Darling 58

Rescue and Restoration of the American Chestnut

The American chestnut was an ecologically, economically, and culturally significant tree species in the Eastern United States. In the late 1800s, the introduction of the chestnut blight fungus (Cryphonectria parasitica) from Asia decimated an estimated four billion American chestnuts. Today, this iconic species rarely reproduces in the wild and is therefore considered functionally extinct.

The American Chestnut Foundation (TACF) is leading an unprecedented mission to restore the American chestnut tree to its native range. By employing complementary methods of traditional breeding, biotechnology, and biocontrol, TACF is working to create a disease-resistant and genetically diverse population of American chestnuts.

One of TACF’s greatest achievements is the establishment of a genetically diverse, reproducing population of thousands of American chestnut trees representing hundreds of breeding lines. These trees are planted in TACF’s expansive network of germplasm conservation orchards and regional breeding and backcross orchards. We have substantially increased blight-resistance and Phytophthora resistance in these populations. The future inclusion of transgenic and/or gene-edited trees will further increase those gains.

Restoration is an incremental process which will require the integration of multiple technologies and institutions. TACF is privileged to work with a diverse range of partners, from private landowners to university research labs, who inform the direction of our scientific inquiry. This collaboration allows TACF to compare results from across the native range of the American chestnut. Through this extensive research network, transgenic methods have shown great promise for generating a blight-tolerant American chestnut tree and play an important role in TACF’s science strategy and restoration efforts.

History of the Transgenic Darling 58 American Chestnut Tree

In 2015, The American Chestnut Research and Restoration Project (TACRRP) at the State University of New York College of Environmental Science and Forestry (SUNY-ESF) created the first line of transgenic American chestnut trees and named it after an early supporter of the project, Herb Darling. Deeming it a promising path toward American chestnut restoration, TACF began formally supporting TACRRP’s work in 2015 through significant funding and research support.

The goal of the Darling line of trees was to confer blight tolerance by inserting a gene from wheat called oxalate oxidase (OxO). The OxO gene detoxifies oxalic acid produced by the chestnut blight fungus and could hypothetically prevent lethal cankers on the tree. Many “events” (random insertions of the gene into the American chestnut genome) were created using the 35s constitutive promoter which causes the OxO gene to always be “switched on” in the tree’s tissues. These different events were given names such as Darling 4, Darling 54, and Darling 58, and were tested for gene placement and OxO expression. One, named Darling 58, was selected for further lab and field testing.

Initial laboratory and greenhouse tests on Darling 58 trees (also known as D58) showed that high disease-tolerance was being conferred by the OxO gene. Extensive safety tests showed no significant difference in the Darling chestnut’s interactions with other organisms compared to American chestnut (read about Nutrition, Wildlife, Plants & Fungi), and the Darling 58 product was poised to play a significant role in American chestnut restoration.

As a result, TACRRP submitted petitions for deregulation to USDA APHIS, EPA, and FDA with the goal that the Darling tree could eventually be used in the wild to restore American chestnut populations. Based on these results and its ongoing support for biotech research, TACF extensively supported TACRRP throughout the research efforts as well as the petitions for deregulation.

Darling 58 research seedlings at Meadowview Research Farms

Watch a recording of the live December 15, 2023 Chestnut Chat webinar about Darling 58 performance issues and TACF’s decision to discontinue developing the Darling line.

Darling 58 research seedlings at Meadowview Research Farms

Watch a recording of the live September 15, 2023 Chestnut Chat webinar about Darling 58 regulatory updates on the Chestnut Chat Archives page.

Why TACF is No Longer Supporting Development of D58 or Deregulation of the Darling Line

Throughout 2023, TACF and its partners observed disappointing performance results from broad-scale field and greenhouse tests of advanced generations of Darling trees across several geographic locations. As discussed in the September 15, 2023 episode of TACF’s Chestnut Chat webinar series (Darling 58 Update), analysis indicated striking variability in Darling trees’ blight tolerance, significant losses in growth competitiveness, reduction in overall fitness including stunted growth, leaf browning and curling, and increased mortality. View an outline of the body of evidence concerning Darling’s performance issues on the Darling 58 Performance page.

In late October 2023, partners at the University of New England and University of Maine informed TACF that there appeared to be a possible mix-up of pollen early in the D58 breeding program and shared with us their results.

