2

Potential Environmental and Human Health Implications of Pest-Protected Plants

This chapter begins with a discussion of risk assessment and its application to pest-protected plants and includes a review of the 1987 National Academy of Sciences white paper. It then considers the array of pest-protection traits and their possible use in transgenic pest-protected plants. The bulk of the chapter discusses potential environmental and human health impacts of conventional and transgenic pest-protected plants, such as human toxicity and allergenicity, nontarget effects, hybridization with weedy relatives, and evolution of pest adaptation to pest-protected plants. Scientific data on the potential for adverse environmental and health effects are presented and discussed. Scientific review in federal agencies is also discussed and will be covered in more detail in chapter 3.

2.1 RISK ASSESSMENT AND PEST-PROTECTED PLANTS

The 1987 National Academy of Sciences (NAS) white paper Introduction of rDNA-Engineered Organisms into the Environment stated that the “risks” posed by transgenic organisms are the “same in kind” as those associated with the introduction of unmodified organisms and organisms modified by other methods. Similar conclusions have been reached by international scientific organizations (FAO/WHO 1996; OECD 1993 and 1997). A clear definition of risk is needed if the committee is to interpret and evaluate that statement appropriately. This section clarifies the meaning of risk and related terms according to well-accepted definitions (NRC 1983).

Risk assessment consists of four steps: hazard identification, dose-



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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 2 Potential Environmental and Human Health Implications of Pest-Protected Plants This chapter begins with a discussion of risk assessment and its application to pest-protected plants and includes a review of the 1987 National Academy of Sciences white paper. It then considers the array of pest-protection traits and their possible use in transgenic pest-protected plants. The bulk of the chapter discusses potential environmental and human health impacts of conventional and transgenic pest-protected plants, such as human toxicity and allergenicity, nontarget effects, hybridization with weedy relatives, and evolution of pest adaptation to pest-protected plants. Scientific data on the potential for adverse environmental and health effects are presented and discussed. Scientific review in federal agencies is also discussed and will be covered in more detail in chapter 3. 2.1 RISK ASSESSMENT AND PEST-PROTECTED PLANTS The 1987 National Academy of Sciences (NAS) white paper Introduction of rDNA-Engineered Organisms into the Environment stated that the “risks” posed by transgenic organisms are the “same in kind” as those associated with the introduction of unmodified organisms and organisms modified by other methods. Similar conclusions have been reached by international scientific organizations (FAO/WHO 1996; OECD 1993 and 1997). A clear definition of risk is needed if the committee is to interpret and evaluate that statement appropriately. This section clarifies the meaning of risk and related terms according to well-accepted definitions (NRC 1983). Risk assessment consists of four steps: hazard identification, dose-

