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

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 21 d; excision of shoot apices; preculture of apical tips on 0.4 M sorbitol for 2 d, followed by incubation in PVS2 (plant vitrification solution 2) for 25 min at 0 °C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1 min 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: 3 d preculture on 0.80 M glycerol, loading treatment with 2.0 M glycerol plus 0.4 M sucrose solution for 20 min, followed by 25 min 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).