Interview: Dr. Thomas Klak on Restoring the Great American Chestnut Trees

Using science and innovation to return the Redwood of the East, the "perfect tree," to its former glory!

Dr. Tom Klak in his lab.

Dr. Klak is a professor in the School of Marine and Environmental Programs at the University of New England in Biddeford, Maine, USA. He is a leading figure in the effort to bring back the great American chestnut trees.

In the interview below, this writer’s questions and comments are in bold, Dr. Klak’s words are in regular text, and extra clarification (links, etc) added after the interview are in bold italics or footnotes.

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It’s good to talk to you. You're doing an impressive job. You're amazingly productive. Great job on the Weekly Anthropocene.

It's become an all-consuming hobby, really, because I just have a burning desire to learn more about some of the positive stuff that's not being covered, like your work, some of the amazing possibilities of building a better relationship between humanity and its biosphere, from clean energy to wildlife conservation to genetic resurrection.

Yeah, right now it’s a key inflection point for genetic engineering for conservation, so we got to be pretty careful. There are people out there that are very against it, you know, the whole anti-GMO thing, right? Well, I just heard recently in the news, you probably saw this, the first child who was deaf from birth, but was basically cured through gene therapy.

Exactly. And you know, something like 85% of the American corn supply is genetically engineered, and that’s worked out more or less okay. I am totally in favor of GMOs.

I've actually written about that in the past, I think that the fact that Greenpeace stopped the vitamin A-enriched golden rice from being distributed because it was a GMO was an absolute travesty¹. It really deeply disappointed me and made me want to try to write to help steer the environmental movement in a pro-progress, less reactionary direction.

For example, I interviewed Ben Novak, who's trying to bring back the passenger pigeon, if you're interested.

I read that. I know Ben. He does parallel science because the extinct passenger pigeon and the American chestnut are the two parallel keystone species of the Eastern U.S. Forest. There's really a lot of connections between them in terms of how they shaped the environment throughout the East. We have an easier time going forward because there are still existing chestnut trees. Unlike Ben's work, he's got to start from scratch. But yeah, super cool stuff that he's doing. I like that organization a lot, Revive and Restore.

Still no commercialization of that [golden rice], that was established by 20 years ago, and can cure child blindness. There's some work, there's some plantations now in the Philippines, but still at the test stage and not the stage where it's commercialized. So it's really unfortunate blockage on that advancement.

Absolutely. Genetic engineering technology brings fixes that have the potential to improve hundreds of thousands of lives! Part of what I'm trying to do with my work is to try to sort of push environmentalism into a less mystic, more data-driven direction in which people accept that, like, a new kind of technology that saves kids from blindness is a good thing.

What about human insulin? I mean, human insulin is genetically engineered, right?

Exactly, exactly.

Millions of people can live very healthy lives through genetically engineered insulin. So there's a lot of obvious examples of what genetic engineering can do. I'm not saying it's always the cure, but it should be a tool. And we have the capacity now to oftentimes reverse things that we've caused the problem of in the first place, right? So that's another part of this, we have the ability to reverse our earlier mistakes.

And not acting is acting. Not taking steps, if we know that the science is sound, sitting back and doing nothing is acting.

In my case, I'm very focused on the Eastern U.S. forest with all of the trees that are in bad shape right now. Go right down the list, the Eastern Hemlock Woolly Adelgid, beech bark disease, beech leaf disease, it's a whole gamut of different hardwoods that are in trouble right now.

So sitting back and not taking steps, if we have the capacity to make a difference, is really, I think, irresponsible.

I absolutely agree. Not making a choice when we could use technology to make things better isn’t neutral, it’s making a choice to not try to make things better!

It's like one story after another, you can just change some of the specific details. It’s how we got the emerald ash borer, having such a devastating effect on the white ash, the green ash and the brown ash, which is a super important Native American plant, the brown ash, for the basket making. That's very relevant in Maine. So all of those things we did is the same pattern, right? Bringing in some pathogen that is going to wipe out a key plant.

So we’ve got to do something.

