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UK's Tobacco and Health Institute Branching Out:
Tobacco as a "Factory" to Grow New Products

by Jeff Worley

There's a new breed of researcher at UK's Tobacco and Health Research Institute (THRI) who might also be thought of as a factory worker. The "factory," in this case, is the tobacco plant.

Photo of tobacco fieldResearchers in recent years have found that tobacco's large green leaves can be an incredibly prolific seedbed, a kind of super-rich petri dish in which to produce everything from antibiotics to sugars to industrial enzymes -- even anti-cancer and AIDS-fighting compounds. "Because its genes and biology are so easily manipulated, tobacco is probably the best white rat in the plant kingdom," says Glenn Collins, UK professor of agronomy, who adds that because tobacco has been the primary plant of choice for genetic bioengineering for a number of years now, there's a large data bank from which to draw information about the plant's genetic structure and its potential for being manipulated genetically.

"In the past year and a half, THRI has undertaken a major refocus of its mission," says Maelor Davies, director of the institute. "We now have in place a comprehensive plant biotechnology program whose goal is to help develop new products based on transgenic tobacco. The purpose is twofold: to explore the use and potential of genetic engineering to create new crop opportunities for farmers, and to seek industrial partners who will collaborate with the institute to grow useful new products in the tobacco plant."

This new commercial mission of the institute follows two decades of work in which THRI was devoted almost entirely to tobacco-related medical research. This previous pure-science mission focused on how tobacco use causes diseases. Projects ranged from work on the effects of tobacco on the human cardiovascular and pulmonary systems, to the effects of environmental smoke and the relationship between smoking and other diseases. "THRI isn't moving totally away from delving into health-related issues, not at all," says Davies. "But presently two-thirds of our efforts are targeted to transgenic tobacco."

This shift of focus to developing new crop possibilities for farmers should be good news for Kentucky, where the farm economy has historically been so dependent on tobacco. Primarily because of growing public awareness of the dangers of smoking, the long-term economic potential for tobacco production remains cloudy, and in recent years there has been considerable interest in developing new crops for Kentucky farmers.

"New crops imply change, and few people like fundamental change because it creates uncertainty," says Orlando Chambers, a newly hired agricultural economist in THRI's Tobacco Biotechnology Group. "Farmers like the crops they know how to produce and which are adapted to their soils and their machinery. Many of Kentucky's small family farms have survived largely because of the nature of tobacco production. The small acerage requirements and the large potential profits make finding alternatives to tobacco very difficult."

There have been several enterprises suggested to farmers as a way to buffer any declines in tobacco income, for example, vegetable production. There have been some successes with alternative crop-growing, but a tobacco farmer who suddenly goes into the tomato business takes a risk that there might not be much of a need for this crop locally, or that the market is overly competitive.

"A major point we need to emphasize is that we're not talking about new plants as much as new markets," says Davies. "What we're really trying to do for the tobacco farmer is to help develop a new commercial opportunity." Here is the scenario Davies envisions: companies that want certain products will contract with farmers to grow a specified acreage of transgenic tobacco. This way, the farmer will have another unique market to call his own.

Photo of Maelor DaviesTHRI Director Maelor Davies: "THRI isn't moving totally away from delving into health-related issues, not at all. But presently two-thirds of our efforts are targeted to transgenic tobacco."

There are sound reasons for developing new crops based on transgenic tobacco, according to Chambers. Tobacco produces a large amount of biomass per acre (some experimental methods have produced as much as 90 tons per acre), it is easy to engineer, and it is well suited to growing conditions in Kentucky. "It's important to note that we're not suggesting to farmers that they quit growing their traditional crops and gamble on transgenic crops; we're suggesting that farmers might want to consider growing some acres of transgenic tobacco as an additional potential income source," Davies explains.

These secondary crops would add to the already-impressive amount of tobacco Kentucky produces. Tobacco is the state's No. 1 cash crop, and Kentucky is second only to North Carolina in tobacco production. In Kentucky the crop is grown by 67,000 farmers in 119 of 120 counties, from the western plains to the steep hillsides in the Appalachians. Tobacco accounts for 25 percent of total farm sales in Kentucky.

Laying the groundwork at UK for tobacco biotechnology
"Here in the College of Ag, we started thinking in the early '80s about harnessing the tools of genetic engineering," says Collins, who is currently marking his 31st year at UK. "Now, as far as putting foreign genes into a tobacco plant using newfound recombinant DNA, we already have a very solid knowledge base."

