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In contrast to many current applications of biotechnology, the intended consequence of the American Chestnut Research & Restoration Project is to produce trees that are well‐adapted to thrive not just in confined fields or orchards, but throughout their natural range. Our primary focus is on disease tolerance, but we believe it will also be critically important that optimal restoration trees should have robust genetic diversity and resilience, which can be supplied by a full complement of their wild‐type genes. Chestnut restoration offers a unique case study because many restoration or intervention options have been attempted: doing nothing, planting non‐native chestnut species, planting hybrids, mutagenesis (exposing seeds to high levels of radiation to induce random mutations), backcross breeding, and now genetic engineering. Any of these techniques may be advantageous independently or in combinations, depending on the specific goals of land managers or restoration practitioners, but genetic engineering offers a unique opportunity to enhance blight tolerance while minimizing other changes to the genome. [ABSTRACT FROM AUTHOR]

American chestnut (Castanea dentata [Marsh.] Borkh.) was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen, Cryphonectria parasitica (Murrill) Barr, functionally eliminated it across its range. One promising approach toward restoring American chestnut to natural forests is development of blight‐tolerant trees using genetic transformation. However, transformation and related processes can result in unexpected and unintended phenotypic changes, potentially altering ecological interactions. To assess unintended tritrophic impacts of transgenic American chestnut on plant–herbivore interactions, gypsy moth (Lymantria dispar L.) caterpillars were fed leaf disks excised from two transgenic events, Darling 54 and Darling 58, and four control American chestnut lines. Leaf disks were previously treated with an LD50 dose of either the species‐specific Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) or the generalist pathogen Bacillus thuringiensis subsp. kurstaki (Btk). Mortality was quantified and compared to water blank controls. Tree genotype had a strong effect on the efficacies of both pathogens. Larval mortality from Btk‐treated foliage from only one transgenic event, Darling 54, differed from its isogenic progenitor, Ellis 1, but was similar to an unrelated wild‐type American chestnut control. LdMNPV efficacy was unaffected by genetic transformation. Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms, this may be due to insertion effects, and variation among different genotypes (whether transgenic or wild‐type) imparts a greater change in response than transgene presence. [ABSTRACT FROM AUTHOR]

We argue that the wild release of genetically modified organisms (GMOs) can be justified as a way of preserving species and ecosystems. We look at the case of a genetically modified American chestnut (Castanea dentata) that is currently undergoing regulatory review. Because American chestnuts are functionally extinct, a genetically modified replacement has significant conservation value. In addition, many of the arguments used against GMOs, especially GMO crops, do not hold for American chestnut trees. Finally, we show how GMOs such as the American chestnut support a reorientation of conservation values away from restoration as it has historically been interpreted, and toward an alternative framework known as rewilding. [ABSTRACT FROM AUTHOR]

Outbreaks of insects and diseases are part of the natural disturbance regime of all forests. However, introduced pathogens have had outsized impacts on many dominant forest tree species over the past century. Mitigating these impacts and restoring these species are dilemmas of the modern era. Here, we review the ecological and economic impact of introduced pathogens, focusing on examples in North America. We then synthesize the successes and challenges of past biotechnological approaches and discuss the integration of genomics and biotechnology to help mitigate the effects of past and future pathogen invasions. These questions are considered in the context of the transgenic American chestnut, which is the most comprehensive example to date of how biotechnological tools have been used to address the impacts of introduced pathogens on naïve forest ecosystems. [ABSTRACT FROM AUTHOR]

Biotechnology offers a new approach for the restoration of tree species affected by exotic pathogens; however, nontarget impacts of this novel strategy on other organisms have not been comprehensively assessed. We evaluated the effect of transgenic American chestnut (Castanea dentata) leaf litter on the growth and survival of larval wood frogs (Lithobates sylvaticus), a forest‐dwelling amphibian species widely sympatric with American chestnut, that forage almost entirely on periphyton and litter detritus that accumulate in temporary vernal pools in forests. We reared wood frog larvae on Castanea leaf litter (American chestnut genetically engineered for blight tolerance, nontransgenic American chestnut, Chinese chestnut [Castanea mollissima], and an American–Chinese chestnut hybrid) and litter from two non‐Castanea, nontransgenic "control" tree species, coupled with two levels of supplementary food. We observed no differences in growth or survival of wood frog larvae reared on transgenic versus nontransgenic American chestnut leaves. Without supplementary food, wood frog larvae provided leaves from American chestnut (both types) developed faster and grew larger than those exposed to other leaf litter treatments. Results of this study provide preliminary evidence that (1) American chestnut may have formerly been an important source of food for forest‐dwelling amphibians and (2) transgenic American chestnut litter generated as part of chestnut restoration efforts is unlikely to present direct novel risks to developing amphibian larvae in the forest environment. [ABSTRACT FROM AUTHOR]

