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. Working in close collaboration with TACF, the State University of New York College of Environmental Science and Forestry (ESF) leads the biotechnology research and testing efforts. This method has proven to be one of the most successful for generating a blight-tolerant American chestnut tree. The most advanced variety, known as Darling 58, retains a majority of the adaptative traits and and ecosystem services once provided by wild American chestnuts in the eastern US. Read more about how Darling 58 will be used in TACF’s restoration efforts on the restoration page.
The Transgenic Darling 58 American Chestnut Tree
ESF has developed a transgenic American chestnut tree with enhanced blight tolerance: Darling 58. The blight tolerance of the Darling 58 tree is a result of inserting a gene from wheat called oxalate oxidase (OxO). The OxO gene detoxifies the acid produced by fungus and prevents lethal cankers on the tree, essentially allowing the tree to coexist with the blight pathogen. ESF researchers chose the OxO gene because it is commonly found in nature as a defense against pathogens, and because enzyme it produced, oxalic oxidase, has prevailing evidence of safety for human and animal health, the environment, and would not pose a plant pest risk. Darling 58 chestnut trees retain a majority of the traits found in the background parent, i.e., those trees bred for diversification with D58, and therefore may perform much like the parents.
As with any genetic trait, however, there is likely to be some variation due to epigenetic, epistatic, and environmental effects. D58 trees produced for outplanting will not be clones, and therefore their performance in the field cannot be guaranteed for either resistance or growth characteristics. Read more about the search for blight-resistant genes and the latest regulatory updates regarding Darling 58.
Watch a recording of the live September 15, 2023 Chestnut Chat webinar about Darling 58 regulatory updates on the Chestnut Chat Archives page.
Deregulation of Darling 58
We do not anticipate Darling 58 trees or its derivatives to be fully deregulated until 2025 or beyond. Here’s why.
Virtually all genetically modified organisms are regulated by the USDA, EPA, and FDA. The process by which a transgenic or genetically engineered organism is granted unregulated status takes an extensive amount of time. In addition, the case of the American chestnut tree is unique in that it will be the first instance of a transgenic plant being produced for the purpose of wide-scale distribution and restoration. For this reason, the regulatory process has been, and will continue to be, longer than most.
The USDA, EPA, and FDA are each charged with ensuring any plant released into the wild under their purview is safe and will not pose a plant pest risk to any element of the surrounding environment. A transgenic American chestnut tree represents a brand new scenario for these agencies and additional work must take place to fit this product into their process.
In order to gain non-regulated status from these federal agencies, ESF has filed a “Petition for Determination of Non-regulated Status for Blight-Tolerant Darling 58 American Chestnut (Castanea dentata)” with the United States Department of Agriculture’s office of Animal and Plant Health Inspection Service (USDA-APHIS). Approval of the petition is a critical step toward not only being able to plant Darling 58 for testing and restoration purposes, but also to create a knowledge base for the regulators to better understand these products and their purpose.
Once a pipeline for transgenic American chestnut has been set with these regulatory agencies, future products will enjoy a less onerous – yet still in-depth – regulatory process. The deregulation of D58 will be precedent-setting. Future genetically modified American chestnut products which also use the OxO gene, varieties now called DarWin and WinWin, will be more familiar to regulators and should require less time to determine plant safety and reach regulatory decision.
We anticipate that new information about the deregulation process will be available in 2023. When that happens, we will share updates with TACF members and the public. Full, non-regulated status will mean that Darling 58 and its offspring can be distributed and planted like wild-type or traditionally bred chestnut trees in unrestricted areas. Read more about the current status of the deregulation process from ESF.
When Can I Have a Darling 58 Tree?
The short answer is, we don’t know.
TACF is thrilled that its members and the general public are excited about obtaining Darling 58 plant material. It is important to understand that once Darling 58 is deregulated (which is not likely to occur until at least 2025), developing and preparing this material for distribution is a process that will take years.
As part of the regulatory process, the EPA requires a limited-release phase. During that phase a D58 can only be planted in limited locations and for limited purposes. TACF will continue research and testing during the limited-release. Eventually this will allow TACF to distribute seeds and seedlings, to its members, supporters, and the public, that are genetically diverse, have improved disease resistance, and are well suited for forest restoration.
As has always been the case, TACF will need volunteers to help with this effort. We encourage everyone who is excited about Darling 58 to become a member and keep an eye out for opportunities to assist with pollination events.
Darling 58 FAQs
When will D58 be deregulated and when can I get a tree?
Our best guess is that the regulatory agencies will make a decision for limited release in 2024 or 2025. Following that initial time period of limited release, which will focus on additional and expanded research and demonstration plantings, more wide-scale release is expected in another 2-5 years. In the meantime, we encourage you to plant wild-type American chestnuts and to become a member of The American Chestnut Foundation to have first access to the most advanced populations of disease-resistant American chestnuts available.
How "pure" do restored American chestnut populations need to be?
Rather than “pure”, the preferred term is “wild-type”. In genetics, the “wild type” represents the phenotype (what an organism looks and acts like) of a species as it occurs in nature. The primary goal of restoration is to recreate the ecosystem and economic services once provided by the species in and close to its native range.
All living chestnut species show evidence of past hybridization; in essence, there are no “pure” chestnut species. Genome sequencing has revealed that there has been gene flow among the ancient ancestors of extant Castanea species. Current day chestnut species, including American chestnut, share an average of 7%, and up to 41%, of their genetic variation with other chestnut species as result of ancient gene flow.
Hybrid swarms of trees occur all the time in nature and there is increasing evidence of hybridization throughout the tree of life. All modern Castanea species are capable of hybridization and the production of fertile offspring. Hybrids should not be immediately dismissed simply because they are hybrids. Their contribution to overall ecosystem function should be the focus of ongoing research and study.