Could you tell me in your own words the history of the American chestnut, its profound ecological, social, and cultural value, how it fell, and how we have a shot of bringing it back? And then we can get to the speed breeding and technical processes in more detail.

Right, right, right. So Europe has its own species of chestnut, it's Castanea sativa, and we [North Americans] have our own native species called Castanea dentata. There are different species of chestnuts also in Asia, but the two that I just mentioned are more related to each other, similar looking kinds of trees.

And they've had better luck in Europe with dealing with the disease situation, so the trees have been able to survive much better than they have in the U.S. We have surviving trees here, but not to the scale that Europe has. The American tree will eventually die from this fungal blight.

There are two [major chestnut tree] diseases in the U.S. In the northern part of the range where I am in Maine, it's still only one. The first is called Cryphonectria parasitica. [Common name: chestnut fungal blight]. Cryphonectria parasitica kills the tree above the ground. It shoots up another sprout, and then eventually the fungus will surround the living tissue of the stem and kill it back. But the roots survive, at least temporarily.

It's a no-win situation, though, because typically with these trees—and there actually are hundreds of millions of these throughout the range that are in this state of growing above the ground and being attacked by the fungal blight—the fungus surrounds the living layer of the stem and then it dies back and then it puts up more shoots. This happens over and over again. There's only a small share of those hundreds of millions of surviving American chestnut trees that grow to the size of being mature trees. A small fraction of those hundreds of millions. So it's not a viable situation. That's why we say the American chestnut is functionally extinct.

It's not extinct in the sense of the passenger pigeon, but it's functionally extinct in the sense that it doesn't play its normal ecological role and only an unusual, fairly small share of the trees live and grow to maturity size where they can reproduce. So that's where we have to intervene.

That's one disease that I mentioned, this above the ground disease called Cryphonectria parasitica, we call it the fungal blight.

There's another disease that is prevalent in the South, and that's why the South is in deeper trouble, and there are way fewer surviving trees. It's a root rot disease in the South, and that's called Phytophthora cinnamomi. Phytophthora cinnamomi kills the tree’s roots. It's creeping northward with everything that's moving northward with climate change, so eventually we're going to need to deal with that particular disease as well. But right now we're not prioritizing it, because we have our work cut out for us with the fungal blight.

A historical image of American chestnut trees

So as you were saying, surviving chestnuts tend to be small. They tend to be understory trees, nothing like they're supposed to be. They were for thousands of years a canopy tree, a hundred foot tall canopy tree, with diameters of five feet, eight feet. ten feet, even more so! A massive tree of the Primeval Forest.

And not just the chestnuts, other trees were very big. We have a really hard time even imagining what the forest was like pre-European settlement.

The chestnut... It was the Redwood of the East, right? That's what it was called?

Yes, Redwood of the East. Not to diminish the significance and size of a lot of other trees as well. The hemlock was massive. The different species of oaks were massive as well. But the chestnut tree, there were up to four billion of them throughout the East. They were not evenly distributed throughout the range, tending to grow in clusters. But in many cases, they could be one out of four trees or more in a particular area. We sometimes use the term redwood of the east, although other trees are massive too. But we can also call it the perfect tree, the American chestnut.

The perfect tree, because it has attributes that no other tree combines, a fast growing tree, a super deep taproot, so it's bringing up nutrients from very deep. And surviving drought, very effectively holding soil. Growing really straight and tall, growing quickly so it could be coppiced easily. And that was the traditional way in which the chestnut was harvested. So you could coppice it. And then, 30 or 40 years later, you could have another chestnut tree that would be a foot in diameter or larger. And you can go through that sustainable cycle. Chestnuts were a sort of preeminent lumber supply that would be sustainable on the landscape.

And then the wood itself is rot resistant, it's got a lot of tannins. It was also really valuable in the leather preservation industry tanning industry during the 1700s, 1800s because of the tannin levels, so it played that significant role especially in the South.

And so it's a rot resistant wood. So there's a lot of examples of chestnut wood today that are being used a second time over, 100 years or more later. It could have been part of a barn or a cabin, something earlier on. What we call wormy chestnut is this second generation of chestnut used to make things out of it. It's actually quite valuable to have it. And when we say wormy, there's one beetle that makes tiny pinholes in the chestnut, but it doesn't affect the integrity of the wood. It just kind of gives it even more character.

