100 YEARS of Bacillus Thuringiensis: a Critical Scientific Assessment BT SMALL REPRINT WEB 10/17/02 10:51 AM Page C2

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100 YEARS OF Bacillus thuringiensis: A Critical Scientific Assessment BT SMALL_REPRINT_WEB 10/17/02 10:51 AM Page C2

This report is based on a colloquium, “100 Years of Bacillis thuringiensis, a Paradigm for Producing Transgenic Organisms: A Critical Scientific Assessment,” sponsored by the American Academy of Microbiology and held November 16-18, in Ithaca, New York.

The American Academy of Microbiology is grateful for the generous support of the following organizations:

American Society for Microbiology Cornell University U.S. Environmental Protection Agency U.S. Food and Drug Administration National Science Foundation Rockefeller Foundation

The opinions expressed in this report are those solely of the colloquium participants.

This report and others from the American Academy of Microbiology are available online: www.asmusa.org BT SMALL_REPRINT_WEB 10/17/02 10:51 AM Page 1

EUGENE W. NESTER LINDA S. THOMASHOW MATTHEW METZ AND MILTON GORDON

100 YEARS OF Bacillus100 Years thuringiensis: of Bacillus thuringiensis ■ 1 A Critical Scientific Assessment AA reportreport fromfrom thethe AmericanAmerican AcademyAcademy ofof MicrobiologyMicrobiology BT SMALL_REPRINT_WEB 10/17/02 10:51 AM Page 2

Board of Governors, American Academy of Microbiology Eugene W. Nester, Ph.D. (Chair), University of Washington Colloquium Participants Joseph M. Campos, Ph.D., Children’s National Medical Center Michael Adang, Ph.D., University of Georgia R. John Collier, Ph.D., Harvard Medical School Illimar Altosaar, Ph.D., University of Ottawa Marie B. Coyle, Ph.D., University of Washington Arthur I. Aronson, Ph.D., Purdue University James E. Dahlberg, Ph.D., University of Wisconsin, Madison Lee A. Bulla, Ph.D., University of Texas, Dallas Julian E. Davies, Ph.D., Cubist Pharmaceuticals, Inc. Christopher Cullis, Ph.D., National Science Foundation Arnold L. Demain, Ph.D., Drew University Peter R. Day, Ph.D., Rutgers University Lucia B. Rothman-Denes, Ph.D., University of Chicago Donald H. Dean, Ph.D., Ohio State University Abraham L. Sonenshein, Ph.D., Tufts University Medical Center Elizabeth D. Earle, Ph.D., Cornell University David A. Stahl, Ph.D., University of Washington David J. Ellar, Ph.D., University of Cambridge, England Judy D. Wall, Ph.D., University of Missouri Brian A. Federici, Ph.D., University of California, Riverside Sarjeet S. Gill, Ph.D., University of California, Riverside Colloquium Steering Committee Milton Gordon, Ph.D., University of Washington Eugene W. Nester, Ph.D. (Co-Chair), University of Washington Peter M. Kareiva, Ph.D., The Nature Conservancy Linda S. Thomashow, Ph.D. (Co-Chair), USDA/ John D. Kemp, Ph.D., New Mexico State University Washington State University Pal Maliga, Ph.D., Rutgers University Luca Comai, Ph.D., University of Washington Michelle Marvier, Ph.D., Santa Clara University Milton Gordon, Ph.D., University of Washington Sharlene Matten, Ph.D., U.S. Environmental Protection Agency Noel Keen, Ph.D., University of California, Riverside Matthew Metz, Ph.D., University of Washington Anne M. Vidaver, Ph.D., University of Nebraska William Moar, Ph.D., Auburn University Carol A. Colgan, American Academy of Microbiology Eugene W. Nester, Ph.D., University of Washington Sheikh Riazzuddin, Ph.D., National Centre of Excellence in Molecular Biology, Lahore, Pakistan Ronald R. Sederoff, Ph.D., North Carolina State University Anthony M. Shelton, Ph.D., Cornell University Allison Snow, Ph.D., Ohio State University Diane Stanley-Horn, Ph.D., University of Guelph Guenther Stotzky, Ph.D., New York University Linda S. Thomashow, Ph.D., USDA/Washington State University