In November 2023, TACF independently verified that information, confirming that the OxO gene of all trees thought to be Darling 58 was on a different chromosome than expected (chromosome 4 instead of chromosome 7). We have subsequently confirmed that all the trees we and other partners have been researching since 2016 are in fact descendants of a different event in the Darling line called Darling 54 (D54).

In Darling 54, the OxO gene has been inserted into a coding region, causing a deletion of 1,069 base pairs in a salinity tolerance gene called SAL1. That research has also indicated that the homozygous state of Darling 54 (when an individual plant inherits a copy of the OxO gene from both parents, which occurs in 25% of offspring) is largely lethal, and that a great majority of homozygous offspring die in the embryonic stage.

The fact that the 35S OxO construct in D54 interrupts a known gene, and has caused a large deletion of that gene, combined with the disappointing performance of the D54 trees in the field and the observed lethal homozygosity, have led TACF to conclude that this product should not be distributed or propagated beyond permitted sites. TACF believes these genetic issues should not be proliferated into current or future restoration or commercial chestnut populations.

In addition to those baseline scientific concerns, TACF is also concerned that further production or development of D54 would decrease public acceptance of the use of biotechnology in forest health and restoration efforts. Knowingly producing and proliferating trees with genetic issues, especially after establishing that these were at least partially the result of an early error, is bound to erode public trust in both the process and the producers of such a product.

Why not then return to the “real” Darling 58? The first reason is that the Darling 58 also uses the 35S constitutive promoter which appears to result in a variety of metabolic costs to the tree and likely causes the majority of performance problems observed in the Darling line. Secondly, there are only a handful, and perhaps even only one, D58 tree(s) in existence. These trees are at either the T0 or T1 stage (the original event and first-generation offspring). If work is to start over at those early diversification stages, it makes sense to focus on new OxO lines that express the gene only in tissues infected with blight. Confining OxO expression to blight infected tissues should reduce the metabolic cost of expressing this gene, thus these new lines are more likely to have enhanced forest competitiveness.

What’s Next?

The Darling transgenic line was one of many branches in TACF’s multi-pronged approach to American chestnut restoration. A diversified portfolio of research initiatives and an extensive partner network ensures that other disease-resistant prototypes, created from breeding and biotechnology methods already in the research pipeline, can be comprehensively vetted and released in a reasonable time frame. Learn more about these projects in the Current Initiatives section below.

We remain hopeful and optimistic that restoration of the American chestnut will be achieved thanks to continued support from your membership and donations and from our dedicated network of volunteers and research partners.

Guiding Principles for Science and Restoration Efforts:

Based on lessons learned through the process of researching and testing Darling lines of trees, TACF has created the following framework to better guide future efforts.

  1. Rigorous testing for efficacy throughout the life cycle of the tree life cycle (both in the lab and greenhouse, and in the field) prior to regulatory submission
  2. Rigorous testing for plant health and environmental risks
  3. Implementation of a tree improvement cooperative structure which can facilitate shared intellectual property, provide full transparency across all members, and ensure the rigor and comprehensiveness of scientific methodology and analysis
  4. Ensuring products remain in the genetic commons as much as is possible, while also protecting the quality and integrity of a given product (for example, through material transfer agreements)

Current Research Initiatives

Traditional Breeding and Backcross Program

TACF continues to make gains in blight resistance with its traditional American chestnut hybrid breeding program by selecting backcross hybrid parents that combine high American chestnut ancestry with above-average blight resistance, and breeding these superior parents together. TACF is working with partners to develop a rapid genotyping method that will allow researchers to more efficiently predict blight resistance and to select offspring that have better blight resistance than their parents.

TACF is also investingating whether blight resistance traits of wild, large surviving American (LSA) chestnut trees can be passed to offspring and combined with other methods of increasing resistance.

Inducible OxO

Insertion of the OxO gene still holds promise as a method to increase blight tolerance in American chestnut. Expressing this gene with inducible promoters, that confine expression to only blight infected tissue, has the potential to reduce the growth and survival penalties observed with constitutive promoters. ESF researchers have developed a prototype inducible OxO line that expresses the gene only in tissues that are wounded or blight infected. In addition, our collaborators at the University of Georgia are developing founders with a blight inducible OxO gene using a variety of inducible promoters to fine tune the expression of OxO. In the next few years, we will be conducting field trials and laboratory experiments to assess the performance of these inducible OxO lines.

