Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra

Black ash (Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloem-feeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators to induce callus and adventitious shoot bud formation. Significant effects of explant, and plant growth regulator interactions were found. The frequency of callus formation ranged from 77.8 to 94.4% and 88.9–100% from single leaflets and intact compound leaves, respectively, with no significant difference between treatments. For adventitious shoot bud induction, however, 22.2 µM 6-benzylaminopurine (BA) combined with 31.8 µM thidiazuron (TDZ) was the best treatment regardless of the initial leaf explant type, showing 21.1 and 28.8% shoot bud induction, with 1.5 and 1.9 adventitious shoots per explant, from single leaflets and intact compound leaves, respectively. The regenerated shoot buds were elongated on MS medium supplemented with Gamborg B5 vitamins plus 2 mg L⁻¹ glycine (MSB5G), 13.3 µM BA, 1 µM indole-3-butyric acid (IBA), and 0.29 µM gibberellic acid. The elongated shoots were continuously micropropagated through nodal stem sectioning until used for rooting. An average of 85.2% of the microshoots were successfully rooted in woody plant medium containing 5.7 µM indole-3-acetic acid plus 4.9 µM IBA with a 10-day initial dark culture, followed by culture under a 16-h photoperiod. Rooted plantlets were acclimatized to the greenhouse and showed normal plant growth and development with 100% survival. This regeneration protocol would be useful for mass propagation for conservation of F. nigra and for use in genetic transformation for EAB resistance.