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Executive Summary The insecticidal proteins produced by Bacillus thuringiensis (Bt) have provided are equally amenable to use in large or a uniquely specific, safe, and effective small scale farming operations and com- tool for the control of a wide variety of patible with other agricultural practices insect pests. Bt has been used in spray and technologies. formulations for over 40 years, where it is considered remarkably safe, in large Concerns associated with the use of Bt part because specific formulations harm include potential for harm to non-target only a narrow range of insect species. organisms, development of resistance in Today, Bt insecticidal protein genes populations of target insects, and, for have been incorporated into several engineered crops, possible ecological major crops where they provide a model consequences of gene flow to non-engi- for genetic engineering in agriculture. neered crops and wild relatives. These concerns merit continued attention on a Effective protection of crops from insect case-by-case basis in order to ensure pests afforded by insecticidal proteins that Bt technologies have the maximum has had a number of positive impacts positive impact with a minimum risk on on agriculture. Reduced insect damage agriculture. Prudent use of Bt technolo- im-proves crop yield, reduces fungal gies will also be key in maintaining their toxins in the food supply, and improves usefulness for a long period of time. the livelihood of farmers. Replacement of toxic chemical pesticides with Bt has reduced hazards to the environment and farm workers. Bt-engineered crops

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Background digm for transgenic plants. A timeline Introduction of transgenic crops has (Figure 1) highlighting important provided new approaches to improving Insecticidal products of Bt were first events in the development of Bt as an crop quality and productivity, but, at commercialized in France in the late insecticide is given. the same time, these crops have 1930s. For over 40 years Bt has been aroused concerns about the safety of applied to crops as an insecticidal spray, The unprecedented and rapid adoption agricultural biotechnology in relation to a mixture of spores and the associated of transgenic crops since 1996 has human health and the environment. protein crystals. By 1995, 182 Bt prod- raised questions among the public, These issues are, for the most part, ucts were registered by the United governmental regulatory agencies, envi- relevant to agricultural practices in States Environmental Protection Agency ronmental groups, and the scientific general, and are embodied in crops (EPA), but in 1999 Bt formulations con- community about the potential effects engineered to produce an insecticidal stituted less than two percent (2%) of of these crops on non-target organisms, protein from the common bacterium the total sales of all insecticides. The human health, and non-engineered Bacillus thuringiensis (Bt). use of Bt has increased as insects have crops or relatives of crop species. The become resistant to chemical insecti- potential toxicity of pollen from Bt corn The insecticidal properties of B. thurin- cides. Recently, the use of Bt in pest on non-target organisms such as the giensis have long been recognized. control has increased substantially monarch butterfly, the prospect of trans- While some accounts indicate that the through more widespread use by both genic plants attaining special allergenic Egyptians were aware of the insecticidal conventional and “organic” food produc- or toxic properties, and possible conse- properties of what probably was Bt and ers, as well as in forestry. quences of gene flow on crop genetic used it to control pests, the organism diversity and weed hardiness are some was first isolated about 100 years ago in In 1987, three reports were published of the safety issues that have received Japan from silkworm larvae suffering that demonstrated that insecticidal attention in recent years. Other con- from the disease “flacherie.” The organ- crystal protein (ICP) genes from Bt cerns are societal, and encompass ism was named Bacillus thuringiensis by could be introduced and expressed in impacts of the technology on interna- Berliner in 1911, who isolated it from a the tissues of tobacco and tomato, tional trade policy and cultural values. diseased flour moth larva. In 1954, Angus resulting in pest-resistant transgenic demonstrated that the crystalline protein plants. By 2001, an estimated 69% of Twenty-five (25) scientists with exper- inclusions produced by B. thuringiensis the cotton, 26% of the corn, and 68% of tise in various aspects of Bt, plant in the course of sporulation were re- the soybeans grown in the United biology, entomology, microbiology, and sponsible for the insecticidal action. States were genetically engineered. For ecology were convened for a 2-1/2 day cotton and corn, the genetic engineer- colloquium to address objectively the ing has involved the introduction of Bt scientific basis surrounding these and genes into these crops to confer insect other concerns about the safety and resistance. Thus, Bt serves as a para-

1901 1911 1938 1954 1983 1987 1996 2001 • • • • • • • •

ISHIWATA E. BERLINER THE FIRST T. ANGUS SCIENTISTS PEST Bt COTTON IN THE U.S. SHIGETANE NAMES THE COMMERCIAL SHOWS THAT DEMONSTRATE RESISTANCE REACHES THE 69% OF THE DISCOVERS ORGANISM PRODUCTS CRYPTALLINE THAT PLANTS OF PLANTS MARKET COTTON, AND THAT A CAUSING THE THAT CONTAIN PROTEINS ARE CAN BE CONTAINING 26% OF THE DISEASE OF DISEASE IN Bt ARE RESPONSIBLE GENETICALLY THE Bt GENE CORN PLANTED SILKWORMS IS SILKWORM— MARKETED IN FOR ENGINEERED DEMON- CONTAIN CAUSED BY A BACILLUS FRANCE INSECTICIDAL STRATED Bt GENES PREVIOUSLY THURINGIENSIS ACTION OF Bt UNDESCRIBED (Bt) BACTERIUM

FIGURE 1. TIMELINE OF IMPORTANT EVENTS IN THE DEVELOPMENT OF Bt AS AN INSECTICIDE.