American chestnuts (Castanea dentata), effectively eliminated from eastern North America by chestnut blight in the twentieth century, are the subject of multiple restoration efforts. Screening individual trees (or tree types) for blight resistance is a critical step in all of these programs. Traditional screening involves inoculating stems of >3-year-old trees with the blight fungus (Cryphonectria parasitica), then measuring resulting cankers a few months later. A quicker, nondestructive, quantitative assay, usable on younger plants, would enhance restoration efforts by speeding the screening process. The assay presented here meets these requirements by inoculating excised leaves with the blight fungus and measuring resulting necrotic lesions. Leaves can be collected from few-month-old seedlings or fully mature trees, and results are measured after less than a week. Leaves from several lines of both American and Chinese chestnuts were inoculated, as well as the congener Allegheny chinquapin, and experimental leaf assay results correlate well with stem assay results from these species. Inoculations with virulent and hypovirulent blight fungi strains also showed relative patterns similar to traditional inoculations. Given the correlations to established stem assay results, this procedure could be a valuable tool to quickly evaluate blight resistance in American chestnut trees used for restoration. [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Over four billion American chestnut trees have been killed as a result of an introduced pathogen, the chestnut blight fungus. Recently, transgenic blight‐tolerant American chestnut trees have been produced by inserting a gene from wheat into the American chestnut genome. Pending federal approval to use these transgenic trees for large‐scale forest restoration, this would be the first instance where a transgenic approach has been used to restore a tree species that has been rendered functionally extinct by an introduced pathogen. With the help of citizen scientists, we estimate that large‐scale forest restoration using blight‐tolerant American chestnut trees is possible within the next few decades. SummaryBreeding transgenic blight‐tolerant American chestnuts with susceptible wild‐type (WT) trees is potentially an efficient method to rescue the genetic diversity and adaptive capacity of the American chestnut population for large‐scale restoration.To develop a breeding plan to diversify a transgenic blight‐tolerant population, we simulated pedigrees to estimate inbreeding coefficients and effective population size in scenarios involving outcrossing 1–4 transgenic founders to a maximum of 1,500 WT trees over 1–5 generations. We also simulated marker‐assisted introgression scenarios to minimize the extent of the transgenic founder genome, especially on the transgene carrier chromosome.Simulations suggest that the effective population size may be increased to >500, and the average inbreeding coefficient reduced to <0.01, by outcrossing a single transgenic founder over five generations to 2, 25, 50, 150, and 450 (677 total) WT parents. Three generations of marker‐assisted introgression is predicted to decrease the length of the founder genome to between 7% and 13% of the transgene carrier chromosome length as compared to 42% with event selection (ES) only. Transgenic outcross selections may be intercrossed to select progeny homozygous for the transgene for planting in seed orchards.A diversified population of transgenic blight‐tolerant American chestnut is estimated to be available for use in large‐scale forest restoration 20–35 years after federal approval to distribute the trees. In contrast, trees from earlier generations would be available almost immediately after federal approval for personal or horticultural plantings. Methods to accelerate pollen production and outcrossing are discussed. [ABSTRACT FROM AUTHOR]

Dependence on wild seed sources is often impractical for large-scale habitat restoration programs. Reliance on commercial seed supplies of unknown provenance and fitness is thereby warranted. Little consideration has been given, however, to how the large volumes of seed required should be sourced. We evaluated commercial and locally collected seed sources for potential use in a New York State-based, landscape-scale program for restoring blue lupine Lupinus perennis. Through analysis of microsatellite markers we determined that 'native' lupine designations by some commercial suppliers were in fact interspecific hybrids and therefore unreliable; at least two commercial sources, however, were genetically as close to native New York populations as native New York populations were to one other. Common garden experiments revealed that seed source influenced first-year overwintering survival and subsequent height growth of surviving plants; seed sources more closely related genetically to native New York populations survived better and produced more stems per individual in the field in the area targeted for restoration. We conclude that (1) commercial suppliers often but not always offer reliably characterized seed sources of sufficient genetic similarity to native populations to warrant their use in restoration projects and (2) genetic affinity of potential seed stock to native populations is positively related to its fitness in the environment targeted for restoration. [ABSTRACT FROM AUTHOR]

Chestnuts are important ecological forest tree species. They can form mycorrhizal connections with a variety of ectotrophic mycorrhizal fungi. Mycorrhizal hyphae can expand the absorbing area to facilitate the intake of nutrients, thereby promoting plant growth and development. In this study, a chestnut-fungus symbiosis system was established in vitro , and typical mycorrhizal structures (root tips bifurcated and apical enlargement) were found after three-month inoculation. Phosphorus content and H + efflux rate of chestnut roots increased significantly after being inoculated with Scleroderma citrinum compared to the non-inoculated control. The increased H + efflux rate provides indirect evidence for the role of ecto-mycorrhizae in increased phosphate uptake. Finally, phylogenetic tree was clustered by MEGA5.0 based on amino acid sequences of five phosphate transporters of Castanea mollissima and known phosphate transporters of other plants. The results showed that CmPT4 was in a clade with known mycorrhizal phosphate transporters, and thus this gene was preliminarily a predicted mycorrhizal phosphate transporter. Further real-time quantitative PCR showed that five phosphate transporter genes ( CmPT1, CmPT2, CmPT3, CmPT4, and CmPT5 ) were upregulated under phosphorus deficiency treatment, and only CmPT4 was induced in the mycorrhizal roots of chestnut. Therefore, It can be concluded CmPT4 is a mycorrhizal phosphate transporter gene in Castanea mollissima . This study provides a foundation for further analysis of chestnut mycorrhizal formation and phosphate uptake improvement by phosphate transporters. [ABSTRACT FROM AUTHOR]