2.2.1 Introduction of Recombinant DNA-engineered Organisms Into the Environment (1987)

In 1987, the NAS published a summary of key issues related to the introduction of recombinant DNA-engineered (rDNA-engineered) organisms into the environment (NAS 1987). This brief white paper outlined the expected risks and benefits associated with all types of transgenic organisms, including bacteria, insects, fish, and crop plants. At the time, commercial field releases of transgenic organisms were still in the planning stages, and the impending “biotechnology revolution” attracted en thusiastic support from some quarters and strong disapproval from others. To address the perception that rDNA techniques might be intrinsically dangerous, the report offered the following conclusions:

• point 1 “There is no evidence that unique hazards exist either in the use of rDNA techniques or in the movement of genes between unrelated organisms.”
• point 2 “The risks associated with the introduction of rDNA-engineered organisms are the same in kind as those associated with the introduction of unmodified organisms and organisms modified by other methods.”
• point 3 “Assessment of the risks of introducing rDNA-engineered organisms into the environment should be based on the nature of the organism and the environment into which it is introduced, not on the method by which it was produced.”

Throughout this report, the committee describes various methods of both conventional and transgenic breeding methods in detail to provide relevant information about their similarities and differences. Some of the similarities and differences in properties of plants produced by varied genetic approaches are presented in box 2.1. Properties of conventional pest-protected plants are discussed, but the committee focuses on risks and benefits that may be posed by growing transgenic pest-protected plants commercially and on their regulatory oversight under the coordinated framework for regulation of genetically engineered organisms.

BOX 2.1

Summary of Genetic Basis of Resistance Traits That Have Been Bred into Cultivated Plants Using Conventional and Transgenic Techniques. Conventionally bred plants only Polygenic traits (controlled by several interacting genes, usually selected without (more...)

The 1987 NAS report noted that the risks associated with rDNA-engineered organisms are “the same in kind” as those associated with unmodified organisms and organisms modified by other methods. The committee agrees with that statement for pest-protected plants in that both transgenic and conventional plants may pose certain risks and the resulting plant phenotypes are often similar. Transgenic breeding techniques can be used to obtain the same resistance phenotype as conventional methods (for example resistance to microbial pathogens, nematodes, and insects). Because both methods have the potential to produce organisms of high or low risk, the committee agrees that

The properties of a genetically modified organism should be the focus of risk assessments, not the process by which it was produced (point 3).

In this regard, the committee found that

There is no strict dichotomy between, or new categories of, the health and environmental risks that might be posed by transgenic and conventional pest-protected plants.

The committee recognizes that the magnitude of the risk varies on a product by product basis. The committee also agrees with points 1 and 2 in the sense that the potential hazards and risks associated with the organisms produced by conventional and transgenic methods fall into the same general categories. As this report discusses, toxicity, allergencity, effects of gene flow, development of resistant pests, and effects on nontarget species are concerns for both conventional and transgenic pest-protected plants.

The committee agrees with the 1987 NAS principles in that the magnitude of quantitative risk does not depend on the genetic-modification process. It depends on the new genes that are expressed in the plant. End points of risk (such as illness in humans and declines in nontarget species) can be the same regardless of whether a specific new gene was transferred by conventional or transgenic methods. For example, if the same alkaloid gene is transferred by sexual hybridization or Agrobacterium-mediated insertion, the risk should be similar. If a gene coding for a novel trait is transferred by transgenic methods, but cannot be transferred by conventional methods, it is the expressed trait that requires scrutiny, not the method of transfer. In summary,

The present committee found the three general principles to be valid within the scope of issues considered by the 1987 paper, and the present report further clarifies and expands on these principles.

Throughout the report, the committee expands on the 1987 principles by describing various methods of both conventional and transgenic plant breeding, and their potential consequences. The greater diversity of genes that can be transferred by transgenic methods, their enhanced effectiveness, and the ability to insert the same gene into many cultivated species have led to concerns about transgenic crops. Does the potential of transgenic methods to expand on the diversity of transferred genes mean that there is a greater chance for unintended risks from transgenic plants than those from conventionally bred plants? That question has been the subject of considerable debate and draws the question away from specific products. Some transgenic breeding results in pest-protective traits that are phenotypically indistinguishable from those conferred by conventional methods. In addition, transgenic methods are based on more complete knowledge of the genes that are being transferred into cultivated plants. In other cases, however, transgenic pest-protection traits may result in plants having new phenotypes, such as novel plant-produced toxins that could potentially affect human or animal health, nontarget organisms, or the weediness of crop relatives. Transgenic methods can also introduce extraneous traits when they involve marker genes, such as antibiotic resistance genes.

An up-to-date assessment of potential problems and advantages of transgenic methods is warranted (see section ES.2). Transgenic methods can improve the precision of plant breeding and lead to many advantages over current pest control methods. With careful planning and appropriate regulatory oversight, commercial cultivation of transgenic pest-protected plants is not generally expected to pose higher risks and may pose less risk than other commonly used chemical and biological pest-management techniques. The committee concludes that

A major goal for further research and development of transgenic and conventional pest-protected plants should be to enhance agricultural productivity in ways that also foster more sustainable agricultural practices and enhance the preservation of biodiversity, and decrease the potential for health problems that could be associated with some types of pest-protected plants.

2.2.2 Field Testing Genetically Modified Organisms (1989)

To expand on the general principles outlined above, NRC published a more detailed report on how genetically modified plants and microorganisms should be regulated for small-scale, experimental field tests (NRC 1989). The recommendations proved useful and remain well-founded with regard to how federal agencies regulate field testing of genetically engineered organisms. One important and widely accepted conclusion of the 1989 report is that genetically engineered organisms should be evaluated case by case. The report also describes many of the same issues that apply to large-scale introductions, such as the potential to create weeds or insects that are resistant to Bt insecticides. However, because the 1989 report did not directly address health or environmental risks associated with commercialization, it has limited utility for providing guidelines for regulation of transgenic pest-protected plants.

2.3.1 Natural Pest-protection Mechanisms

2.3.2 Transgenic Pest-protection Mechanisms

Plant biologists and breeders have developed a number of plants that have pest-protection conferred by transgenes. Transgenic pest-protection strategies generally depend on expression of novel genetic resources or transfer of natural plant resistance or defense genes. Transgenic pest-protection based on novel genetic traits involves the introduction of genes that interfere with a specific pest but that are derived from organisms in which the gene's natural function is not that of plant protection. The application of transgenic resistance should be most useful where natural conventional breeding has failed due to lack of resistance genes in sexually compatible plants or due to undesirable agronomic traits in conventional pest-protected crops. For example, the oat Pc-2 resistance gene which controls crown rust disease caused by the fungus Puccinia coronata is coinherited with a trait that confers sensitivity to an unrelated fungal pathogen, Cochliobolus victoriae, so it would not be useful to deploy this gene in oat cultivars by conventional breeding methods (Walton 1996). Transgenic pest-protection can also reduce the time required for cultivar development in some crops. Release of conventionally bred varieties of winter wheat that have the eyespot-disease-resistance gene Pch1 required 13 years from the initial crosses, mainly because of time-consuming selections of lines with acceptable agronomic and disease-resistance characters (Jones et al. 1995).

It is important to recognize that transgenic resistance programs do not displace traditional breeding because transgenes alone cannot currently provide the full spectrum of agronomic traits necessary in commercial varieties. Furthermore, use of transgenes for resistance does not circumvent the normal process of agronomic quality assurance and testing that occurs throughout a well-managed breeding program.

2.4.1 Health Effects Associated with Different Types of Gene Products

Different types of transgenic pest-protected plants that might be developed have the potential to cause adverse health effects. The degree of risk is related to the chance that potentially hazardous toxic or allergenic compounds are produced and to the magnitude of exposure of such compounds. The chance that hazardous compounds will be produced by either intended or unintended modifications is related largely to the specific type of transgene used.

