Genetic transformation and regeneration of green ash (Fraxinus pennsylvanica) for resistance to the Emerald Ash Borer

Green ash (Fraxinus pennsylvanica; Oleaceae; Section Melioides), is a widely distributed native tree species, planted for timber production and popular for landscaping in North America. However, the Emerald Ash Borer (EAB) is attacking all North American ash spp. and it has become the most important pest of ash trees in North America. The objectives of this project were to develop an adventitious shoot regeneration and rooting system, and an Agrobacterium-mediated transformation system to produce green ash plants constitutively expressing a Bt toxin gene (Cry8Da) for resistance to the EAB. To achieve this goal, three experimental approaches were taken. First, an adventitious shoot regeneration and rooting protocol was developed for green ash from in vitro explants. Adventitious shoots were induced on in vitro-derived hypocotyl and cotyledon sections using Murashige and Skoog (MS) medium supplemented with 13.3 µM 6-benzylaminopurine (BA) plus 4.5 µM thidiazuron (TDZ), and 22.2 µM BA plus 4.5 µM TDZ. Second, a genetic transformation protocol for green ash hypocotyl explants using an Agrobacterium tumefaciens vector system was developed. Stably transformed plants of green ash were obtained after 12 wk from culture initiation. This transformation and regeneration protocol provides a foundation for green ash genetic improvement via genetic transformation, and will facilitate achievement of EAB-resistant ash. Studies are underway using a construct containing the cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. Finally, in an effort to reduce concerns for transgenic plant release, we are working to develop reproductively sterile green ash through manipulation of a flower development gene, the AGAMOUS (AG) homolog. An AGAMOUS homolog (FpAG1) was isolated from green ash using a RT-PCR method. RNA accumulation was higher in reproductive tissues than in vegetative tissues. Ectopic expression of FpAG1 in Arabidopsis plants strongly suggests functional homology between FpAG1 and AGAMOUS