Source: Robert L. Anderson, USDA Forest Service, Bugwood.org
(Creative Commons Attribution 3.0 License)
Four different options have been pursued for chestnut restoration:
finding the few naturally-resistant trees still surviving in the woods, with just American chestnut genes, then growing new stock from their nuts (and grafts) to ensure the resistance is transmitted to future generations
using the latest laboratory techniques to insert blight-resistant genes into the Castanea dentata genome, developing a genetically-modified organism (GMO) that could live in the forest and survive the fungus
breeding a blight-resistant hybrid tree, mixing genes of the Chinese chestnut species (Castanea mollissima) with the American chestnut
biocontrol by introducing a virus, bacteria, or other fungi that weakens the Cryphonectria parasitica fungus, allowing chestnuts to resist the attack
First Option: Finding Naturally-Resistant "All American" Trees
The effort to find an all-American tree, naturally resistant to the blight, has been led by the American Chestnut Cooperators' Foundation based at Virginia Tech in Blacksburg. Seeds and grafts from relatively-healthy trees throughout the natural range of the chestnut have been planted near Mountain Lake, at the Blacksburg Airport, in Lesesne State Forest (Nelson County) and in other locations near Virginia Tech. In 2012, the foundation also had growers in 28 states and Canada.1
However, it appears those trees lack the ability to pass along their resistance to future generations. The largest "champion" chestnut in Virginia, located on a pasture fenceline in Amherst County, apparently lacks the ability to transmit its good fortune to its offspring. The healthy trees may have been located in areas that received fewer-than-average fungal spores, perhaps because of wind patterns. If that is the case, then trees may not be resistant, but instead are trees that by chance were not heavily infected.
An alternative explanation: the trees may have been infected by a hypovirulent (weakened) form of the fungus, so natural resistance was sufficient to fight off the blight. A virus does affect the fungus, weakening it - and chestnuts in Europe have recovered somewhat, as a result of this pattern. One dream of chestnut restoration specialists is that a hypovirulent form of the Cryphonectria parasitica fungus could out-compete the current form in the wild, allowing American chestnuts with pre-1904 genes time to grow wound tissue and resist the attack. In such a scenario, the native chestnut could recover naturally, without a massive re-introduction effort.2
A third possibility is that the healthy trees have some genetic pattern that provides resistance to the blight, but the genes are not expressed fully in the offspring (F1) generation. In that case, breeding healthy trees to each other will not create a new generation of blight-resistant chestnuts.
old chestnut tree, in full growth form
Source: US Department of Agriculture, PLANTS Database (Image by John Foley. Provided by National Agricultural Library. Originally from US Forest Service. United States, MD. 1901)
Second Option: Creating a New Genome
The second option - direct intervention in the genetic pattern of the chestnut - is being pursued at the State University of New York, College of Environmental Science and Forestry and with numerous cooperators. National Science Foundation grants are supporting the research, with the expectation that the results will assist in tackling other diseases in other species within the Fagaceae plant family, including beeches and oaks.3
Genetic modification requires far less time than standard plant breeding practices, since it takes at least seven years to grow a chestnut to maturity and collect pollen/nuts for a new generation. However, the chestnut tree turned out to be a difficult species to manage in a laboratory setting. It is relatively challenging to generate a seedling from a mass of chestnut cells, genetically modified or not.
The genes located naturally within the chestnut nucleus that allow Asian trees to resist the fungus are still not well understood. Isolating the gene in Chinese chestnuts which is expressed and creates blight resistance was an option, but scientists chose another path. Instead of transferring those Asian genes into the American species, much chestnut GMO research is focused on transferring a well-understood natural gene from wheat that produces the oxalate oxidase enzyme. The resulting enzyme is not a pesticide, and any species that eats wheat (including humans) is already are well-adapted to the enzyme.
The oxalate oxidase enzyme disarms the blight fungus by converting its oxalate into carbon dioxide and hydrogen peroxide. The fungus shifts from being a deadly pathogen which attacks live tissue to a saprophyte which lives only on already-dead tissue. Slowing the rapid growth of the Cryphonectria parasitica fungus allows the chestnut tree enough time to create a sufficient amount of wound tissue, blocking further invasion of the tree by fungal hyphae. Several transgenic American chestnut lines have been developed at the State University of New York, College of Environmental Science and Forestry, and a new assay process allowed scientists to identify blight-resistant trees within one year of growth in a greenhouse.