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environmental consequences of Bt crops, particularly as compared with alternatives, such as conventional Bt sprays and synthetic insecticides. This report summarizes the conclusions reached in their discussions.

Bt’s Family Tree Bt is a member of the genus Bacillus, a diverse group of gram-positive, aerobic, spore-forming bacteria consist- ing of more than 20 species that differ in their basic biological properties. In addition to Bt, the most important species are B. subtilis, a source of industrial enzymes; B. cereus; and B. anthracis, the causative agent of anthrax. Members of the genus Bacillus generally are considered soil bacteria, and Bt is common in FIGURE 2. THE INSECTICIDAL BACTERIUM, BACILLUS THURINGIENSIS. A. CELL OF terrestrial habitats, including soil, living B. THURINGIENSIS IN THE PROCESS OF SPORULATION, DURING WHICH INSECTI- and dead insects, insect feces, granaries, CIDAL PROTEINS ARE PRODUCED AND CRYSTALLIZE. B. PARASPORAL CRYSTALS and on the surfaces of plants. Bt occurs ISOLATED FROM THE HD1 ISOLATE OF B. THURINGIENSIS SUBSP. KURSTAKI (BTK). in nature predominantly as spores that THE HD1 ISOLATE OF PRODUCES FOUR MAJOR CRY PROTEINS, CRY1Aa, can disseminate widely throughout the CRY1Ab, CRY1Ac, AND CRY2Aa, AND IS USED WIDELY IN AGRICULTURE AND environment. B. anthracis also survives FORESTRY TO CONTROL CATERPILLAR PESTS. C. A PARASPORAL CRYSTAL OF B. as spores, but there is no evidence of THURINGIENSIS SUBSP. ISRAELENSIS (BTI). BTI PRODUCES FOUR MAJOR INSEC- toxin gene transfer from B. anthracis to TICIDAL PROTEINS, CRY4Aa, CRY4Ab, CRY11Aa, AND CYT1Aa, AND IS USED TO B. thuringiensis or B. cereus. CONTROL THE LARVAE OF MOSQUITOES AND BLACKFLIES. E = EXOSPORIUM MEMBRANE, SP = SPORE, AND PB = PARASPORAL BODY. BAR IN A EQUALS 250 B. thuringiensis, B. cereus, and B. anthra- NM, OR ONE 40,000TH OF A CM. cis are closely related. Until recently they were differentiated based on bio- chemical, nutritional, and serological presence in B. thuringiensis of parasporal each other than to isolates in other clus- analyses, on the presence in Bt of insec- crystals, but this is now considered too ters, these analyses indicate that B. ticidal parasporal crystals visible by narrow a criterion for taxonomic pur- anthracis is genetically distinct from B. microscopy (Figure 2), and on patho- poses. A clearer understanding of the thuringiensis and B. cereus. This emer- genicity to mammals. Insecticidal relationships among these organisms ging picture of genetic relationships is activity and pathogenicity in animals has begun to emerge, based on the consistent with what is known of the depend, in part, on the presence in application of high-resolution analyses ecology of these species: B. thuringien- B. thuringiensis and B. anthracis of plas- of the genetic variability among large sis and B. cereus are indigenous to mids, but the plasmids in the two numbers of isolates. Isolates of B. thurin- habitats in and around soil and insects, species differ functionally and geneti- giensis and B. cereus are highly diverse, whereas B. anthracis is restricted to cally, and those present in B. anthracis whereas very little genetic variation growth in animal hosts because of its have never been found to be transferred exists among isolates of B. anthracis. obligately pathogenic lifestyle. to other species of Bacillus. Thus, when isolates are grouped in a phylogenetic tree on the basis of genetic Accumulated molecular evidence sug- similarity, isolates of B. anthracis are gests that B. thuringiensis and B. cereus tightly clustered. In contrast, isolates of should be considered a single species. B. thuringiensis and B. cereus form sev- They have been distinguished by the eral different clusters that contain isolates from both species. Because isolates within a cluster are more similar to

