American chestnut (Castanea dentata) was transformed with a wheat oxalate oxidase (oxo) gene in an effort to degrade the oxalic acid (OA) secreted by the fungus Cryphonectria parasitica, thus decreasing its virulence. Expression of OxO was examined under two promoters: a strong constitutive promoter, CaMV 35S, and a predominantly vascular promoter, VspB. Oxo gene transcription was quantified by RT-qPCR. Relative expression of OxO varied approximately 200 fold among events produced with the 35S-OxO. The lowest 35S-OxO event expressed approximately 3,000 fold higher than the highest VspB-OxO event. This was potentially due to the tissue-specific nature of the VspB-controlled expression, the strength of the CaMV 35S constitutive promoter, or position effects. Leaf assays measuring necrotic lesion length were conducted to better understand the relationship between OxO expression level and the blight fungus in planta. A threshold response was observed between the OxO expression level and the C. parasitica lesion length. Five events of the 35S-OxO line showed significantly reduced lesion length compared to the blight-susceptible American chestnut. More importantly, the lesion length in these five events was reduced to the same level as the blight-resistant Chinese chestnut, C. mollissima. This is the first report on enhanced pathogen resistance in transgenic American chestnut.

Many hardwood tree species are being threatened by exotic pests, and for some, only genetic engineering can offer a solution before functional extinction occurs. An example of how genetic engineering can be a useful tool for forest restoration is the transgenic american chestnuts, which contain a wheat oxalate oxidase gene conferring resistance to the chestnut blight. Many hundreds of these trees are needed for field trials and eventual restoration plantings throughout its natural range, but production is bottlenecked because of the difficulty of making hardwood trees produce roots through micropropagation. The presence of roots and living shoot tips precede successful acclimatization of tissue culture-produced american chestnut plantlets. In these experiments, we attempted to improve the post-rooting stage of our american chestnut propagation protocol. We examined vessel type, hormone, and activated charcoal concentrations, and using a vermiculite substrate. For plantlets with the best combination of roots and living shoot tips we recommend using semisolid post-rooting medium containing 2 g·L−1 activated charcoal and 500 mg humic acid in disposable fast-food takeout containers. When using vermiculite as a substrate, adding 2.0 g·L−1 activated charcoal to post-rooting medium without a gelling agent was the preferred treatment. Improving the survival rates of the american chestnut plantlets will benefit the american chestnut restoration project by providing more plant material for both ecological studies and eventual restoration, since pursuit of a nonregulated status for these transgenic trees will require extensive field testing. These procedures may also be applicable to other difficult-to-root hardwood trees in transgenic programs, such as american butternut, white oak, and black walnut.