Collins says a recent major event was an international symposium on plant engineering held at UK on October 1-4, 1995. John Diana, former director of THRI, and the THRI board of directors committed resources to sponsor this symposium with the specific focus of engineering plants for possible commercial products and applications.

"This gathering was tremendously important, a huge success," Collins recalls. "We focused on five topical areas and attracted 425 participants from 23 countries. This was the first symposium ever held specifically devoted to engineering plants to make commercial products."

One result of this conference was the publication of a special issue in the Annals of the New York Academy of Sciences titled Engineering Plants for Commercial Products and Applications. The book, edited by Collins and former UK agronomy professor Robert Shepherd, contains 20 articles on a wide range of plant genetic engineering technology.

A nearly concurrent event, Collins says, was the hiring in February 1996 of Davies, who came to UK from industry with a solid background in the genetic engineering of plants. Davies, originally from Wales, had worked at the Calgene company in California since 1981, where he focused on genetically engineering canola, or rapeseed, which has traditionally been used to make margarine. (Acres of rapeseed, also known as mustard, make an indelible visual impression -- a field of flowing, brilliant gold.) The purpose of Calgene's work with this crop was to alter the composition of the oil for new applications, for making detergent products, soaps and shampoos, for example.

"This work at Calgene was very much a collaborative effort," Davies explains. "I managed the project with one other scientist, and we were supported by several technical staff." The goal of the research was to cause the rapeseed plant to manufacture lauric acid, an important ingredient in detergent products and soaps. This acid is currently obtained from palm kernel and coconut oil.

"A million tons of lauric acid are imported by the U.S. every year, and as the demand for various soap products was rising worldwide, U.S. companies were interested in finding a domestic source for lauric acid," Davies says. Huge new markets for personal care products were then opening, he explains, in developing countries like China, where more and more people could suddenly afford to buy products like laundry detergent.

In their work, the Calgene team used a local wild plant -- the California bay -- which also produces lauric acid. The researchers put a gene from the bay plant into the rapeseed plant to see what changes would occur in oil composition. Once they had a base measurement of the changes that resulted, the group, by increasing the number of bay genes inserted, "tuned the process up" to get higher lauric yields. The result, after eight years of work with the rapeseed plant, was a commercially viable product that is now in successful production.

"Maelor was hired here primarily on the basis of his very successful work at Calgene," Collins says. "There's a commonality of course in using the techniques of biotechnology with various plants, and Maelor was eager to take up the challenges here of bioengineering the tobacco plant for commercial potential."

New Faces at THRI
"When I saw the ad for this job, it just spoke to me," says Assistant Professor Deane Falcone, who joined the plant biotechnology group at THRI in January of last year. Falcone, who has a Ph.D. in microbiology from Ohio State University, came from the Carnegie Institution of Washington at Stanford University, where he worked as a postdoctoral fellow for four years.

"At Stanford I worked with Chris Somerville, one of the top plant biologists in the country," Falcone says. "This was a tremendous experience for me, since Chris is one of the pioneers in using mutagenesis to solve problems in plants." Mutagenesis is a process of generating stable, genetic changes in plants.

Falcone's research plan is focused on a new way of genetically engineering tobacco to make various compounds, some of which may have commercial uses. Falcone explains that plants already make a diverse array of compounds, that 25 percent of all medicines, in fact, are made by plants. Falcone characterizes the tobacco plant as a "willing factory"; his work specifically involves tinkering with tobacco's genetic structure in order for the plant to make its less abundant compounds in much greater amounts.

"Although tobacco makes hundreds of compounds naturally, in order for them to be useful commercially we have to cause the plant to over-accumulate these substances," says Falcone. By "useful," Falcone specifically means useful to firms in the business world that might be interested in striking up a partnership with UK, companies which would use THRI's technology in order to "grow" their products in tobacco.

Falcone describes the work he does in the lab as "complicated science but simple procedure," admitting that what he does isn't exactly a spectator sport.

Photo of Orlando ChambersOrlando Chambers is involved in identifying companies that might be interested in using THRI's expertise in plant biotechnology in order to "grow" specific products in tobacco.

