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Gene Transformation
Gene transformation: high-tech hope to solve scab
By Tracy Sayler
Some crop scientists conducting research under the U.S. Wheat and Barley Scab Initiative are employing a high-tech defensive strategy in the fight against scab in wheat and barley, called gene transformation.
Under the same umbrella as biotechnology and genetic engineering, gene transformation is the process of introducing genes into plants by methods which by-pass the sexual seed production process. Essentially, it is a process by which genes (the parts of a cell that provide blueprints for inherited traits) are "cut" from the cells of one organism and "pasted" and integrated into the cells of another organism. Once the cells are transformed, they are grown into new plants capable of "expressing" a desired characteristic.
The process is still relatively new to wheat and barley research. However, more research attention, on several fronts both public and private, is being placed on using genetic engineering to: 1) identify genes in wheat and barley that are involved in the scab defense response, such as mapping scab resistance genes with molecular markers - road signs or tags to mark regions of the plant chromosomes that carry scab resistance genes; 2) identify and insert antifungal genes in wheat and barley from other wheat and barley germplasm or other organisms, including bacteria and fungi; 3) identify and insert genes that can detoxify deoxynivalenol (DON) a contaminating byproduct of scab.
Nancy Alexander, microbiologist at the National Center for Agricultural Utilization Research (NCAUR), the U.S. Department of Agriculture's Agricultural Research Service lab near Peoria, Ill., was involved in the discovery of one promising gene now under study at several research labs across the country.
Several years ago, researchers at the NCAUR lab inoculated the heads of wheat with several strains of Fusarium to evaluate their effects. They noticed that a genetically-engineered strain of Fusarium sporotrichioides that did not produce DON. The strain also had less scab. "There's reason to believe that the toxin has an important role in the virulence and life cycle of the disease," says Alexander. "So we thought, can we make a plant that's resistant to the toxin, and therefore resistant to Fusarium head blight?"
Further research involving genetic engineering led to the trichothecene-resistant gene, or TRIr gene, now called the TRI101 gene. Trichothecene is the generic name for the toxins produced by Fusarium. The TRI101 gene chemically modifies DON, rendering it nontoxic. DON is a protein inhibitor: the TRI101 gene reduces protein inhibition, allowing a plant's defensive mechanism to be fully active.
Patricia Okubara and Ann Blechl, crop scientists at the USDA-ARS Western Regional Research Center, Albany, Calif, are propagating transgenic plants with anti-fusarium genes. They have developed at least 3 independent lines carrying one of several anti-fungal genes, one being PDR5, a gene that comes from yeast and affects DON. It serves as a molecular pump; it protects yeast from toxins by transporting the toxins out of the cells.
Sufficient seed of the lines containing the anti-fungal genes is now ready for planting in greenhouse resistance trials to be conducted by Ruth Dill-Macky and Bob Busch at the University of Minnesota, St. Paul. Okubara and Blechl's WRRC research group is collaborating with U of M wheat breeder James Anderson to perform genetic crosses to generate germplasms carrying combinations of transgenes.
Gary Muehlbauer, molecular geneticist at the U of M, just started working with the TRI101 gene from the fungal strain Fusarium graminearum. Muehlbauer and other crop scientists are also involved in collaborative research on the transformation of proteins (thionin, thaumatin) and enzymes (glucanase, chitinase) that attack the cell walls or punch holes in the cell membranes of Fusarium fungi, thus inhibiting growth of Fusarium and production of DON. Transforming the genes that encode these proteins into spring wheat lines and other classes will enable the ultimate goal, says Muehlbauer: transgenic wheat and barley plants that are scab resistant.
Lynn Dahleen, research geneticist at the USDA-ARS Northern Crop Science Laboratory in Fargo, N.D, is working on biotech barley. Dahleen's lab began its gene transformation experiments on barley in the fall of 1998, with limited success. Experiments begun in 1999 using TRI101, with technical improvements, are showing more promise.Plants are being regenerated and evaluated through molecular testing to determine if the introduced gene is present.
