Grape Expectations

MTSU undergraduate research grant recipients Nolan Jolley, front, Amanda Uhls and Aimee Wilson

The story of a dogged research team, a cantankerous plant, and wine that could change the world

by Allison Gorman

Wine has been integral in human culture for thousands of years, from the Last Supper to the works of Shakespeare to Hannibal Lecter’s “nice Chianti.” But, despite its global reach, the flow of commercial wines begins in a few distinct regions within two narrow latitudinal bands—one in the Northern Hemisphere, most famously including Tuscany and parts of California, and one in the Southern Hemisphere, encompassing parts of Australia, New Zealand, South Africa, Argentina, and Chile.

The wines most of us recognize, whether or not we drink them, come from one species of grape, which flourishes in the cool nights and warm days specific to those fertile regions.

“The chardonnays, the cabernet sauvignons, the merlots, the pinot noirs, the sauvignon blancs, those are all Vitis vinifera,” MTSU Agriculture Professor Tony Johnston said. “The global industry is built on that genus and species—from 95% to 99% of commercial vines. There are just a handful of other species that are commercially grown for wine production around the world.”

Zion Market Research projects that the wine industry will reach $423.6 billion in global revenues by the end of 2023. Finding a grape that could flourish outside Vitis vinifera’s 20-degree latitudinal range could give whole swaths of the world, many of them quite poor, access to that lucrative market—or at least provide one other means of economic self-sufficiency.

“If another variety of grape can be shown to be viable and produce good-quality product, we can open up the whole equatorial range of the earth to grape production,” Johnston said.

In other words, Johnston is not crazy for spending the last 25 years mildly obsessing over Vitis aestivalis, a North American grape commonly known as Norton/Cynthiana.

Norton/Cynthiana is not on anybody’s wine tour. It’s the official grape of Missouri. But like Mark Twain and Harry Truman, it’s notoriously scrappy. Unlike its delicate cousin in Napa Valley, it shrugs off little things like drought, humidity, diseases, and pests.

Grown primarily in the Mid-Atlantic and Midwest U.S., Norton/Cynthiana is traditionally used for table grapes, juices, jams, and jellies. It makes for delicious wine too, Johnston said. He first worked with it in the mid-1990s as a research assistant at the University of Arkansas, and he believes it has “enormous economic potential”—if it can be propagated.

In the words of a certain Shakespearean prince (and almost certainly a wine drinker), “Ay, there’s the rub.”

Frustration by the Bucket

Amanda Uhls is not an expert at propagating Norton/Cynthiana. But having spent an entire fall, winter, and spring trying unsuccessfully to get the darn thing to root, she can tell you a thing or two about not propagating it.

Twice a month from September 2015 through August 2016, Uhls, an Honors Biology student from Kingston, Tennessee, grabbed a 5-gallon bucket and drove to the Rutherford County Agricultural Extension property west of I-24, about 20 minutes from campus. From there she’d take a dirt road to a small plot where Johnston had arranged to plant 200 Norton/Cynthiana vines donated to him by an east Tennessee farmer who’d bought too many. When the dirt road got too muddy to drive on, she had to park her car and make the last part of the trip by foot—a 10- to 15-minute slog.

She’d take maybe 45–50 cuttings, enough to fill the bucket, haul it back to the car, drive to the greenhouse behind the Science Building, and put the new cuttings in a growing medium. Then she’d check all her previous cuttings for roots and record the results.

MTSU Biology Professor John DuBois, a plant physiologist, gave Uhls the project. His goal was to find out when Norton/Cynthiana propagated best.

DuBois had heard horror stories from Johnston about how hard the vine is to propagate in the traditional manner. Johnston still has vivid memories—or maybe they’re flashbacks—of being a doctoral student in Arkansas, trying to root enough cuttings to replace the occasional Norton/Cynthiana vine that failed.

“With every other variety of grapes, we could just take a few cuttings and put them in the greenhouse in the winter, and in the spring go back and plant them, and we’d have plenty of vines,” Johnston said. “With this variety, we couldn’t take enough cuttings. We literally had greenhouses full [of them] to get a handful of plants in the spring.”

After hearing those stories, DuBois had tried to propagate the grape himself because how hard could it be? He took cuttings in March and tried to root them. They died. He got better results from cuttings he took in June, but the majority of those died too. So when he gave Uhls her project in September, he warned her not to get her hopes up, especially before June.

