Shaping the Future of Farming

Thirty-five years ago, when CALS bacteriologist Winston Brill and his colleagues set out to exploit science’s newfound ability to manipulate genes to confer new traits on crop plants, the technology was, literally, a shot in the dark.

Working in a facility in Middleton, just west of Madison, Brill and his team blasted plant cells using a gene gun—a device that fired microscopic gold beads laden with DNA.

The idea was to introduce foreign genes that could confer new abilities on the plants that would ultimately be grown from the altered cells. First as Cetus of Madison, Inc., later as Agracetus and still later as a research and development outpost of Monsanto Company, the Middleton lab was, by all accounts, a hub of plant biotechnology innovation.

“Agracetus was the first in the world to engineer soybean, first in the world to engineer cotton, first in the world to field-test a genetically engineered plant,” recalls Brill, who was recruited by Cetus to establish the lab in the early 1980s. “Thus, the Madison area and the UW influence led to historically important events.”

In December 2016, the $10 million,100,000-square-foot facility—a warren of labs, greenhouses and growth chambers—was donated to UW–Madison by Monsanto to become the Wisconsin Crop Innovation Center (WCIC).

The hope, according to agronomy professor Shawn Kaeppler BS’87—now WCIC’s director—is that the center will add to its string of plant biotechnology achievements as one of just a few public facilities in the country dedicated to plant transformation, where genetically modified plant cells are taken from tissue culture and regenerated into large numbers of complete fertile plants.

The advent of the WCIC “is an unprecedented opportunity to add capabilities and capacity we couldn’t afford otherwise,” says Kaeppler, an expert on corn. Its acquisition by UW–Madison, he and others note, comes at an opportune time as powerful new techniques in synthetic biology are poised to make the development of plants with new or improved traits much more than a shot in the dark with a gene gun.

WCIC will function very much like a core facility, providing cell culture, phenotyping and plant transformation services for researchers at UW– Madison and other universities. It is also coming online at a time when the need for such resources is acute.

“There is a recognized need nationally,” explains agronomy professor Heidi Kaeppler BS’87, an expert in plant transformation who is serving as WCIC’s transformation technology director. “There are just a few public facilities around the U.S. and demand is outpacing the abilities of those facilities. It is a bottleneck.”

For researchers like bacteriology and agronomy professor Jean-Michel Ané, a member of the WCIC scientific advisory board, the new center means he will be able to devote more time to exploring such things as the genetic interplay that occurs when plants and bacteria collude to draw nutrients from the air through the act of nitrogen fixation.

Nitrogen-fixing plants such as soybean, alfalfa and clover are staples of modern agriculture. They are essential to the crop rotation practices that prevent exhaustion of soil from crops such as corn. Ané and many other scientists have long dreamed of engineering the ability to fix nitrogen into plants like corn to transcend the need for expensive and environmentally harmful chemical fertilizers.

However, engineering complex traits such as nitrogen fixation in plants that don’t have that innate ability is a monumental scientific and technological undertaking. To begin with, there are two organisms—the plant and a bacterium—working cooperatively. Each has its own genome, and many different genes from each organism are in play to accommodate the act of drawing life-sustaining nutrients from the air.

To confer that trait on corn, for example, is an exercise far more complicated than tinkering with one or a few genes, notes Ané. “The goal is to create maize that has this association. However, modifying a single gene will not be sufficient,” he says. “We modify many genes at a time. There is a lot of trial and error. We need to try many combinations.”

Those combinations come about in the lab as scientists alter individual plant cells by adding or subtracting genes of interest. Today, scientists can harness new techniques such as CRISPR– Cas9—a fast, cheap and accurate genome editing tool—and potent new cloning technologies that allow scientists to easily assemble multiple DNA fragments and their assorted genes into novel sequences.

Even with potent new tools like CRISPR–Cas9, engineering plants is a big, difficult task. A gene needs to be dropped in the right place on the genome and be in association with the right “promoters,” segments of DNA that initiate gene transcription, the first step toward expressing a new gene in an organism. Once plant cells are genetically altered, they must be transformed into large numbers of actual plants for further testing in the lab and, ultimately, the field. It is essential to know, for example, that the new genetic construct is stable, that the new genes are passed from generation to generation, and what effects they may have on plant growth or yield.

The promise of WCIC, Ané believes, will be the opportunity to work through all of those steps more efficiently and cost-effectively, and carry projects from the lab to the field much faster.

“We can focus on really doing science instead of growing plants,” Ané says. “We can now make genetic constructs very quickly. Within a month we can make hundreds of constructs. The limiting aspect is plant transformation. However, the scale of transformation we can do at WCIC allows us to think seriously about applying synthetic biology to plants.”

To begin with, WCIC is providing plant transformation services for corn, soybean and sorghum, big commercially important crop species. But Shawn Kaeppler envisions WCIC playing a role, as well, with crop plants that have not yet risen to the top of commercial research agendas.