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION response evaluation, exposure assessment, and risk characterization. 1 The definitions of those and other terms in the National Research Council's (NRC's) “Red Book” (NRC 1983) are widely used and generally accepted. Hazard identification is “the determination of whether a particular chemical is or is not causally linked to particular health effects” (NRC 1983). Hazard is usually determined experimentally in controlled experiments with known doses. In the case of pest-protected plants, hazard would be the effect of a gene product (such as Bacillus thuringiensis (Bt) toxin, or a secondary plant product, such as a glycoalkaloid) which is expressed or changed as a result of genetic modification. The effects of gene flow or the effects on nontarget organisms could be considered potential hazards for ecological risk assessments. Dose-response assessment is the determination of the relationship between the magnitude of exposure and the probability of occurrence of the adverse effect in question. Dose-response assessment can address the potency or severity of the hazard. For example, many substances lead to adverse effects only at high doses and might be regarded as posing less severe hazards. The relationship between dose and adverse effects for a particular hazard is reflected in the dose-response curve. In the case of pest-protected plants, some proteinase inhibitors require very high concentrations to cause adverse health effects (Ryan 1990). On the other hand, some plant glycoalkaloids cause adverse health effects at relatively low doses. This allows toxicants to be ranked according to “relative hazard” which is not the same as “relative risk.” Overall risk is the product of the likelihood of an adverse consequence and the severity of that consequence. Hazard severity, and probability and magnitude of exposure all contribute to the overall risk. The risks that may be posed by proteinase inhibitors and glycoalkaloids could be similar depending on the probability and magnitude of exposure. Exposure assessment is the determination of the extent of exposure to a toxicant under any stated set of circumstances. In the context of pest-protected crops, exposure of nontarget species to a plant-pesticide might be considered for ecological risk assessment, and exposure of humans to a plant-pesticide for human health risk assessment. Exposure assessment of pest-protected plants should deal with such questions as how much of the toxicant humans consume, concentrations in the edible portions of the crop, and how often and how much nontarget insects consume. Risk characterization considers all the above and is often reported as a quantitative assessment of the probability of adverse effects under de- 1   Note that these essential steps may be categorized and/or termed differently in various risk assessment frameworks.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION fined conditions of exposure—for example, one in 10,000 humans will become ill given a certain set of circumstances. Hazard identification, dose-response assessment and exposure assessment are all essential elements of a risk assessment. Standard toxicological human health risk assessment, despite problems of uncertainty and variability and the consequent difficulty in extrapolation, is science-based. Variability is the range of differences implicit in a natural population (such as the genetic variability in sensitivity to allergens); uncertainty is based on incomplete knowledge or data (such as inadequate surveys of genetic variability to allergens) or on measurement error. Quantitative risk assessment is being used for not only cancer or toxicological risk assessment, but also for ecological risk assessment, microbial risk assessment, and other diverse types of assessment. In principle, quantitative risk assessment of transgenic pest-protected plants could be based on the methods of quantitative risk assessment if a hazard is detected. If adequate data were not available, the assessment could use uncertainty analyses, ranges of values, and extrapolation. However, until methods are adapted and applied to quantitative risk assessments for pest-protected plants, “relative hazard” ranking may be the best approach, recognizing that this is an interim solution and that quantitative risk assessment is the desired goal. Because the fundamental elements of risk assessment, such as hazard identification, dose-response assessment, exposure assessment, and risk characterization, can also be applied to risk assessments for transgenic pest-protected plants, the committee found that Health and ecological risk assessments of transgenic pest-protected plants do not differ in principle from the assessment of other health and ecological risks. 2.2 REVIEW OF PREVIOUS NATIONAL ACADEMY OF SCIENCES AND NATIONAL RESEARCH COUNCIL REPORTS 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-

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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. 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.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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 traits2 (controlled by several interacting genes, usually selected without knowledge of which genes are involved) Both Conventionally bred and transgenic plants Single-gene traits2 from the same species or a related species Several single-gene traits that are not genetically linked and are therefore inherited independently Several single-gene traits that are physically linked and inherited as a unit; occasionally possible with conventional breeding, as when a chromosome segment bearing more than one resistance gene is transferred to the cultivar usually accompanied by extraneous DNA; transgenic methods allow several single-gene traits to be tightly linked without extraneous DNA Single-gene traits expressed only in particular tissues or at particular developmental stages because of specific promoters; occasionally possible with conventional breeding, but more flexible and precise with transgenic methods Transgenic plants only Single-gene traits found in the same species or a related species and modified by changes in the nucleotide sequence of the structural gene or the promoter to improve the plant's phenotypic characteristics Single-gene traits obtained from unrelated organisms (such as viruses, bacteria, insects, vertebrates, and other plants); sometimes modified by a change in the nucleotide sequence of the structural gene or the promoter to improve the plant's phenotypic characteristics Single-gene traits that can be induced by a chemical spray or by specific environmental conditions (such as threshold temperature), based on the action of specific promoters; (these traits may also occur naturally in nontransgenic plants, such as those with systemic acquired resistance, but have rarely been selected intentionally by conventional breeding) 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 2   A molecular technique known as marker-assisted selection can speed the identification of polygenic or single-gene traits in the plant 's own genome, and rapid advances in genomics are expected to speed the identification of additional single-gene resistance traits in plants and other organisms.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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 FORMS AND MECHANISMS OF GENETICALLY CONTROLLED PEST-PROTECTION Use of genetically controlled pest-protected germplasm for pest management is widely perceived as providing a number of benefits. First, crop losses or damage can be eliminated or minimized resulting in improvement of both yield and quality. Second, resistant germplasm constitutes a low-input option for pest management that often reduces the need for chemical pesticides and their associated financial costs. Third, by reducing the use of traditional pesticides, pest-protected plants can increase the safety of the food supply and reduce environmental impacts. An example of reduced pesticide use and costs as a direct result of planting conventional pest-protected crops is the case of winter wheat bred for resistance to eyespot disease caused by the fungus Pseudocercosporella