Stacking Inducible OxO With Backross Hybrids

TACF is also conducting experiments to combine inducible OxO expression with additional genes for blight resistance through breeding. For several years, TACF has been breeding OxO transgenic trees with American chestnut backcross hybrids and with large surviving American chestnut parents selected for increased blight resistance. Results obtained in the coming years will show whether or not there is an additive effect of stacking OxO with resistance genes from chestnut.

Inserting Resistance Genes From Other Chestnut Species

If it is determined that resistance in Chinese chestnut is additive with OxO, another potential pathway is to insert genes from Chinese chestnut directly into American chestnut using methods similar to OxO insertion. TACF has made substantial progress in identifying candidate genes from Chinese chestnut that may contribute to blight resistance. We are collaborating with researchers from Virginia Tech to validate whether these genes play a role in blight resistance.

Darling 58 FAQs

How did the Darling 58 mix-up happen?

This happened at the very beginning of the program, likely in 2016. It is a “switched at birth” scenario, where pollen which was thought to have been gathered from a Darling 58 (D58) tree was actually gathered from a Darling 54 (D54) tree. Pollen from those two originating D54 first-generation trees were the basis for all research moving forward on what has since been assumed to be Darling 58.

Why not start over with the "real" Darling 58?

The first reason not to start over with the “real” Darling 58 is that Darling 58 also uses the 35S constitutive promoter which appears to result in variety of metabolic costs to the tree and causes some or most of the performance issues we are seeing. Secondly, there are only a handful, and perhaps even only one, D58 tree in existence. These trees are at either the T0 or T1 stage (the original event or first generation). If work is to start over at those early diversification stages, it makes the most sense to focus on lines that are likely to provide better competitive performance, for example, with wound-inducible promoters.

Does TACF still support transgenics as a restoration tool?

Yes, TACF still supports transgenics as a restoration tool. The technology itself remains safe and represents one of several branches of research TACF is investigating for American chestnut restoration. While TACF believes the Darling line to be ineffective, the trees are not unsafe for the surrounding ecology. Our concerns are that we do not want the underlying genetic issues to be passed on to future generations of American chestnut restoration populations.

We also do not want a defective product released into the public domain. During this deregulation process, we have seen what appears to be an increase in overall public acceptance of these technologies. To bolster that trust, our organization, its scientists, and its advisors, therefore, must sometimes make difficult decisions which will ensure that the safest and most effective product will be what is deployed for restoration.

Does TACF continue to support transgenic research at SUNY-ESF?

TACF remains open to transparent collaboration with partners who employ safe and effective scientific methods for American chestnut restoration, including ESF.

TACF is privileged to work with many different partners throughout the native range of the American chestnut, all of whom inform the direction of our scientific inquiry. As TACF continues to strive to fulfill our mission, to restore the iconic American chestnut to its native range, it is imperative to include data and input from many credible sources, from the work of private landowners to university-level, ongoing projects.

Does this mean Darling 58 or transgenic technologies are unsafe?

Not at all. Extensive ecological and other testing has demonstrated that the Darling prototype trees do not present plant pest risks different from native chestnuts. While Darling trees would not adversely affect the natural environment, TACF has concluded that these trees are unsuitable for restoration due to their performance issues (decreased growth and fitness, increased mortality, and variable resistance). Premature distribution of this or other inferior varieties may also unfairly skew public perception against the use of biotechnology solutions to save threatened forest tree species. TACF is confident that our path will eventually yield safe and effective restoration trees.

Why did TACF find out the problems with D58 so far into the regulatory review process?

The regulatory review is focused exclusively on safety and not efficacy (i.e. how well the tree would perform in restoration populations). The studies performed on environmental safety, as well as data regarding American chestnut spread and silviculture, continue to suggest that the researched Darling line will not constitute a plant pest risk.

However, it is the producer who is in charge of ensuring that the product is effective for its intended purpose, not the regulatory agencies. This means that research into the ability of Darling 58 to survive blight and effectively compete and reproduce in a forest setting has been ongoing throughout the regulatory review. The accumulation of data concerning performance is a long-term process and can continue long after a prduct is deemed environmentally safe.

