FUNCTIONALLY EXTINCT CHESTNUTS


“…in comparison to the trees’ towering ancestors, which used to reach 80 to 100 feet tall with trunk diameters growing around 10 feet”

BACKCROSS BREEDING
https://sciencefriday.com/american-chestnut-resurrecting-a-forest-giant
https://modernfarmer.com/2021/the-great-american-chestnut-tree-revival
The Great American Chestnut Tree Revival
by Shea Swenson / December 20, 2021

“As she walks amongst the sea of green, yellow and orange leaves of a chestnut tree orchard, carefully collecting chestnut burrs from the trees, Sara Fitzsimmons, director of restoration for the American Chestnut Foundation, is aware she won’t be around to see the full fruits of her labor. The lifecycle of a tree is much too long for that. For almost two decades, Fitzsimmons has been working to revive the American chestnut (Castanea dentata), a foundational species that once dominated the Eastern forests of the United States and southern Ontario, Canada.


chestnut blight or canker (Cryphonectria parasitica) (Murrill) M.E. Barr” photo Richard Gardner. Bugwood, Creative Commons Attribution-Noncommercial 3.0

But more than a century ago, the trees were exposed to chestnut blight, an invasive pathogen that was accidentally introduced by imported Asian species of the trees, used mainly for ornamentals and in orchards. American chestnut trees, vulnerable to the fungal disease, were devastated by the blight, leaving behind forests full of toppled trees or stalks with bare, dead branches. Now, 84 percent of chestnut trees in America remain small and are unable to bear fruit.

At one point, there were anywhere between three and five billion American chestnut trees. Today, there are, at most, 435 million still alive. Organizations like the American Chestnut Foundation are working to develop a new, blight-resistant chestnut tree to reintroduce and help revive the population. The timeline? “It’s going to take us between 150 to 200 years to make an ecological impact with millions of seedlings on the landscape,” Fitzsimmons says.  Before blight, American chestnut trees bore importance not just to the ecosystem, but to humans and their way of life.

When chestnut trees were abundant, farmers could rely on their nuts as a source of nutrition for their pigs or turkeys. They also often foraged for the nuts to eat as their own food or even trade with. The wood from the trees proved sturdy for building structures, and was used for shingles, beams and flooring in homes, as well as for railroad ties and telegraph or telephone poles. “The wood was very rot resistant, we had many uses for it. It’s very good at carbon sequestration, which is important in these days with climate change. It’s a really nice habitat for wildlife. And people used to harvest chestnuts this time of year,” says William Powell, director of the American Chestnut Research and Restoration Project at the State University of New York College of Environmental Science and Forestry (SUNY-ESF).

And, of course, they were good for roasting. “You hear that song, ‘chestnuts roasting [on] an open fire,’” Powell says, referencing the classic Christmas song written in 1945 by Robert Wells and Mel Tormé. “That’s American chestnuts. So that was all lost.” City streets were once lined with carts serving up the sweet, toasty holiday treat. Although chestnuts’ association with the holidays can be traced to sixteenth century Europe, chestnuts grown in North America were known for having a richer, sweeter flavor and were especially suited as a warm treat during winter months. That all changed when blight diminished American chestnut numbers to nearly nothing.

Now, any chestnut roasting over an open fire is likely an imported one, as the US is responsible for less than one percent of the crop’s total world production. Despite the massive loss, chestnut trees aren’t technically extinct. In fact, they aren’t even on an endangered list. The blight fungus can’t attack the trees’ root systems due to microorganisms in the soil that act as a protectant. This results in a unique ability for the American chestnut trees to survive at the roots. But today’s version of a chestnut tree pales in comparison to the trees’ towering ancestors, which used to reach 80 to 100 feet tall with trunk diameters growing around 10 feet. “The problem is that 84 percent of them are only an inch in diameter at breast height,” Fitzsimmons says. “And they’re only about 15 feet tall. They’re not serving the services and ecosystems that the species is supposed to. So we call them functionally extinct.”