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How Does Bt Work? budworm in forests; the cabbage looper and diamondback moth in vegetable Various strains and may contribute to its virulence. One crops; and certain insects with chewing insecticidal proteins hundred and twenty six (126) Bt micro- mouthparts such as beetles. While Bt The diversity within B. thuringiensis is bial insecticides are currently registered sprays are used on many crops, only reflected in the fact that more than 60 in the US, but these are based on only cotton, corn, and potatoes expressing Bt serotypes and hundreds of different sub- four subspecies of B. thuringiensis. Only ICPs are commercially available. Mos- species have been described. The two five ICP genes have been engineered quitoes and blackflies targeted with Bt most widely used in commercial insecti- into commercial Bt crops, but many sprays and aquatic treatments include cides are B. thuringiensis subsp. kurstaki others are available for future use. the blackfly vectors of Onchocerca (Btk), which kills a wide range of lepi- volvulus, the etiologic agent of river dopteran species that are important Susceptible organisms blindness; mosquito vectors of viral pests in agriculture and forestry, and B. Insecticides are generally sought that encephalitis and West Nile virus; and thuringiensis subsp. israelensis (Bti), target high-impact agricultural pests, immature forms of many nuisance mos- used primarily for the control of mos- disease vectors, or nuisance pests. quito, blackfly, and midge species. Table quito and blackfly larvae. Whether applied as a commercial spray 1 gives a sample list of organisms that or deployed in crops, Bt is highly spe- are susceptible to Bt. Most of the insecticidal activity of Bt is cific, with toxicity limited to only some due to intracellular crystal inclusions species of one of the major groups of MECHANISMS OF produced during the process of sporula- insects—typically lepidoptera (butter- ACTION AND RESISTANCE tion. These parasporal crystals (Figure 2) flies/moths), coleoptera (beetles), or Bt insecticides, whether in the form of a are comprised of one or more related diptera (flies/mosquitos). New Bt pro- spray or a Bt crop, do not function on ICPs encoded by cry (crystal) and cyt teins are being discovered which are contact as most chemical insecticides (cytolytic) genes located mainly on plas- active against other orders of insects do, but rather, as midgut toxins. They mids. Like B. thuringiensis itself, the cry and other pests, such as mites and must be ingested by the target organ- and cyt genes are highly diverse; more nematodes. The number of susceptible ism to be effective, and killing takes than 30 types of Cry proteins and 8 organisms is expanding to include virtu- hours to days, longer than is required for types of Cyt proteins have been ally all invertebrate plant pests, as well synthetic insecticides. described, and over 100 genes have as insects that transmit human and been cloned and sequenced. Target animal pathogens. Key agricultural pests In the case of Bt sprays, parasporal crys- insect specificity is determined mainly currently targeted with Bt insecticides tals ingested by insect larvae feeding on by the ICPs, although B. thuringiensis include bollworms, stem borers, bud- plant surfaces dissolve and the insectici- also produces a variety of other worms, and leafworms in field crops dal proteins are activated by proteases enzymes and insecticidal proteins that and grains; the gypsy moth and spruce in the juices of the midgut, which typi-

FIGURE 3. ACTIVATION OF Bt ICP IN AN INSECT GUT. THE CONTENTS (BLUE BALLS) OF THE INSECT GUT EPITHELIAL CELLS ARE SEPARATED FROM THE ALKALINE CONTENTS (YELLOW BALLS) OF THE GUT LUMEN BY THE CELL MEMBRANE. A. ICP IN THE PRO-TOXIN FORM (RED SHAPE), ONCE DISSOLVED IN THE GUT LUMEN IS PROCESSED BY PROTEASE (YELLOW SHAPE) IN THE GUT. B. THE TOXIN FORM OF THE ICP (RED SHAPE) BINDS TO SPECIFIC RECEPTORS (GREEN SHAPE) IN THE CELL MEMBRANE AND INSERTS INTO THE MEMBRANE. C. SEVERAL ACTIVE ICPS (RED SHAPES) IN THE CELL MEMBRANE WILL COME TOGETHER, FORMING A PORE. MIXING OF THE CONTENTS OF THE GUT LUMEN AND THE GUT EPITHELIAL CELL KILL THE CELL. THIS WILL EVENTUALLY RUPTURE THE LINING OF THE GUT AND KILL THE INSECT.

