Agrobacterium-mediated co-transformation of American Chestnut (Castanea dentata) somatic embryos with a wheat oxalate oxidase gene

Abstract

Background American chestnut is a tree of great historical, ecological, and economical importance. It once dominated forests in eastern United States until the introduction of chestnut blight fungus (Cryphonectria parasitica) in the late 19th century. Within 50 years, C. parasitica killed almost all of the 4 billion American chestnut trees in the eastern United States. The fungus first infects wounded stem, secretes oxalic acid to decrease the pH of the infected tissue to toxic levels for the tree, but optimum for fungal enzymes, and then mycelia fans spread forming a canker which when it girdles a branch prevents water and nutrient transport, eventually killing the tree above the canker. The fungus does not infect the roots, thus allowing the growth of adventitious shoots to keep the tree alive. However, this survival is only temporary because these spouts will again get infected by the fungus and die back to the ground. It is this continuing circle that made American chestnut, once a great canopy tree, to no more than an early-succession-stage shrub today. Efforts to restore American chestnut back to its native range are currently being made. In our labs, we use an Agrobacterium-mediated co-transformation system to deliver potential resistant genes into somatic embryos of American chestnut together with a selectable marker gene. The gene of interest we used is an oxalate oxidase gene from wheat (Triticum aestivum). Oxalate oxidase (OxO) can degrade the fungus secreted oxalic acid to carbon dioxide and hydrogen peroxide. This action has a duel-effect: bringing up the pH of the infected sites thus neutralizing the virulent effect of oxalic acid, and increasing the expression of defense related genes induced by hydrogen peroxide byproduct. The selectable marker we used is Green Florescent Protein (GFP) from Aequorea victoria, which allows us to have a direct visual selection of successful transformants.