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.

The American chestnut (Castanea dentata) was once an integral part of eastern United States deciduous forests, with many environmental, economic, and social values. This ended with the introduction of an invasive fungal pathogen that wiped out over three billion trees. Transgenic American chestnuts expressing a gene for oxalate oxidase successfully tolerate infections by this blight fungus, but potential non-target environmental effects should be evaluated before new restoration material is released. Two greenhouse bioassays evaluated belowground interactions between transgenic American chestnuts and neighboring organisms found in their native ecosystems. Potential allelopathy was tested by germinating several types of seeds, all native to American chestnut habitats, in the presence of chestnut leaf litter. Germination was not significantly different in terms of number of seeds germinated or total biomass of germinated seedlings in transgenic and non-transgenic leaf litter. Separately, ectomycorrhizal associations were observed on transgenic and non-transgenic American chestnut roots using field soil inoculum. Root tip colonization was consistently high (>90% colonization) on all plants and not significantly different between any tree types. These observations on mycorrhizal fungi complement previous studies performed on older transgenic lines which expressed oxalate oxidase at lower levels. Along with other environmental impact comparisons, these conclusions provide further evidence that transgenic American chestnuts are not functionally different with regard to ecosystem interactions than non-transgenic American chestnuts.

American chestnut (Castanea dentata[Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight,Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance againstCryphonectria parasiticawithout changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.

American chestnut (Castanea dentata (Marsh.) Borkh) was almost completely wiped out by the fungal pathogen, Cryphonectria parasitica (Murrill) M.E. Barr. Another invasive pathogen, Phytophthora cinnamomi Rands, is devastating American chestnuts in the southern region of the United States. An alternative approach for controlling these pathogens is to use genetic engineering or gene editing. We successfully transformed American chestnut with a detoxifying enzyme, oxalate oxidase, to enhance blight tolerance and more recently with the Cast_Gnk2-like gene, which encodes for an antifungal protein, to be tested for P. cinnamomi putative tolerance. Eight somatic embryo lines were transformed using three methods of selection: semisolid medium in Petri plates, liquid medium in RITA^® temporary immersion bioreactors, or liquid medium in We Vitro containers. No significant differences were found between the treatments. These methods will allow for further testing of transgenes and the development of enhanced pathogen resistance in chestnut. It can serve as a model for other tree species threatened by invasive pests and pathogens.

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.

Limited rooting and acclimatization success when micropropagating certain hardwood tree species may hinder conservation efforts of certain threatened and endangered species. Restoration efforts for such trees, such as the American chestnut [Castanea dentata (Marsh.) Borkh.], require a massive number of plantlets to be produced by micropropagation for testing, initial distribution, and orchard establishment. Therefore, increasing the number and quality of lab-produced plantlets is a key research focus. After previously determining that an ex vitro rooting system produced significantly more robust plantlets, we examined extending the time in elongation medium, rooting substrates, exogenous auxin applications, root-promoting substrate soaks, submerging the cut site, and light intensity. The most effective methods included seven weeks in elongation medium, using Jiffy peat pellets soaked in water as the rooting substrate, cutting off callus while submerged, then dipping in 0.31% IBA rooting gel, and placing plantlets in low light of 60 μmol·m-2·s-1 after rooting. By increasing the number of roots and improving acclimatization success, we can ensure that many more blight-tolerant American chestnuts will be available for field studies and eventual public distribution. Demonstrating the ecological safety and blight survival of these trees will help restore this foundational tree species and assist future restoration efforts for other threatened species. Index words: Rooting, ex vitro, American chestnut, Castanea dentata, IBA, substrate. Species used in this study: American chestnut, [Castanea dentata (Marsh.) Borkh.]. Chemicals used in this study: IBA (indole-3-butyric acid).