At TACF, we want to ensure that the product (trees destined for restoration efforts) are effective before they “hit the market”. We value greatly the trust granted to us by supporters which has allowed us to devote 40 years to the process, and which will take many more decades for successful restoration to be realized.

The process of science leads to learning. One of the major lessons learned from this process is the need to have field test data and results, not just lab and greenhouse data, available prior to plans for deployment and release of a given product.

Tree life spans and cycles necessitate longer-term timelines than those of most crop plants. Despite a perception that modern transgenic and other genetic engineering technologies are a “quick fix”, their application and testing remains subject to “tree time”.

What would you have done if these trees had already been deregulated and released to the public?

TACF has two tools that allow us to track and implement quality control on trees we produce and plant. The first is called the Germplasm Agreement (GPA). The GPA is a type of material transfer agreement (MTA) which is signed between TACF and someone using material from our research programs. The GPA ensures that the cooperator understands that, because the germplasm is still in a research trial, the material is not to be used for commercial purposes, and cannot be distributed beyond land which that person owns. Second, the GPA has provisions on guarantees. These research materials cannot be guaranteed for performance, and we reserve the right to recall the materials should they be found not suitable for restoration.

The second tool we have is a customized, online database called dentataBase. This tool allows us to track not only the progress of our breeding and research programs, but also the locations of trees that we have distributed as part of our research programming.

TACF is committed to ensuring any product it distributes and deploys is both safe and effective for long-term restoration goals.

Has TACF contacted regulatory agencies?

Yes, TACF has provided information to regulatory agencies regarding its concerns with Darling performance, as well as the organization’s new stance that these trees should remain under permitted research status.

What does the future of American chestnut restoration look like?

In the immediate future, American chestnut restoration will involve forest plantings of trees that incorporate various approaches to disease resistance (backcross, transgenic, gene-editing, etc.) in scientifically selected sites, and testing various treatments to overcome obstacles to growth and survival These plantings  will require collaboration with diverse partners who are prepared to take chestnut restoration through the 21st century.  

In the longer term, American chestnut restoration will involve monitoring forest plantings for flowering, reproduction, regeneration, recruitment, and population dynamics over one to two decades. It will also include monitoring sustained impacts on other plants, animals, and microbes, on soil health, and on other important ecosystem processes. We must continue to partner with diverse stakeholders who are committed to the long haul and to incremental improvement through the rest of this century and beyond.

What do we need for successful restoration?

American chestnut restoration is a much broader and more complex endeavor than developing blight-resistant varieties of trees and mass-producing them.

Successful reintroduction of self-sustaining and growing populations of our target species requires a whole-ecosystem approach. This means not only tree improvement for disease resistance and competitive ability, but also identifying and targeting optimal habitats and improving establishment techniques that protect from animal predation, novel pests and diseases, climate change, habitat degradation, and anything else that is likely to stand in the way of a seed becoming a seed-bearing tree.

The forests of eastern North America have changed drastically in a multitude of ways and will continue to change rapidly through this century and beyond. We will have to discover the role that disease-resistant American chestnuts will play in these new forests through a combination of predictive models and long-term in-forest performance trials.

Restoration is also a socio-ecological exercise, not solely biological or technical. Just as forests have and continue to change, so will the relevance of the American chestnut tree to current and future generations of people who will carry this work forward.

What has TACF learned and gained from this experience?

As in all scientific endeavors, TACF has gained much from the experience with the Darling lines. Enthusiasm for this project has expanded an already large network of engaged people with a passion for the species, both within our chapters and across the range. Their efforts have conserved dozens of diverse, wild American chestnut populations, and public support for integrated solutions to forest health issues is at an all-time high. Our diversified portfolio of research initiatives and the partners implementing them ensures that other disease-resistant prototypes – those created from breeding and biotechnology methods that are already in the research pipeline – can be comprehensively vetted and released in a reasonable time frame.

The following achievements, gained through the process of studying the Darling line, will continue to have a positive impact in our research and restoration programming.

  1. Creation of additional, better performing transgenic lines which can be used for research and deployment in the next few years (see What’s Next section above)
  2. Ongoing expansion of research and implementation partnerships
  3. Field tests with large sample size, wide geographies, and 12-month+ direct inoculations of research trees are essential to gathering appropriate performance indicators
  4. Increased acceptance of transgenic and biotechnologies for forest health applications
  5. Greater understanding of the regulatory process and its application for forest health concerns
  6. Expanded and ongoing conservation of wild-type American chestnut germplasm
  7. Deployment & improvement of high light methods for “speed breeding”
  8. Increased standardization of processes between TACF and external research locations which minimize confounding variables

I have given money to TACF specifically for D58 research. Was my money wasted?