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TABLE 1. SOME AGRICULTURAL AND FORESTRY PESTS CON- TROLLED BY Bt SPRAYS

ALFALFA CATERPILLAR GYPSY MOTH Current Uses of Bt ALFALFA LOOPER HORNWORM BAGWORM IMPORTED CABBAGE WORM Sprays BEET ARMYWORM JAPANESE BEETLE Viable bacterial spores constitute the BLACK-HEADED FIREWORM LEAFWORM active ingredient in Bt spray formula- BLISTER BEETLE LINDEN LOOPER tions. Cry and Cyt ICPs are the primary BRUCE SPANWORM OBLIQUE-BANDED cause of insect death. However, other BLUEBERRY SPANWORM LEAFROLLER components in sprayed Bt pesticides CABBAGE LOOPER OMNIVOROUS LEAFROLLER can contribute, especially in insects CITRUS CUTWORM PEACH TWIG BORER that are not very sensitive to Cry pro- COLORADO POTATO BEETLE PEAR CANKERWORM teins. For example, the Bt spore is CORN EARWORM REDHUMPED CATERPILLAR important to lethality to both gypsy COTTON BOLLWORM SALTMARSH CATERPILLAR moth larvae and to such pests as the CRANBERRY FRUITWORM SOD WEBWORM beet armyworm and the cotton boll- CRANBERRY LEAFROLLER SPINY LOOPER worm. The Bt spore germinates after DIAMONDBACK MOTH SPRUCE BUDWORM the gut is damaged and then begins EUROPEAN CORN BORER TENT CATERPILLAR vegetative growth, producing other FALL ARMYWORM TOBACCO BUDWORM insecticidal toxins and synergists. These FALL CANKERWORM TOMATO FRUITWORM include vegetative insecticidal proteins FALL WEBWORM TWIG GIRDLER (MACADAMIA) (VIPs), β-exotoxin, zwittermicin A, chiti- FRUITTREE LEAFROLLER WESTERN GRAPELEAF nases, and phospholipases. FRUITWORM SKELETONIZER FUNGUS GNAT WESTERN TUSSOCK MOTH Bt sprays comprise one to two percent GRAPE LEAFFOLDER WESTERN YELLOWSTRIPED of the global insecticide spray market, GREEN FRUITWORM ARMY WORM estimated to be U.S. $8 billion per annum. At one time, Bt sprays consti- tuted $100 million in annual sales, but with the advent of transgenic plants cally are akaline (pH 8-10.5). In Bt crops, Stability, persistence, and uniformity of engineered with ICP genes, sales have the plant tissues produce specific ICPs coverage are major factors in determin- decreased to $40 million. Half of current in a soluble form. In either case, the ing the probability that insects will sales are used in Canadian forests to active ICP then traverses the peritrophic develop resistance to Bt. When applied control gypsy moths, spruce budworm, membrane and binds to specific recep- as a spray, Bt is relatively unstable; it and other lepidopteran pests. tors on the midgut epithelium, forming can be washed off by rain or broken pores and leading to loss of the trans- down by ultraviolet light and may need Bt sprays are used sporadically and typi- membrane potential, cell lysis, leakage to be reapplied as frequently as every cally over small areas. In tropical and of the midgut contents, paralysis, and two to four days. This instability, along subtropical areas (e.g., Hawaii), popula- death of the insect. ICP activation and with variable, low-dosage residues on tions of diamondback moth have pore formation is shown in Figure 3. One the plant may contribute to the emer- become resistant to Bt sprays following level of specificity in this interaction is gence of resistant insect populations. their intensive use. Crops sprayed with provided by the midgut environment of These problems are avoided by higher traditional Bt formulations include vari- the insect, and a second by the binding and more uniform doses that can be ous vegetables, tree fruits, artichokes of ICPs only to membranes carrying the obtained in Bt plants. In soil, the stabil- and berries. Sprays are chosen by appropriately matched receptors. ity of ICPs is affected by microbial organic farmers to meet guidelines for Insects that develop resistance to Bt degradation. In general, the drier the using strictly non-synthetic materials. most commonly exhibit decreased or soil, the longer an ICP will be stable, as An additional use of Bt is in the protec- altered receptor binding, although there is less microbial degradation. tion of stored commodities (e.g., wheat) altered proteolytic activation also has from pest infestation. been reported.

