Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

Abstract

An efficient vitrification procedure was developed and successfully applied to cryopreserve six endangered West Australian species (family Haemodoraceae: Anigozanthos humilis ssp. chrysanthus Hopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectans Hopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopper and C. wonganensis Hopper). Species were initially evaluated for cryostorage using a basic vitrification protocol involving: culturing plantlets in vitro for 21d; excision of shoot apices; preculture of apical tips on 0.4M sorbitol for 2d, followed by incubation in PVS2 (plant vitrification solution 2) for 25min at 0°C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1min in a 40°C water bath. Using this protocol five of the six species exhibited low post-storage survival, while the sixth species, A. viridis ssp. terraspectans posted higher survival (61.1%). Using A. viridis ssp. terraspectans as an indicator species, the initial protocol was modified to include: 3d preculture on 0.80M glycerol, loading treatment with 2.0M glycerol plus 0.4M sucrose solution for 20min, followed by 25min exposure to a modified PVS2. Survival was significantly improved in the test species, and in further experiments three other species also showed significant improvements with the new protocol. Key findings include: effectiveness of glycerol in the preculture medium; the effect of preculture duration; the importance of a loading stage for these species; and the successful use of modified PVS2 solutions with reduced or zero dimethyl sulfoxide (DMSO).