Abstract
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 OD
600 = 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.
Keywords
Black ash,
Emerald ash borer,
Fraxinus,
Genetic transformation
Citation
Lee, Jun Hyung; Pijut, Paula M. 2018. Optimization of
Agrobacterium-mediated genetic transformation of
Fraxinus nigra and development of black ash for possible emerald ash borer resistance. Plant Cell, Tissue and Organ Culture (PCTOC). 134(2): 217-229. https://doi.org/10.1007/s11240-018-1414-9.