Agrobacterium -mediated genetic transformation of Fraxinus americana hypocotyls

An Agrobacterium tumefaciens -mediated genetic transformation system was successfully developed for white ash ( Fraxinus americana ) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (BA) and 0.5 µM thidiazuron (TDZ) were transformed using A . tumefaciens strain EHA105 harboring the binary vector pq35GR containing a fusion gene between neomycin phosphotransferase ( nptII) and gusA , as well as an enhanced green fluorescent protein (EGFP). Explants were transformed in a bacterial suspension with 100 µM acetosyringone using 90 s sonication and 10 min vacuum infiltration. Putative transformed shoots representing seven independent lines were selectively regenerated on MS medium with 22.2 µM BA, 0.5 µM TDZ, 50 mg L −1 adenine sulfate, 10 % coconut water, 30 mg L −1 kanamycin, and 500 mg L −1 timentin. Timentin at 500 mg L −1 was optimal for controlling excess bacterial growth, and transformed shoots were selected using 30 mg L −1 kanamycin. The presence of GUS (β-glucuronidase), nptII , and EGFP in transformed plants was confirmed by polymerase chain reaction (PCR). Reverse transcription-PCR and fluorescence microscopy confirmed the expression of EGFP. Transgenic microshoots were rooted (80 %) on woody plant medium supplemented with 4.9 µM indole-3-butyric acid, 2.9 µM indole-3-acetic acid, and 500 mg L −1 timentin, and subsequently acclimatized to the culture room. This transformation protocol provides the framework for future genetic modification of white ash to produce plant material resistant to the emerald ash borer.