2.4.2 Indirect Effects Associated with Different Breeding Methods

To understand the risks posed by genetically modified pest-protected plants, it is important to understand that, in addition to the direct effects of the pest-protective gene (section 2.4.1), breeding can lead to indirect effects, such as the effects of extraneous genes linked to the protective genes and pleiotropic effects caused by the protective genes.

The potential for inadvertent changes caused by the addition of extraneous genes that are physically linked to protective genes depends on the breeding method used and the source of the protective gene. The breeding method and the source of genes used for breeding determine the amount of new DNA moved into the cultivar and the number of novel genes linked to the pest-protective gene. Therefore, genetic modification methods, both conventional and transgenic, are discussed below with regard to their potential for adding novel extraneous genes and their potential for causing unanticipated pleiotropic effects.

2.5.1 Toxicity and Allergenicity Tests During EPA Review

To provide a cross section of recent activity under the general guidance that EPA informally provides to prospective registrants, the committee examined EPA pesticide fact sheets and data provided by the registrant for three registered Bt toxins that are regulated as plant-pesticides: Bt Cry3A delta endotoxin in potato (EPA 1995a), Bt subs. kurstaki Cry1Ac delta endotoxin in cotton (EPA 1995b), and Bt Cry1Ab delta endotoxin in corn (EPA 1997a and 1998a). The committee also reviewed EPA pesticide fact sheets for Bt subs. tolworthi Cry 9C protein in corn (EPA 1998c).

In general, oral toxicity testing for Bt endotoxins is based on the presumption that there is unlikely to be a problem inasmuch as a number of Bt toxins have been widely used for many years in microbial sprays without human toxicity. A variety of Bt proteins have been subjected to toxicological testing, starting with testing conducted on microbial Bt products, which typically contain multiple Bt proteins. This testing included acute, subchronic and chronic toxicology testing of products during the 1960s and 1970s (EPA 1988b; McClintock et al. 1995). However, it should be noted that most previous field uses resulted in minimal toxin ingestion by humans because sprayed microbial Bt toxin only remains effective for an average of 1.5 days (largely because of ultraviolet degradation), and therefore, residues are rare on fruits and vegetables. Also, sprayed microbial Bt toxins are protoxins, while some Bt plants produce activated toxins.

Information in peer reviewed studies indicates that plant-expressed Bt proteins are probably without human health risk. Nevertheless, a minimal number of properly defined tests are needed to determine if based on plant modification of the proteins, or if based on use of more novel Bt toxins or Bt toxins not found in currently registered microbial products (for example Cry9C), there is a potential impact on human health. Post-transcriptional modification is known to occur in plants and such characteristics as the degree of glycosylation might also affect stability and other physiochemical properties of proteins. Tests should preferably be conducted with the protein as produced in the plant (see also section 3.1.3). However, the committee recognizes that it is often difficult to obtain enough plant-expressed protein; in these cases, the committee recommends that

The EPA should provide clear, scientifically justifiable criteria for establishing biochemical and functional equivalency when registrants request permission to test non plant-expressed proteins in lieu of plant-expressed proteins.

The strong likelihood that gene products currently found in commercial transgenic pest-protected plants are not allergens does not remove the need for a minimum of properly planned and executed tests. For example, allergenicity is assumed to be unimportant for many Bt endotoxins, more because of the common characteristics of food allergens than because of rigorous testing. The Cry 1Ab pesticide fact sheet (EPA 1998a) states that

current scientific knowledge suggests that common food allergens tend to be resistant to degradation by heat, acid and proteases, are glycosylated and present at high concentrations in the food. The delta endotoxins are not present at high concentrations, are not resistant to degradation by heat, acid and proteases, and are apparently not glycosylated when produced in plants.

In the case of Cry3A in potatoes (EPA 1995a), the company demonstrated that the endotoxin is not a major component of the food, is apparently not glycosylated in plants, and is digested by gastric enzymes. However, Cry9C toxin, unlike the Cry1A and 3A toxins, does not degrade rapidly in gastric fluids and is relatively more heat-stable (EPA 1998c); these characteristics of Cry9C raise concerns of allergenicity. It is important to note that levels of gastric enzymes may vary among individuals and that those variations may need to be considered.

Although the standard tests indicate nonallergenicity for Cry3A, they were not all carried out on the endotoxin produced in potatoes, and none involved testing the immune system itself. Allergenicity is difficult to test, in part because prior exposure is a prerequisite to an allergic reaction, but tests for allergenicity ideally should involve the immune system or use an allergic endpoint. Useful guides to protein allergenicity include a supplement to Critical Reviews in Food Science and Nutrition (Metcalfe 1996a) and the proceedings of a workshop held at the 1998 Society of Toxicology annual meeting and recently published (Kimber et al. 1999). They make it clear that food allergy is relatively common and can have numerous clinical manifestations, some of which are serious and life-threatening. Furthermore, it is well established that allergenic proteins can be found in many food plants, of which some, such as soybeans and potatoes, have been genetically modified for pest-protection, and many others are or will be candidates for this type of genetic modification.

Those two sources also summarize the problems in protein allergenicity testing. Some, such as the double-blind placebo-controlled food challenge and the skin-prick test, although they involve adverse toxicologic endpoints and are carried out directly on human volunteers, nevertheless provide questionable results, in that they require volunteers who are already sensitized and that they are difficult to implement with novel proteins that have not traditionally been consumed. Tiered tests involving protein homology and stability comparisons with known food allergens and immunoassays for specific classes of antibodies are also proposed in these documents and are currently used by the agencies as a screen for allergenicity (figure 2.1). However, the tests in figure 2.1 either are indirect, do not involve adverse effects, or are otherwise problematic for testing of novel proteins that have not previously been components of the food supply. Indeed, figure 2.1 starts with a decision based on whether or not the protein is derived from a source that is known to be allergenic. This decision can usually be made clearly if the source is a food plant. For transgenic proteins such as Bt endotoxins making such an assessment would be complicated. If we conservatively choose the “yes” decision, then it would be extremely difficult to complete all of the tests listed because test materials and previously exposed human subjects are not readily available.

FIGURE 2.1

Tests for Potential Allergenicity. Source: Adapted from Metcalfe (1996b).

It is clear from the report of Nordlee et al. (1996) on the expression of Brazil nut protein in soybeans that genetic engineering can result in the expression of an allergenic protein in a food plant, but this is not known to be the case with any commercialized transgenic pest-protected plant. However, some testing of pest-protected plants and purified gene products is appropriate in many cases inasmuch as allergenicity is one of the possible adverse effects. The possibility that proteins in spore-crystal formulations of Bt can interact with the human immune system was suggested by a recent study on workers in crops topically treated with Bt sprays (Bernstein et al. 1999). The response seen in this study could have been due to proteins other than the Bt toxins in the formulation, so similar studies should be performed with purified Bt toxins.

2.5.2 Questions Concerning Health Impacts

The potential for transgenic pest-protected plants to pose a threat to human or animal health must be considered against the background of existing information. To date no such effects have been shown with commercialized transgenic crop plants. The work of Ewen and Pusztai (1999) hints of some possible interaction between a lectin expressed in potato and alterations in the potato caused by the genetic engineering process. According to the study, diets containing genetically engineered potatoes expressing the lectin, Galanthus nivalis agglutinin (GNA), showed some effects on different parts of the rat gastrointestinal tract. Those effects fell into two categories, ones caused by the GNA transgene itself and others caused by pleiotropic effects of expressing the transgene. However, analysis of the work of Ewen and Pusztai by the Royal Society (Royal Society 1999) and by Kuiper et al. (1999) indicates that the study lacked scientific rigor. For example, data concerning the biochemical composition of the potatoes used in the study show that the nontransgenic variety differed significantly from the transgenic variety. These differences could be attributable to natural variations in potato lines and are not necessarily due to the genetic modification (Kuiper et al. 1999).