"The hope is that we can incorporate this gene into a new barley variety that is more acceptable to the malting industry," she says.
Ironically, although six-row malting barley varieties are the desired target for gene transformation, they are the most difficult to transform.Thus, Dahleen is using the two-row barley cultivar Golden Promise as a host plant for gene transformation, a two-row barley variety with a genetic makeup that's easier to manipulate than other varieties.
The goal is to insert anti-fungal genes, such as the TRI101 gene, in Golden Promise, then back cross or breed it with other barley lines to come up with a new variety that not only has scab resistance, but has agronomic traits desired by growers and quality traits desired by maltsters. "Golden Promise is an old English variety from the 1960s. It doesn't meet our agronomic or malting needs, but it works well for transformation. We've been screening some of the breeders' material to see if they have better lines to work with, and there are some possibilities."
PDR5 is another anti-toxin gene included in Dahleen's research. "We are using that one now (PDR5) in barley and others are looking at it in wheat. I think we need these different research angles, so we don't duplicate our efforts."
Bill Bushnell, USDA-ARS Cereal Disease Lab, St. Paul, Minn., and Ron Skadsen, USDA-ARS Barley and Malt Lab, Madison, Wisc, are also employing high-tech methods of researching barley.The GFP gives a green fluorescence to the fungus when viewed with a microscope under blue light. In preliminary trials, the GFP has greatly improved their ability to trace development of the fungus in infected head tissues.
Bushnell and Skadsen are now comparing fungus development in inoculated head tissues (the palea and lemma) to development in inoculated leaf and coleoptile tissues. Their ultimate objective is to determine the principal pathways of infection in head tissues of wheat and barley. The GFP mutant was prepared by Dr. Thomas Hohn, formerly of the National Center for Agricultural Utilization Research lab, USDA-ARS, Peoria, IL.
ARS research geneticist Prem Jauhar, of the ARS-NCS lab in Fargo, is employing high-tech research to help develop scab-resistant durum germplasm.
In 1994, he initiated research work on biotechnology at Fargo, acquiring a gene gun and developing the facilities for direct gene transfer into plants by microprojectile bombardment. Using this facility, his lab produced the world's first transgenic durum wheat, incorporating a gene conferring herbicide resistance.
Having standardized the technique of direct gene transfer into durum wheat, Jauhar's lab now hopes to incorporate Fusarium head blight resistance in durum wheat by engineering it with anti-fungal, pathogenesis-related (PR) protein genes.
Jauhar's research lab has identified two types of wheatgrasses (wild relatives of wheat) as excellent sources of resistance to scab. To transfer this resistance to durum, they crossed two cultivars, Lloyd and Langdon, with the wild grasses. Some of these crosses show high resistance to scab. With further backcrossing and selection, this research success may help boost efforts to produce durum cultivars with scab resistance.
"The in-vitro approaches to gene transfer are developing rapidly and promise to become an integral part of plant breeding programs. This technology and conventional plant breeding should go hand in hand to accelerate the genetic improvement of wheat and other cereal crops," says Jauhar.However, he points out that the technology, which is highly focused and applicable for well-defined genes, will benefit and complement, but not replace, conventional breeding and cytogenetic approaches to crop improvement.
Stephen Baenziger, agronomist at the University of Nebraska-Lincoln, might be described as an intermediary between winter wheat breeding efforts at UNL and molecular scab work at the Plant Transformation Core Facility at the University of Nebraska's Center for Biotechnology.Research work there includes soft red winter wheat, grown in the east central U.S., but studied at UNL because of the biotech facilities there that specialize on winter wheat.
"We just started our transformation efforts.We want to make sure that the best genes move into lines people need most."
With funding from the National Scab Initiative, the UNL is hoping to step up its research of inserting anti-fungal genes into viable winter wheat varieties. "What are the genes to use? If the TRI101 or PDR5 genes fail, what next? Those are key questions. We need to try these and other genes to see if they work.