His genetic tinkering takes place in a petri dish, in which a cut tobacco leaf is exposed to a culture of bacterium commonly used as a transport system for any genetic material Falcone wants to put into tobacco to cause it to over-accumulate already-present substances. The gene is introduced into the bacteria which, in turn, invade and infect the exposed cells of the plant, inserting the introduced genes into the plant's cells. "We can then grow an entire tobacco plant from just one of these cells," Falcone says. "We call the process 'regeneration'; the payoff is that every cell of the new plant will contain the new gene."

"But don't the bacteria kill the plant cells?" Falcone asks rhetorically, in anticipation of that question. "No — because any 'bad genes' have been taken out of this particular bacterium."

He explains that plant geneticists commonly use a species of bacteria called Agrobacterium, which causes galls -- large tumor-like protuberances -- in plants. This bacterium introduces part of its own DNA into a plant cell's DNA and overwhelms the infected cells; the galls are the dramatic evidence of the bacteria's success.

"The tumor-causing genes have been removed, though, from the bacterium we use; it is disarmed. What's left is the DNA necessary to do the transformation," Falcone says.

In this process, Falcone doesn't know exactly which substances will over-accumulate in the altered plant. The important thing, he says, is that the over-produced compounds can now be identified (by biochemical assays or analytical methods) and then isolated.

This basic science is not only important in furthering knowledge of plant genetics, Falcone says, but is also a starting point to interest business and industry in the possibility of using UK-developed technology to make naturally derived materials ranging from insecticides to pharmaceuticals.

As a result of work on the basic science of plant bioengineering, another aspect of the process will be better understood — the development of "promoters." Promoters are sections of DNA which must be coupled with a gene in order for the gene to "express," or function, in plant tissue.

"If we want to put a plant or animal gene into tobacco, we have to attach it to a promotor for the gene to work," says Davies. "It kick-starts the gene into action, you might say." The gene itself, Davies explains, is like the software on the computer: it can't be activated without an operating system. What's important about a promoter is that it activates the gene and also tells it when to turn off.

Part of the swirl of activity at THRI now involves the development and, hopefully, patenting of new genetic promoters and "switches" which will serve as the starting motors in the process of turning tobacco plants into gene-making sources of new products. This research focus is what led another new hire -- Susheng Gan -- to THRI.

"I came to UK from the University of Wisconsin because of the unique, new program here in biotechnology," says Gan. "I like to do both basic and applied research, so this is a perfect setting for me." While at the University of Wisconsin, Gan invented a molecular technique that delays plant aging. According to Gan, this technology can be used to create new crops that will have higher yields as well as vegetables that will remain fresh much longer.

"This technology has been patented and licensed out to several biotechnology companies," says Gan. "I am very happy to see my research being put to practical use."

It was through several years of intense work on plant aging at Wisconsin that Gan discovered and identified six "aging genes" from a weed called Arabidopsis. He isolated the promoters of these genes and coupled one of the promoters to a well-established anti-aging gene and, in a sense, turned the aging process against itself.

"When we put the new hybrid gene into a plant at the onset of aging, the aging promoter is activated to direct the anti-aging gene to manufacture cytokinins (a type of plant hormone that inhibits aging). In doing this we, in a sense, re-program the plant's aging process," Gan says.

The results of the genetic inducement are dramatic. A non-engineered control tobacco plant begins to show some yellowing leaves after six weeks; at 20 weeks, the plants leaves have nearly all turned brown and have wilted. The 20-week-old transgenic plant is vibrant and green, its leaves still sturdy.

Gan is continuing his research on what he calls "leaf senescence" at THRI, work which will not only contribute to basic knowledge regarding how and why plants age, but will also allow plant geneticists to devise more sophisticated ways to genetically manipulate senescence for agricultural improvement.

"Senescence research will lead to the development of new tools for tobacco biotechnology," Gan says.

"Susheng and Deane were hired for their expertise in specific areas of plant bioengineering research," Davies says. "And Orlando Chambers was an important new addition, since he has a very relevant background as an ag economist." Indu Maiti, who has a solid background in developing promoters, and Quinn Li, who has considerable experience developing transgenic tobacco plants, complete the program team. "These are the most ideally suited individuals you could imagine to move our work to the next level," says Davies. An additional scientist with expertise in disease resistance will join the group in the near future.