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION herpotrichoides. Resistant cultivars, which were introduced in 1988 and are now grown on nearly 1,000,000 hectares in the Pacific Northwest United States, have reduced midseason fungicide treatments to roughly half of that needed with susceptible cultivars (Jones et al. 1995). Estimates from 1994 indicate that genetic protection from eyespot disease reduced growers' production costs by $40 per hectare. Plants with pest-protection properties can inhibit growth, reproduction, or survival of a particular pest or group of pests, or they may tolerate a pest infestation with minimal or acceptable levels of damage. Pest-protected plants that reduce pest populations can exhibit pest-protection characteristics through structural mechanisms. Trichomes on leaf surfaces, for example, present a structural barrier that reduces feeding activity of some insects. Pest-defense systems can also involve intracellular or biochemical mechanisms. These defense mechanisms can work through the action of preformed defensive compounds, and through induced defensive compounds, reactions, and signaling pathways that are triggered specifically or nonspecifically by an invading pest. To understand the rationale of current and future directions of transgenic breeding for pest-protection and to assess risks of transgenic pest-protected plants relative to those that may be posed by conventional pest-protected plants, this section reviews mechanisms of conventional and transgenic resistance to insects and pathogens. 2.3.1 Natural Pest-protection Mechanisms Preformed Chemical Defenses Plants constitutively produce a variety of antimicrobial or insecticidal chemicals that are known or suspected to provide pest-protection (Mansfield 1983; Rosenthal and Berenbaum 1991). The chemicals are often sequestered in specialized cells or expressed in particular organs. Chemicals having antibiotic or suppressive activities against pathogens and insects include saponins, glycoalkaloids, terpenoids, and phenolic compounds. They can have acute or chronic toxic effects and some compounds can have behavioral effects on insects that reduce insect feeding, reproduction, or colonization. The saponin avenacin A-1, for example, is a glycosylated triterpene that is toxic to fungi by perturbing membrane structure and function (Osbourn 1996). It is found in the roots of some cereals. Avenacin A-1 in oats confers resistance to a number of root-infecting fungal pathogens, such as Gaeumannomyces graminis. Like other chemical defenses, avenacin A-1 is effective as an antibiotic in proportion to its accumulation in roots, the inherent sensitivity of the fungus, and the ability of the pathogen to detoxify the compound. Some compounds have relatively broad

