Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

Abstract

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.