“A man is dwarfed by a large surviving American chestnut in Kentucky”

In its mission to restore the American chestnut by creating a blight-resistant tree, the foundation uses a system that incorporates breeding techniques and biotechnology. One method utilized by the American Chestnut Foundation is known as backcross breeding. For this method, scientists select and move desirable characteristics from one variety to another. The goal is to isolate the blight-resistance genes from another species and incorporate them into the genetic makeup for American chestnut trees. Leila Pinchot, a research ecologist for the US Forest Service who specializes in reintroducing chestnut trees into the forest, explains backcross breeding as an “approach to incorporate the genes for resistance from Chinese chestnut with the American Chestnut because what we want is a tree that looks and acts American.”

Pinchot explains that this method, as shown by geneticist Jared Westbrook’s research for the American Chestnut Foundation, turned out to not be the solution in the case of the chestnut tree. The goal of backcross breeding is to isolate two or three genes, but in the case of the chestnut, “there are so many genes for resistance in the Chinese chestnut, that it’s just not feasible to combine those with the American chestnut and produce a tree that’s mostly American, but still incorporates the genes for systems from Chinese,” says Pinchot. Even so, the backcrossed trees do have higher blight tolerance than a wild tree, and are still planted in forests to supplement the tree population for the time being.

Backcross breeding is not the tree’s only hope. In Powell’s lab at SUNY-ESF, he recently used genetic engineering to develop a blight-resistant tree by combining a single strand of wheat DNA with the DNA of American chestnut. Powell, who has worked on the project for more than 30 years, isolated a gene from wheat, targeting it for its ability to deal with the blight fungus. “The nice thing about this gene is it counteracts how the fungus attacks the tree,” he says.  Powell explains that when blight infects a tree, it starts making acids and enzymes that work in those acids. The main acid it produces, known as oxalic acid, is a toxin that kills the plant cells. The fungus then feeds on those dead cells, forming a structure called a canker that eventually girdles a tree and kills everything above the point of infection. “What this [wheat] gene will do is actually make an enzyme that detoxifies that acid. It breaks it down into hydrogen peroxide and carbon dioxide, two things the plant uses anyway,” Powell says. “So basically we are taking the weapon away from the fungus.” The genetically engineered, or transgenic, trees are known as Darling 58 and are the first of their kind. And while the new trees can still become infected with the same chestnut blight that killed their cousins more than a century ago—and potentially even be slightly damaged by it—the tree will survive.


“Planting transgenic seedlings at an orchard in New York”

Because of Darling 58’s status as a genetically engineered plant, the next step for getting the trees into forests is a strict regulatory process through the USDA, EPA and the FDA. SUNY-ESF introduced a petition to the USDA in 2020, seeking to deregulate the trees in order to plant them in the wild. This process, Powell hopes will conclude sometime in 2023. In the meantime, more than 2,000 of the Darlings are planted in highly regulated fields, prohibited to flower or spread pollen into the wild. While they await regulations needed to plant trees in forests, Powell’s lab and the American Chestnut Foundation are at work crossing the Darling trees with wild chestnut DNA, hoping to create stronger trees that will grow across the country.

In this way, even with their functionally extinct status, wild chestnut trees are of vital importance to the process of restoring the American chestnut. The wild trees, prominent across regions with vastly different climates—spanning from Georgia all the way up to southern Canada—have adaptive diversity that allow them to thrive within their ecosystems. “Darling 58 is a clone. We can’t restore the American chestnut or any species with a clone,” Fitzsimmons says. “The tree wouldn’t be able to persist in all types of environments. Say I take a chestnut tree from Alabama, and I try to plant it in Maine, at least at this moment, that tree would not survive.” Without adaptive cold tolerance, the chestnut tree would not be viable in a cold region. Crossing the Darling 58 trees with wild chestnut trees allows the diversity the species will need to rejuvenate nationwide.

The foundation relies on impassioned individuals, or citizen scientists to find wild American chestnut matter they can use in the breeding and biotech programs. And for the next steps in the restoration process, when the time comes to start introducing blight-resistant trees into the American forests, the process is going to be no easier than developing the new tree. Pinchot notes potential ecological challenges in the next steps of revitalizing the species. “We need to know how much light the trees need to grow, and what types of treatments are appropriate for chestnuts to encourage their growth without, again, giving the competitive advantage to other species. That’s really where my research interests lay,” she says.

