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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.

Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA® Temporary Immersion Bioreactors and We Vitro Containers