Still, Uhls found it disheartening, making the bi-weekly slog from vineyard to greenhouse and then to DuBois, only to deliver the same one-word report: “Nothing.”

“It was very hard, to say the least,” she said. “There was one month where I got maybe three or four roots, and it was the biggest deal ever—like, ‘I got something!’—and then literally right after that it went right back to zero for the next few months. Every time I would tell Dr. DuBois I had nothing, he’d have to remind me, ‘Nothing is still a result.’ ”

The fact this was Uhls’ Honors thesis project at MTSU made everything worse. She was supposed to graduate in December 2016. As the months ticked away with no discernible progress, she started feeling panicky.

“By the end of May, I knew there wasn’t enough time to meet with a new advisor to change projects or even do an entire project,” she said. “It was kind of scary, wondering whether or not I was going to finish my thesis.”

Johnston compares research to that classic definition of war — “long periods of boredom punctuated by sheer terror.”

MTSU undergraduate research grant recipients Nolan Jolley, front, Amanda Uhls and Aimee Wilson

MTSU undergraduate research grant recipients Nolan Jolley, front, Amanda Uhls and Aimee Wilson check the status of their grape projects in a special Science Building laboratory. (MTSU photo by Andy Heidt)

Surprising Successes

In June, nothing turned to something. Some of the cuttings rooted. Not most of them, but significantly more than zero.

Uhls got enough data to write her thesis and graduate as planned. Undeterred, she accepted a job as a microbiologist at a state of Tennessee public health lab, where she is still happily employed.

DuBois got enough data to know that he needed more data.

He and Johnston agreed that the study was publishable, but not with just one year’s worth of numbers.

So DuBois repeated the experiment with another undergraduate Honors thesis student, Nolan Jolley, making the bi-weekly slog—this time from March to October, since DuBois knew the cuttings wouldn’t root in winter, the way most grapes do.

Jolley’s data supported what DuBois and Johnston suspected: Unlike every other commercially produced grape in the world, Norton/Cynthiana propagates best in June.

“That’s not a good time to propagate grapes because they’re going to be setting fruits,” DuBois noted. “You don’t want to be cutting plants at that point.”

To summarize: The worst time to propagate Norton/Cynthiana is when it propagates best.

“And even when it’s doing well, it’s still poor,” Johnston offered.

The best propagation rate he and DuBois ever achieved with their cuttings was 30%, he says. That’s compared to 90%–95% for most commercial grape varieties.

So the study was bad news for anyone wanting to propagate Norton/Cynthiana through traditional farming methods.

But it was good news for Uhls and Jolley, whose efforts were ultimately rewarded with a publication. The two MTSU students are listed as first and second authors, followed by Johnston and DuBois, of “The Effect of Sample Date and Timing of Cuttings for Maximum Propagation Efficiency of the Grape, Vitis aestivalis ‘Norton/Cynthiana’” in the March 2018 issue of Food and Nutrition Sciences.

Being published before graduate school isn’t as rare as it used to be, especially for Biology majors at MTSU, DuBois said. But, as more undergraduates get published, the pressure grows on all undergraduates to have research experience if they want to compete for spots in the workforce or graduate school.

Uhls did a good job as an undergraduate setting herself up to succeed. In addition to working in DuBois’ lab, she interned two years in the state lab where she eventually was hired. Upon graduation, the state paid her to work part-time and earn her certification as a medical technologist in microbiology. Now as a full-time employee, she’s pursuing her master’s degree in Epidemiology.

While Uhls had already landed a great job by the time her research was published, she says having principal authorship of a publication was an important addition to her résumé.

“MTSU has a lot of students that get published, so we’re all fortunate to have gone there and to have research-oriented professors who incorporate their students into their work,” she said. “But when you do research, it’s a big deal whose name goes first on the paper, because that’s who is [considered] most responsible for it. It’s still surreal to me that there’s a published paper with my name listed first.”

A Fungus among Us

It was a trip Johnston took to Honduras 14 years earlier—a study abroad trip totally unrelated to grapes—that eventually led to Uhls getting top billing on a published paper. A fairly new faculty member at MTSU at that point, Johnston hadn’t worked with Norton/Cynthiana in ages, although he still puzzled about the irascible plant from time to time.