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Transgenic plants vide alternatives to Bt. Although regis- Approximately 12 million hectares of tered based on extensive safety testing, insect-protected transgenic crops incor- controlled in any growing season to these chemicals are viewed as being porating Bt ICPs are now planted annual- meet specific pest pressures. The draw- less environmentally benign than Bt ly worldwide. The vast majority of these backs are that spray can drift during microbial insecticides and Bt crops. crops are grown in the United States. application, cannot be applied uniformly Their total acreage has increased tenfold to all parts of the plant, and cannot be Farm workers have benefited from the since 1996 and is expected to continue delivered to pests that are inside plant use of Bt sprays and Bt plants in place of to increase along with other transgenic tissues. Further, insecticidal crystals and hazardous insecticides. The high speci- crops. In 2001 the number of farmers spores of Bt exposed on plant surfaces ficity of Bt ICPs not only leaves humans planting genetically engineered crops is are highly sensitive to degradation by UV and other animals unaffected, but also expected to have exceeded 5 million, light and removal by water runoff. Multi- makes these proteins harmless to the and global areas planted in transgenic ple applications are therefore required to wide array of insects outside of their tar- crops are expected to have increased by provide extended pest protection. get range. A testament to its safety is 10% or more since 2000. In March of that Bt is the only insecticide for which 2002, the Indian government announced Bt transgenic crops also have strengths there are no mandated tolerance limits that it would join the ranks of nations and weaknesses. ICPs are present at for residue in food. Bt transgenic plants allowing commercial use of genetically high concentrations in most or all tis- more frequently displace synthetic engineered crops by licensing Bt cotton. sues in current transgenic plants. This insecticides than Bt sprays. Both uses of feature eliminates difficulties in targeting Bt can contribute to agriculture. pests that burrow into plant tissues, as Bt crops currently are engineered to well as the labor and expenses associ- Insect pest management strategies that produce a single ICP continuously ated with applying sprays. A potential are available to agriculture need not be throughout all parts of the plant. The limitation of engineered plants is that considered strictly as alternatives to Bt. ICP gene is placed under the regula- the ICP specificity cannot be changed Biocontrol through insect parasitoids or tion of a promoter that is highly active within a growing season if resistance fungal pathogens, mating disruption, in plants, and engineered so that begins to develop. Engineered plants crop rotation, adjustments in date of codon preference is optimized for also currently lack the components planting, polyculture, and conventional expression in plants. The most widely found in bacterial formulations that act chemical sprays are all compatible with deployed transgenic crops expressing synergistically with the ICP to kill par- Bt use. In particular, indigenous insect Bt ICP genes are Bt corn, which is tially resistant insects. In the event that populations and introduced biocontrol grown in the U.S., China, Argentina, a sexually compatible relative is near a agents that are harmed by broad spec- Canada, South Africa, Spain, Germany, cultivated Bt crop, the risk that the Bt trum chemical pesticides are not and France; and Bt cotton, grown in transgene could be transferred via pollen generally harmed by Bt. Thus, Bt is well- the U.S., China, Australia, Argentina, also is an issue of concern. suited to Integrated Pest Management South Africa, and Mexico. Many addi- programs that preserve large segments tional Bt crops are close to release, Differences between Bt and of indigenous invertebrate predator and including rice, canola, and various veg- other pest control methods parasitoid populations. etables and fruits. Bt technologies—sprays or transgenic plants—will not control all insect pests. Bt crops can significantly reduce the use As with any pesticide, the potential of synthetic insecticides and are highly Evaluation of exists for target insects to develop cost-effective in controlling economi- Bt Technologies resistance. Both Bt sprays and Bt crops cally important pests compared to can only harm close relatives of the existing insecticides. Bt crops provide Comparing Bt sprays target pest, and only if eaten. The pri- ease of management by reducing the and transgenic crops mary alternatives to Bt insecticides are need for repeated pesticide applications, There are advantages and disadvantages synthetic chemical pesticides with the labor costs for scouting to deter- to the use of Bt in spray form. Like much broader toxicity, impacting many mine when to spray, the energy and chemical pesticides, the timing, dosage, non-target organisms including benefi- equipment costs for applications, and and formulation of the application can be cial insects, fish, birds, and human the danger of pesticide exposure to farm beings. In the future, existing chemical workers. Conventional cotton is the pesticides, as well as new chemicals coming to market, will continue to pro-