It is important to ask whether any such threats have resulted from more conventional genetic modification of agricultural crop plants (conventional pest-protected plants) and, if so, whether they can serve as examples for assessing the risks of transgenic pest-protected plants. The questions outlined below can be asked, with equal validity, of transgenic pest-protected or conventional pest-protected plants. It should be noted, however, that conventional breeding usually selects for endpoints that are almost always controlled by several genes. It could be reasoned that such selection because of genetic linkage would be more likely to select simultaneously and inadvertently for an additional undesirable characteristic than would the introduction of a single gene or even a small number of genes with transgenic techniques (section 2.4.2). Both animal and plant breeding have yielded examples of inadvertent selection of undesirable characteristics.

2.5.3 Summary

The above questions can be used to guide the review process for the potential health effects of transgenic pest-protected plants. In reviewing toxicity testing relevant to human health for currently commercialized transgenic pest-protected plant products (that is, Bt toxins and viral proteins), the committee found that,

When the active ingredient is a protein, short-term oral toxicity and potential allergenicity testing are currently appropriate, inasmuch as the testing protocols are the ones currently available. However, testing protocols, particularly for allergenicity, should be improved with additional research.

The committee recommends that

When the active ingredient of a transgenic pest-protected plant is a protein and when health effects data are required, both short term oral toxicity and potential for allergenicity should be tested. Additional categories of helath effects testing (such as for carcinogenicity) should not be required unless justified.

Additional categories of toxicity testing do not appear justified for currently commercialized products (such as products with Cry1A and 3A Bt endotoxins and viral coat proteins). However, in these cases, it important that the tests that are performed be rigorous, logical, and scientifically sound (see section 2.5.1, section 2.5.2, and section 3.1.3). Novel, or less familiar plant-pesticides (that is, in comparison to viral coat proteins and Bt toxins) may require additional toxicity testing.

In addition, given the difficulties with determining the potential allergenicity of proteins not currently in the food supply, the committee recommends that

Priority should be given to the development of improved methods for identifying potential allergens in pest-protected plants, specifically, the development of tests with human immune-system endpoints and of more reliable animal models.

Protocols for toxicity- and allergenicity-testing, whether of gene products or plants, should be reconciled across agencies (see section 4.3). Variations among EPA, FDA, and USDA toxicity-testing protocols, other than those dictated by legislative authority, should be minimized or abolished. It should also be noted that the use of “familiarity” as a guideline to minimize testing can sometimes be inappropriate and warrants caution. Familiarity can be defined as indirect knowledge or experience obtained from similar gene products, plant varieties, or progenitor varieties grown under similar conditions and used for the same purposes in the same way.

2.6.1 Definitions

It is useful to divide the effects of conventional and transgenic pest-protected plants on nontarget species into direct and indirect effects. Direct effects include the adverse effects of the toxic plant components on nontarget herbivores, omnivores, and microorganisms that feed on live plants (Hare 1992) and on detritivores that feed on dead plants (Horner et al. 1988). When toxic substances are on the surface of a plant, there could also be direct effects on organisms that do not feed on the plant solely caused by contact toxicity or repellence (Farrar and Kennedy 1993).

Indirect effects include adverse effects of genetically modified pest-protected (GMPP) plants through an intermediary species (Bergman and Tingey 1979; Hare 1992; Price et al. 1980). For example, if an herbivore is tolerant of toxic substances in a plant, these substances could be found in the herbivore's digestive system (Price et al. 1980) or even be sequestered in the herbivore's tissues (Brower et al. 1984; Duffey 1980; Tallamy et al. 1998). Such a herbivore can be unpalatable or toxic to some of its predators or parasites because of this biological magnification of plant-defense compounds (Brower et al. 1984; Ferguson and Metcalf 1985; Hoy et al. 1998). If a pest-protected plant causes dramatic decreases in some herbivore or omnivore populations, there will be less nutrient material for the next level in the food chain. It is theoretically possible that a specialized predator, parasite, or pathogen of an affected herbivore could become locally extinct (Riggin-Bucci and Gould 1997).

The few data sets on nontarget impacts of transgenic pest-protected plants come mostly from crops that express Bt toxins (Hoy et al. 1998). However, a considerable number of studies have examined direct and indirect effects of conventional pest-protected crops and of wild host plants that differ in their pesticidal properties. Some of these studies are reviewed in the following sections (2.6.2 and 2.6.3).

2.6.2 Direct Effects

Effects of Pollen Ingestion by Nontarget Herbivores

Pollen from wind-pollinated pest-protected plants can be deposited on nearby vegetation (figure 2.2) and inadvertently ingested by nontarget leaf-eating insects. For example, a small fraction of corn pollen is known to disperse up to180 ft from the edge of the crop (Raynor et al. 1972) and can be deposited on milkweed (Asclepias sp.), which are common in and along edges of corn fields in the midwestern United States where about half the population of US monarchs spend some of the summer (Wassenaar and Hobson 1998). Milkweed is the only food of monarch butterfly larvae. Laboratory studies showed that young monarch larvae were killed by ingesting high doses of Bt corn pollen that had been experimentally dusted onto milkweed leaves (Losey et al. 1999). Another study by Hansen and Obrycki (1999c) obtained similar results when monarch larvae were placed on milkweed leaves that had been collected at the edges of Bt and non-Bt corn fields in Iowa and brought into the laboratory. Although both studies should be viewed as preliminary and actual negative impact, if any, on the population densities of monarch butterflies is yet unknown, they have focused attention on the question of whether widespread dispersal of insecticidal pollen can occur if entire regions are planted with wind-pollinated Bt plants (such as corn, poplar, and pine). It should be noted, however, that some Bt corn cultivars apparently do not produce any Bt in their pollen (EPA 1998b). In those cases, Bt toxin would not be a potential hazard to nontargets even if pollen dispersed great distances.

FIGURE 2.2

Examples of pollen and gene dispersal as a function of distance from edge of crop. Dispersal distances are expected to depend on local conditions and relative sizes of donor and recipient groups of plants.

One recent field test indicates that at least 500 Bt pollen grains per square centimeter is necessary to sicken monarch caterpillars and that milkweed plants growing adjacent to corn fields had only an average of 78 grains per square centimeter (Kendall 1999). Eighty-eight percent of milkweed within one meter fell below the level of toxicity to caterpillars. On the other hand, laboratory tests by Hansen and Obrycki (1999b) demonstrated that when monarch caterpillars consumed milkweed leaves experimentally dusted with 135 grains per square centimeter of pollen (comparable to pollen concentrations they found in the field), there was 46% mortality if the Bt pollen source was insertion event Bt11 and 65% if the source was event 176. Moreover, research suggests that wind direction, rainfall, and other factors can significantly affect pollen concentration (Weiss 1999). Further field-based research is needed to determine whether dispersed Bt pollen could have detectable effects on the population dynamics of nontarget organisms.

2.6.3 Indirect Effects

Studies in which predators were fed insects that developed on pest-protected cultivars have often produced adverse effects on the predator. For example, the compound DIMBOA is found in lepidopteran-resistant grain crops, including corn. Ladybugs that consumed aphids that had fed on diets containing DIMBOA had slower development (Martos et al. 1992). The tobacco hornworm parasitoid Cotesia congregata is adversely affected when the hornworm feeds on tobacco or artificial diets with high nicotine concentrations (Thorpe and Barbosa 1986). Cucumbers that are resistant to spider mites typically have higher concentrations of the triterpenoid cucurbitacin-C than nonresistant cultivars (DaCosta and Jones 1971; Gould 1978). Spotted cucumber beetles that feed on the resistant cultivars sequester cucurbitacin-C in their bodies and eggs (Ferguson and Mecalf 1985). The cucurbitacin-C in their eggs keeps the eggs from being killed by the entomopathogenic fungus Metarhizium anisopliae (Tallamy et al. 1998).

