Application of somatic embryogenesis for development of emerald ash borer-resistant white ash and green ash varietals

Emerald ash borer (Agrilus planipennis; EAB) has devastated populations of ash (Fraxinus spp.) trees in dozens of U.S. states and Canada over the past few decades. The continued survival of scattered ash trees known as "lingering ash" in heavily infested natural stands, however, offers evidence of genetic resistance or tolerance to EAB. These surviving or "lingering" ash individuals may form the basis for reforestation programs in EAB-impacted areas, and clonal mass-propagation of these genotypes can help accelerate these efforts. Between 2013 and 2018, we initiated embryogenic cultures by culturing immature zygotic embryos from open-pollinated (OP) seeds collected from several surviving white ash and green ash trees in Michigan and Pennsylvania. In addition, in 2018, we initiated cultures from crosses made between lingering green ash parents from the USDA Forest Service ash breeding program in Ohio. Somatic embryos were produced by growing cultures in liquid suspension, followed by fractionation and plating on semisolid medium to produce developmentally synchronous populations of somatic embryos. Somatic embryo germination and conversion were enhanced by a combination of pre-germination cold treatment and inclusion of activated charcoal and gibberellic acid in the germination medium. Ash somatic seedlings derived from OP explants grew rapidly following transfer to potting mix and somatic seedlings representing nine ash clones were acclimatized, grown in the greenhouse and planted in a preliminary field test, along with EAB-resistant Manchurian ash (F. mandshurica) and EAB-susceptible control seedlings. Somatic seedlings have now been produced from cultures that originated from seeds derived from the progeny of lingering green ash parents and an ex vitro germination protocol has shown some promise for accelerating early somatic seedling growth. Results of this research could provide the basis for scaled-up production of EAB-resistant ash varieties for seed orchard production for forest restoration and cultivar development for urban tree restoration.