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most pesticide-intensive crop in the mental impacts of Bt on related nontar- U.S., requiring upwards of 10 treat- get organisms or their predators. ments per season. In Arizona, the logical and health concerns. However, Bt Because chemical insecticides are gen- deployment of Bt cotton has reduced corn that targets both corn borer and erally used less frequently on Bt crops, it chemical insecticide applications from corn rootworm will eliminate these risks is likely that such crops will benefit non- 12 or 14 treatments to 2 to 6 treat- to farm workers and the environment. target populations, especially those of ments per season, with the number of parasitoids and predators that control applications depending on the growing Impacts on insect pests. In fact, studies in the U.S., region and the season. In 1999, Bt cot- Non-target Organisms China, and elsewhere have documented ton in the U.S. resulted in a reduction of Bt has a long history of use in spray larger populations of predatory bugs, 1,200 metric tons of active ingredient of formulations and is generally considered spiders, and ants, and enhanced biodi- insecticides, and in China, a reduction of a safe insecticide because it is highly versity of beneficial insects in Bt cotton 15,000 tons of formulated insecticide. specific. It directly affects only target fields as compared with conventional From 1996 to 2000, U.S. farmers have organisms and closely related species, fields treated with chemical insecti- saved $100 million annually when although the biotic community may be cides. Similar results have been reduced pesticide costs and increased affected indirectly because of perturba- reported in Bt versus sprayed conven- yields are weighed against the added tions in insect populations. Populations tional potato fields. Studies examining purchase cost of Bt cottonseed. A fur- of non-target insect species belonging to the impact of Bt formulations in aquatic ther benefit of Bt crops is reduction of the same order as the target pest may environments have failed to show certain food-contaminating toxins such be vulnerable. Initial concerns about the adverse effects. Similarly, field studies as the fungal toxin fumonisin in corn. negative effects of pollen from Bt corn on of forest applications of Bt sprays have the larvae of the monarch butterfly, shown that the impact is restricted to Danaus plexippus, have been allayed by moths and butterflies and appears to be Environmental additional laboratory and field studies. transient for many non-target organ- Considerations isms. Laboratory tests also have failed Despite concerns about the use of Bt Field studies of transgenic crops have to show toxicity of Bt to birds, fish, and sprays and Bt transgenic crops, both failed to demonstrate significant detri- invertebrates, including earthworms. technologies have advantages over standard chemical insecticides. Unlike The Monarch Butterfly, An Issue? some insecticides that accumulate in biological systems, there are no data Non-target insects closely related to a targeted pest can be affected by ingestion that Bt proteins do so. Bt sprays and Bt of ICPs. However, the risk that any pesticide will affect a non-target organism crops only kill the target pests and some depends on both the dose that is toxic and the probability that the organism will closely related species, and only after be exposed to that dose in its natural environment. ingestion of the Bt spray or plant. Bt cot- Bt ton that targets the budworm and A controversial laboratory study raised concern about the impact of on the bollworm kills only these pests and monarch butterfly because monarch caterpillars were found to experience 44% related Lepidoptera when the plant is mortality following ingestion of milkweed dusted with transgenic corn pollen. eaten. Bt corn is currently under devel- However, the levels of ICPs or pollen ingested in that study were not quantified, opment to control both the European and the study did not address the probability that monarchs would be exposed to corn borer and the corn rootworm. Corn toxic doses of pollen containing ICPs in the field. rootworm costs growers sums in excess More recent assessments indicate that the actual risk to monarchs is negligible. of $2 billion annually in crop losses and These studies took into account the toxicity of Bt to monarchs in laboratory and insecticide expenditures. Most chemical field studies, the amount and distribution of corn pollen on milkweed plants, and insecticides have a relatively broad the temporal and spatial overlap between patterns of monarch larval feeding and range of activity against nontarget pollen shed from Bt corn. Although monarch larvae may experience less than 2% organisms including spiders, insect para- mortality by ingesting pollen from Event 176 Bt corn, other varieties produced no sitoids and predators, as well as fish, detectable harm. This variety, a minor proportion of commercial plantings, is now birds, and humans. Lorsban®, now used being withdrawn from the US market. In contrast, the studies showed that the to control corn rootworm, is broadly widely used corn insecticide, lambda cyhalothrin, has an adverse effect on toxic to invertebrates and presents eco- monarch butterflies. While research on potential long-term impacts of Bt pollen on monarchs is ongoing, the experience to date illustrates that laboratory studies alone cannot adequately evaluate the impact of a new technology at the field level.