So you’ve got a wood that is preserving itself because of its ability to be pretty much insect free, then it's capturing carbon for centuries. So it's a massive carbon sequestration machine. I'm not saying that it's going to solve the problem that we have in terms of pulling carbon out of the air, but it can contribute significantly to that. So that's a really valuable component.

Gathering Chestnuts, painting by Ernest Smith

And then we get into the value of the tree to Native American people. There were a lot of medicinal values to the leaves and the food itself is unprecedented. The nuts fall very consistently, not 100% the same each year, there's going to be some fluctuations in quantity, but pretty much guaranteed that there's going to be a very significant chestnut crop. Unlike the typical mast years you have for oaks. This year is a red oak mast year here in Southern Maine. So the mast quantity of oak acorns is everywhere. You feel like you're rolling around on ball bearings. Well, that's good this year for animals, but what about next year and the year after? Because it's like a four-year cycle for red oaks, whereas chestnuts would be every year providing food.

It was early October, the nuts would drop. The burrs with the nuts in them would drop around October 1st here, a little earlier in the south, of course, warmer there. And then you'd have the food supply going into the winter. So timing wise, in terms of food, it was really great. Once your gardens were dropping back because of the weather change, then you can have the chestnuts kicking in as a supply for people, for animals. People would let their pigs out into the forest and they would fatten up on chestnuts. And the quantity, just the sheer quantity of chestnut mast is like nothing else.

We're trying to piece together that information. Back when the chestnut was abundant, the science and data collection wasn't like it was today. But studies have been done to show that the quantity of chestnut mast is many times more than any replacement tree, say the different species of oaks that have filled in where the chestnut has died back.

So all of that would be some of the ingredients of why we call it the perfect tree. It's got more positive features than any other tree in combination.

Right, that sounds like the perfect tree! It's got wood, it's got nutritional value, it supports the ecosystem. I mean, right off the top of my head, I've read that there's often way higher human-wildlife conflict in bad mast years. There's not a lot of acorns so bears and deer get more desperate and are more likely to come to human gardens or dumps for food.

So if there was just a consistent supply of chestnuts, then that could really, really reduce human wildlife conflict.

Right, and the ability to support wildlife continually is unprecedented, right? That was the key point about, say, the oak. Okay, now this year the squirrels are super happy, they've got all the food they can imagine with the acorns that are available. So they're doing great now, but what about next year? You see this sort of this massive up and down fluctuation of wildlife populations because of the variability of mast production [oaks’ acorns] for all the other species. Not so for chestnuts. You can guarantee their availability, so it can sustain wildlife populations at a much higher level than we really can even imagine today.

Chestnutting, by Winslow Homer, 1870

And even villages. There are a lot of villages in Appalachia that went belly up when the chestnuts were decimated with the fungal blight. That was a key ingredient of their economies.

We haven't gotten into so much of the economics of it, but that would be the key thing for poor Appalachian villages where chestnuts were a dominant species. They can go and collect quantities in bags, bring them to the country store, buy their shoes for school, their school books, trade them for everything in late September. A major source of cash for these communities. And then those chestnuts would be moved by train to the big cities and then you'd have the chestnut vendors on the streets, like you still now see in Europe and in Asia, because those species of chestnut have survived. So we've lost that ingredient of the urban landscape that these other regions of the world still continue to enjoy. Nothing like a little snack of roasted chestnuts.

I actually had the opportunity in France of gathering wild chestnuts and roasting them, just in a little apartment oven. And it was amazing. I've been following the American Chestnut Foundation's work just as an interested amateur for ages, but it was that experience that really inspired me actually to come to come interview you because it was it was amazing. It was delicious. It was this ready to eat delicious protein-rich fat-rich nut. You could just pop the whole spiky shell in the oven and pop out the roast nut when it’s done. It was like a natural pre-packaged food. And this seems like it could be really useful in Eastern North America to help soften the effects of climate change by providing some more resilience, a more consistent food source in the ecosystem.