Will pollen only be sent to those with trees proven to be wild-type American chestnuts?
Current priority for pollen distribution will be given to those who:
1. are knowledgeable and trained on pollinations; and
2. have the tree(s) logged in dentataBase
“Purity” of the American chestnut is not a requirement for pollen distribution.
Rather, trees with higher genetic diversity will be prioritized. In many cases, TACF backcross trees will be prioritized over wild-type simply because they represent high CADE background (85% – 99.9%) and have a wide range of diversity from across the native range.
Often, these trees have additional valuable genes for resistance to both chestnut blight and Phytophthora root rot. When Darling 58 pollen is crossed with a backcross tree that inherited a high percentage of its genome from American chestnut, some of the progeny will inherit all of their genome from American chestnut. TACF is currently developing cost-effective genotyping methods to select for 100% genome inheritance from American chestnut when desired.
Will only New York Chapter members have first access to Darling 58 plant materials?
There are 316 NY Chapter Members who joined prior to 2017. Those 316 NY Chapter members have been notified via multiple announcements that they will receive a seedling free-of-charge from ESF as soon as regulatory decisions make it possible to do so.
NY Members joining after 2017 will have no special access to Darling 58 material.
We encourage TACF members to join their local chapter. There are many advantages to joining a local chapter including having access to a greater diversity of wild-type American germplasm, building and expanding community and knowledge of important restoration work, and gaining knowledge regarding events coming up in your area.
Will Darling 58 trees get the blight and to what degree?
On average, the inheritance of OxO significantly increases blight resistance above the background in which it is placed. We have observed that there is significant variation in the level of resistance when OxO is bred into different genetic backgrounds.
Some D58 trees that inherited the OxO gene die from chestnut blight. Many trees appear to be highly resistant. Those which are highly resistant appear to have levels equal to that of highly-resistant Chinese chestnuts.
Will Darling 58 alone restore the American chestnut?
As described in the 3BUR framework, TACF encourages the implementation and deployment of multiple methods for American chestnut restoration in order to create the most robust and successful restoration populations. D58 is a milestone for both research and the regulatory pathway, but it is not a “final product” which will be the sole solution to the problem of American chestnut restoration.
Restoration of the American chestnut will take decades, perhaps even more than a century. During that time, current and future scientific breakthroughs will allow for integration of more robust, disease-resistant reintroduction populations of American chestnuts.
Because D58 has some limitations in both disease-resistance and growth competitiveness, additional technologies are already in the research pipeline and will be deployed once proven safe and effective.
What material is distributed via TACF's wild-type seedling sales and seed sponsor program?
A “wild-type” American chestnut tree is one that has not been intentionally hybridized with other chestnut species. Because these trees are open-pollinated, seedlings from wild-type American chestnuts may have been naturally pollinated by other chestnut species. TACF can offer no guarantees, but to the very best of our knowledge, our wild-type seedlings are American chestnuts.
TACF’s seed sponsor program distributes the best performing lineages of disease-resistant American chestnuts available. These seeds are primarily sourced from TACFs backcross seed orchards at Meadowview Research Farms and the Arboretum at Penn State. Resistance is not guaranteed, but the parents of the seed/seedlings have been tested to have measurable blight-resistance above wild-type American chestnuts.
Eventually, upon regulatory approval and under formalized agreements with SUNY-ESF, TACF intends to integrate D58-based materials into this program.
Visit the seeds & seedlings page for more information about how to get American chestnuts from TACF.
How can I help with Darling 58 and American chestnut restoration?
There are a few ways that you can support American chestnut conservation and restoration. You can become a member of The American Chestnut Foundation. You can also plant wild-type and/or backcross American chestnuts to participate in our tree breeding efforts as a citizen scientist.
Are Darling 58 trees safe?
We are continuously studying interactions between our transgenic trees and the environment and potential impacts on ecosystem and human health. A series of articles summarizing various safety tests relevant to the regulatory process were published by The American Chestnut Foundation (read about Nutrition, Wildlife, Plants & Fungi).
Is Darling 58 safe for celiacs?
People with celiac disease or other forms of gluten intolerance can eat nuts from Darling 58 chestnuts safely. The OxO gene in Darling 58 is not related to gluten and is also found in many gluten-free grains and other foods (sorghum, rice, bananas, etc.) that are regularly consumed by celiac patients. ESF used the amino acid sequence (specific arrangement of the protein) to screen against a large database of known toxins and allergens as well as theoretically predicted ones, both resulting in no matches. In addition, Darling 58 chestnuts have been tested by a commercial testing lab for both wheat allergens in general and gluten specifically. Those tests all came back negative.
Are Darling 58 trees hybrids?
No, Darling 58 American chestnuts are not hybrids. They retain a majority of the background tree into which they are bred, along with an extra gene, OxO, which confers some amount of blight tolerance. Hybrid chestnuts, like those produced through The American Chestnut Foundation’s backcross breeding program, are a cross between Chinese and American trees. Chestnut species freely hybridize and other hybrid varieties exist. Crossing D58 with both backcross trees and wild-type American chestnuts will be an essential part of utilizing this product for ongoing species restoration efforts.
How did Darling 58 get Its name?
The Darling American chestnut was named after Herb Darling, the founder of the New York Chapter of TACF. Herb, along with Stan and Arlene Wirsig, were the people who approached Dr. William Powell and Dr. Charles Maynard, the co-founders of this project, back in 1989 about taking a biotech approach to develop a blight-resistant American chestnut tree. Herb Darling was instrumental, providing the project with original funding and supporting ESF through the NY-TACF Chapter for the next 25 years.
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.
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. A recent development by 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.