Regeneration of Plants from Fraxinus nigra Marsh. Hypocotyls

Abstract

Fraxinus nigra Marsh. (black ash) is a native North American hardwood tree species that is ecologically important and has ethnobotanical significance to American Indian communities of the eastern United States. Black ash has immature embryos at seed set, combined with complex stratification requirements, making natural regeneration difficult. This, combined with the threat and devastation being caused by the emerald ash borer, makes an in vitro adventitious shoot regeneration and rooting protocol imperative for mass propagation, conservation, and genetic improvement of this species. Hypocotyls were cultured for 4 weeks on a modified Murashige and Skoog (MS) medium containing 13.3 μM 6-benzylaminopurine (BA) and 4.5 μM thidiazuron for adventitious shoot induction. Shoots continued to regenerate when explants were then cultured on MS medium supplemented with Gamborg B5 vitamins plus 0.2 g·L−1 glycine (B5G), 6.7 μM BA, 1 μM indole-3-butryic acid (IBA), and 0.29 μM gibberellic acid (GA3) for 4 weeks, followed by transfer to MSB5G medium with 13.3 μM BA, 1 μM IBA, and 0.29 μM GA3 for shoot elongation. Once elongated, the microshoots were continuously micropropagated through nodal sectioning, and cultured on MSB5G medium supplemented with 13.3 μM BA, 1 μM IBA, 0.29 μM GA3, and 0.2 g·L−1 casein hydrolysate. Rooting of elongated microshoots was successful using woody plant medium supplemented with 4.9 μM IBA and 5.7 μM indole-3-acetic acid with a 10-day initial dark culture for root induction followed by culture under a 16-h photoperiod. Rooted plantlets were successfully acclimatized to the greenhouse with 100% survival.