Right. Yeah. And you can think of two things for the [chestnut tree] landscape that I want to see in coming decades. Certainly reintroducing the chestnut to the forest where it was for thousands of years until we introduced the fungal blight, that for sure.

But then also the other part of it would be on a permaculture landscape. It's an ideal permaculture tree where it would be mixed in together with other kinds of sustainable, ideally native species, food producing species. Here in Maine, things like hazelnuts would be not a tall tree like a chestnut, and blueberries, of course, things like that could be part of this landscape that would be sustainably harvested. Beach Plum comes to mind. These would all be native plants that are going to be valuable in terms of the ecosystem relationships, but they would also be a sustainable harvestable landscape.

So I think chestnuts would be the perfect dominant tree-level component of a permaculture landscape. That's sort of a different story of where we see it going and why we're so excited about bringing back this particular tree, why this particular species of all the different issues that we could take on if we're trying to do ecological restoration, which is what I've been doing for decades. Why did I settle on the chestnut?

Because there's no other species that can compare in terms of the contribution that it made, and will in the future make again, to the landscape writ large.

That's spectacular. That is incredibly powerful.

So can we get into the details of exactly how you're doing this? Like you mentioned, you're doing back crossing and crossbreeding with Asian chestnut species, and genetic engineering. You mentioned speed breeding. How are you doing that?

Right, yeah, so there's two different approaches that have been worked on for decades now. A little bit of overlap now between the two, but mostly operating separate from one another. One is the Chinese hybrid crossbreeding and backcross program. And the other one is the transgenic or biotech program. So those generally have moved forward separate from one another. There's some thinking now about the ways in which they potentially could combine, but generally there's been practitioners that have been involved, scientists, researchers, volunteers that are in one camp or the other.

You asked, how did I get involved with this? I just answered it partially, to say that there's no other species that I can contribute more with, as somebody that's interested and committed to ecological restoration, than the chestnut.

I've always been interested in reintroducing native plants, helping to heal the landscape, putting it back more closely to what it was like before the Great Clearing and before the idea of these kinds of lawn and cultivar and ornamental plant landscapes that have come to dominate the U.S. yards and people's private property. Trying to reverse that. So that's something I've been involved with for a long time.

The historical native range of the American chestnut

And having moved to Maine 13 years ago, I got involved with the project here because it [chestnut] is a native plant here. Where I lived before, near Cincinnati, Ohio, it's actually not, it doesn't grow in the local landscape there because the soils are alkaline and the chestnut, like the blueberry and the azalea and the rhododendron that are so common here in Maine, they like an acidic soil. The cranberry, of course, would be another local plant that really enjoys an acidic soil. So the chestnut is one of those.

In our lab, when we grow them in the speed breeding process, we're always reducing the pH level² of the soil or of the water. The water out of the tap comes through at 7.2 [pH] and we drop it to 5.5 [pH] before we give it to our trees and fertilize our trees in the lab, because that's what they prefer. That's what they enjoy, a 5.5 level.

It’s sort of a different story on the Chinese hybrids. There are three species of chestnut that are in Asia, but it's particularly the Castanea mollissima that the effort has been put into breeding. It's a larger tree than the other two ones, the Japanese chestnuts, Castanea crenata, and then there's one from southern China as well, called Castanea henryi. So those are the three species of chestnut in Asia. The focus has been on the mollissima.

I think a really important point to stress in terms of what we can do now and what we can learn and how quickly science and medicine can move forward today is the genomics revolution.

Is CRISPR Cas-9 being used in chestnut restoration efforts? “Copy and paste” for the genome?

Yes, it's being done. CRISPR is definitely being used in trying to understand the genome better, not only of the chestnut, but also of the fungal blight. So that's going on now.

The method that has been used with the genetically engineered trees that I work with in my lab is using a little bit more traditional approach. It's the agrobacterium vector approach. That's a naturally occurring bacteria that has the amazing capacity to invade the genome of a plant.

Horizontal gene transfer!