Although a pest-protected plant could have adverse effects on beneficial insects in the agroecosystem, the value of the cultivar is generally based on the balance between adverse effects on beneficial and detrimental species. For example, Kauffman and Flanders (1985) found an adverse effect of pest-protected soybean on a parasitoid of the Mexican bean beetle, but the effect on the beetle pest was greater than on the parasitoid; it was therefore concluded that overall the cultivar would be useful. Hare (1992) reviewed the published data on overall effects of pest-protected host plants on biological control. Of the 16 case studies he reviewed, there were antagonistic interactions in six, positive synergistic interactions in two, neutral effects in five, and a concentration-sensitive relationship in three.

There have been a few published studies of the indirect effect of transgenic Bt-producing crops on natural enemies. Depending on the species involved , some studies reported no significant effects (for example, Hough-Goldstein and Keil 1991), but others have found adverse effects. Giroux et al. (1994) reported that the ladybug predator of the Colorado potato beetle consumed fewer potato beetle eggs when potato Bt levels were high. Hillbeck et al. (1998a, b) found that when chrysopid larvae were reared on prey that were fed Bt-producing corn, they suffered 62% mortality. When they were reared on prey that were fed non-Bt corn, mortality was only 37%. Those results were found with two prey species, one sensitive to Bt and the other insensitive. The results indicate that it was the Bt toxin in the bodies of the prey, and not simply unhealthy prey, that caused the heightened mortality.

It is important to ask whether such indirect effects will have a harmful effect on the agroecosystem. In some cases, the use of conventional pest-protected plants has lead to decreased foraging efficiency of predators and parasitoids (Boethel and Eikenbary 1986). A few field studies have attempted to measure the effect of transgenic Bt potatoes on the diversity of insects in crop fields (Hoy et al. 1998). In one 4-year study at five locations in the Wisconsin potato-production system, it was found that populations of predators were, on the average, 63.8% lower in fields where non-Bt potatoes were managed with typical insecticides than in fields of transgenic Bt potatoes. Parasitoid populations in the chemically treated potato fields were, on the average, 58.4% lower than in Bt potato fields. Insecticidal treatment of potatoes to control Colorado potato beetles with conventional insecticides often results in secondary outbreaks of aphids because the aphids are released from biological control. In three of the four years of the study, aphid populations (which are not affected by Bt toxins) were lower in the transgenic Bt potato fields than in chemically treated potato fields, presumably because of greater biological control.

A study of potatoes in Ohio reported similar results (Hoy et al. 1998). In potato fields treated with pyrethroid insecticide to manage Colorado potato beetles, natural enemies were reduced to undetectable levels. This resulted in aphid outbreaks that could be only marginally controlled by insecticides (253 aphids per meter of row in late July). In the transgenic Bt potatoes where natural enemies were always detectable (1 to 4 per meter), the aphid density was very low (below 10 per meter).

Those two studies compared the impacts of chemically-intensive potato farming and farming with transgenic Bt-potato. No comparison with a no-chemical control treatment was presented, but it might have been inappropriate. Another study provided to the committee indicated that there were no significant differences in beneficial arthropods between plots in which transgenic Bt potatoes or microbial Bt sprays were used (Feldman et al. 1994). Both had similar densities of beneficial arthropods, while plots in which chemical insecticides were used had much lower densities of some of these arthropods (Feldman et al. 1994).

In most of the areas where Bt potatoes will be used commercially, the crop is usually protected against the target Colorado potato beetle with conventional pesticides. In the corn system, most of the acreage has not been treated for the European corn borer, the major target pest of the Bt corn cultivars, because the insect feeds mostly within the plant stem where pesticides are typically not effective. In addition, pesticide treatment is more expensive than the yield losses due to the borer. In this case, the appropriate comparison of environmental impacts of the transgenic Bt cultivars would be with a system where non-Bt corn was not treated with pesticides aimed at the European corn borer.

It is useful to ask what will happen to the long-term biodiversity of agroecosytems if biotechnology provides us with crops that are constitutively toxic to almost all insect herbivores. A study of insecticide effects on quail populations in soybean fields provides interesting insight. Palmer et al. (1998) found no physiologic effects of the pesticide residues on the quail. However, quail in sprayed fields had lower weight gain and lower survival than those in control fields because there were fewer insects to feed on (Palmer 1995). If in the future we continue to commercialize pest-protected crops that constitutively express more diverse insect-specific toxins, we could inadvertantly produce crops that lower general herbivore abundance. This could result in lower biodiversity of species at higher trophic levels that depend on herbivorous insects as food.

2.6.4 Summary

Conventional and transgenic pest-protected crops can adversely affect nontarget organisms through direct contact with or ingestion of the plant or pollen by the nontarget organisms and through indirect contact when the pest-protective substances (or their effects) are passed to other trophic levels that are consumed by nontarget organisms. However, the committee found that

Both conventional and transgenic pest-protected crops could have effects on nontarget species, but these potential impacts on nontarget organisms are generally expected to be smaller than the impacts of broad-spectrum synthetic insecticides, and therefore, the use of pest-protected plants could lead to greater biodiversity in agroecosystems where they replace the use of those insecticides.

Current criteria for commercialization of transgenic pest-protected plants includes several laboratory toxicity tests for nontarget organisms (see appendix B and section 3.1.2). In light of the above discussions, more field evaluations should be conducted to determine the impacts of specific pest-protected crops on nontarget organisms, compared to impacts of standard and alternative agricultural practices. The committee recommends that

Criteria for evaluating the merit of commercializing a new transgenic pest-protected plant should include the anticipated impacts on nontarget organisms compared with those of currently used ⁵ pest control techniques.

2.7.1 Pollen Dispersal and Outcrossing

The distance that pollen moves varies widely among species. Some crops—such as rice, wheat, and soybean—are mostly self-pollinated (selfed). In sorghum, most seeds result from self-pollination, and the rest come from outcrossing or cross-fertilization among plants via wind pollination (Arriola and Ellstrand 1996; Ellstrand and Foster 1983). Oilseed rape (canola) also produces a mixture of selfed and outcrossed seeds, whereas corn, carrot, sunflower, poplar, radish, strawberry, clover, and many other species produce most of their seeds by outcrossing rather than selfing (for example, Brown et al. 1985; Free 1970; Richards 1986). By definition, outcrossers disperse pollen to other plants and therefore need greater isolation distances than selfing species in situations where pollen dispersal is considered undesirable (table 2.1). Wind-pollinated outcrossers, such as many trees and grasses, tend to produce larger quantities of pollen (causing problems for people with pollen allergies) than similar-size plants that are pollinated mainly by insects and other animals. Pollen from animal-pollinated plants is not as buoyant in air currents, and very little is shed into the air; these types of pollen adhere to the pollinator's body. Pollen grains of a few species, such as sugar beet and oilseed rape, are dispersed by both wind and insects (Cresswell et al. 1995; Free et al. 1975; McCartney and Lacey 1991).

TABLE 2.1

Isolation distances required by USDA for producing foundation seed used for seed increase. Note that this is not a complete list of all crops.

The amount of pollen dispersed from outcrossing plants declines rapidly as a function of distance from the source (section 2.6.2, figure 2.2). In the corn example, the amount of pollen deposited at 60 m was only 0.02% of the amount deposited 1 m from the edge of the crop, but this still represents 2,500 pollen grains per square meter (Raynor et al. 1972). Most pollen is deposited within a few meters of its source, but a small proportion can be carried more than 1 km away (for example, Arias and Rieseberg 1994; Kirkpatrick and Wilson 1988; Klinger et al. 1992). Such long-distance gene flow is not easy to measure, because very large samples are needed to detect low-probability events. In addition, the extent of long-distance gene flow is highly variable and depends on local conditions, the relative sizes of donor and recipient populations, and synchrony of flowering. Once pollen from a crop has spread to wild plants, further gene flow occurs in a ripple effect through both pollen and seed dispersal.

2.7.2 Crop-to-Wild Gene Flow

The escape of novel resistance traits into free-living populations of wild relatives is often cited as an undesirable consequence of growing transgenic crops on a commercial scale (for example, Bergelson et al. in press; Ellstrand and Hoffman 1990; NRC 1989; Parker and Kareiva 1996; Raybould and Gray 1998; Rissler and Mellon 1996; Snow and Morán-Palma 1997; Tiedje et al. 1989; Van Raamsdonk and Schouten 1997). Concerns arise mainly when novel, beneficial traits have the potential to spread to wild populations and cause them to become more invasive and difficult to control. Naturally-occurring damage by insects and diseases can regulate the density and distribution of populations of wild relatives, so release from these pressures by genes from a cultivated relative could potentially make weeds more troublesome in agricultural fields. Moreover, many wild or weedy relatives also occur in other habitats, including rangeland, roadsides, forests, wetlands, and other natural areas where they are not controlled with weed-management techniques, such as the use of herbicides, mowing, and tilling.

Conventional crop genes have spread to wild populations in the past (for example, Arriola and Ellstrand 1996; Ellstrand et al. 1999; Linder et al. 1998; Small 1984), but this process has not been studied by weed scientists, and it is not known whether it has facilitated evolutionary adaptations in weedy relatives (Small 1984; Snow et al. 1998). Circumstantial evidence suggests that several weedy species such as Johnson grass have become more robust and abundant as a result of hybridization with crops (NRC 1989). Known problems have involved hybridization between a weed and a crop, often due to range expansions of one species or the other (Ellstrand et al. 1999; Rissler and Mellon 1996), rather than a transfer of few novel crop genes within an existing crop-weed complex. A well-documented example of a new weed problem occurred in France: a wild gene for early bolting spread from sugar beet seed nurseries. Seeds from those nurseries were then grown by farmers and new weed populations resulted. This led to serious economic losses (Boudry et al. 1993). Scientists have viewed wild relatives mainly as sources of genes that could affect crops, either as useful germplasm or as undesirable pollen contamination; thus, there is little empirical evidence of whether wild species have benefited or not from crop-to-wild gene flow.

Recently, weed scientists have redoubled their efforts to quantify genetic diversity in weed populations and to gain a better understanding of how weeds adapt to changing conditions. The commercialization of transgenic crops has provided added incentives for studies on crop-to-wild gene flow because some transgenic phenotypes have never occurred in wild relatives of certain crops. For example, wild sunflowers lack protection against their seed-eating insects and a fungal disease known as Sclerotinia rot (Seiler 1992). If transgenic methods introduced protectants against these pests into cultivated sunflowers, the genes that code for these protectants could move into wild sunflowers, perhaps increasing their ecological fitness and abundance. Likewise, genetic resistance to the herbicide glyphosate (Roundup^TM) has not been found in the germplasm of crop relatives, but this trait could be transmitted to wild or weedy relatives of transgenic, glyphosate-resistant crops. The use of a wider and more effective variety of transgenic methods could increase the rate at which weedy species acquire novel types of strong, single-gene resistance via crop-wild hybridization.

A first step toward assessing the consequences of crop-to-wild gene flow is to determine which cultivated species are capable of crossing with wild relatives (table 2.2). Corn, soybean, tomato, and cotton do not escape cultivation, nor do they have many close wild relatives within the continental United States (except for cotton in southern Florida). In contrast, some cultivated species survive in feral populations or cross with wild plants of the same species (for example, carrot, sunflower, squash, rice, poplar, certain grasses, oilseed rape, radish, and beet). Many crops have weedy ancestors or occur as weeds in portions of their worldwide distribution (Colwell et al. 1985; Ellstrand et al. 1999; Small 1984). Hybridization between different species or genera is sometimes possible, especially if they share a close, common ancestry. Spontaneous hybridization can occur even when the two taxa have unequal chromosome numbers, which typically creates problems during cell division (for example, Mikkelsen et al. 1996; Zemetra et al. 1998). In fact, new molecular methods have shown that gene flow among related species is much more common than was previously thought (for example, Arnold 1997). Genes often spread among species without being detected by visible indicators, so genetic markers, such as unique DNA fragments are needed to identify current and historical patterns of crop-to-wild gene flow. Recent studies have augmented the number of plant groups in which interspecific hybridization is known to occur naturally, as in oaks, birches, orchids, irises, and wild gourds. The information derived from studies of ongoing gene flow could also be used to understand what types of genes and traits move between these species, thus providing a basis to predict whether specific traits would raise concern if the genes/traits were introduced and transferred.

TABLE 2.2

Examples of Commercially Important Species That Can Hybridize with Wild Relatives in the Continental United States.

Early generations of crop-wild hybrids generally have lower fertility than their parents, but there can be high variability among the fertility of the hybrids, with some having high fertility. Even those which produce few viable gametes can transmit crop genes to later generations (a process known as introgression). With each successive generation of backcrossing with wild genotypes, the crop's contribution to the plant's total genome is reduced by 50% (figure 2.3) and the progeny become more similar to wild genotypes. After two or three generations of backcrossing, plants with a crop ancestor can be just as competitive and successful as wild plants (for example, Snow et al. 1998). Moreover, the fitness of first-generation crop-wild hybrids is sometime as great as the fitness of wild plants (for example, Arriola and Ellstrand 1997; Klinger and Ellstrand 1994).

FIGURE 2.3

Dilution of crop alleles in plant genotypes after spontaneous hybridization and backcrossing with wild plants. Two parent plants are shown on each line (for example, crop x wild), with an arrow pointing to their offspring.

The frequency of a given crop gene in a wild population depends on many factors, including the rate at which it is introduced into the population; temporary fitness barriers, if any, in the first and early backcross generations; possible fitness costs associated with the gene itself; and possible benefits of the gene for the plant's survival and reproduction. Fitness costs associated with transgenes may be small or absent when a seed is ready to be sold, because seed companies choose breeding lines with the best transformation events available (for example, Fredshavn et al. 1995). When several transgenes are inserted together as tightly linked traits (inherited as a unit), the combined ecological costs and benefits of these traits will determine whether and to what extent a wild relative's fitness is enhanced. If one transgene confers resistance to a common herbicide, such as glyphosate, selective pressures favoring plants with the transgene will be very strong when that particular herbicide is used, thereby increasing frequencies of the linked transgene in weed populations. Eventually, wild species will be able to acquire additional beneficial transgenes that are released in different cultivars, and the new traits could accumulate in wild populations.

The ecological and evolutionary benefits conferred by crop genes that enter wild populations are difficult to evaluate. This is an issue that lies at the crux of concerns about gene flow from transgenic and conventional crops. We know little about the extent to which insects and diseases limit wild, weedy populations that are sexually compatible with cultivated species. Critics of biotechnology argue that the spread of beneficial traits could quickly lead to the spread of weeds; advocates of transgenic crops maintain that this risk is small or nonexistent. Empirical data with which to address the question are lacking. Many publications describe proposed methods for evaluating the effects of beneficial crop genes on the dynamics of wild, weedy populations (for example, Crawley et al. 1993; Kareiva et al. 1996; Rissler and Mellon 1996). However, inadequate funding and restrictions on trial releases of transgenic pest-protected plants have hampered opportunities to carry out this important research before commercialization (Wrubel et al. 1992; Purrington and Bergelson 1995). A few studies have focused on how novel genes affect plant fitness, (that is, the relative survival and reproduction of new genotypes) (for example, Bartsch et al. 1996; Stewart et al. 1997), but it is much more difficult to determine how new traits will affect the geographic distribution and local abundances of a given species. The latter requires a combination of field studies, field experiments, and mathematical modeling (for example, Rees and Paynter 1997).

Until better data are available, it will be necessary to rely on general ecological and agricultural knowledge to predict the consequences of com mercial-scale, crop-to-wild gene flow from pest-protected plants. The common perception that most wild plants are adapted to tolerate or resist pests is misleading in that even species that appear to be protected by mechanical or chemical defenses can be decimated by specialist herbivores or new diseases. Many studies have demonstrated that herbivores, especially insects that specialize on flowers and seeds, can regulate wild plant populations (for example, Guretzky and Louda 1997; Rees and Paytner 1997; Waloff and Richards 1977). In contrast, much less is known about the extent to which diseases affect the population dynamics of wild plants (Burdon 1987; Burdon and Jarosz 1988). The impact of diseases is much more obvious in cultivated species, especially genetically uniform plants grown at high densities. However in natural populations, where nonspecific plants are often scattered and genetically diverse, disease could still be important. Wild plants are susceptible to many native, introduced, and newly evolved diseases, but we know little about how often they are exposed to these pathogens and whether such exposure constrains population growth. Although general surveys of disease frequencies in wild populations are helpful, they might miss infections that occur inconspicuously (in seeds, seedlings, or roots) or infrequently, and those which are so virulent that they cause local extinctions.

Because of the uncertainties described above, it is premature to predict the ecological impacts of gene flow from transgenic pest-protected plants. Meanwhile, regulatory decisions must be made in a timely fashion. It seems unlikely that the transfer of one or two novel crop genes for pest-protection would transform a wild species into a problematic weed, although in some cases unwanted population increases of weedy species could result. Moreover, the cumulative effects of beneficial crop genes could potentially lead to expensive and ecologically damaging problems in weeds that are already difficult to control, such as Johnson grass (Sorghum halepense). In the future, additional phenotypic traits might include broad-spectrum resistance to insects or diseases and greater tolerance of cold, drought, salinity, nutrient scarcity, or acidic soils. Such traits could be more advantageous to wild relatives than those now in use.

Consequences of gene flow other than weediness are also perceived to be detrimental to preserving biodiversity. For example, the spread of transgenes to wild relatives that are rare or endangered is sometimes considered as a potential ecological risk, especially in regions that are centers of diversity for crop relatives (for example, Rissler and Mellon 1996). However, if a portion of wild species's genome is being exchanged for new genetic material by hybridization, this process will occur whether or not the crop is transgenic. The transfer of resistance traits to rare wild relatives is unlikely to exacerbate population declines or lower genetic diversity in these species, given that transfer of traits between crops and wild relatives is an ongoing process. However, researchers and regulators should be aware of these and other potential unintended consequences of gene flow from transgenic pest-protected plants (for example, the hypothetical spread of a Bt gene from an ornamental species to the only host plant of a rare beetle).

Eventually, it could be possible to reduce gene flow from cultivated plants with various containment methods. If a transgene is inserted into the chloroplast genome, which is usually not transmitted by pollen, spread of the transgene into natural populations could be delayed (Daniell et al. 1998) but would not be avoided entirely (Stewart and Prakesh 1998). Gressel (1999) suggested linking transgenes to genes for traits that are detrimental to weeds but not crops (for example, a gene for lack of seed dormancy). Again, this would slow down the process of introgression but would not necessarily provide complete containment of ecologically beneficial transgenes. A more effective method of confining gene flow may be to use a system in which transgenic seeds are killed just before they mature, so that they can still be harvested but will not germinate (US Patent No. 5,723,765, “Control of plant gene expression”). Popularly known as “terminator” technology, this potential method of containment is highly controversial and has not yet been used commercially. In the future, biotechnology companies might develop transgenic plants with inducible pest resistance traits that require a chemical spray to be “turned on.” If these systems work as predicted, resistance genes that spread to wild species would not be expressed in the wild species unless the spray was used, and therefore, would be extremely unlikely to contribute to the wild species' invasiveness. Because of the concerns raised about using chemicals to turn on genes in the environment, it is unclear whether or not such technology will be acceptable. However, if this strategy is pursued on a commercial scale, it could greatly reduce the need to regulate transgenic crops based on weedy-relative considerations.

2.7.3 Crop-to-Crop Gene Flow

Crop breeders try to minimize gene flow into their breeding lines by using crop-specific isolation distances (for example, Bateman 1947a, b, c). Seeds used for commercial sale are often mandated to have less than 0.1% contamination from phenotypically different sources (table 2.1). Less stringent genetic isolation is required for producing certified seeds to sell to farmers, and only rarely is any isolation required when farmers grow a crop for harvest. Now, however, organic farmers are concerned about gene flow from transgenic plants as organic standards may forbid the use of transgenic products. Under proposed standards, organic farmers are not allowed to sell transgenic food, and a tolerance for very low levels of contamination by transgenic DNA has not been established (NOSB 1996). The committee knows of no scientific evidence that crop-to-crop gene flow has caused health or environmental risks to date, but we recognize that consumer choices are involved. Nonorganic farmers might also face problems with crop-to-crop gene flow and undesirable seed characteristics—for example, if transgenic crops are commercially developed for pharmaceutical or industrial compounds, or when farmers' fields are inspected in search of the unauthorized use of patented transgenic genotypes. Clearly, these hypothetical concerns apply to a variety of transgenic crops. Contamination with pollen from other farms is likely to be very low in most cases, as illustrated in figure 2.2 and table 2.1 and described further below.

Crop-to-crop gene flow is unlikely to occur over long distances in species with high selfing rates, although even rice, sorghum, and wheat are known to hybridize occasionally with nearby plants (Arriola and Ellstrand 1996; Langevin et al. 1990; Seefeldt et al. 1998; Zemetra et al. 1998). Pollen from outcrossing species could easily move 600 ft or more among plantings (table 2.1). In a study of oilseed rape fields in Scotland, researchers documented a small amount of pollen dispersal as far as 3 km from the crop, indicating that farm-to-farm spread of transgenes was common (Thompson et al. 1999). In that region, over 60% of the crop plantings occurred within 100 m of another crop planting. Gene flow into row crops can also occur via volunteer crop plants that survive to reproduce and via feral plants that start new populations, for example, when sunflower or oilseed rape seeds are inadvertently spilled along roadsides. Even for selfing species, total containment of crop genes is not considered to be feasible when seeds are distributed and grown on a commercial scale. The inevitability of gene escape has been recognized by federal agencies, and regulatory decisions are based on it.

In some situations, unwanted levels of contamination can be avoided by altering the distances among plots, using border rows to intercept unwanted pollen, or planting large acreages to minimize edge effects (for example, figure 2.2 and table 2.1; Hokanson et al. 1997; Morris et al. 1994). These approaches have been discussed and recommended by the USDA for field testing of transgenic plants products prior to the regulatory approval of these products (USDA 1993; USDA 1996a). Figure 2.4 illustrates the general principle that small populations are more susceptible to contamination from long-distance pollen sources than are large populations (for example, Ellstrand and Elam 1993; but see Klinger et al. 1992). For outcrossing cultivated plants, contamination of less than 1% of the seeds in a given crop can often be prevented by isolation distances of over 1 km, but the threshold distance depends on the species, the relative sizes of donor and recipient plantings, use of border rows, and overlap in flowering times.

FIGURE 2.4.

Number of border rows recommended to keep “off-type” seeds in corn fields under 0.5% as a function of isolation distance from other sources in corn fields. These requirements apply to contaminating pollen from sources with kernels that (more...)

2.7.4 Summary

In light of the above discussions, the committee found that

Pollen dispersal can lead to gene flow among cultivars and from cultivars to wild relatives, but only trace amounts of pollen are typically dispersed further than a few hundred feet.

The transfer of resistance traits to weedy relatives could potentially exacerbate weed problems, but such problems have not been observed or adequately studied.

The committee recommends that

Criteria for evaluating the merit of commercializing a new transgenic pest-protected plant should include whether gene flow to feral plants or wild relatives is likely to have a significant impact on these populations.

In order to study the effects of gene flow, the committee recommends areas for research in chapter 3 and the executive summary (section 3.4 and section ES.5.2).

2.8.1 Evolution of Resistance to Pest-Protected Crops

The emergence of strains of pathogens that overcome plant-genetic resistance or other disease-control methods has been and probably always will be a problem in agriculture. Indeed, this problem is common to all plant and animal hosts for which pathogens exist. Traditionally the problem has been managed by development of multiple strategies for disease control (genetic and other control measures), surveillance of pathogen activity and strain development, deployment of new pest-protected germplasm in response to emerging pest strains, and development and use of longer-lasting forms of pest-protection. With transgenic pest-protected plants that express a pest-protection gene transferred from another plant, the selective pressure for development of resistance-breaking strains should be qualitatively similar to the selective pressure associated with conventionally bred pest-protected plants. In section 2.9, these issues are discussed.

2.8.2 Risks Posed by Virus-Derived Transgenes

2.8.3 Summary

In light of the above analysis, the committee found that

Most virus-derived resistance genes are unlikely to present unusual or unmanageable problems that differ from those associated with traditional breeding for virus resistance.

Case studies of virus resistant squash and papaya are presented in chapter 3 (section 3.1.4).

2.9.1 Pest Resistance to Control Tactics

The history of agricultural pest management has demonstrated that insects, weeds, and microbial pathogens have the evolutionary potential to overcome or circumvent most control tactics imposed by farmers (Barrett 1983; Green et al. 1990; Gould et al. 1991). That over 400 insect species have become resistant to at least one insecticide (Georghiou 1986) is often cited as evidence of the genetic ability of arthropods to evolve resistant strains. In addition, weeds and pathogens also have an impressive record of successful adaptation to control measures (Green et al. 1990). Although the number of cases of resistance by weeds to herbicides is smaller than that of insects, the percentage of weed species that has developed resistance is greater than that of insects (Gould 1995). It is well documented that microbial pathogens can successfully adapt to crop cultivars that are bred to resist specific diseases (Lamberti et al. 1981). In examining a random selection of 63 cases of viruses that live in association with specific plant hosts, Fraser (1990) found that in 28 of the cases there were good data indicating that adapted isolates of the virus existed (in only five cases was there good evidence that there had been no adaptation by the virus). Fungal and bacterial adaptation to pest-protected cultivars has caused serious crop losses. In many cases, pathogen resistance has occurred less than 5 years after a classically bred resistant cultivar was released for commercial use (see section 3.1.1). Experience with both insect and pathogen adaptation to genetically modified pest-protected (GMPP) ⁶ plants indicates that the more intensively a control tactic is used, the more rapidly pests will adapt to it (Gould et al. 1991). History also indicates that pests adapt more rapidly to some types of GMPP plants than to others (Lamberti et al. 1981).

If a GMPP cultivar is lost because the target pest adapts to the cultivar, replacing the cultivar with a new GMPP cultivar can have a number of associated costs. Even if new GMPP genes are available, moving those genes into a modern cultivar is expensive. Although the health and environmental safety of the plant protectant in the new cultivar can be tested in laboratory experiments, the new cultivar will need to be monitored for impacts that could not have been detected in the laboratory experiments. If new pest-protection genes are not available, farmers might need to move back to reliance on broad-spectrum pesticides. Decreasing the rate at which target pests adapt to GMPP cultivars can therefore produce societal benefits.

2.9.2 Decrease in the Rate of Resistance Evolution

Concern over the risk of pest resistance to conventional pesticides led to development of a relatively new field of applied science, called pest-resistance management (NRC 1986). The goal of this field is to determine approaches for developing and deploying pest control tactics in ways that maximize long-term benefits. Pest-resistance management is grounded in concepts and empirical findings from the basic sciences of quantitative genetics and population genetics (NRC 1986). In this regard, it is very similar to the applied science of classical crop breeding. These fields of inquiry rely heavily on statistical inference. A theoretical population geneticist or a crop breeder is therefore unlikely to make a deterministic prediction about the outcome of a natural evolutionary event or the exact characteristics of his or her next new cultivar. For the same reason, scientists investigating pest-resistance management tactics are reluctant to provide regulatory officials or farmers with exact predictions about how many years it will take for a specific pest to adapt to overcome a proposed resistance management plan. However, they can provide information on which of a number of approaches to development and deployment of transgenic and conventional pest-protected plants is likely to be most successful in decreasing the rate of pest evolution to adapt to those plants.

Quantitative comparisons of resistance management approaches for crops protected against insect damage began in 1986 (Cox and Hatchett 1986; Gould 1986a, b). A list of potential approaches has since been developed (Gould 1988a; McGaughey and Whalon 1992; Roush 1997; Tabashnik 1994). Some of the general approaches for resistance management for insect pests are as follows:

• High dose of a single contained toxin in most plants, with some plants producing no toxin at all and thus serving as a refuge (approach 1).
• Multiple toxins at high (or in some cases moderate) doses in most plants, with some nontoxic plants serving as a refuge (approach 2).
• GMPP plants with low doses of a toxin that only slow the growth of the pest, so that pest population growth decreases and natural enemies can become more effective (approach 3).
• Development of GMPP plants that produce the toxin only when and where it is most critical to protecting the plant (approach 4).

Much of the research aimed at developing these approaches and assessing their expected impacts has focused on transgenic pest-protected plants that produce Bt toxins.

A high dose of a toxin has been defined by the EPA Science Advisory Panel (SAP 1998) as an amount that is 25 times the amount needed to kill 99% of susceptible insects. Empirical information on development of insect genetic adaptation to Bt toxins indicates that such a high dose will kill most partially adapted insects in a pest population (such as heterozygotes). The result is interruption of the typical stepwise process of evolving from susceptible populations, to one that is partially adapted, to fully adapted to the GMPP plant. An analogy can be made to the use of antibiotics to treat human pathogens. The utility of decreasing the survival of partially adapted human pathogens has long been recognized by medical researchers and physicians who routinely recommend that their patients continue to take antibiotics past the period when most of the infectious organisms have been killed. The prolonged treatment period ensures that partially adapted target pathogens will also be killed and so not be transmitted to other people.

Many researchers have examined field and laboratory insect populations in an attempt to understand the mechanisms of insect adaptation to Bt toxins (Tabashnik 1994; Gould and Tabashnik 1998). Results indicate that either multiple genes or single recessive genes are needed to confer full adaptation to a high dose of Bt although partial resistance can be confered by a dominant gene. A very low proportion (1 out of a million, to 1 out of 1000) of fully adapted insects is expected to exist in a population before the population is exposed to Bt. If all host plants on a farm produced the high dose of Bt, only those few insects with the right gene combinations would survive. If they then mated with each other, their offspring would be fully adapted, and the pest population would no longer be affected by the GMPP crop. The planting of nontoxic host plants (refuges) is designed to make sure that a relatively large number of totally susceptible insects are produced on each farm, compared with the few fully adapted insects produced. As long as this refuge is maintained, almost all fully adapted insects produced in the Bt crop are expected to mate with susceptible insects. The offspring of these matings will not have the proper combination of genes needed to be fully adapted, so the evolutionary process is again interrupted. Many researchers expect use of the refuge in combination with plants that produce a high dose of the toxin to increase the time needed for insect adaptation by a factor of 10 (for example, from eight years to more than 80 years) if properly implemented (Gould 1998; Roush 1997; Tabashnik 1994).

