Fraxinus nigra Marsh. (black ash; Oleaceae) is a native hardwood species in northeastern North America occurring in Newfoundland west to Manitoba and south to Iowa, Illinois, West Virginia, and Virginia (Wright and Rauscher, 1990). Black ash has significant ecological and ethnobotanical importance. The tough, durable wood is valued commercially for paneling, flooring, doors, cabinets, interior finish, and various specialty products such as traditional snowshoe frames, baskets, barrel hoops, canoe ribs, and chair seats. The seeds of black ash are an important food source for wildlife such as quail, songbirds, and waterfowl (Leopold et al., 1998), and twigs and foliage are also used as food for white-tailed deer and moose. The strongly ring-porous wood affected by ecosystem site factors (Benedict and Frelich, 2008) is preferred by Native Americans for making splints for basketry (Benedict, 2001). Black ash populations have been declining as a result of pests, disease, loss of habitat, inadequate natural regeneration, and various interacting (climatic, edaphic, and physiographic) factors (Benedict and David, 2000; Palik et al., 2011, 2012; Ward et al., 2006). The emerald ash borer (EAB; Agrilus planipennis), an aggressive exotic phloem-feeding beetle from Asia, also threatens native populations of black ash. The adult beetles feed on the foliage, but the larvae bore through the bark and into the cambial region, feeding and producing galleries that disrupt the flow of water and nutrients, eventually girdling and killing the tree. The pest is fatal to an infested tree, and at present, there are no effective means of complete eradication because the EAB is spreading throughout North America (Emerald Ash Borer, 2013; Poland and McCullough, 2006).
Changes in global climate and increased global trade of wood products also have negative impacts on forest ecosystems. Forest dynamics are constantly being challenged by the invasion of exotic pests and diseases, changes in land use, and human encroachment. Changes in disturbance regimes (i.e., flooding and fire) can also affect the dynamics of a forest ecosystem. The decline of F. nigra populations will have negative impacts on ecosystem function, because it would open up the forest floor to invaders and disrupt the hydrology of an area, since black ash is found on moist sites, such as floodplains and along streams. Crown dieback and mortality, and radial growth, of black ash have been reported in recent years and may be related to spring droughts, wetter sites, temperature, site factors, tree age, and distribution (Palik et al., 2011, 2012; Tardif and Bergeron, 1993). Flooding appears to play an important role in the ecology of F. nigra, and changes in flooding regimes and timing may impact black ash population dynamics and decrease the resilience of the ecosystem (Tardif and Bergeron, 1999).
Black ash is difficult to propagate because of irregular seed production intervals [1 to 8 years] (Gucker, 2005), its immature embryo at seed set (Vandstone and LaCroix, 1975), and complex stratification and germination requirements (Benedict and David, 2003). In vitro culture is a feasible tool for conserving, propagating, and genetically improving threatened and endangered tree species. Adventitious shoot regeneration and rooting is a prerequisite for use in genetic modification technology. Adventitious shoot regeneration has been reported for common or European ash (F. excelsior) (Hammatt, 1996; Mockeliunaite and Kuusiene, 2004; Tabrett and Hammatt, 1992), narrow-leaved ash (F. angustifolia) (Tonon et al., 2001), white ash (F. americana) (Bates et al., 1992; Palla and Pijut, 2011), green ash (F. pennsylvanica) (Du and Pijut, 2008), and pumpkin ash (F. profunda) (Stevens and Pijut, 2012). To our knowledge, there has been no in vitro propagation research published for F. nigra. The development of adventitious shoot regeneration and rooting protocols would provide a means for conservation, mass propagation, and genetic improvement of black ash. Thus, the aim of the present study was to develop a complete protocol for plant regeneration of black ash from hypocotyls for further use in genetic transformation studies.
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