To date, commercial interest has focused primarily on just a handful of traits—insect and herbicide resistance—in a handful of widely planted crops. Uncharted territory, Kaeppler says, exists in the full range of crop plants and their many different traits.

A ready example is switchgrass, a native perennial that is under the microscope at the Great Lakes Bioenergy Research Center (GLBRC), a U.S. Department of Energy- funded multi-institutional research center headquartered on the UW–Madison campus. The grass is seen as a potential feedstock for converting its biomass to liquid fuel. However, efficient conversion of plant materials to energy remains a challenge, and plant genetics will play a big role in refining the traits that will make that possible.

“WCIC will help lead us to the next generation of crop breeding and plant genetics,” explains Kate VandenBosch, the dean of CALS, referencing, broadly, the genetic makeup of the crop plants in play. “Scientific agencies at the federal level have invested a lot in understanding genomes, but we still have a lot of work to do to understand how those genes function.”

Indeed, genetic sequencing technologies have advanced to the point where new plant genomes are sequenced with increasing regularity. The genomes of crop plants like watermelon, cucumber, potato, soybean, wheat, corn and many others have been sequenced, but as VandenBosch notes, exploring those sequences to identify the genes that govern plant traits is an unexplored frontier.

Shawn Kaeppler’s own research, for example, is a window to both the complexity and opportunity that lurk in the genomes of plants. One of his interests is the complex of genes—involving anywhere from tens to hundreds of genes—that governs the root architecture of corn. Knowing more about the combination of genes that directs the plant to send shoots into the soil, it might one day be possible to engineer a plant that can send its roots deeper into the earth, providing farmers with a hedge against drought.

“Fifty to 70 percent of all maize genes are expressed in roots,” Kaeppler says. “Some control processes in all parts of a plant, and some specifically control root development and response to environmental stimuli.”

A gene of interest for Kaeppler and his team is one that influences root angle. “Altering root angle even five to 10 degrees can dramatically increase the rate that roots get deep in the soil,” as well as how much root biomass a plant lays down at depth, he explains.

Identifying those candidate genes and mutations of those genes means they can be selected and manipulated in the laboratory to generate plants with different root structures. At WCIC, those plants can be grown in quantity, their new qualities studied and, if promising, tested in the field. The goal, of course, is to provide a practical outcome that is useful to growers.

In plant science, numbers matter. The more plants you can grow to test a new genetic combination, the better, as there are so many variables in play.

“In many aspects of science, doing things on a large scale is critical,” says biochemistry professor Rick Amasino, an expert on flowering in plants. “To have WCIC in our capability is great. Large-scale transformation opens up a lot of possibilities.”

Amasino, who is also a member of WCIC’s scientific advisory board, views the center as an important new national resource. Individual labs, he explains, do not have the same capacity.

“This has the potential to be on a scale greater than any other university’s,” Amasino says. “Individual labs can’t generate the hundreds or thousands of transgenic plants needed to fully test certain hypotheses. Labs around the country and, hopefully, around the world can now do experiments they couldn’t otherwise do. There are so many opportunities out there.”

A Facility With Deep CALS Roots

The name is new, but the Wisconsin Crop Innovation Center (WCIC) holds a prominent place in the young history of agricultural biotechnology. The facility also has long and deep ties to CALS researchers and alumni.

Originally known as Cetus of Madison, Inc., the Middleton facility—owned by
the Cetus Corporation of Emeryville, California—opened in 1981 under the direction of CALS bacteriology professor Winston Brill. The Wisconsin Alumni Research Foundation (WARF) played a key funding role in the early days of the company.

Cetus of Madison, Inc. initially focused on evaluating and testing a wide variety of natural rhizobia species to better understand their role in nitrogen fixation and nodulation in legumes, with the hope of someday enabling maize to have that capacity.

As interest in biotechnology grew in the early 1980s, the facility’s focus changed to inventing and innovating ways to introduce genes into plants. In 1984, Cetus Corp. sold half of its interest in Cetus of Madison, Inc. to the WR Grace Co.—and thus the company name “Agracetus” was born.

Great discoveries followed. An electric “gene gun” and transformation methods developed at Agracetus revolutionized the plant transformation process. Many plant species were subsequently transformed, including tobacco, peanut, sunflower, soybean, maize, cotton, cranberry, canola, poplar, wheat and rice. CALS researchers Kenneth Raffa, Brent McCown PhD’69 and Elden Stang, as well as WCIC associate director Michael Petersen BS’87 (then still an undergraduate) and Richard Heinzen MS’74, collaborated with Agracetus scientists during that period. But that wasn’t the only significant research taking place. Other studies critical to agricultural improvement focused on cotton fiber quality, transformation process improvements, polymerase chain reaction (PCR) method development, insect and disease resistance and herbicide tolerance. A number of CALS faculty, including Michael Sussman, Richard Amasino and Andrew Bent, were highly involved in consulting with Agracetus in many of these areas.