The Commercial Payoff
Even though the THRI program is in its early stages, the business community has already shown an interest in the growing UK expertise in genetic manipulation of plants. One company that is collaborating with THRI is Biosource Technologies Inc. of Vacaville, California. Biosource has been operating a 22-acre tobacco field in Owensboro, Kentucky, for two years, producing genetically engineered proteins for use as infection-fighting drugs.

Photo of Deane Falcone Deane Falcone characterizes the tobacco plant as a "willing factory." His work involves tinkering with tobacco's genetic structure in order for the plant to make its less abundant compounds in much greater amounts.

"We ground up 165 tons of tobacco leaves last summer," says Barry Holtz, vice president of process development for the company, who adds, "This isn't a drill -- this is the real thing." The real thing in this case, the end product, is an antibiotic that kills bacteria in a new way — by creating a hole in the cell membrane of bacteria.

The tobacco institute can not only offer such companies the facilities to carry out this kind of high-tech agriculture, but can also provide the technical and academic expertise to support it, Davies says.

"One reason BioSource decided to establish its field operation in Owensboro was the expertise at the University of Kentucky in plant biotechnology," Holtz says. "The fact that Maelor was brought in (he used to work just down the road from us in Vacaville) and the work we've done with Scott Smith in agronomy and with others at UK added up to a nice confluence of factors that helped us make up our minds."

Another THRI research collaboration is with InterLink Associates Inc. of Princeton, New Jersey. This relationship, which began last July, combines InterLink's developments of novel, bioactive peptides with THRI's expertise in plant transformation and the development of genetic promoters. "This is an ideal relationship," says Davies. "We have the technology and knowhow; they have the marketing ideas. They can develop commercial possibilities, which is difficult for universities to do, and which would be outside THRI's mandate."

The objective of this work will be to see if the InterLink peptides introduced into tobacco by THRI scientists will raise resistance to plant diseases such as tobacco blue mold, and to assess the potential for large-scale production of such peptides using the tobacco plant.

Blue mold is a major nemesis of tobacco, and currently all breeders can do to fight it is use the natural defenses of the tobacco plant and try to breed in characteristics from other tobacco varieties to strengthen this resistance. "If we had some agent that could kill blue mold, we could introduce that into the plant, even if it's an antifungal agent made by spiders—or giraffes," says Davies. Last year in Kentucky, blue mold caused an estimated loss to farmers of $165 million.

A third THRI industry relationship was established last November. This agreement between THRI and United Agi Products (UAP) of Lubbock, Texas, is enabling the company to develop new crop varieties containing a genetic promoter which was developed at THRI by Robert Shepherd and Indu Maiti of UK's agronomy department. UAP is interested in developing a series of major agricultural crops with improved farming practices. The soybean will be the initial crop of interest.

While THRI's focus remains firmly on tobacco, Davies is pleased to see THRI-developed technology used by others to improve other crops farmed in Kentucky. "And by establishing these relationships with companies, we have their attention when we make newsworthy developments with tobacco," he says.

The Future of Plant Bioengineering
Many questions remain unanswered concerning this new tobacco production. Intellectual property rights, the cost of crop development, and how this new tobacco production will coexist with the production of traditional tobacco must be considered.

"For commercialization to be feasible, it will be necessary for the production to be profitable for the farmer and for the processor," says Chambers. "Markets for the products will need to be available and long term to ensure an adequate return on the cost of R&D."

And if this is to be the growing area of research and development that it appears to be, the question could be asked where the plant biotechnicians of the future will come from.

Photo of Susheng GanSusheng Gan has invented a molecular technique that delays plant aging. This technolgy can be used to create new crops that will have higher yields as well as vegetables that will remain fresh much longer.

"We'll be supplying our fair share from right here at UK," is Collins' answer. "We started a new undergraduate degree program in 1988 in agricultural biotechnology," he says, "and this program has taken off tremendously." The program currently has about 110 students enrolled, and it graduates from 16 to 20 a year, Collins says.

In looking back at his year and a half of working to get the THRI plant biotechnology program off and running, Davies says he is pleased that UK has built several new relationships with companies and has revitalized others.

"Things have moved along faster than I would have thought," he says. "I believe this will be a snowballing kind of thing: the more companies that we can form partnerships with -- and who realize the benefits of such a partnership -- the more will jump on the bandwagon. These next few years are going to be very exciting."