Right. So the sweet potato is your classic example of agrobacterium transforming a naturally occurring plant. Now we have these tubers thanks to the agrobacterium invasion and we enjoy that thanks to something that happened something like 8,000 years ago. So that method that happens in nature was piggybacked to move a gene to protect the chestnut. And the gene that was identified [to be moved into the chestnut genome by the agrobacterium] was one from wheat. It's very a common gene in many dozens of plants, food plants as well as wild plants. Any grass has this same gene. It's an enzyme protein called oxalate oxidase, and it detoxifies the oxalic acid that the fungus uses to kill the chestnut tree. It reduces the acidity because that's one of the chief mechanisms by which the fungus will attack, find its way through the bark, attack the living layers, and then consume the insides of the cells.

So that's the method we use and it's genetic engineering, but using an earlier method [earlier than CRISPR]. I work with collaborators at the SUNY Environmental Science and Forestry College in Syracuse, ESF for short, they did the transformations of the chestnut trees that I have in my lab. So it's agrobacterium transformed. The thing about it is that the agrobacterium decides where the gene is located. It's less precise than CRISPR. That then gets us into a little bit of the issues that have been in the news lately about where the gene is within the chestnut genome. We can talk about that in a second if you'd like.

But that's the method that I work with. And the selective speed breeding that I do in my lab with my many students is to do three things, mainly.

Source

One is to produce transgenic pollen. I've been a major supplier of pollen throughout the native range to other collaborators, who also work under U.S. Department of Agriculture permits. Right now with this genetically engineered plant, we're restricted by the USDA as to what we can do. We have permits that specify the locations both in the lab and in the field where we can experiment and test out the plants.

So we've been producing pollen in vast quantity in the lab and then shipping it throughout the native range, to collaborators from Georgia, Virginia, Penn State, ESF in Syracuse, New York, Vermont. Our lab’s pollen has been a key element of the experiment.

We have a lot of trees in what we call germplasm conservation orchards. That means that we found some surviving wild trees out there, and my lab and others have grown up the seedlings from those trees. So in Maine here we have still surviving trees in different places throughout the southern half of the state, and then we put them in these orchards. Typically these orchards will have about 100 trees in them, but they'll represent maybe 20 different original mother trees. So you can have just one location, very concentrated, you can take care of it, that represents a really wide range of genetic diversity, even trees that are the offspring of other parts of the range. I've got some from Georgia, for example, and those are rare because, as I mentioned earlier, two diseases are affecting the chestnuts in the southern part of the range.

So that's one major thing we do, producing pollen. And that would be pollen, up till the present, from the Darling54 line.

The second thing we've been doing is, with the speed breeding, we can advance generations indoors. So we can move through generations. The original transformation, I mentioned, begins as a clone, so all of the first offspring are exactly the same with each other. Completely the opposite of what nature does and what nature needs. So we need to then work hard to genetically diversify those lines, always inheriting the transgene but mixing up the genes through crosses so that you get more and more of nature's diversity.

So we can move through generations in my lab. Now we have plants that are the fifth generation post-clone and those in terms of modeling are genetically diverse and suitable for outplanting, comparable to what you would find in nature in terms of genetic profile.

The third major thing we do in my lab is that we, and we're just early on the process of this, but we can breed chestnuts where the mother has a copy of the transgene, the father pollen also has a copy of the gene, and we can breed the flowers of the mother in the lab with pollen from other plants in the lab, and produce what are called homozygous chestnut offspring that would have two copies of the gene on both sides of the chromosome. [We’re still talking here about the gene from wheat, introduced via agrobacterium, that protects the chestnut trees from chestnut blight fungus].

An example Punnett square from Wikipedia. In this example, both parents are heterozygous for this trait, with a dominant purple-flowers allele (“B”) and a recessive white-flowers allele (“b”). Heterozygous parents, each with a “hidden” recessive allele, can result in offspring with all-recessive alleles, as shown in the lower right corner of this Punnett square. Dr. Klak is breeding homozygous chestnut trees so that pollen from those trees, will put on the flowers of any wild-type chestnut, will guarantee that 100% of the resulting chestnuts will inherit the gene for blight tolerance.

