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.