By using transgenic or conventional pest-protected plants that contain high levels of multiple toxins with high doses (approach 2), the chance that insects will have the proper gene combination to be fully adapted is further decreased compared to the case where only one toxin is produced by the plant. It also increases the efficiency with which refuge-produced insects can break up combinations of resistance genes from the few pests that happen to carry the proper gene combination (Gould 1986a, b; Roush 1997). The development of resistance may be delayed by the use of several toxins with different modes of action (Zhu et al. 1994; Jach et al. 1995). The toxins could arise from a combination of conventional breeding and transgenic techniques. However, even if the target sites of two toxins differ, there is still the possibility of cross resistance if the two toxins can be detoxified by the same enzymes. The new high-dose refuge approach (approach 1) has been the most widely accepted tactic for resistance management of target pests of transgenic or conventional pest-protected plants. Approaches 3 and 4 also have potential applicability. Approach 3, which relies on an interaction between the GMPP plant and natural enemies, is expected to decrease the rate of evolution of adaptation because it does not result in a major decrease in the fitness of either susceptible or adapted pests. Companies have not embraced this approach, because it cannot always be relied on to protect the crop, and they may have liability for control failures. There have also been concerns that the approach might not always inhibit evolution of adaptation to the pest-protected plant (for example, Johnson et al. 1997a, b). Approach 4 would also decrease the rate of evolution of resistance because only the fraction of the pest population that feeds on the protected-plant parts would be killed. This general approach could be useful if correctly implemented, but technological and ecological problems must be solved before it can be used (Roush 1997).

The high-dose approach is feasible with Bt toxins because even at high doses no health or environmental problems have been reported in commercially grown varieties. Also, crop yield has not been reduced by production of high doses. That might not be the case with some other plant-protection mechanisms.

Much of the theory of resistance management for GMPP plants has been developed for diploid, sexually reproducing organisms (NRC 1986; Roush and Tabashnik 1990). The theory is therefore only partially applicable to viruses, bacteria, and even a large group of insects that have different means of reproduction.

Plant pathologists have long been concerned with viral, fungal, and bacterial adaptation to conventional pest-protected plants. In the 1950s they developed the concept of GMPP plants having either vertical or horizontal resistance to pathogens (Van der Plank 1963). Vertical resistance typically involved single plant genes that were initially very effective at mitigating a disease but were expected to be evolutionarily overcome by rapid genetic shifts in the pathogen (Lamberti et al. 1981). Horizontal resistance was typically controlled by many genes, offered lower but adequate suppression of the target pathogen, and was expected to be more durable (recalcitrant to pathogen adaptation). This system for stereotyping GMPP plants had some predictive power, but later investigators found that there were too many exceptions (Lamberti et al. 1981). Durability of a specific GMPP plant is now typically judged in retrospect on the basis of its long term performance (Johnson 1981).

There have been some recent attempts to use population-genetics theory for developing and deploying conventional pest-protected plants in ways that slow pathogen adaptation (Burdon et al. 1994; Lannou and Mundt 1996 and 1997; Mundt 1990; Zeigler 1998), but it has not become common practice. Instead, many current pathology programs for production of GMPP plants emphasize continual discovery of new resistance genes so that breeding programs can stay a step ahead of an evolving pathogen (McIntosh and Brown 1997).

Researchers developing engineered pathogen-resistant plants have also been concerned with pest adaptation (Beachy 1997). Although new molecular approaches could lead to plants that offer a greater evolutionary challenge to pathogens (Beachy 1997; Bendahmane et al. 1997), little empirical or theoretical work has been aimed at determining how to produce durable engineered pathogen resistance (but see Qiu and Moyer 1999).

2.9.3 Future of Resistance Management for GMPP Plants

EPA has been active in developing resistance management plans for Bt crops. It has developed an internal group of staff to work on the issue and has consulted formally and informally with researchers (Matten et al. 1996; Matten 1998). Researchers and EPA regulatory officials will probably learn a lot of general principles about how to develop and implement resistance management of transgenic pest-protected plants from the continuing work on Bt crops. Much has already been learned from the Bt system regarding theoretical and practical aspects of developing and implementing a resistance management program, but the EPA policy is still evolving (Matten 1998). Each year, new empirical results should provide information on better ways to optimize resistance management for these crops. Therefore, plans implemented today will need to be periodically reviewed for their continued usefulness.

Although EPA has instituted programs and regulations that demonstrate serious concern about insect adaptation to Bt crops (Matten 1998; SAP 1998), it has not indicated concern about virus adaptation to transgenic pest-protected plants with plant-produced viral coat protein. In general, EPA has not commented in formal documents about when it considers pest adaptation to pest-protected plants to be an important public or social problem and when it considers resistance to be only a business problem or an insignificant public or social problem.

There has been considerable public debate about this issue. One opinion is that there is no more reason to institute resistance management for transgenically produced Bt than there is to institute resistance management for conventional pesticides. Others argue that the use of Bt toxins in transgenic pest-protected crops is fundamentally different from the use of chemical pesticides, for a combination of the following reasons:

• Insecticides are typically used only when pest populations increase to the point where substantial yield-losses could occur, so refuges are already present. With transgenic Bt crops, the toxin is selecting for resistance all season long, even during weeks when the pest cannot feed on plant parts that affect crop yield, or during years when pest number are too low to cause yield loss.
• Bt toxins are seen as benign to the environment and public health, and no equally benign replacement product is available.
• Bt toxins are the active component in Bt spray formulations that have been used sustainably by organic and conventional farmers for many years, and this tool could be lost if transgenic Bt crops are not managed correctly.

Many transgenic pest-protected plants of the future may be protected by novel mechanisms and therefore not compromise the utility of plant protectants that are already being used by farmers. In such cases, the company that produces the plant protectant can be seen as the major party affected by pest evolution of adaptation to the company's product. However, there could be cases in which a new transgenic pest-protected plant cultivar is produced by transferring a plant protectant that is already in use to another crop species. The new use could increase the risk that pests will involve adaptation to the plant protectant in all uses. An example might be moving a pathogen-resistance gene from tomato into cotton. If the same pathogen is now controlled by this resistance-mechanism in both crops, the intensity of selection for adaptation could be substantially increased. A resistance management program could be developed in such a situation to ensure that adaptation does not evolve at a rate or in a manner that causes environmental, economic, or health problems.

2.9.4 Summary

In light of the above discussion, the committee found that

Evolution of pest strains that can overcome the pest-protection mechanisms of plants can have a number of potential environmental and health impacts.

For example, adaptation of a pest to an environmentally pesticidal substance produced by a transgenic plant may cause farmers to return to or begin the use of a conventional chemical pesticide with toxic effects on nontarget organisms. Also, adaptation of a pest to one type of transgenic pest-protected plant could result in its replacement with a novel transgenic pest-protected plant for which there is less information regarding health and environmental impacts.

Our understanding of the evolution of adaptation to pest-protected plants is still limited, but there is reasonable expectation that specific approaches to the development and deployment of transgenic pest-protected crops can substantially delay the evolution of pest adaptation. The committee found that

Although EPA has worked actively to develop useful resistance management plans for crops containing Bacillus thuringiensis (Bt) toxins, the agency has not articulated a general policy indicating when it believes it should require the development of resistance management plans for specific transgenic pest-protected crops.

The committee recommends that EPA continue to deal seriously with Bt resistance management (section 1.6.1), and it should also begin to consider resistance management strategies for other transgenic pest-protected plants. Specifically,

If a pest protectant or its functional equivalent is providing effective pest control, and if growing a new transgenic pest-protected plant variety threatens the utility of the existing uses of the pest-protectant or its functional equivalent, implementation of resistance management practices for all uses should be encouraged (for example, Bt proteins used both in microbial sprays and in transgenic pest-protected plants).