He had stopped to check out a small, state-run agricultural school in central Honduras when he saw what appeared to be grapevines growing in the distance.

“It was like walking down Main Street and seeing an elephant,” he said. “Everything was wrong. The latitude, the environment. I didn’t even know they had grapes growing in Central America.”

Johnston couldn’t stop wondering about what he’d seen, and so on a return trip to Honduras, he went back to that school and asked how and why they were growing grapes there. It turns out that a faculty member had traveled to Italy, collected cuttings from a vineyard there, brought them back to Honduras, and established a small vineyard. Proceeds from wine sales were helping fund school operations.

Growing grapes where they do not want to grow is not easy.

Over the course of several years, as Johnston kept up his relationship with the school, he heard stories about their struggle to keep the vineyard going in a tropical climate. The humidity and hot nights were a big problem, and the insects were an even bigger one, controlled only through pesticides.

While the school managed to make it work, Johnston was told the cost of chemicals alone would be prohibitive for the typical Honduran farmer.

Johnston thought about that, then said, “What you guys need is a better grape.”

In 2013, Johnston began hand-delivering Norton/Cynthiana cuttings to the school, bringing down a few hundred cuttings at a time in duffle bags. In 2014, with an International Foundation grant aimed at improving the lives of poor people in developing communities, Johnston purchased new trellis posts and other infrastructure for the little vineyard. He also continued hauling grapevine cuttings down to the school—about 1,500–2,000 total.

Only 12 rooted.

Meanwhile, the school had hired a new director, Mario Turcios, a graduate of Zamorano Pan-American Agricultural School, the flagship agricultural institution for Central and South America. Turcios developed an alternative plan, asking Zamorano Assistant Professor Maria Bravo to clone the cuttings instead.

Although not a traditional farming method, using biotechnology to clone a plant is a tried-and-true process: You take a piece of a live plant, surface sterilize it, remove a tiny piece of tissue, and put that tissue in a sterile dish with a growth medium—a special formulation with nutrients and hormones that will direct the tissue to become callus. From the callus—the plant equivalent of stem cells—you grow a plantlet.

Except not with Norton/Cynthiana.

“I visited Maria’s lab several times and saw firsthand the overgrown plates she was encountering,” Johnston said. “She actually kept some rooted grape plants in her greenhouse to have a supply of tissue to cut from and literally tried until there were no plants left to work with. Mold would overtake the tissue culture plate within 24 hours—fast.”

Johnston hadn’t grappled with the grape since 1995, but Norton/Cynthiana had found a new way to confound him.

Big-Picture Problem

When Johnston first worked with Norton/Cynthiana, the grape was simply a puzzle in an Arkansas greenhouse—an interesting agricultural aberration. Nearly 20 years later, it had developed into a biotechnological conundrum in Honduras, with potentially global economic implications.

That’s the sort of big-picture problem the U.S. government wants universities to solve. A project spanning disciplines would almost certainly have broader real-world applications, and interdisciplinary research is now far more likely to attract federal grants, which constitute most research funding for large public universities like MTSU, Johnston said.

“I’m highly encouraged that the federal grant-making agencies have endorsed the idea of collaboration,” he said. “I think it reflects the
reality of the problems that we’re trying to solve.”

The trend has been most obvious with major grant-funding organizations within the federal government, such as the National Science Foundation, Food and Drug Administration, and U.S. Department of Agriculture (USDA), he says.

“They now require that you present collaborative proposals, not individual researchers focused on their research,” Johnston said. “They want inter-institutional proposals, they want interdisciplinary proposals, so it’s become formalized, the recognition that ‘I can’t answer all the questions myself; I need other experts to come put their heads on this problem.’ And I think it’s great. It’s taken us a while to come around to the idea that more heads are a lot better than one.”

In the spirit of that philosophy, when Johnston got back to MTSU from Honduras, he stopped to talk to Shannon Smith, then a new Molecular Biosciences Ph.D. student working in the plant tissue culture lab in MTSU’s new Science Building. Johnston knew that Smith, who earned an Agriculture bachelor’s degree before moving to Biology for graduate work, was smart.

Johnston told him about Norton/Cynthiana and how the lab in Honduras was trying to get callus out of tissue culture but was getting nothing but mold. Smith offered to give it a shot himself.