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION specificities. Cyclic hydroxamic acids, such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), have been shown to confer protection against both fungal pathogens and insect pests (Frey et al. 1997). Although many preformed chemicals, such as avenacin A-1 and DIMBOA, have been shown to provide pest-protection, the great majority of natural plant chemicals that have antibiotic properties in vitro have not been proved to be active defensive compounds in vivo. The array of compounds with potential defensive capability is vast, and it includes a large number of potential animal and human toxins. For example, 49 natural products or metabolites found in cabbage are known toxins in microbial or animal models (Ames et al. 1990a). Additionally, a number of natural products in the food supply do have acute human toxicity; the cholinesterase inhibitors solanine and chaconine in potato are well-documented examples. Ames et al. (1990b) estimated that the typical American consumes such compounds at roughly 1.5 g/day, primarily in fruits and vegetables, but diets rich in fruits and vegetables are associated with lower, not higher, risks of illnesses such as certain forms of cancer and heart disease (NRC 1982). Therefore, there is not necessarily a correlation between consumption of fruits and vegetables containing compounds with toxicity in experimental systems and adverse health effects. Resistance Genes Although the term resistance gene is sometimes used to describe any gene that encodes a plant-protection mechanism, it is most commonly applied to a gene that triggers a defense response to a specific pest or pathogen. In this report, these pathogen-specific resistance genes will be referred to as race-specific R genes, or simply, R genes. The more general term, defensive genes, will be used to describe natural plant genes specifying antibiotic or insecticidal factors that have broad specificity. The identification and deployment of R genes have been among the most important factors in the development of high-yielding conventional crop varieties. Genes have allowed the continued cultivation of many crops in areas where virulent pathogens and detrimental pests are common (for example, leaf stem, and stripe rust in wheat) (Knott 1989; Line 1995; McIntosh and Brown 1997). In many cases, the use of R genes has permitted a reduction in reliance on externally applied chemical pesticides (Jones et al. 1995). Genetic interactions between flax and the flax rust pathogen indicated that many R genes are effective against only particular races of a pathogen (or types of a pathogen with specific virulence properties) (Flor 1971). The races that are suppressed by a given R gene are known to

