Hypocotyl derived in vitro regeneration of pumpkin ash (Fraxinus profunda)

Abstract

Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0-22.2 lM 6-benzyladenine (BA) and 0-6.8 lM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L -1 glycine (B5G) containing 6.7 lM BA, 1 lM indole-3-butryic acid (IBA), and 0.29 lM gibberellic acid (GA3). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 lM BA, 1 lM IBA, and 0.29 l MG A 3 for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 l MB A and 10 lM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 lM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod. Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.