“Shannon did the classic new-graduate-student thing,” Johnston recalled. “He said, ‘Oh, I can solve that problem. Why don’t you just give me some cuttings?’ And I laughed and laughed, and I said, ‘You know what? You’re on.’ ”

Eight weeks later, Johnston got an update from Smith: “Nothing but mold.”

“He’d just learned the first lesson of graduate school,” Johnston said. “Don’t think you can solve problems in the first swing.”

Digging In

To be fair, DuBois had the same response when he heard that Norton/Cynthiana was practically impossible to propagate: He had to try it himself to believe it. And even then, he wouldn’t take no for an answer. That kind of tenacity is a prerequisite for a career in research, especially when the researcher’s hopes are regularly dashed by the whims of nature.

Beyond any one academic lesson, Uhls says that’s the most important thing she learned from her research experience at MTSU—especially those long slogs with the 5-gallon bucket, those nine months of “nothing.”

“I learned that not everything is going to happen right away,” she said. “A lot of my friends who did research projects ended up with so much data that they had to run statistical analyses, and I would think, ‘I have one data point that I’m excited about.’ It was kind of nice to see a real-world project rather than a lab-created one. It taught me perseverance and patience.”

Through such perseverance and patience, what began as a casual conversation among scientists about a hard-to-propagate grape evolved over the course of a few months into a two-pronged interdisciplinary research project.

Smith, whose doctoral and other lab work had nothing to do with Norton/Cynthiana, nevertheless spent much of the first part of 2015 watching mold overrun tissue culture plates. Meanwhile, DuBois was on a parallel track, taking cuttings from Johnston’s little vineyard off campus and watching them not root.

Slicing the Pie

DuBois is particular about the undergraduates he invites to work in his research lab. When he teaches freshman Honors biology, he notices the students who show up to class, who raise their hands, who make good grades, who show intellectual curiosity. Those are the students he seeks out for project work. When students he doesn’t know seek him out, he gets a scouting report from their professors.

He’s looking for other qualities too—like reliability and the ability to work both independently and as part of a team. Biology students in the University Honors College, who’ve committed to completing a research-based thesis project, tend to have those qualities.

DuBois and his Honors thesis students have a symbiotic relationship. The students need a research project and a mentor; DuBois needs help with the myriad sub-projects emerging from complex research problems like Norton/Cynthiana.

He cuts up those big projects “like a pie,” he said. “Every student takes a slice and works on it.”

Thanks to a USDA grant, the Norton/Cynthiana project has been divided into about a dozen slices. No two slices have been alike.

For example, while Uhls was in the vineyard and the greenhouse, slogging and fretting, Aimee Wilson was in the tissue culture lab, fighting fungi. DuBois had brought Wilson, another Biology Honors thesis student, onto the project to help Smith, who had his own research
to do.

In his solo attempts to clone the grape tissue, Smith had made a critical discovery: Norton/Cynthiana had a fungal endophyte. The fungus was coming from inside Norton/Cynthiana. Smith had succeeded in taking pictures of it with an electron microscope.

A fungal endophyte isn’t necessarily a bad thing, Johnston says. Who knows? It could turn out that some of the qualities that make Norton/Cynthiana so resilient—its tolerance for drought and humidity, its resistance to pests and disease—are attributable to the fungus that naturally lives inside it.

Lesson Learned

Even before she received her slice of grape project pie, Wilson had distinguished herself in DuBois’ lab. Like most newbies, she’d spent time under the tutelage of more experienced undergraduates and grad students, learning basic procedures like sterilization and
note-taking—as Smith puts it, “the hundred little persnickety things that protect you in your research.”

As a lab assistant under Smith and graduate student Matthew Fuller, Wilson “stepped up to every challenge we set for her,” Smith said. So he wasn’t surprised when, given her own project, she stepped up again.

Wilson picked up where he’d left off, trying to get callus tissue without the mold. Except now the job seemed impossible, given that they’d established the mold lived inside, rather than on, the plant—and that the growing medium was 3% sugar.

“You put fungi together with anything containing sugar, and the fungi will go crazy,” DuBois said.

The goal was to find a workaround. The idea the team came up with—putting fungicide in the culture medium—flew in the face of basic laboratory protocol.

“There should be no reason to use an antifungal agent if you’re using sterile techniques,” Johnston said.

DuBois and Smith worked up various procedures for Wilson to try, all involving fungicides. She plugged away at it.