Other research is based on an artificially-created gene, mimicking the capability of frogs to survive despite all the fungi surrounding them in their moist habitats. The gene manufactures antimicrobial peptides, small proteins that kill the fungus.4
If this approach is successful in creating a genetically-modified chestnut that resists the blight, there is still a major constraint in planting this new version of the chestnut in the wild. All the laboratory products will share a common genome, and would lack much of the natural genetic diversity that allows a forest to survive attacks by other microbes. In a natural setting, genetic variations provide varying resistance to a number of threats. Some trees in a forest survive different attacks, while other trees succumb - but there are always survivors. However, if chestnuts from the laboratory share resistance to blight and almost all their other genes too, then some pathogen other than Cryphonectria parasitica could wipe out all of the chestnuts at once.
In 2013, the Forest Health Initiative planted three experimental patches of chestnuts in the wild, including one location in Virginia. The three-year experiment assessed the resistance of the genetically modified chestnuts and the response of the ecosystem to the reintroduction. Experimenters speculated that using chestnuts that are all-American, without Asian genes, will minimize surprises that might develop during a wide-scale introduction, where potential impacts of "alien genes" from Asia might creating problematic interactions with other species in an American forest.
By 2019, the Darling 54 and Darling 58 transgenic lines had demonstrated resistance and the New York University was working with the American Chestnut Foundation to grow 10,000 trees. Transgenic pollen was placed on the flowers of blight-susceptible mother trees, with the expectation that perhaps half of the offspring would inherit the resistance. That mixed the genes and minimized the risk of planting a monoculture of new chestnuts with the same genome, so all the trees could be susceptible to a different pest or disease other than Cryphonectria parasitica.
In 2020, the State University of New York College of Environmental Science and Forestry sought Federal approval to plant transgenic trees from the Darling58 line in the wild. The petition asked the US Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) to declare that the blight-resistant plants did not have to be regulated, because the genetic engineering to add the capacity to produce the oxalate oxidase enzyme posed no risk.
The Animal and Plant Health Inspection Service included in its Notice of Intent to conduct an Environmental Impact Statement that the "preferred alternative" is to allow planting of Darling 58 transgenic trees in the wild, without regulations.5
A new line of transgenic, disease-resistant trees was announced in 2022. The "DarWin" line had a different version of the gene that produced oxalate oxidase (OxO), with the gene activated when blight infections occur. Getting Federal approval to plant DarWin trees in the wild will require submitting a separate application to the U.S. Department of Agriculture Animal and Plant Health Inspection Service; the request for approval of the Darling 58 kine continued without modification.
The expectation was that Federal approval for the DarWin line would be swift, once the Darling 58 approval was granted:6
...the USDA will already be familiar with OxO expressing American chestnut trees, and therefore likely only go through step one of the review process. It is unlikely they
would find a higher plant pest risk than Darling 58, since the "DarWin" trees are producing less and more regulated OxO production. So, the time for completion is expected to be 180
days post Darling 58.
The American Chestnut Foundation, founded in 1983, has been the leader in efforts to cross-breed the Chinese chestnut (Castanea mollissima) with the American chestnut (Castanea dentata). Two parent trees (named "Clappper" and "Graves") located at the Connecticut Agricultural Research Station have been the primary sources of pollen with Chinese chestnut genes that appear to provide resistance, though other parents (including "Nanking" and "Mahogany") are also involved in testing.7
Seven generations of chestnuts have been raised since the 1980's. Volunteers and staff working for the foundation climb mature trees (or get a lift in a bucket from a utility truck...) to gather the male pollen from specific trees and bag female flowers to isolate them from wild pollen. The volunteers go back up again to pollinate the female flowers with pollen from specific desired father trees, so the tree will grow a nut with genes from pre-determined parents. After a summer of growth, the nuts are harvested by volunteers/staff who go up the tree a third time - ideally, before squirrels decide the chestnuts are ripe.
By interbreeding (backcrossing) offspring of Clapper and Graves with various American chestnut parents, the American Chestnut Foundation has produced a final genome that is 15/16ths American and 1/16th Chinese, including the all-important genes for blight resistance from the Chinese ancestors but showing other characteristics (such as height at maturity) from the American ancestors. Resistance to Phytophthora pathogen is also desired.
Breeding a Chinese/American hybrid is a long-term (perhaps 100-year) project, requiring continued private support and cooperation from universities and government agencies. Creating the BC3F2 generation that is 15/16ths American and 1/16th Chinese took until 2005, when the "restoration chestnut" was finally born. Further efforts will include two more generations, one to be bred in 2014 and another in 2021, before reintroduction on a large scale starting in 2028.
