Bumble bee (Bombus impatiens) survival, pollen usage, and reproduction are not affected by oxalate oxidase at realistic concentrations in American chestnut (Castanea dentata) pollen.

Transgenic American chestnut trees expressing a wheat gene for oxalate oxidase (OxO) can tolerate chestnut blight, but as with any new restoration material, they should be carefully evaluated before being released into the environment. Native pollinators such as bumble bees are of particular interest: Bombus impatiens use pollen for both a source of nutrition and a hive building material. Bees are regular visitors to American chestnut flowers and likely contribute to their pollination, so depending on transgene expression in chestnut pollen, they could be exposed to this novel source of OxO during potential restoration efforts. To evaluate the potential risk to bees from OxO exposure, queenless microcolonies of bumble bees were supplied with American chestnut pollen containing one of two concentrations of OxO, or a no-OxO control. Bees in microcolonies exposed to a conservatively estimated field-realistic concentration of OxO in pollen performed similarly to no-OxO controls; there were no significant differences in survival, bee size, pollen use, hive construction activity, or reproduction. A ten-fold increase in OxO concentration resulted in noticeable but non-significant decreases in some measures of pollen usage and reproduction compared to the no-OxO control. These effects are similar to what is often seen when naturally produced secondary metabolites are supplied to bees at unrealistically high concentrations. Along with the presence of OxO in many other environmental sources, these data collectively suggest that oxalate oxidase at field-realistic concentrations in American chestnut pollen is unlikely to present substantial risk to bumble bees.

Transgenic American Chestnuts Do Not Inhibit Germination of Native Seeds or Colonization of Mycorrhizal Fungi.

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.

Chestnut, American (Castanea dentata (Marsh.) Borkh.).

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable markers, a very robust DNA delivery system, and a reliable regeneration system. The most important components of this transformation protocol for American chestnut are the following: starting out with rapidly dividing somatic embryos, treating the embryos gently throughout the Agrobacterium inoculation and cocultivation steps, doing the cocultivation step in desiccation plates, and finally transferring the embryos into temporary-immersion bioreactors for selection. None of these departures from standard Agrobacterium transformation protocols is sufficient by itself to achieve transgenic American chestnut, but each component makes a difference, resulting in a highly robust protocol. The average transformation efficiency that can be expected using the described protocol is approximately 170 stable embryogenic transformation events per gram of somatic embryo tissue, a considerable improvement over the 20 transformation events per gram we reported in 2006 (Maynard et al. American chestnut (Castanea dentata (Marsh.) Borkh.) Agrobacterium protocols, 2nd ed., 2006). We have regenerated nearly 100 of these events, containing 23 different gene constructs, into whole plants. As of the fall of 2013, we had a total of 1,275 transgenic chestnut trees planted at eight locations in New York State and one in Virginia. Based on a combination of field-trial inoculations, greenhouse small-stem inoculations, and detached-leaf assays, we have identified three transgenes that produce stronger resistance to chestnut blight than non-transgenic American chestnut. Depending on the transgene and the event, this resistance can be either intermediate between American chestnut and Chinese chestnut, approximately equal to or even higher than the resistance naturally found in Chinese chestnut.

Comparisons of ectomycorrhizal colonization of transgenic american chestnut with those of the wild type, a conventionally bred hybrid, and related fagaceae species.

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 against Cryphonectria parasitica without 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.

A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic American chestnut (Castanea dentata) leaf bioassay.

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.