Optimization of Agrobacterium-mediated genetic transformation of Fraxinus nigra and development of black ash for possible emerald ash borer resistance

Emerald ash borer (EAB; Agrilus planipennis Fairmaire) is the most devastating insect pest of North American ash species, including black ash (Fraxinus nigra Marsh.). As a first step in an effort to develop transgenic black ash plants resistant to EAB, we successfully established an efficient Agrobacterium-mediated transformation system for black ash hypocotyls. Kanamycin and timentin at 40 and 300 mg L-1, respectively, were most effective to select transformed explants and control excess Agrobacterium growth. Using a plant transformation vector harboring the enhanced green fluorescent protein (eGFP) gene, the effects of Agrobacterium strain, bacterial density, and the concentration of Silwet L-77 on transformation efficiency were evaluated. The best result was obtained when Agrobacterium strain EHA105 was used at a density of OD600 = 1.0. Silwet L-77 failed to promote transformation frequency and showed an adverse effect at higher concentrations (> 0.015%). Using this optimized transformation system, transgenic black ash shoots expressing a synthetic Bacillus thuringiensis toxin gene (cry8D2) were regenerated. Although no morphological abnormality was observed, transgenic shoots showed severe growth restriction. Three independent transgenic lines were selected for further assessment. All selected lines contained two copies of the cry8D2 gene, and the expression of the transgene was verified in transcript and protein levels. These transgenic shoots can be used for future bioassay to evaluate its efficacy against EAB.