Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda.
Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35 GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7 days on Murashige and Skoog (MS) medium with 22.2 µM 6-benzyladenine (BA), 4.5 µM thidiazuron (TDZ), 50 mg L-1 adenine hemisulfate (AS), and 10% coconut water (CW) prior to transformation. Hypocotyls were transformed using 90 s sonication plus 10 min vacuum infiltration after Agrobacterium was exposed to 100 µM acetosyringone for 1 h. Adventitious shoots were regenerated on MS medium with 22.2 µM BA, 4.5 µM TDZ, 50 mg L-1 AS, 10% CW, 400 mg L-1 timentin, and 20 mg L-1 kanamycin. Timentin at 400 and 20 mg L-1 kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (β-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through fluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB.