NRC HOLDS WORKSHOP ON ECOLOGICAL MONITORING OF GM CROPS

The National Research Council's (NRC) Standing Committee on Biotechnology, Food and Fiber Production, and the Environment sponsored a Workshop on Ecological Monitoring of Genetically Modified Plants on July 13-14 in Washington, D.C. Workshop presentations collectively described the current state of knowledge regarding ecological effects of GM plants, limitations of current knowledge, and potential future directions for ecological monitoring. The workshop was attended by over 100 academics, government officials, representatives of non-governmental organizations, and private citizens.

The workshop included 16 presentations by individual researchers and four panel discussions involving 18 speakers. Many speakers made multiple points. Hence, only highlights can be presented here. Barbara Schaal (Washington University), co-chair of the Standing Committee, introduced the workshop by discussing the context for short- and long-term ecological monitoring of GM plants. Paul Waggoner (Connecticut Agricultural Experiment Station) described ongoing programs for monitoring plant pathogens, noting relevant aspects of how to organize, finance, and maintain effective monitoring programs. Peter Day (Rutgers University) considered the issue of the baseline for assessing benefits and risks posed by production of GM plants, arguing that agricultural production of non-GM plants was the most appropriate baseline.

Allison Power (Cornell University) pointed out that most monitoring research has focused on estimating the probability of an event as opposed to the extent of the associated hazard. For example, concerning GM plants expressing viral genes to achieve virus resistance, the probability of recombination between transgenic and wild-type viruses is known to be high, but the extent of the hazard posed is unknown, and should become the focus of monitoring. She and several other speakers discussed the need for designing experiments robust enough to avoid Type II experimental error, the error of failing to detect a significant effect.

The role of ecological monitoring within a framework of adaptive management was discussed by Anne Kapuscinski (University of Minnesota). She suggested that results of monitoring should inform an iterative process of public policy design and implementation. In a panel on international perspectives on monitoring, Jeremy Sweet (National Institute of Agricultural Botany, UK) pointed out that monitoring should address gene flow from GM crops not only to wild relatives, but also to non-GM crops of the same species in nearby plantings. Rob McDonald (Aventis, Canada) noted that design of a monitoring study should be specific to the crop, trait, environment, and event of interest. He described a concept of product stewardship in which a company makes a commitment to realize the benefits of GM crops in a safe and sustainable manner. Monitoring for gene flow from GM canola to weeds using the green fluorescent protein gene as a biomarker for pollen dispersal and gene flow was described by Neal Stewart (University of North Carolina - Greensboro).

Presentations on the second day of the workshop addressed the role of modeling in establishing a monitoring program, and monitoring for pest resistance in target pests, herbicide tolerance in weeds, effects on non-target species, and evolution of pathogen resistance. Ecological modeler Steve Bartell (Cadmus Group) discussed use of models to determine tradeoffs among the frequency, scale, and extent of a monitoring program, and described the structure of a spatially explicit model of a farm using Bt corn. Fred Gould (North Carolina State University) identified major issues in monitoring for resistance in insect pests targeted by Bt in GM corn, concluding that mobility of the insects affected both the desired proportion of non-Bt refuge areas and the spatial extent of monitoring for novel Bt resistance. Guenther Stotzky (New York University) presented laboratory results showing slow degradation of the Bt toxin, slowed degradation of crop residues, and no significant indirect effects of Bt toxins on soil microbes, earthworms, or nematodes.

Two panel discussions focussed on monitoring for effects on ecological communities and changing farm practices. Mark Lipson (Organic Farming Research Foundation) questioned the extent of farmers' compliance with requirements for refuge areas within Bt plantings and the reliance on farmers to report unexpected ecological effects of GM plants. William Hallman (Rutgers University) discussed public perception of risk posed by GM plants and the implications for development of an effective risk communication strategy. He pointed out that people are averse to perceived unfairness or lack of control over their lives, and that they want their questions answered, not to be educated about plant biotechnology.

In a wrap-up session, a panel considered criteria and priorities for monitoring. Fred Gould pointed out that it is difficult to be sure what to monitor a priori. Steven Duke (USDA - Agricultural Research Service) emphasized the importance of baseline monitoring to support distinction of what ecological effects are attributable to a GM plant and what effects track baseline changes. Steve Bartell suggested selecting ecological endpoints that are scaled to modeling resources, use of sensitivity analysis to identify key uncertainties, focusing on ecological function as well as structure, and practicing monitoring within an adaptive management framework. Max Carter, a farmer from Georgia, noted that farmers grow GM plants in order to realize a profit and, for purposes of establishing criteria and priorities for monitoring, suggested gathering farmers to ask them what they see in terms of adverse ecological effects. Key issues identified by Barbara Schaal in her concluding remarks included questions on post-commercialization monitoring needs, experimental design when planning monitoring, consequences of ecological effects of GM plants, who should monitor and who should pay for monitoring, and engaging the public in respectful dialog.

CHLOROPHYLL FLUORESCENCE IN GM CROP ANALYSIS

Space is apparently not the only "final frontier" for NASA researchers. Much of their R&D has earth-based applications as well, ranging from automobile technology to medical devices. NASA has recently announced the development of an optoelectronic instrument with promise for traditional and transgenic crop research. Paul Kebabian, Herman Scott, and Andrew Freedman, of Aerodyne Research Incorporated in Massachusetts, recently designed a plant fluorescence sensor for remotely and accurately monitoring photosynthetic efficiency of crops in the field¹.

The fluorescence sensor technology exploits chlorophyll's ability to fluoresce at 660 to 800 nm of light when exposed to solar or artificial light. Previous fluorescence detectors could not completely discriminate against scattered sunlight. The new unit applies a discrimination technique that uses the spectral absorption lines of oxygen to remove inaccuracies introduced by background solar radiation and does so without the need for expensive optical components found in other spectral-line discriminators.

Chlorophyll fluorescence is directly related to the photosynthetic efficiency of plants. When light excites chlorophyll's photosystems, they produce fluorescence within the red and infrared range. The magnitude of that fluorescence, which represents only 3-9% of the light energy absorbed by the photosystems, is correlated with production of NADPH, needed for driving the photosynthetic dark cycle. Thus the degree of chlorophyll fluorescence provides a rate value that can be translated into a photosynthesis efficiency measure. Various studies dating back to 1978 on aquatic and terrestrial plants have confirmed the accuracy of this measure.

The NASA researchers point out that the utility of accurately measuring chlorophyll fluorescence is that it can be mathematically correlated, relative to the photosynthetic rate, with plant stress, growth rate, and physiological status². For example, they suggest that a plant stressed by lack of adequate fertilizer responds by limiting chlorophyll production, because the light capture efficiency of plants is dependent on the quality and quantity of pigment molecules, which in turn is determined by nutrient availability. A measure of the degree of that physiological stress can be made by comparing the magnitude of chlorophyll fluorescence and its shift in spectral distribution in the stressed plant to that of healthy plants (refer to NASA Tech Brief SSC-00050, Spectral reflectometer for quantifying stress in plants). The ability to measure stress is useful for researchers studying GM crops, as stress can distract plants from adequately expressing characteristics introduced into genomic DNA or extrachromosomal sites. Measurements of stress can also allow researchers to make more timely applications of resources such as water, fertilizer, or pesticides.

Other methods of evaluating photosynthetic rate and stress in crops include plant tissue biomass analysis and CO² assimilation. Biomass analysis is an indirect measure of photosynthesis and does not provide immediate results. Researchers do not have the luxury of being able to monitor fields and make immediate modifications to growth conditions when using the biomass analysis technique, as they would using chlorophyll fluorescence detection. Measurements of the assimilation of CO² in plants have been directly correlated to fluorometric measures in the laboratory, as reported by Agu Laisk of the Tartu University Institute of Molecular and Cellular Biology in Estonia. However, unlike chlorophyll fluorescence, CO² assimilation cannot be monitored using remote instrumentation in large fields of crops.

The use of fluorometry for measuring photosynthesis is well accepted in forestry and traditional crop research². Success with the technique has been shown in forest canopy biomass research and in crop yield studies on apples, bananas, beets, mangos, peppers, and spinach. Much of the research using fluorometry has also been used to evaluate traditional crops and wild plants under a variety of stresses³, and it is showing promise in evaluating GM crops as well. Researchers at the National Institute for Biology in Okazaki, Japan have used chlorophyll fluorescence to measure photosystem II activity in Arabidopsis thaliana transformed with the codA gene for choline oxidase. The researchers were able to monitor real-time photosystem II activity of the control and experimental plants in salt tolerance and cold stress trials. They also used the technique in subsequent studies of heat tolerance in Arabidopsis engineered to accumulate glycinebetaine. In addition, researchers at the Institute of Molecular Biology in Barcelona, Spain recently used fluorometry to investigate the role of a carotenoid-associated protein in photosystem II modulation in bioengineered plants.

The NASA plant fluorescence sensor can discern plant stress at very early stages. The sensor's unique ability to passively monitor field conditions by making remote, precise, and immediate measurements of the physiological status of green plants could make valuable contributions to traditional and GM crop research.

[Further information about the instrument can be obtained by calling Herman Scott or Laurie Dean at Aerodyne Research, Inc. at 978-663-9500.]

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MITOCHONDRIAL DNA HETEROPLASMY IN CLONED CATTLE

During cloning by nuclear transfer, mitochondrial DNA is transferred along with the donor nuclear DNA into the enucleated egg. What is the fate of these donor mitochondria among the mitochondria already present in the host egg? If the donor mitochondria replicate along with the host mitochondria, then both mitochondria will be present resulting in a condition termed mitochondrial heteroplasmy.

In 1999, the mitochondrial DNA population was analyzed in ten nuclear transfer-derived sheep, including Dolly. In all ten sheep, the mitochondrial DNA was found to be derived exclusively from the recipient enucleated oocytes, with no detectable mitochondrial DNA contribution from the donor cell. The authors speculated that the absence of donor mitochondria resulted from an active mechanism that destroyed donor mitochondria in the recipient oocyte. This mechanism may be similar to that which is thought to lead to the elimination of sperm-derived mitochondria during normal fertilization.

In the July 2000 issue of Nature Genetics, however, researchers in Austria and Germany report that mitochondrial DNA heteroplasmy is present in cloned cattle. The mitochondrial DNA population was examined in ten cattle clones generated from primary fetal fibroblasts, adult mammary epithelial cells, or adult skin fibroblast cells. Identification of donor and recipient mitochondrial DNAs was based on the presence of single nucleotide polymorphisms.

In seven out of ten cattle clones analyzed, the percent of the mitochondrial DNA that was derived from the donor cell ranged from 0.4%-4%. This mitochondrial DNA heteroplasmy was detected in a number of different tissues, and the donor to recipient mitochondrial DNA ratios remained constant throughout embryogenesis or development to term. In the other three cattle clones, there was a significant reduction or absence of donor mitochondrial DNA. The mechanism that leads to mitochondrial heteroplasmy in some cloned cattle and not others is unknown. The presence of two normal and healthy cattle clones with a mixed mitochondrial DNA population demonstrates that heteroplasmy is not deleterious to normal development.

Currently, it is not clear why cloned sheep contain only recipient mitochondria, while some cloned cattle contain both donor and recipient mitochondria. One possibility is that in these cloned sheep the particular combination of recipient nuclear and donor mitochondrial DNA is incompatible, whereas in the cloned cattle the recipient nuclear and donor mitochondrial DNA are compatible. A second possibility is that regardless of the recipient nuclear and donor mitochondrial DNA combination, sheep cells are intrinsically different from bovine cells and contain a mechanism that prevents mitochondrial heteroplasmy.

This finding of mitochondrial heteroplasmy will force a reevaluation of the use of nuclear transfer to correct mitochondrial genetic disorders. Nuclear transfer was proposed as a method of transferring nuclear genes without the accompanying mutant mitochondrial genes. This would have been possible if no mitochondria are cotransferred with the donor nucleus. However, the observation that mitochondria are transferred from the donor makes this approach less attractive.

Sources

1. Evans MJ et al. 1999. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nature Genetics 23: 90-93.

2. Steinborn R et al. 2000. Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nature Genetics 25: 255-257.

A GROWTH FACTOR GENE THAT INCREASES OVULATION RATE IN SHEEP

Multiple ovulations are uncommon in cattle and certain breeds of sheep. Inverdale sheep carry a naturally occurring X-linked mutation that causes increased ovulation rate and a high incidence of twins and triplets in heterozygous animals. A second family of sheep, Hanna, which is unrelated to the Inverdale sheep, also displays the same X-linked trait of increased ovulation rate and multiple births. Genetic studies have shown that the Inverdale and Hanna phenotypes are likely due to mutations in the same gene. Interestingly, sheep homozygous for the Inverdale or Hanna genes are infertile due to a block in follicular development. Ovarian follicles in these homozygous animals do not normally grow beyond the primary stage of development.

In the July issue of Nature Genetics, researchers from New Zealand and Finland reported they have mapped the mutated gene in Inverdale sheep to a chromosomal region that contains the gene encoding a bone morphogenetic protein, BMP15. BMP15 is a member of the transforming growth factor b gene family and is expressed exclusively in oocytes. The BMP15 gene product likely plays an important role in supporting the growth of oocytes. In Inverdale or Hanna homozygous sheep, the oocytes can grow for a short period in the absence of BMP15 but then degenerate. It is not clear why 50% of normal BMP15 levels in heterozygous sheep results in an increase in ovulation rate.

Analysis of the BMP15 gene in Inverdale and Hanna sheep revealed point mutations in the gene. The mutation in the Inverdale BMP15 gene results in a substitution of a valine with an aspartic acid in a highly conserved region of the protein, whereas the mutation in the Hanna sheep introduces a premature stop codon and causes the synthesis of a truncated BMP15 protein. These results demonstrate that natural mutations in the BMP15 gene can result in increased ovulation rate in heterozygotes and infertility in homozygotes.

THE PRECAUTIONARY PRINCIPLE: LEGAL DOCTRINE OR RORSCHACH INKBLOT TEST?

The precautionary principle may have first appeared in 1976 in the national law of former West Germany as "Vorsorgeprinzip," a view that the government should avoid environmental damage by cautious planning. Some have suggested that the precautionary principle first entered international law in the World Charter for Nature, adopted by the United Nations General Assembly in 1982, while others point to the Ministerial Declaration of the Second Conference on the Protection of the North Sea (1987). The principle was clearly recognized during the U.N. Conference on Environment and Development in Rio de Janeiro in 1992. The principle also resides in the Treaty Establishing the European Community and can be found in the preamble of the Cartagena Protocol on Biosafety to the Convention on Biological Diversity (2000). But, what is the precautionary principle?

What it is, is . . .
According to the Commission of the European Communities, the precautionary principle is a full-fledged, general principle of international law. This is a significant position for the European Commission to take. Three primary sources of international law are treaties and conventions, customary practices, and general principles. The latter are principles that are intrinsic to legal systems of the world, or are principles derived from the nature of international community, such as territorial integrity. An example of a general principle common to many national legal systems is res judicata, which asserts that a matter is settled once a final judgment has been made.

If the precautionary principle is a general principle of international law, then it can be used as a subsidiary source of law to complete a treaty. However, not all nations recognize the precautionary principle as such. US officials, for example, have recently referred to it as the "so-called" precautionary principle, an outlook that probably reflects the principle's lack of a precise definition.

A principle that is polysemous and abstruse, and yet not totally cryptic
To some, the precautionary principle implies that precautions should be taken even if a cause and effect relationship between an activity and its potential harm to the environment, or to human health, has yet to be scientifically established. According to one commentator, the first documented use of the precautionary principle occurred in 1854 when Dr. John Snow found an association, but not a causal connection, between drinking water from a London pump and a cholera epidemic. That is, Dr. Snow apparently decided that the potential cost of being wrong in removing the water pump handle was likely to be much smaller than the potential cost of not removing the handle.

The conceptual core of the precautionary principle seems to be that if a regulatory inaction permits environmental risks that are in some way uncertain, but non-negligible, then regulatory inaction is unjustified. Elements woven into the principle include a willingness to take precautions in advance of formal scientific proof, and to consider of the cost-effectiveness of action, the intrinsic value of non-human life forms, and concerns for future generations. An underlying mandate of the precautionary principle is that, in the face of scientific uncertainty, a party should refrain from actions that might harm the environment, and that those who oppose this prohibition have the burden of proof for assuring the safety of the proposed action.

A significant problem in implementing the precautionary principle as a policy tool arises from the extreme variability in its interpretation, with approaches ranging from eco-centric and risk averse to utilitarian and risk-taking. For example, certain formulations of the precautionary principle require that actions must be taken in advance of scientific certainty, while others contend that deliberate inaction is not justified by a lack of scientific certainty. Some interpretations allow cost-benefit analysis and discretionary judgment, and yet others call for clear proof of safety before new technologies can be adopted.

A basic unresolved question about the precautionary principle concerns the amount of evidence (or lack thereof) needed to invoke it. Should evidence of "likely harm" or "serious or irreversible harm" trigger the principle? As one observer notes, the precautionary principle has deviated from a strong mandate for precautionary action toward a universal sentiment, with little guidance on practical implementation.

Despite the variability in its interpretation, the precautionary principle gets a workout. For instance, France banned imports of British beef based on the precautionary principle. Earlier this year, the German government used the precautionary principle as the rationale for banning the commercial scale cultivation of Bt corn by Novartis. Additional examples include decisions by the EU to ban American and Canadian beef produced with growth-promoting hormones, and to delay approval of genetically engineered crops for sale in European markets.

Aside from its possible use as an excuse for trade protectionism, one reason for the popularity of the precautionary principle is that it reflects the current mood of distrust of technologies that are perceived to be risky and forced on a naive public by commercial interests aligned with governments. The precautionary principle also reflects misgivings over perceived manipulations of cost-benefit analysis by powerful commercial interests.

A more certain way to deal with uncertainty?
The Commission of the European Communities issued a report outlining a definite approach for implementing the precautionary principle. At the outset, the Commission explained that the precautionary principle presupposes that the potential dangers of a product or process are known; but that current scientific evaluation does not allow the risks to be precisely determined. In the European Commission's view, action based on the precautionary principle should be proportional to the chosen level of protection, nondiscriminatory in application, and consistent with measures taken under similar circumstances. Furthermore, precautionary measures should be subject to cost-benefit analyses and reviewed in light of new scientific evidence. The Commission also stated that, if action is deemed necessary, the measures based on the precautionary principle should contain the capacity to assign responsibility for producing new scientific evidence when required for a more comprehensive risk assessment.

Certain manifestations of the precautionary principle present a challenge to the scientifically based process of risk assessment. The European Commission's approach, which explicitly blends precautionary measures with scientific analysis, may well lay a foundation for the future development of the precautionary principle. It is unclear, however, whether any particular formulation of the precautionary principle, no matter how balanced and well reasoned, could become accepted on an international level.

Can one size fit all?
One of the prerequisites for effective implementation of a treaty that includes a precautionary principle directive is the definitive interpretation of the principle in terms of practical measures. Without such interpretation, the principle would remain as a token statement of belief. However, the perception of risk, the very trigger for invoking the precautionary principle, varies between populations of various countries, and between groups within a particular country. To take one example, several studies indicate that it is the qualitative factors of risk, rather than the magnitude of the probability of an adverse outcome, that influence consumer acceptance of foods produced using biotechnology.

The upshot is that an implementation of the precautionary principle may only be feasible when stakeholders collaborate at the national level to make a decision in a particular context, trading costs against benefits, and identifying those levels of damage deemed tolerable to that society. In other words, the precautionary principle will be put into practice according to the predominant national values. Since the presumption of the precautionary approach is that a precautionary action must be taken despite a lack of full scientific information, policy decisions must be based upon ethical, moral, or political grounds, as well as the science. As the European Commission notes, establishing an acceptable level of risk for society is a political responsibility.

Both the European Commission and US officials contend that decision-making procedures should be transparent and should involve all interested parties. A process that treats uncertainty in an open manner, rather than dismissing or downplaying it, may begin to address the concerns that are fueling the popularity of the precautionary principle. In the long run, dealing with the underlying apprehensions that have fostered the precautionary principle may be more practical than attempting to devise a formulation intended to fit all nations.

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BIOPHARMACEUTICALS FROM TOBACCO
Profiling CropTech Corporation

CropTech was established in 1992 with a mission to develop and commercialize genetically engineered plants for production of high-valued proteins and biochemicals. CropTech has demonstrated that plants have surprising promise to provide large-scale, cost-effective production of complex bioactive recombinant proteins. In fact, plants may offer the only effective means to manufacture proteins and biochemicals at the scale and cost that will be required for many biopharmaceutical applications, such as anti-cancer drug treatments. We currently have seven sites in Virginia where tobacco with human genes encoding pharmaceutically useful proteins is being grown. CropTech has approximately 40 employees and is located in Blacksburg, VA.

The biopharmaceutical product candidates that CropTech has initially chosen to pursue were selected because the market demand for them is large, the products address significant medical needs, or because they are relatively expensive to manufacture using current techniques. Cost is a special concern in vaccine therapy; CropTech's technologies could feasibly put the vaccine products in the hands of the Third World countries where many effective vaccines cannot be employed due to the expense associated with their production, storage, and distribution. All of the Company's product candidates are in various stages of pre-clinical evaluation.