In 1990, WR Grace Co. acquired full ownership of Agracetus. During the early 1990s, Agracetus ventured into research in DNA vaccines—using an improved “gene gun”—and contracted plant transformation services to others within the industry, including, most notably, the Monsanto Company. Collaborating with biological systems engineering professor Richard Straub PhD’80 (now CALS senior associate dean) and other CALS researchers, the company also worked on producing industrial enzymes in plants.

After successfully generating plants that eventually became commercial products
for Monsanto, including Roundup Ready Soybeans and Bollgard Cotton, the facility was acquired by Monsanto in 1996.

Over the next 20 years, Monsanto used the facility as its primary site for soybean and cotton transformation. Other R&D at the site included corn, canola, wheat, rice and alfalfa transformation, gene expression, molecular testing and seed chipping/genotyping.

The site was considered a “center of excellence” for Monsanto due to its highly innovative employees, high throughput transformation capabilities and ability to consistently perform above and beyond expectations.

In July of 2016, Monsanto relocated a number of remote functions back to its St. Louis headquarters in the interest of business consolidation. In the hope that the Middleton facility would continue to work toward the betterment of agriculture, Monsanto the following December donated it to longtime collaborator the University of Wisconsin– Madison, along with University Research Park.

Not surprisingly, given the long history of CALS involvement, agronomy professor Shawn Kaeppler BS’87 was chosen to serve as facility director.

The Shocking Truth

It is well known that a certain kind of fish swims the world’s waters protected, as it were, by its very own stun gun.

Unknown, until now, is how electric fish evolved such a defense. A team of researchers led by CALS biochemistry professor Michael Sussman has established the genetic basis for the electric organ, an anatomical feature found only in fish. It evolved independently half a dozen times in environments ranging from the flooded forests of the Amazon to murky marine environments.

“These fish have converted a muscle to an electric organ,” says UW Biotechnology Center director Sussman, who began this research almost a decade ago. The study, recently published in the journal Science, provides evidence to support the idea that the six electric fish lineages used essentially the same genes and developmental and cellular pathways to make the electric organ, which fish use to communi- cate with mates, navigate, stun prey, and as a shock- ing defense. The jolt from an electric organ can be several times more powerful than the current from a standard household electrical outlet.

Worldwide, there are hundreds of electric fish in six broad lineages. Their taxonomic diversity is so great that Darwin himself cited electric fishes as critical examples of convergent evolution, where unrelated animals independently evolve similar traits to adapt to a particular environment or ecological niche.

The new work includes the first draft assembly of the complete genome of the South American electric eel. “A six-foot eel is a top predator in the water and is in essence a frog with a built-in five-and-a- half-foot cattle prod,” says Sussman. “Since all of the visceral organs are near the face, the remaining 90 percent of the fish is almost all electric organ.”

Electric fish have long fascinated humans. The ancient Egyptians used the torpedo, an electric marine ray, in an early form of electrotherapy to treat epilepsy. Much of what Benjamin Franklin and other pioneering scientists learned about electric- ity came from studies of electric fish. In Victorian times, parties were organized where guests would form a chain to experi- ence the shock of an electric fish.

All muscle cells have electrical potential. Simple contraction of a muscle will release a small

amount of voltage. But at least 100 million years ago some fish began to amplify that potential by evolv- ing from muscle cells another type of cell called an electrocyte—larger cells, organized in sequence and capable of generating much higher voltages than those used to make muscles work.

The “in-series alignment” of the electrocytes and the unique polarity of each cell allows for the “sum- mation of voltages, much like batteries stacked in series in a flashlight,” says Sussman.

In addition to sequencing and assembling DNA from the electric eel genome, the team produced protein sequences from the cells of the electric organs and skeletal muscles of three other electric fish lin- eages using RNA sequencing and analysis.

“I consider exotic organisms such as the electric fish to be one of nature’s wonders and an important gift to humanity,” says Sussman. “Our study dem- onstrates nature’s creative powers and its parsimony, using the same genetic and developmental tools to invent an adaptive trait time and again in widely disparate environments.”

And the findings may be useful to humans. “By learning how nature does this, we may be able to manipulate the process with muscle in other organ- isms and, in the near future, perhaps use the tools of synthetic biology to create electrocytes for generating electrical power in bionic devices within the human body or for uses we have not thought of yet,” says Sussman.

Sussman’s collaborators include Harold Zakon of the University of Texas at Austin and Manoj Samanta of the Systemix Institute in Redmond, Washington. The study was funded by the National Science Foundation, the W. M. Keck Foundation and the National Institutes of Health.