Current plans are to establish numerous stands of "new" chestnuts widely throughout the Appalachians, by planting trees in each of the areas defined by the US Geological Survey 1:24,000 scale quadrangles. Planting of test plots began in 2008, with expansion to 14,000 trees planted on Appalachian Strip mines in 2014. Details of the genetics of planted samples are carefully planned and tracked. (When being grown, female flowers of key trees in the American Chestnut Foundation orchard were covered with bags - "chastity belts" - so researchers could fertilize with pollen from a specific father.) Wide distribution may lead to natural escape and growth of chestnut trees with American characteristics and blight resistance.8
The American Chestnut Foundation has distributed pollen and nuts to cooperators throughout the eastern United States, but their focus is on growing genetically-resistant trees on a research farm in Meadowview, Virginia (near Abingdon). There, the Wagner and Price research farms are stocked with thousands of chestnut trees that are watered, weeded, fertilized, studied. In many cases, the trees that have been planted are destroyed and replaced, once the evidence is clear that a particular mix of genes is not a success.
The US Forest Service has signed a Memorandum of Understanding with the American Chestnut Foundation, to govern the restoration in the forests. Starting in 2009, crossbred chestnuts were planted in National Forests in Virginia, Tennessee, and North Carolina. Though the locations are not made public in order to protect the planted seedlings, it is possible to guess at one character of each site: they are protected from deer. Excessive deer browsing will kill chestnuts faster than the blight, so it is likely that restocking efforts will be concentrated on rocky outcrops and fenced areas.
If plantings are successful over the next 50 years and the chestnut is able to regain its once-dominant position in the Appalachian forest, then there will be substantial ecological effects. Restoration may be a good thing for the chestnut species in particular and perhaps for the southern forest ecosystem as a whole, but other species will be diminished in their significance. One concern is that a genetically-improved chestnut would become an invasive species comparable to the Bradford pear, or some insects that normally feed on chestnuts may have a toxic response to the hybrid Chinese/American chestnut trees.
The American Chestnut Foundation (TACF) relies upon volunteers to plant chestnuts at its nursery on Blandy Experimental Farm, the State Arboretum of Virginia
Fourth Option: Biocontrol by Weakening the Blight Fungus
If a virus, bacteria, or other fungi weaken the Cryphonectria parasitica, then a chestnut tree might constrain canker growth and survive into maturity. Creating a "hypovirulent" fungus would alter its lethal character. At a minimum, such a virus could be introduced in breeding orchards to facilitate growing trees which might be resistant due to genetic engineering or cross-breeding.
Today, The American Chestnut Foundation is pursuing the last three strategies to breed a blight-resistant tree, in its "3BUR: Breeding, Biotechnology and Biocontrol United for Restoration" program.
The first option is not viable; no one has found naturally-resistant trees which can pass their resistance along to the next generation. The search still continues, however. In 2019, a healthy 50-year-old chestnut tree was found in Delaware in an isolated part of the Delaware Nature Society's Coverdale Farm Preserve.9
Because planting a transgenic tree in the wild could have environmental impacts, the U.S. Department of Agriculture Animal and Plant Health Inspection Service (APHIS) had to complete an Environmental Impact Statement (EIS). The State University of New York - College on Environmental Science and Forestry (ESF) triggered the process in 2020 by filing a petition to deregulate the blight-tolerant Darling 58 American chestnut.
Chestnuts are not the first tree species to go through the approval process. In 2015 the Federal government approved planting genetically-modified apple trees, after the public review process stimulated 175,000 comments. An enzyme was modified so the "Arctic apples" would be less likely to turn brown when bruised or sliced open.
Researchers also planned to obtain approval to plant chestnuts with the DarWin trait, and were examining the potential to genetically modify chinkapin, elm, and ash trees to resist diseases better.
Those opposed to introducing the genetically-modified trees into the wild argued that the potential risks were not understood well enough. The preferred alternative was to focus on restoration using offspring of surviving pure American chestnuts.
Opposition was not limited to just the Darling 58 chestnuts. The Global Justice Ecology Project objected to plans by Living Carbon to grow poplars that had genes inserted to enhance photosynthesis and thus sequester carbon faster. Living Carbon, a public benefit corporation, bypassed the review requirements of the US Department of Agriculture by using the "gene gun method" to incorporate into the tree chromosomes a trait for faster growth.
There was also opposition to a proposal to grow genetically-modified eucalyptus trees in Brazil, but by 2004 China had already planted more than a million genetically modified trees.10
Though development of chestnuts with a blight-resistance genome is a great challenge to reintroduction, ink disease caused by Phytophthora cinnamomi could still limit the ability to restore the chestnut as a major component of the Eastern deciduous forest. Modifying the complex genetic makeup of the chestnut species to address both internal threats is not yet accomplished.
One external factor is probably the most significant constraint on replanting chestnuts in the 21st Century. Deer browse so heavily on planted trees that the genome of the seedlings may not matter. If deer eat all the planted chestnuts, there will be no opportunity for new trees to demonstrate resistance to blight or ink disease.11