Logistical challenges like growing enough seedlings in nurseries and then successfully transferring them to the forests are also top of mind. As are boots-on-the-ground volunteers, who will be integral in getting the tens of millions of trees planted. “It’s going to take lots of people, lots of money, lots of energy, lots of time. You know, with trees, nature does it a lot better. But nature can’t do this on her own, and we’re going to have to give her a lot of help,” Fitzsimmons says. “It’s really poetic that I’m helping to work on a project that my forbearers started 100 years ago and it’s gonna take my progeny another 100 years plus to finish. I think that there’s something really cool about that.”


“Historic picture of a large American chestnut tree”

GENE STACKING
https://apsnet.org/ChestnutBlightDisease
https://theconversation.com/genetically-engineered-american-chestnut
New genetically engineered American chestnut will help restore the decimated, iconic tree
by William Powell  /  January 19, 2016

“American chestnut trees were once among the most majestic hardwood trees in the eastern deciduous forests, many reaching 80 to 120 feet in height and eight feet or more in diameter. The “then boundless chestnut woods” Thoreau wrote about in Walden once grew throughout the Appalachian mountains. They provided habitat and a mast crop for wildlife, a nutritious nut crop for humans and a source of valuable timber. Because of their rapid growth rate and rot-resistant wood, they also have significant potential for carbon sequestration, important in these days of climate change. The species has a sad story to tell. Of the estimated four billion American chestnut trees that once grew from Maine to Georgia, only a remnant survive today. The species was nearly wiped out by chestnut blight, a devastating disease caused by the exotic fungal pathogen Cryphonectria parasitica. This fungus was accidentally introduced into the United States over a century ago as people began to import Asian species of chestnut. It reduced the American chestnut from the dominant canopy species in the eastern forests to little more than a rare shrub.


“Chestnut blight canker”

After battling the blight for more than a century, researchers are using the modern tools of breeding, bio-control methods that rely on a virus that inhibits the growth of the infecting fungus, and direct genetic modification to return the American chestnut to its keystone position in our forests. To restore this beloved tree, we will need every tool available. It’s taken 26 years of research involving a team of more than 100 university scientists and students here at the not-for-profit American Chestnut Research and Restoration Project, but we’ve finally developed a nonpatented, blight-resistant American chestnut tree. My research partner, Dr. Chuck Maynard, and I work with a team at the SUNY College of Environmental Science and Forestry (ESF) that includes high school students, undergraduate and graduate students, postdoctoral fellows, colleagues from other institutions and volunteers. Our efforts focus on direct genetic modification, or genetic engineering, as a way to bring back the American chestnut. We’ve tested more than 30 genes from different plant species that could potentially enhance blight resistance. To date, a gene from bread wheat has proven most effective at protecting the tree from the fungus-caused blight.


“A ghost forest of blighted American chestnuts in Virginia”

This wheat gene produces an enzyme called oxalate oxidase (OxO), which detoxifies the oxalate that the fungus uses to form deadly cankers on the stems. This common defense enzyme is found in all grain crops as well as in bananas, strawberries, peanuts and other familiar foods consumed daily by billions of humans and animals, and it’s unrelated to gluten proteins. We’ve added the OxO gene (and a marker gene to help us ensure the resistance-enhancing gene is present) to the chestnut genome, which contains around 40,000 other genes. This is a minuscule alteration compared to the products of many traditional breeding methods. Consider the techniques of species hybridization, in which tens of thousands of genes are added, and mutational breeding, in which unknown mutations are induced.

Genetic engineering allows us to produce a blight-resistant American chestnut that’s genetically over 99.999 percent identical to wild-type American chestnuts. For some, this raises a question: isn’t moving genes between species unnatural? In short: no. Such movement has been essential to the evolution of all species. Researchers are discovering that horizontal (between-species) gene transfer happens in nature and even in our own bodies. In fact, the same organism (Agrobacterium) that we use to move blight-resistance genes into chestnuts has also permanently modified other plants in the wild. For example, all the sweet potato varieties on the market today were genetically engineered by this bacterium around 8,000 years ago.