Tech Transfer Showcase

When CALS biochemistry professor Harry Steenbock experimented with vitamin D in the early 1920s, his work proved groundbreaking in more ways than one.

Steenbock’s discovery that he could increase the vitamin D content of foods through irradiation with ultraviolet light eventually eliminated rickets, a then-common and often deadly disease characterized by softening of the bone due to vitamin D deficiency.

With his own $300, Steenbock patented his discovery and offered it to the University of Wisconsin. When the university declined, Steenbock conceived of the idea to form a foundation to collect, invest and distribute money earned through research-based discovery—
a pivotal step in establishing the Wisconsin Alumni Research Foundation (WARF), the nation’s first university technology transfer office. WARF’s first licensing agreement with Quaker Oats in 1927 led to the fortification of breakfast cereals with vitamin D.

Since then WARF has patented nearly 2,000 university inventions. And—in the grand tradition of Steenbock—many of them stem from the labs of CALS scientists and alumni. Here we present some highlights from recent years.

Deltanoid

Though the term biotechnology was little known in his time, Steenbock was one of the world’s first biotechnologists—and he passed on that torch to his gifted graduate student, Hector DeLuca.

The path was not always smooth, and DeLuca hit some obstacles when his own seminal work on vitamin D in the 1960s led him to WARF. When he discovered the active form of vitamin D and chemically identified its structure, he was unable to file a patent due to unwieldy government restrictions. DeLuca eventually obtained a patent with the help of WARF patent attorney Howard Bremer and some influential people in Washington. That same group worked with federal legislators on the 1980 Bayh-Dole Act, which allowed nonprofit organizations to obtain patents spurred by federally funded research. As a result, WARF now holds more than 200 active patents from the DeLuca lab.

DeLuca is the founder of three spin-off companies, each stemming from his vitamin D work. Bone Care International, a maker of drugs to treat dialysis patients, was sold in 2005 to the biotech firm Genzyme for nearly $600 million. A second company, Tetrionics (now SAFC Pharma), was acquired by Sigma Aldrich Fine Chemicals in 2004 for close to $60 million.

Now DeLuca’s main focus is Deltanoid Pharmaceuticals, which he founded nearly 10 years ago with his fellow biochemistry professor (and wife) Margaret Clagett-Dame. The company is testing various vitamin D derivatives against osteoporosis, psoriasis, and kidney and autoimmune diseases, as well as other types of compounds to treat kidney failure. In clinical trials one vitamin D derivative seems to be highly effective in stimulating bone growth, and a number of other Deltanoid products are nearing the human testing phase.

With a business office located on Madison’s Monroe Street and about 10 employees, DeLuca describes Deltanoid as small but tenacious. “Our plan is to keep the company lean and mean until it has an income of its own,” he says.

TRAC Microbiology

Food contamination outbreaks generate headlines, especially when they result in illness or death. Virginia Deibel, while still a graduate student in food science and bacteriology at CALS, combined her interest in both subjects by forming TRAC Microbiology, a company that helps keep our food supply safe.

Deibel describes how it felt when TRAC played a pivotal role in identifying the type and location of bacteria that forced a shutdown in a large meat processing plant. The culprit turned out to be Listeria monocytogenes, the same bacteria that recently killed several dozen people who ate contaminated cantaloupes.

“We went in and found where the bacteria were harboring, removed it and tested that it was effectively gone. We then rewrote the client’s food safety programs, retrained all their employees and presented our corrective actions to the USDA,” Deibel recounts. “During the retraining phase I had employees coming up to me and thanking me for reopening the plant, which impacted entire families. That made me realize what we could do for a community.”

Deibel founded TRAC (for Testing, Research, Auditing and Consulting) 12 years ago. She was less than 18 months away from completing her Ph.D. when she began redirecting her energy toward writing a business plan and securing a start-up loan of $400,000.

“I knew from my work as a food scientist that there were many smaller companies that needed help with food safety,” says Deibel. “They simply did not have the necessary infrastructure to implement food safety systems.”

Initially TRAC services included helping food plants develop and update their food safety systems, train their quality assurance personnel and provide scientific justification for such practices as freezing, packaging and adding preservatives.

“Our original goals were to conduct research projects and provide food safety consultations,” says Deibel. But she soon discovered that many small food companies needed testing to meet customer requirements. That need inspired Deibel to expand its testing services, and TRAC, which eventually grew to 30 employees, soon succeeded in attracting larger clients from around the region.

Last fall Covance, one of the nation’s leading bioscience companies, announced the acquisition of TRAC Microbiology. Covance had paid close attention to TRAC and tapped Deibel to head development of its own food safety consulting division.

“Covance has excelled in so many different arenas—drug development, nutritional chemistry. I’m enjoying the challenge of helping such a respected company develop and grow a food microbiology arm,” says Deibel.