:Genotype; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D012150:Polymorphism, Restriction Fragment Length; D058989:Vitrification
chemical_list: D000464:Alginates; D015415:Biomarkers; D006603:Hexuronic Acids; D020723:Glucuronic Acid