Conservation of Green and White Ash Germplasm Using the Cryopreservation of Embryogenic Cultures

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2-3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (p < 0.05) between experiments and with genotype (p < 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (p > 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and restoration.