And that's super valuable because then there's no recessives that are still vulnerable. I just remember my Punnett square biology from high school. Then there's no recessives such that, like a quarter of the offspring in the classic Punnett square would lack copies of the gene.

Right. And basically, when we have bred my pollen from the lab with wild trees, about half inherit the gene. In practice, it's a bit less than that, somewhere between 40 or 50%.

But using homozygous pollen, then you would have 100% of the nuts inheriting the gene, so that would be vastly more efficient. And so we can imagine into the future, chestnut orchards that would be comprised of homozygous plants. You'd have all of that going on and producing all offspring that would have inherited the transgene.

So that's the third major effort that's going on in my lab. And we're just beginning to produce these homozygous plants.

This is brilliant, this is spectacular. Thank you so much for sharing it.

Back to your Punnett table, which is so crucial here. You know, they say people die twice, right? Once when they actually die, and once when their name is no longer used. So we gotta give credit to Gregor Mendel, who continues to be relevant today with the Punnett table, and Punnett himself, the scientist.

A chestnut tree in Italy that’s over two thousand years old

I can say a few more words about the speed breeding operation. It's pretty remarkable. Let's back up for a second. We're lucky if we can live a century, right? There are producing chestnut trees in Italy that are over a thousand years old.

Wow, that's amazing.

Yeah! And so we know that the American chestnut can live at least 500 years. So we've got a time discrepancy here between our lives and theirs. And we want to make a difference, so how do we intervene? We use selective speed breeding.

Blue Jay

It’s the Blue Jay who's our key partner here in terms of future restoration. The Blue Jay is going to be the biggest contributor to restoring the forested landscape once we get to the point where we have a tree that we're comfortable with, having sufficient blight resistance.

Okay, so if that Blue Jay takes the nut from the mother tree, and we know that people have been looking at this and seeing what Blue Jays do even with surviving wild trees, the Blue Jay can move a nut up to one kilometer away from the mother tree. So you got a two kilometer radius all around that tree. And they only put one or two nuts in the ground at once, so they're perfect for planting. If a squirrel caches the nuts it'll put 20 or 30 nuts in the same hole, well that's not so useful as a planting strategy, right? The Blue Jay is the way to go.

We got to give credit to these other species that can do things that we absolutely can't do. Everybody should go out in their backyard and bury 20 nuts and come back the next day and then try to find them. I tried it. I couldn’t find any of them. And yet blue jays can come back and find most of them.

But they won't find them all. Or some Blue Jays won't make it till when they need to eat them. So then they're the classic planter of the chestnut.

But this is why I'm making the contrast between nature, which is amazing, but why speed breeding is so valuable. In nature, that nut then would take many years to get to a point when it's a mature tree. Usually going to be planted in the understory, right? It's not going to have an opportunity for a break in the canopy to bring a sufficient amount of light. So that nut could grow as a seedling for decades on the forest floor before it would eventually get a chance to grow up and become a mature tree. I mean, we're talking a very long time for that tree to become a mature tree in nature.

Now we could speed it up by putting it in a place outdoors with more light and give it all kinds of nutrients to make it grow faster. But even if you're doing that, to produce female flowers in nature, you're typically talking probably around a decade in many cases, certainly in Maine, probably faster in the South. So it's a long process for that to happen outside.

But what we can do in the lab, we can take a nut that we've harvested, and then it requires a stratification period. So there's going to be a two to four month period between when you harvest the nut, whether you harvest it from nature or you harvest it from the lab before it's ready to grow a radicle, which is the early component of the root that eventually becomes the root. Okay, so once we get that radicle on the nut, we've been able to produce male pollen five months later!

[A radicle is the embryonic root of a plant, the first part to emerge from a seed. Dr. Klak is saying that his lab can get a chestnut tree from “seed with a tiny root-let poking out” to “pollen producing adult” in five months!].

So it becomes a mature tree in five months after we've planted it under the speed breeding conditions. Basically in the time frame of around a year's time, we can produce mature trees, which would take many years to happen in nature.

