If you drive through Central Washington’s mint-growing country in mid-summer, you’re likely to be overwhelmed by the scent of mint rising like an exhalation—at once delightful and inescapable—from the surrounding fields. In fact, your senses might deceive you into believing that not much has changed in the last 30 years or so. But during that time Rod Croteau, professor at the Institute for Biological Chemistry at Washington State University, has been doing research that has helped make Washington mint plants produce more and better peppermint.
Peppermint plants produce menthol, which is a terpene, as are all the other compounds Croteau researches. Terpenes are chemicals put together in specific ways from units containing five carbon atoms. Menthol is a small terpene, with just 10 carbons—small enough to evaporate, which is why we can smell it, says Croteau. Rubber is a terpene, a huge one with hundreds of thousands of carbons. Taxol, used in the treatment of breast cancer, is a terpene, as are some of the resin compounds made by trees to protect themselves from bark beetles.
“Nature uses only so many tools,” says Croteau, so what he and his fellow researchers have learned through the work with mint has been applicable to work on these and other terpenes.
Peppermint flavoring is made from oil produced by the peppermint plant, oil that is a mixture of at least 25 different components. The primary ingredient is always menthol, however. “It’s what gives you that cooling sensation,” says Croteau. The oil mix produced by a given plant varies with location and growing conditions. Yakima oil is easy to tell from Flathead Valley oil or oil from other countries. American peppermint oil is a high-end, quality product, says Croteau.
The taste of a given peppermint product depends on the mix of oils used to flavor it, a mix that usually results from a blending of oils from plants of different regions.
Croteau began his work with peppermint in 1976, when he received the start of what has become continuous funding from the Washington Mint Commission. The Commission originally was looking for ways to improve the state’s production of mint. The costs were high, there was competition from less expensive foreign oil, and synthetic oils could be made from petrochemicals. Croteau chose to concentrate on increasing yields and improving the composition of the oils.
At the start, the research involved classic biochemistry and the manipulation of agronomic practices. “We had little idea of practical molecular techniques then, for they didn’t exist,” says Croteau. Nor was it possible to use breeding programs or mutants to improve production. The mint plant is a sterile hybrid created hundreds of years ago in nature.
The biochemical research was aimed at determining the pathway the plant uses to make menthol. The experiments were tedious and time consuming—it took the laboratory a decade to elucidate the nine steps in the main pathway the plant uses to produce menthol. Understanding that pathway was important in that it helped the researchers understand the effects of environmental and agronomic conditions on oil production and indicated agronomic manipulations that might alter yield or composition.
The lab studied those manipulations primarily in the greenhouse, where conditions could be standardized, and determined that both water use and irrigation method were important. Most irrigation of mint had been done by furrow irrigation but is now done mostly by overhead systems, a more economical method that unfortunately reduces yield. The plant’s oil glands, which are where peppermint oil is made, are on the leaf surfaces, and overhead sprinkling disturbs them. In addition, the lab found that moderate, well-timed water stress increased the yield of oil with good composition.
Croteau’s group also looked at harvest timing, traditionally based on word of mouth, and found that harvesting when 10 percent of the plants are in flower maximizes the yield of peppermint oils with good composition.
In the early 1990s, the lab developed a method for isolating oil glands from the leaves, providing a highly enriched source of material not only for biochemical studies but also for what would follow, studying the genes responsible for making the proteins active in the pathway that produces menthol.
The lab’s approach to genetic manipulation was different than most, however. To assuage the public’s fear of moving genes from one organism to another, the lab used only mint genes to improve mint.
Once the genes active in the oil glands were isolated, they were compared to already characterized genes. Several genes were further studied and manipulated, and two were chosen to move into mint plants. One improves yield and was overexpressed so that the plant would make more than a normal amount of the protein it coded for. The other reduced undesirable oil components and was knocked out so that the plant would make less than normal amounts of these components. The two plants that resulted showed roughly a 50 percent higher yield and 50 percent fewer undesirable oil components, respectively.
Both of these genes have been incorporated into a plant that is the focus of much of the lab’s current work. This plant will be used as parental stock, and a variety of other genes will be moved into individual parental stock plants. The goals for this work are to further increase the yield, up to double that of the parent plant, and to improve the quality of the peppermint oil produced. Differences that, again, you won’t see or smell, but that will make Washington mint growers even more competitive.