Your donation and membership was and continues to be vital for our capacity to research transgenic technologies. That funding is what has allowed us to comprehensively review the field performance of advanced-generation Darling lines, the analysis of which has led to this unfortunate, but science-based conclusion. 

The beauty of science is that we learn and then apply those lessons to future work. See the ‘What has TACF learned’ FAQ above and the ‘What’s Next’ section for more information about how your funding will continue to be used to further restoration of the American chestnut. 

Why should I continue donating to TACF or continue my membership?

TACF is grateful for the support of their thousands of members, donors, and volunteers. We have all embarked on this journey with an understanding that working with trees requires timespans that will surpass our own lifetimes.

As outlined in our “What’s Next” section above, TACF follows a multi-pronged approach to American chestnut restoration and pursues a range of viable options simultaneously. With the disappointing realization that Darling 58 is not an effective pathway, we must now focus our efforts toward other research initiatives that show promise.

Your membership and donation dollars are more important than ever in allowing TACF and its research collaborators to develop safe and effective methods toward resoration of the American chestnut. We hope you will continue to support the exciting new and ongoing research that TACF and its partners have in the pipeline. 

 

The NY Chapter promised me Darling 58 trees... what now?

Distribution is dependent upon deregulation and then whether SUNY-ESF still chooses to distribute Darling 54/58 material at that time. TACF does not support further development or deregulation of the Darling line of American chestnuts because, in our opinion, they have unacceptable performance and underlying genetic issues that make them unsuitable for release and restoration.

Can I still get a Darling 58 tree?

The Darling tree is still pending approval by regulatory agencies. There is currently no information about whether trees from the Darling line will be distributed if they reach deregulated status.

TACF’s position is that the Darling line is ineffective as a restoration tree, and may compromise future generations of disease-resistant American chestnut populations. We are researching other transgenic lines that show promise and, as always, we encourage you to plant wild-type American chestnuts, assist with the planting and research efforts of our Chapters as a volunteer, and to become a member of The American Chestnut Foundation to have first-access to the most advanced populations of disease-resistant material.   

Can I still get pollen from D58? 

Due to performance concerns, likely caused by a constitutive promoter used to express the OxO gene in the Darling transgenic American chestnut, TACF is no longer planning to incorporate any Darling trees in our species restoration efforts.

The Darling tree also has yet to be deregulated (a step that TACF no longer endorses) and the original D58 event is at a very early stage of production. If we have to start at such an early stage of research and diversification of a transgenic product, we believe that our efforts would be much more efficiently placed on events with different genetic constructs. TACF looks forward to focusing on current and new alternative approaches.

What do we do with all these GCOs/wild-type American chestnut plantings?

One of the greatest achievements of The American Chestnut Foundation (TACF) is the establishment of a broadly diverse, sexually-reproducing population of American chestnuts containing thousands of trees and representing hundreds of breeding lines. 

Please continue to plant and care for these trees! Our ongoing efforts are dedicated to achieving enhanced disease-resistant American chestnuts. These wild trees and germplasm conservation orchards (GCOs) will continue to play a pivotal role in testing and research, as well as the ultimate reintroduction of a robust and diverse population back into the native range. Along with their potential outreach benefits, these plantings are vital for preserving genetic diversity, which is essential for the long-term survival and adaptation of the American chestnut. For more detailed information on our current scientific strategies, please refer to the Science Strategy page.

Glossary

Expand the glossary below to see a list of terms and acronyms relating to Darling 58 research and deregulation.

Glossary of Darling 58-related Acronyms and Terms

APHIS – Animal and Plant Health Inspection Service – A unit of the U.S. Department of Agriculture responsible for protecting animal welfare, animal health, and plant health.