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION contain specific “avirulence” genes; races that are not suppressed lack a functional corresponding avirulence gene. In at least some cases, pathogen avirulence genes encode proteins that are required for infection of susceptible plant hosts (Kearney and Staskawicz 1990). The “gene-for-gene” concept was proposed to explain the interaction between a plant R gene and a pathogen avirulence gene, and this concept is used in agriculture to develop pest-protected crop varieties that are resistant to damage by pathogen races that have known virulence properties. A feature of race-specific R genes, and one of the major limitations associated with their use, is the occurrence of pathogen races that are unaffected by a given plant R gene; these can be pre-existing races that lack the corresponding avirulence genes or new races that have lost avirulence gene function. Study of numerous R genes isolated over the last few years has shown that many have a common evolutionary origin (Baker et al. 1997). Furthermore, race-specific R genes appear to function by triggering a cascade of molecular signaling and biochemical reactions that arrest pathogen spread at the initial site of infection, regardless of whether a particular R gene specifies resistance to a virus, fungus, or bacterium. Several other types of disease-resistance genes that do not fit the gene-for-gene concept have also been identified. The HM1 gene of maize encodes a reductase that inactivates HC toxin, a cyclic tetrapeptide required for virulence of the fungus Cochliobolus carbonum race 1 (Johal and Briggs 1992). The recessive mlo gene in barley confers resistance to all races of the powdery mildew fungus, Erisyphe graminis f. sp. hordei, by priming the onset of several defense pathways (Buschges et al. 1997). Polygenic traits that confer quantitative pest-protection can also provide durable protection. Although the basis for this type of pest-protection is not entirely clear, cumulative effects of plant R genes that have been overcome by virulent pathogens might play a role in some systems (Li et al. 1999). Genes for controlling insect and other invertebrate pests have also been identified and deployed, although they might be less common than plant R genes for viral, fungal, and bacterial pathogens. Some encode enzymes that catalyze synthesis of insecticidal or insect-deterrent compounds, whereas others trigger localized defense responses. Several nematode R genes are chemically related, or sequence-related, to race-specific pathogen R genes (Cai et al. 1997; Milligan et al. 1998); this suggests that the signaling mechanisms leading to resistance to nematode are similar to those for resistance to pathogens. The tomato Mi gene for resistance to the root-knot nematode, Meloidogyne incognita, also confers resistance to the potato aphid, Macrosiphum euphorbiae (Rossi et al. 1998; Vos et al. 1998); thus, some insect resistance genes could have broad specificity.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION Induced Resistance Responses A number of resistance responses by plants are induced by pathogen invasion or insect attack (Hutcheson 1998). The hypersensitive response (HR) results after R-gene-mediated, race-specific recognition of a pathogen. The HR in a natural infection is often limited to relatively few cells around the initial infection site. It can also be triggered nonspecifically by various elicitor compounds, such as fungal cell-wall components. The HR involves a cascade of reactions that result in production of reactive oxygen intermediates, antimicrobial compounds (termed phytoalexins), and degradative enzymes; alteration of cell membranes and cell walls; and ultimately cell death. The result of the HR in infected tissues is usually localized necrosis, inhibition of pathogen growth, and limitation of the disease. The HR can occur in plants that contain race-specific R genes effective against all types of viruses, fungi, and bacteria. The HR leads to a number of other localized and systemic processes that result in increased generalized resistance to a wide array of pathogens. The systemic-acquired-resistance response results in activation of genes that encode defensive proteins, such as glucanases and chitinases, and antimicrobial biosynthetic pathways throughout the plant (Ryals et al. 1996). Defensive proteins can also be induced during the natural course of development of some plants; for example, pathogenesis-related proteins (such as several chitinases and osmotin) with antifungal activity are the predominant proteins that accumulate in the ripening fruit of grape plants (Salzman et al. 1998). Insect herbivore activity can lead to a systemic defense response (Ryan 1990). This response can be triggered by biotic damage, such as that caused by chewing insects, or by mechanical damage. Insect feeding on a single leaf can result in production of defensive chemicals in all of a plant's leaves (Rosenthal and Berenbaum 1991). An important component of this wound-induced response is activation of genes that encode proteins, such as proteinase inhibitors, that have insecticidal activity. Proteinase inhibitors prevent digestion of plant material in the insect gut, and so result in starvation. Thus, plants exposed to chewing insects gain resistance to additional insect feeding through the wound response. Viruses activate a defensive response that resembles post-transcriptional gene silencing (PTGS) (Carrington and Whitham 1998). PTGS response is adaptive in providing a customized antiviral response to each new virus that the plant encounters. Silencing in response to viruses with a RNA-based genetic code involves degradation of the genome itself. For viruses with a DNA-based genetic code, the PTGS results in degradation of the transcription products (mRNA). In either case this results in lower virus accumulation or in recovery of the plant. PTGS response can be

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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 AGRONOMIC RISKS ASSOCIATED WITH VIRUS-RESISTANT CROPS Agronomic risks are defined as those related to quality or productivity of a modified crop. A number of issues and concerns emerge when agronomic consequences of using transgenic pest-protection strategies against viruses are considered. These center on emergence of new or novel viral strains, introduction of new transmission characteristics, and changes in susceptibility to heterologous viruses. Some of the concerns, such as the question of new virus emergence, also have relevance to ecological risks. 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 Recombination Between Transgenes and Viral Pathogens Recombination between a virus-derived transgene and a virus during plant infection has been suggested as a potential source of novel virus strains with enhanced virulence characteristics. From an evolutionary perspective, new viruses emerge through gradual accumulation of point mutations and major acquisition or deletion of genetic material. New genetic material can be incorporated by recombination with nucleic acids