Finally, success: mold-free callus tissue.

“I had a couple of failures at the beginning, but once I got it, I got it,” she said. “Sometimes negative results are a good thing, because it tells you what you’re doing wrong, so you can change it and try to get better. That’s the biggest lesson that I learned from the whole project.”

Now, as a first-year medical student at Edward Via College of Osteopathic Medicine in South Carolina, Wilson challenges herself to adopt a new mindset when she’s faced with a difficult concept.

“I try to change the way I’m looking at it or the way I’m thinking about it and see if that helps a little bit,” she said. “I have to step back
and think, ‘OK, what am I doing wrong?’ and then try to fix it and see if that works. That’s something I learned through research.”

Sharing the Spotlight

Wilson, like Uhls and Jolley, got a publication for her efforts. And, because her research wasn’t forced into a seasonal timeframe, it was published significantly earlier than theirs—months before Jolley even began his vineyard slog.

“A Protocol for Endophyte-Free Callus Tissue of the Grape Vitis aestivalis ‘Norton/Cynthiana’ (Vitaceae)” appeared in the October 2016 issue of Agriculture Science. Once again, an undergraduate was first on the author list: Wilson, Fuller, Smith, Johnston, DuBois.

The Norton/Cynthiana paper wasn’t Smith’s first publication, “but it’s one of my prouder ones,” he said, “simply because I was in a mentorship position with that one. I trained Aimee, and to see her get that accomplished—I’m more proud of that than I am of some of my
other publications.”

Norton/Cynthiana was Fuller’s first publication, although he’s now a doctoral student with two more under his belt. But he also deflects to the undergraduates on the grape project.

“They spent hours and hours and hours getting that experiment to work,” Fuller said. “Shannon and I were there too, but we had our own experiments, so there was a fair amount of initiative they had to put in, and they did a really fantastic job. We watched them start off as students and end up as scientists.”

The team culture of DuBois’ lab stays the same even as the team itself is continually changing.

At the undergraduate level, the student-to-scientist cycle begins anew each year. Smith, who hopes to wrap up his own research project in a year or two, is overseeing a new group of lab assistants, as well as new Honors thesis students working on their own slices of grape project pie. Some are trying to learn more about the mold living inside Norton/Cynthiana; others are trying to grow new Norton/Cynthiana plants that don’t carry the mold at all.

As of October, Hannah Hall (’18), a former undergraduate Honors thesis student and now a lab technician for DuBois, had found small roots on some of the callus plates, and current Honors thesis students Rebekkah Riley and Sara Moore have been trying to generate plantlets a different way, using callus from flower tissue.

“Once we get a plantlet from this callus and actually get it to grow, we can watch that plant and see whether it takes up the mold,” Johnston said. “Does it survive without the mold if it doesn’t take it up? How does it affect the fruit quality or the fruit flavor or the viability of the plant? As science does, one question leads to more.”

Time to Shine

In research, you’re playing the long game. But there are wins all along the way.

In 2016, just before Wilson graduated from MTSU, Johnston invited her and DuBois to go back with him to Honduras. He was taking a group of students on an agricultural tour there through the Tennessee Louis Stokes Alliance for Minority Participation (TLSAMP) program, and Zamorano University would be one of their stops. Bravo was interested in seeing the new protocol for getting mold-free callus from Norton/Cynthiana.

When the group arrived at Bravo’s lab, Wilson turned to DuBois.

“So, you’re going to do this, and I’ll assist you?” Wilson asked.

“No,” he said, “you’re going to do it, and I’ll assist you.”

Wilson looked surprised, DuBois recalled, but she stepped up.

As Zamorano faculty and students looked on, she demonstrated the procedure with DuBois handing her instruments, “like she was the surgeon and I was the attending nurse,” the professor said.

When they walked out of the lab, they were both glowing. DuBois says one of the highlights of his job is watching students mature
as scientists. And that’s something he’s uniquely positioned to do.

“When Johnston told us that he wanted us to do this presentation, I told myself that Aimee was the one who did the work, that this was her project, her thesis,” he said. “She knew this stuff, and it would be a good experience for her. So I just thought she needed to take the
lead, and I needed to step back and let her shine.”

Thanks to DuBois, Johnston, and Norton/Cynthiana, many more MTSU students will get a chance to shine too.