Biopharmaceutical Product Candidates
Urokinase: Urokinase-type plasminogen activator is a thrombolytic enzyme used to lyse acute thrombi obstructing coronary arteries; these occlusions are associated with evolving myocardial infarction. CropTech has cloned and expressed active urokinase at high levels in transgenic tobacco. The Company estimates that production using tobacco will cost much less than current methods. Cost estimates for the currently used animal-cell production systems are above $1,000 per gram. At this time, urokinase therapy is not available in the United States because of concerns with the safety of the production method.

Glucocerebrosidase: This molecule is a lysosomal enzyme used in replacement therapy for Gaucher's disease, a rare genetic disorder. Current treatment requires extensive processing of cells isolated from human placenta or Chinese hamster ovary tissues. Between 400 and 2000 placentas are required to supply a standard dose, which is a major factor in the extreme cost to patients, $100,000 to $400,000 annually. CropTech has successfully cloned and expressed active glucocerebrosidase in tobacco. More importantly, the synthesized protein is enzymatically active. Further testing is required before pre-clinical trials can begin.

Human Serum Albumin (HSA): HSA is a major product of the blood processing industry, used primarily to replace blood loss from surgery, burns, shock, and other types of physical trauma. The current market for HSA, valued at $1.4 billion, requires about 100 metric tons of the serum annually. HSA is now obtained from blood donors and, for that reason, periodic shortages occur. In addition, serious safety concerns related to contamination issues are raised regarding a blood product obtained from human donors. CropTech has planted, harvested, and assessed HSA production levels from multiple field sites this year. We have also initiated scale-up of processing and purification of this product.

Vaccine Product Candidates
The recent worldwide upsurge in the occurrence of serious infectious disease has renewed concern over the adequacy of currently available methods to deliver protection to at-risk human populations through immunizations. While many vaccines are effective, problems associated with their cost, distribution, and safety suggest the need for new approaches. The World Health Organization estimates that over 12 million children under the age of five die each year from infectious diseases. Vaccines already on the market could have saved at least two million of these children. The goal of CropTech's vaccine research program is to develop transgenic plants for the cost-effective production of new recombinant vaccines for both oral (food-based) and traditional delivery systems. These vaccines will be based on isolated DNA sequences coding for highly immunogenic protective antigens. One of the first candidates will use plant-based expression of a viral coat protein antigen for HIV, the pathogen for AIDS. AIDS infection has reached pandemic proportions in the world. A safe, cost-effective vaccine will provide for worldwide relief from this disease.

PROGRESS MADE IN USING BIOTECH TO SOLVE FHB IN WHEAT AND BARLEY

Biotechnology is still relatively new to wheat and barley research. The first genetically engineered wheat variety to be available commercially is expected in 2003, with the rollout of Roundup Ready^® wheat. Genetic engineering is also being used as a high-tech approach to help solve Fusarium head blight (FHB, commonly called scab and caused mostly by the fungus F. graminearum), which has plagued wheat and barley production to a varying degree worldwide.

In the US, FHB was particularly problematic in the 1990s, inflicting yield and quality losses on farms in at least 18 states. The disease is responsible for over 500 million bushels of wheat lost in the US since 1991, conservatively valued at over $2 billion in farm-gate losses alone, according to industry and university estimates.

The fungal disease not only stings farmers with yield and quality losses, but is a serious trade and food safety issue as well. The toxin that may be produced from FHB called deoxynivalenol (DON or vomitoxin) can make barley unacceptable for malting and brewing, and wheat unacceptable for milling.

In 1997, recognizing the threat of scab to the viability of the US wheat and barley industries, leaders representing producers, millers, brewers, food processors, and state and federal crop scientists began to organize a national, multi-disciplinary, and multi-institutional research system to focus on FHB research. The US Wheat and Barley Scab Initiative (USWBSI) was created. Current research supported by the Initiative involves 104 projects in six research areas carried out in 23 states by over 70 scientists from 22 land grant universities and the USDA's Agriculture Research Service (ARS).