That's why UNL is also looking at bacterial sources for gene transformation, including lactoferrin and oxylase, that would help winter wheat plants ward off the scab fungus. Researchers are using the spring wheat variety Bobwhite as the model for wheat gene transformation. Like Golden Promise for barley, Bobwhite does well in tissue culture, has a genetic makeup that is more "moldable" than other wheat varieties, and is conducive to greenhouse crossing.
New and valuable genes can be introduced into cereals via transformation that are not available through traditional breeding practices. However, improvements in the technology are needed to provide efficient and useful wheat and barley transformation which can be easily employed in applied breeding programs, says Olin Anderson, supervisory research geneticist at the USDA-ARS Western Regional Research Center, Albany, Calif.
A project leader for wheat bioengineering efforts, Anderson says the transformation procedure is still too inefficient, and requires too much expertise for routine use. Another challenge is simply finding the appropriate anti-fungal gene or genes to insert.
More research is also needed on the general knowledge of transgenic gene expression. Scientists must be mindful about when and where in the plant the gene will be expressed. The gene needs to be expressed in the plant tissue where disease resistance is needed, via genetic promoters that 'turn on' genes in response to infection.
Thus, if a gene is inserted into a wheat plant that offers more resistance against the spread of scab within the wheat head, the gene would "kick in" when the genetically-engineered wheat plant needs this resistance most, during head development.
"We need promoters that are tissue specific.The plant is wasting resources when we only need the gene turned on in the head," says Dahleen.
"Research on isolating a promoter specific to scab and DON in wheat and barley is limited thus far," says Anderson. It's a good candidate for the U.S. Scab Initiative. Making wheat transformation more efficient is indeed a priority of the U.S. Scab Initiative, say leaders involved with the research effort.
"Another transgenic challenge: researchers can't select just any wheat and barley variety as a host for transformation. As a result, most elite breeding lines (the best candidates for varietal release) cannot be used for gene transformation."
"Some lines simply won't transform very well. It's still a process that's limited to a few lines."
Increasing patents and intellectual property laws involved with gene transformation technology worries some crop scientists.
However, these genes are owned by various entities: The herbicide-resistant gene is owned by a private company. The overall transformation method is owned by another. "You can see that there might be problems," says Dahleen, or at the very least, potential legal hoops to clear for transgenic plants or processes developed by both the public and private sectors.
Some private companies including Novartis and Monsanto are researching anti-fungal genes in wheat and barley as well. But most company researchers are tight-lipped about their progress.
"I can't be very specific right now about our disease control program but I would like to let it be known that work on this disease is continuing," says Jack Berg, a wheat breeder with Monsanto, one private researcher willing to offer a research update. "Monsanto is investigating multiple avenues of biotech research against Fusarium head blight, including anti-fungal proteins, molecular enhancement of breeding for resistance, and functional genomics aimed at understanding the infection processes. We believe that multiple mechanisms may be required to provide a high level of sustainable control of this complex disease."
There has been some debate among crop scientists involved with transgenic scab research on how soon to incorporate transgenes into a breeding program. On one hand, it could be argued that the effectiveness of engineered genes should be confirmed under conventional study procedures that usually involve repeated steps of evaluation. Thus, time and breeding resources won't be lost if engineered varieties from genes deployed quickly prove to be inadequate.
On the other hand, scab is an industry-threatening problem that is demanding accelerated results. This fact weighs in favor of early deployment.
"I don't see any problem with doing crosses right away." The traditional way in most cases is fine, but this is such a huge problem that it's worth the extra effort to get the materials advanced."
Anderson agrees, as long as the caveats are kept in mind.
"Viewing the TRIr gene and possibly a few others as an experiment in speed of deployment certainly has merit, as long as we all understand that any specific gene may fail. But, I think I agree that the TRIr gene or some others coming along in different labs could be used to test transgene utilization so we have a clearer idea how all of this will actually work."
"It's always risky when transforming plants as to whether the desired genes will be expressed. But this is a new attack on the problem, and we're hoping it'll give positive results," says Alexander.