Backcross breeding – a technique to develop disease resistance wherein American and Chinese chestnuts are bred together and their progeny are bred with American chestnuts to produce a mostly American chestnut with traits of resistance

B3F3 (or similar conventions) – common nomenclature to refer to 7th generation backcrossed progeny of American and Chinese chestnuts by TACF

BRAG – Biotechnology Risk Assessment Grant – A funding program of the U.S. Department of Agriculture’s National Institute of Food and Agriculture intended to support generation of scientific evidence needed to assess affects of introduction of genetically engineered organisms

CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats – a technique for gene editing and insertion

Cryphonectria parasitica – The scientific name of the fungus that causes chestnut blight

Common garden experiment – A type of experiment used to compare how organisms from geographically or environmentally distinct sources respond to living and growing in the same environment. This helps parse their apparent differences into those driven by genetics and those driven by their environment.

Constitutive promoter – A promoter is a DNA region that “switches on” a gene. Technically it initiates “transcription” which is the process of creating a complimentary strand of RNA which then makes a protein (for example, the enzyme oxalate oxidase). A “constitutive” promoter is one that is always “switched on” meaning the gene is expressed at all times in all tissues.

D58 – Darling 58 – transgenic variety of American chestnut developed at SUNY-ESF

DarWin – Darling with Wound Inducible promoter. A transgenic chestnut tree which expresses the same OxO gene as D58, but only when wounded and only at the site of wounding, developed at the Powell lab at SUNY-ESF

Deregulation – the current process of submitting appeals to the USDA, EPA, and FDA to allow wider dispersal and expanded trials of the transgenic D58 chestnut tree

EPA – Environmental Protection Agency

FDA – Food and Drug Administration

FHI – Forest Health Initiative

GCO – Germplasm Conservation Orchard – an orchard where seeds from wild-type American chestnuts are planted to preserve their genetic diversity

Hypovirulence – a phenomenon wherein the blight fungus is weakened by infection with a virus. Depending on the interaction and relative virulence of the fungus and virus, the fungus may exhibit slower growth, reduced virulence and overall reduction in fitness.

MOU – Memorandum of Understanding – an agreement between TACF and a partner organization which documents the goals and general terms of that partnership

OxO – Oxalate oxidase – The terms commonly applied to a gene which produces the enzyme oxalate oxidase, which neutralizes oxalic acid. In American chestnut restoration context, the OxO gene is one of the most promising to confer resistance to the chestnut blight fungus.

PRR – Phytophthora root rot/ink disease

Phytophthora cinnamomi – the scientific name for the organism which causes Phytophthora root rot

Pure/purity – a commonly misconceived quality attributed to wild-type American chestnuts. All chestnut species have experienced hybridization events

Reciprocal transplant experiment – A set of replicated common garden experiments where organisms from all source populations are compared in each other’s and in their own habitats of origin. This helps uncover whether particular populations are well adapted only to their own habitats or in other habitats as well.

RNAi – In the context of American chestnut restoration, this is a method whereby partial sequences of important pathogenicity genes from the chestnut blight fungus are inserted into American chestnut. The partial fungal sequences are expressed in the chestnut tree as double stranded RNA, which are then degraded by the chestnut trees through a pathway called RNA interference. The degradation of the key fugal pathogenicity genes by the host reduces the chestnut blight fungal virulence. Thus far ESF researchers have used this technology to silence chestnut blight genes involved in the production of oxalic acid, which reduces the chestnut blight fungus’s ability to kill tissues in chestnut bark.

ROC – Regional Outreach Coordinator – employee of TACF responsible for oversight of chapters and volunteer initiatives in their respective region

RSC – Regional Science Coordinator – employee of TACF responsible for oversight of scientific endeavors in their respective region

RSC – Resistance Screening Center – a US Forest Service facility at Bent Creek Experimental Forest, Asheville, NC

S&T – Science & Technology Committee of the Board of Directors of The American Chestnut Foundation.

SSA – Small stem assay. An inoculation trial performed on seedling trees wherein the pathogen is artificially administered in order to compare differences in apparent response for early screening of disease resistance.

Stacked resistance – a technique utilizing the best progeny of the backcross breeding program to then cross with transgenic American chestnut to yield an offspring with resistance characteristics from both techniques

SUNY-ESF – State University of New York College of Environmental Science and Forestry
Transgenic – modification of an organism using an inserted gene that comes from a different organism that is not sexually-compatible with the target organism

TACF – The American Chestnut Foundation

USDA – United States Department of Agriculture

Wild-type American chestnut – a tree which is either a member of the remnant population of American chestnuts in the wild, or progeny thereof.