The Initiative has six major research emphasis areas:
• Variety development and coordinated screening nurseries
• Chemical and biological control
• Epidemiology and disease management
• Food safety, toxicology, and utilization
• Germplasm introduction and enhancement
• Biotechnology

Biotechnology is being applied to three distinct emphasis areas of FHB research conducted under the USWBSI: 1) To identify genes in wheat and barley that are involved in the scab defense response, such as mapping scab resistance genes with molecular markers; 2) To identify and insert antifungal genes in wheat and barley from other wheat and barley germplasm or other organisms, including bacteria and fungi; 3) To identify and insert genes that can detoxify DON.

Advancements on several fronts
Progress is already being made on several fronts. For example, Bill Bushnell, USDA-ARS Cereal Disease Lab, St. Paul, Minn., and Ron Skadsen, USDA-ARS Barley and Malt Lab, Madison, Wisconsin, are conducting research using a genetically transformed strain of F. graminearum containing a gene for green fluorescent protein (GFP). GFP gives a green fluorescence to the fungus when viewed with a microscope under blue light. GFP has greatly improved the ability to trace development of the fungus in infected head tissues. Bushnell and Skadsen's 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, Illinois.

The University of Nebraska is making progress as well in its goal of inserting anti-fungal genes into viable scab tolerant winter wheat varieties. The U of N has had success during the past year in using two different types of genes—lactoferrin and IAP, which has antiapoptic qualities—in its goal of developing resistance to the scab fungus. Lactoferrin is a type of protein found in mammals that has long been reported to be active against a wide range of microorganisms, including fungi, explains Stephen Baenziger, agronomist at the U of N. Antiapoptic, in this case, refers to a gene's ability to help prevent plant cells from being killed by the scab fungus.

The genes are among several chosen for scab research in small grains, since they have also demonstrated success in combating economically important fungal diseases in transgenic tobacco plants. Transgenic wheat plants with adequate levels of expression of these genes are currently being grown in U of N growth chambers for inoculation with Fusarium graminearum. This will help determine if the anti-fungal genes are being successfully expressed in the transgenic wheat plants.

Transgenic spring wheat and barley plants carrying antifungal protein genes have been developed, and will be tested to determine if the plants convey scab resistance, says Gary Muehlbauer, molecular geneticist at the University of Minnesota, one of several crop scientists involved in biotechnological research approaches of the USWBSI. If resistance is successfully confirmed, these plants will provide the germplasm needed to help breed wheat and barley varieties that have enhanced resistance to scab, he says.

Muehlbauer and others have developed a cDNA library from wheat spikes inoculated with F. graminearum, and have initiated the sequencing of genes from this library, with the goal of sequencing 2500-3000 genes. "This work will provide the genetic tools to further study the interaction between the fungus and wheat in addition to [identifying] novel genes for resistance," says Muehlbauer.

Biotech researchers have characterized some of the early events in F. graminearum infection, and the molecular response in wheat, says Muehlbauer. They have found that F. graminearum can infect through multiple pathways, and that wheat responds to infection by inducing the expression of defense response genes. The expression of these genes can be found in colonized as well as uncolonized portions of the spike. Completed characterization of a strain of F. graminearum that expresses the GUS reporter gene will be useful for investigating the infection process in more detail, he says.

Mapping scab resistance genes in barley also continues, says Muehlbauer. Mapping involves using molecular markers to identify regions of the barley genome that carry scab resistance genes. This work will provide the genetic tools to conduct marker-assisted selection and enhance scab resistance in the barley breeding project.

Rick Ward, Michigan State University wheat breeder and co-chair of the USWBSI, says the mapping and sequencing work, along with other facets of biotech research conducted under the USWBSI, will create a groundwork of knowledge that will prove beneficial for research applications beyond FHB.

"Biotechnology as it applies to FHB research is in its infancy in many ways. The component technologies are still more abstract than proven, as opposed to Roundup Ready^® and Bt, where the right component of the engineered gene is well proven and characterized," says Ward. "But it is an avenue that must be pursued. The investments are appropriate. We don't know how fast or whether biotechnology will help yield a solution, although I think it will. But the timeframe is difficult to predict."

Ward says biotechnology cannot be depended upon as the "silver bullet" solution to FHB. Alternative research solutions of controlling and managing the fungal disease should (and are) being undertaken. Still, the potential for a research breakthrough using genetic engineering is there. "All sorts of proteins are being tested, and one of them just might work. The key thing then is implementing the system for deployment, which is in need of greater focus," he says.