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.
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).
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