There is another logical question: what about unintended consequences? Of course undefined questions are impossible to answer, but logically the method producing the smallest changes to the plant should have the fewest unintended consequences. We have not observed nontarget transgene effects – that is, changes that we didn’t intend – on our trees or on other organisms that interact with our trees, for example with beneficial fungi. And at any rate, unintended consequences aren’t constrained to the genetics lab. Chestnut growers have seen unintended consequences resulting from their hybrid breeding of chestnuts. One example is the internal kernel breakdown (IKB) seen in chestnut hybridization, caused by crossing a male sterile European/Japanese hybrid (“Colossal”) with Chinese chestnut. By mixing tens of thousands of genes with unknown interactions through traditional breeding, occasionally you get incompatible combinations or induced mutations that can lead to unintended outcomes like IKB or male sterility.


“Butterfly on male flowers of an American chestnut”

One of the key advantages of genetic engineering is that it’s far less disruptive to the original chestnut genome – and thus to its ecologically important characteristics. The trees remain more true to form with less chance of unforeseen and unwanted side effects. Once these genes are inserted, they become a normal part of the tree’s genome and are inherited just like any other gene. They have no more chance of moving to other species than do any of the approximately 40,000 genes already in chestnut. One of the challenges of genetic engineering that is not faced by any other methods of genetic modification also serves as a safeguard. We must shepherd these trees through federal regulatory review by the U.S. Department of Agriculture, the Environmental Protection Agency and the Food and Drug Administration. Our plan is to submit these applications as we finish collecting the necessary data; we expect the process to take three to five years. Once we receive (anticipated) approval, we will quickly make the trees available to the public. This project is unique because it is the first to seek approval of a transgenic plant to help save a species and restore a forest’s ecology. Our forests face many challenges today from exotic pests and pathogens such as Emerald Ash Borer, Hemlock Wooly Adelgid, Sudden Oak Death, Dutch Elm Disease, and many more. The American chestnut can serve as a model system for protecting our forest’s health.


“Transgenic American chestnut ‘Darling 54.’”

Direct genetic modification will likely not be used in isolation. Integration might improve the outcomes of both the conventional hybrid/backcross breeding program of the American Chestnut Foundation and our genetic engineering program. Allowing crosses between the best trees from both programs will allow gene stacking – having multiple and diverse resistance genes in a single tree – with each working in a different way to stop the blight. This would significantly decrease the chances that the blight could ever overcome the resistance. The two programs working together would also allow the addition of resistance genes for other important pests, such as Phytophthora, which causes a serious root rot in the southern part of the chestnut range. And combining methods increases the chances that the resistance will be long-lasting and reliable, which is very important for a tree that in good health can live for centuries.


“Thirty days after infection with chestnut blight, the wild-type American chestnuts on the left are wilted, while the ‘Darling 54’ transgenic trees are doing well”

A unique aspect of the genetically engineered American chestnut trees is their ability to rescue the genetic diversity in the small surviving population of American chestnut trees. When we cross our blight-resistant transgenic trees to these surviving “mother” trees, directly in the wild or from nuts gathered from them and grown in orchards, we’re helping preserve the remaining wild genes. Half the resulting offspring will be fully blight-resistant, while also containing half the genes from the mother tree. By making these crosses, the restoration trees will be ecologically adapted to the diverse environments in which they’ll grow. These trees could also be used to boost the genetic diversity of the hybrid/backcross breeding program, or used directly for restoration and left to fend for themselves, allowing natural selection to make the final determination of the effectiveness of our efforts. The American chestnut was one of the most important hardwood tree species in the eastern forests of North America, and it can be again. This tiny change in the genome will hopefully be a huge step toward putting the American chestnut on a path to recovery.”

PREVIOUSLY

 

CLONING the SUPER-TREES
https://spectrevision.net/2011/03/18/cloning-super-trees/
DE-EXTINCTION
https://spectrevision.net/2013/05/09/de-extinction/
GENETIC RESCUE
https://spectrevision.net/2021/05/19/genetic-rescue/