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION from the host or from other viruses during coinfections (Koonin and Dolja 1993). Several laboratory studies have shown that viruses can recombine with homologous transgene sequences (Borja et al. 1999; Greene and Allison 1994; Wintermantel and Schoelz 1996). Those studies required moderate or heavy selection pressure to detect the recombinant viruses. In all cases examined, only homologous sequences were exchanged. No experimental data indicate that recombination can occur between virus genomes and transgene sequences that are derived from distantly related or unrelated viruses. Two points should be considered in assessing risks that may be posed by virus-transgene recombination. First, will large-scale plantings of transgenic material increase the risk of recombination above the preexisting risk due to the widespread occurrence of mixed infections? Mixed infections by related and unrelated viruses are common in natural and agricultural ecosystems. For example, mixed infections by two or more viruses were detected in 64% and 90% of peppers surveyed in three California counties in 1984 and 1985, respectively (Abdalla et al. 1991). Mixed infections provide a continuous opportunity for intervirus recombination. Either because intervirus recombination occurs with such low frequency or because new recombinants so rarely have a competitive advantage, the new viruses have not been detected in agricultural settings. One could argue that past and current agricultural practices have provided a fertile environment to spawn novel recombinant viruses with virulent properties, but these viruses have not been observed. Second, can transgenes be engineered to reduce or eliminate the risk that recombination will spawn new pathogens? Evidence suggests that elimination of genome replication-control sequences from transgenes can limit recombination and therefore risk (Greene and Allison 1996). Furthermore, strategies to produce resistance-mediating transgenes that encode nonfunction proteins or no protein can be used effectively against viruses. For example, resistant plants that express nontranslatable RNA can confer immunity through induction of post-transcriptional gene silencing (Kasschau and Carrington 1998; Lindbo and Dougherty 1992). Transcapsidation and Gain-of-Transmission Characters In the process of encapsidation, a virus genome is packaged in a shell of self-encoded coat proteins after it is replicated. Although encapsidation of one virus genome by the coat protein from a different virus (transcapsidation) is well documented (Matthews 1991), it is highly unlikely that functional coat proteins expressed in transgenic plants pose a significant risk of expansion of host range to new crop or non-crop hosts. Transcapsidation does not involve exchange of genetic material, meaning

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION that any unique insect vectoring properties of a transcapsidated virus genome will not be inherited. Synergism Between Viral Transgenes and Heterologous Viruses Mixed infections by viruses can sometimes lead to a synergistic disease syndrome that is more severe than that caused by either virus individually (Matthews 1991). In cases of synergism involving the potyvirus family of viruses, the region of the viral genome that causes exacerbation of disease codes for a protein termed HC-Pro (Pruss et al. 1997). The synergism effect appears due to the natural role of HC-Pro as a suppressor of the gene-silencing response (Scheid 1999). Thus, plants are unable to mount an effective defense response to infection. Indeed, transgenic plants that express potyvirus genome segments that include HC-Pro exhibit more severe symptoms when inoculated with heterologous viruses (Pruss et al. 1997). No data indicate that expression of viral coat protein or replicase proteins enhances the virulence of heterologous viruses. The problem of synergism is manageable through avoiding the use of functional transgenes that encode defense-suppressor substances or pathogenicity-enhancer substances. In addition, the normal process of testing in breeding programs that seek to incorporate natural or transgenic resistance traits will reveal the extent to which the virulence of heterologous viruses is exacerbated. Thus, it is highly unlikely that transgenic plants with general hyper susceptibility characteristics will pass through a breeding program to commercialization. 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 PEST RESISTANCE TO PEST-PROTECTED PLANTS AND RESISTANCE MANAGEMENT In this section, the ability of pests to adapt and develop resistance to transgenic or conventional pest-protected plants will be discussed, and resistance management strategies to abate this development and their scientific basis will be presented.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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)6plants 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. 6   As a reminder to the reader, GMPP plants include both transgenic and conventional pest-protected plants. See section ES.3.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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). 2.10 RECOMMENDATIONS 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 categori.es of health effects testing (such as carcinogenicity) should not be required unless justified. The EPA should provied 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.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 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. 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 pest control techniques 7 and whether gene flow to feral plants or wild relatives is likely to have a significant impact on these populations. 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 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). 7   Includes both chemical and non-chemical methods which are currently used.