So you can see the value of that for breeding, combinations together, genetic diversity, advancing the generations away from the original clone, overcoming what's called the founder effect, and then producing the homozygous plants, which is something we've been able to do and work on, and that led to some of the major new revelations that have come out in the last few months. That was part of that story that came out in the Washington Post in December 2023.

Detail of Gathering Chestnuts by JW Lauderbach, 1878. We can have this again!

Yeah, that was a mislabeling story, wasn't it? Darling54 and 58 were labeled as each other, two different strains.

Right. Everyone thought we were working with the Darling58. And in genetics, the location of the gene is guaranteed across the generations. So if [the gene] is where it is on the original clone, Darling58 clone, in the first post-clone generation, it was on the seventh chromosome, well, it's going to be there for the next generations.

That's just standard genetics. So no one bothered to look to see exactly if that were true.

It was submitted for deregulation to the U.S. Department of Agriculture. There are USDA permits that we operate under, the hope would be that they would deregulate. It wouldn't be completely open to anybody doing whatever they want, because the Environmental Protection Agency is also involved and they would be monitoring the next round, if and when deregulation happens. So that's what was thought to be the case. The pollen that we've been producing, pollen we've been sending throughout the native ranges was thought to be Darling58.

What happened was when we were beginning to produce these potentially homozygous plants, that had potentially two copies of the gene, at least 25% of them, we started to build up a quantity of that kind of seedlings. And we needed to test to see if they indeed had two copies. You can do different kinds of PCR tests. And we can do what's called a histochemical assay ourselves in my lab. And that just tells you whether the transgene is present.

Okay, so we can test them. We sent our first 38 leaf samples to my plant geneticist collaborator at the University of Maine, Dr. Ek Han Tan. He and I have been working closely together on this project.

We sent him 38 leaf samples to see which of those inherited two copies of the gene on the seventh chromosome where Darling58 transgene is located. And we already knew that a very significant number of them had the gene because we did the histochemical assay, that, again, detects the presence of the gene.

Well, what he found when Dr. Tan was looking for the location of the gene, he didn't find it on Darling58's location toward the end of chromosome 7. It just wasn't there. We then did some more investigation and realized that we knew about the Darling54 transformation because, in fact, in the petition to the Department of Agriculture, much of the data in terms of off-target impacts was done with Darling54 plants because those were an earlier generation than Darling58 and they'd grown up bigger. You can do soil mycorrhizae tests, you can do tests with how the bees use the pollen to make honey and things like that. So much of the work in the petition for deregulation was done with Darling54.

So we knew that Darling54 existed, but it had been put aside because Darling58 was better because of where agrobacterium inserted the gene. It's inserted in a good non-coding location for Darling58, and for Darling 54 it is located on chromosome four inside of a coding gene. It's a coding gene called SAL1. So it was decided earlier that it wasn't an advantageous location. But in fact, that's where we discovered the gene to be located when we were trying to sort out which ones were homozygous.

The other thing we found out in that homozygous research is that only one out of the 38 plants leaf samples we sent to Dr. Tan’s lab had two copies of the gene. Way lower than your Punnett table and way different than Gregor Mendel would have predicted.

Was that just random chance?

No. Statistically, it's extremely unlikely.

Okay, so not due to chance.

It's due to embryo lethality. So with the gene located inside of a coding gene, there's a disruption of that native gene. We haven't had time to study what exactly the SAl1 gene does, but it's related to desiccation and salinity, based on some work that's been done with other plants like Arabidopsis and oak. No one studied it in chestnut because we didn't need to until now. So the Darling54 transgene is at a disadvantageous location on the chestnut genome.

We're still working with the Darling 54s. We want to know more about them. We have reproduced a lot of them with one copy of the gene. They seem to be doing okay, but there's a lot more data to be collected as to their performance over the longer term.

Again, what we're working with is just very, very young plants compared to trees that grow for 500 years or more. So it's like a baby in the hospital and you're trying to decide what they're going to be like when they're going to be a 60-year-old person. Extremely early on. So you've got to be really careful with our assessments.

But there's another transformation we're also working with that we're excited about. Its transgene is not located in a coding gene. And it's a different promoter as well. We're starting to breed those in the lab and produce fertile offspring.

It's one with everything we talked about with the Darling lines that have a promoter that turns on the protection all the time. It's called a constitutive promoter. We have another line again from ESF, which is a wound-induced promoter. And that means that it's on only when the tree senses an attack or wound.

So it’s a more efficient promoter and we’re beginning to breed those, and we have some offspring of those already in the lab. We want to produce homozygous plants and homozygous pollen out of that line.

It'll take us a little while but it won't take us anywhere near as long as if we were doing it in nature.

I see. So this is fascinating. Thank you so much for this really, really in-depth explanation. I can't wait to transcribe it and illustrate it and show it to you and really build this into an article to share this story you're sharing with me with the world.

Last question, and then I can let you go on with your amazing work.

What is your vision for the future? What will the 2100 forest landscape of New England and Eastern North America look like once your work has reached its best possible long term outcome?

Source

Well, it's going to look as close as possible to what a naturally occurring landscape would have looked like.

There's no going back to 1600. Let's not fool ourselves. But we can do much better than what we have been doing, and bringing back this crucial component of the landscape is going to make a really big difference. For all the kinds of reasons we talked about early on, in terms of the value of this particular tree.

Also, it's just a beautiful tree in itself. Let's not forget that just the aesthetic value of a chestnut is really something!

The other thing about it is a little bit of the silver lining, and why I've gotten so deeply involved with this project over the last eight and a half years here in Maine is that the native range for the chestnut would only have reached about the southern half of Maine. That's basically where we still find, hit and miss, a few trees around. But now nearly the entire state of Maine is habitat suitable [due to global warming].

I have worked with a collaborator up at the University of Maine, Fort Kent. His name's Professor Neil Thompson, and he's been growing American chestnut trees, the wild type. Which again, I don't want to diminish that. That's absolutely half of the story, one side of the coin, the native, natural, genetic components of the 35,000 genes that the American chestnut has, so far as its survival and its future.

So he [Professor Thompson] grows those up in the same kind of germplasm conservation orchards up in the Crown of Maine, up in Fort Kent. That kind of positions us here in Maine as really being in the center of the action for that restoration.

We can help. Animals can move, plants can't move on their own, unless the Blue Jays help them. So we can help move that forward to produce a much more sustainable, productive landscape than we have now. So that's the vision.

And then there's the old adage that society grows great when old men plant trees they know they won't sit in the shade of. And that's where I think we are. We're not going to see the full restoration by any stretch of the imagination, but if we can plant the seed, pun intended, to get it rolling during our lifetimes, then that's the exciting legacy we can leave behind.

Thank you. Thank you so much for planting these trees. This is amazing. This is an incredible, incredible work you're doing.

You're doing a great job with your Weekly Anthropocene.

The fact that I get to talk with people like you is one of the reasons I write The Weekly Anthropocene. Thank you so much.

That's great. Thank you, Sam.

1. The golden rice story is one where a genetically modified vitamin-supplemented rice that could have prevented millions of deaths in developing countries was delayed indefinitely due to anti-GMO activism. To my mind, the golden rice episode means that the net impact of anti-GMO activism on the global agriculture system has been overwhelmingly negative. The environmental community would be much better off and more morally sound to embrace GMOs as a way to help feed more people.

2. pH measures the ratio of loose hydrogen ions (H+) to hydroxyl ions (OH-) in a liquid, with a 0 pH solution having lots of extra hydrogen ions (very acidic), a 7 pH having a roughly equal ratio (H+ and OH- react with each other to form H2O, plain water, so that’s just “neutral”), and a 14 pH solution having lots of extra hydroxyl ions (very alkaline, aka basic). Notably, it’s a logarithmic scale, not a linear one.

Interestingly, the H in pH stands for hydrogen, but, amazingly, nobody on Earth knows exactly what the “p” in pH stands for, as Sørensen, the chemist who developed the pH scale, didn’t explain it in his notes. It got adopted with multiple countries assuming that it stood for a “p” word in their language.