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Toxicity tests on a standard set of protozoa, fungi, and bacteria. Because organisms including avian and aquatic Bt is a natural inhabitant of soil, we also species, beneficial insects, soil inverte- Bt crops have only recently been know that soil organisms have a long brates, and mammals are required deployed and until now relatively small evolutionary association with Bt. before pesticide registration, providing a sample sizes have been used in labora- basis for assessing potential toxicity to tory and field studies. Thus the data in Resistance in Target Pests non-target species. Fact sheets summa- support of the safety of Bt crops to non- Insects have the potential to develop rizing results from the testing of target organisms is still limited. resistance to insecticides. Based on commercial Bt crops can be found at: However, additional data are rapidly laboratory selection experiments and www.epa.gov/pesticides/biopesticides/ accumulating from field programs moni- models derived from these studies, the factsheets. toring the actual impact of Bt use on potential exists for development of resis- ecosystems. Studies using large sample tance to Bt ICPs when these products Laboratory and field tests can provide sizes of non-target organisms are under- are overused or used incorrectly. Several information about an organism’s suscep- way to determine the potential of both strategies are available for resistance tibility to Bt, the dose of Bt that is toxic, short- and long-term effects of Bt crops. management to Bt in spray formulations. and the fate of Bt in the environment. Initial results have failed to reveal signifi- These include the use of nontreated However, these tests say little about the cant non-target effects, in contrast to refuges, high dosage, mixtures of insec- risks to non-target organisms. Risk use of standard chemical pesticides. ticidal toxins, and rotation or alternation assessment measures both the degree of Bt toxins. After 40 years of use there of toxicity and the level of exposure and Will soil-dwelling non-target organisms is only one known example (the dia- attempts to determine what effects Bt be affected by Bt ICPs? Laboratory stud- mondback moth) of control failure due to will have on a population under the ies have shown that actively growing Bt resistance to Bt sprays having developed conditions in which exposure will occur. plants can increase the levels of ICPs in under field conditions, although other For example, Bt sprays affect some non- soil, and that soil can bind active Bt pro- species have developed high levels of target lepidopterans in the laboratory, teins for extended periods of time. resistance through laboratory selections. but often the time of spraying does not However, there are no known toxicologi- coincide with the presence of suscepti- cal effects of Bt ICPs on nontarget soil Because ICPs expressed in plants will ble non-target species, reducing their invertebrates or microbes. Root exu- see increasing use, concern that insect exposure to Bt in the field. dates from transgenic Bt corn appear to populations will develop resistance is be nontoxic to earthworms, nematodes, justified. It is anticipated that resistance could arise if management strategies are not carefully followed, especially in Measuring Effects on Non-target Organisms crops based on single-gene technology. Two main approaches are used to predict the impact of Bt on susceptible As with conventional pesticides, the non-target organisms: evolution of resistance to ICPs will depend on the genetics of resistance, ■ Toxicity tests conducted in the laboratory and field determine what doses the competitiveness of resistant individ- Bt of cause lethal or sublethal effects on representative non-target organ- uals in the field, and the implementation isms. Ideally, field studies to quantify exposure are combined with feed- of resistance management strategies. ing studies to determine if there is relevant exposure. The species that require attention are those related to the target pest, close relatives of The resistance management program endangered species, and beneficial species indigenous to habitats practiced most frequently for commer- where exposure may occur. cialized Bt crops is the “high dose/ ■ Species diversity and abundance in a biotic community are measured structured refuge strategy,“ in which the and compared to communities within similar agricultural lands. Analyses crop produces an ICP at concentrations should detect a negative or positive impact of Bt sprays or transgenic with a goal of more than 99.9% lethality plants on biodiversity. in the target pest. High ICP concentrations in the tissues on which insects In either case, exposure studies should take into account persistence of the feed serve to counter incremental devel- ICP in the environment, direct and indirect exposure (food chain effects). opment of resistance. At the same time, Both short-term and long-term population effects should be considered. from five to 50 percent (5-50%), Finally, the impact of the spray or transgenic crop must be considered in light of alternative pest control measures in a cost/benefit analysis.

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depending on the crop, of the acreage is Gene flow of Bt ICP genes from Bt crops planted with a conventional version of to wild relatives could occur where sex- the crop as a refuge for target insects. very low frequency. The probability that ually compatible wild relatives are within These conventional plants serve to sus- such transfers will occur in nature, and the pollination range of the crop. Ecolog- tain susceptible alleles within the insect involve functional genes is even lower. ical consequences could result if the population. Through random mating, There is no evidence that genes from gene confers resistance to a pest that is rare recessive resistance genes are non-engineered crops have been trans- a significant challenge to the wild rela- diluted out of the insect population and ferred to microorganisms and there is tive. This might provide a selective prevented from providing a selective no basis to suggest that transgenes advantage to the hybrid and its progeny advantage in offspring of the matings. would transfer more readily than any through introgression of the gene into This strategy will not be effective if cases other genes. Microbes are so much the wild relative’s gene pool. Negative develop where resistance is dominant. more likely to obtain genes, such as ecological impact—predicted on a those for antibiotic resistance, from case-by-case basis—should be Numerous studies have indicated that other microbes in the environment that weighed against impacts of the separate refuges are superior to seed the probability for such genes to be accepted alternative practices to Bt mixtures in delaying the development of acquired from genetically engineered technology, such as use of conventional resistance in insects that can move plant DNA is insignificant. pesticides. In the U.S., the EPA has con- between plants in the larval stage. Care cluded that there is not a reasonable must be taken in managing the insect Gene transfer between closely related possibility of gene flow from Bt cotton, population within the refuge to ensure species of plants occurs via pollen corn, or potatoes to wild relatives that enough susceptible individuals will under natural conditions all the time. except in certain areas of Florida and exist to breed out resistant individuals. Hybrids of conventional crops and wild Hawaii, where the use of cotton is The effectiveness of a refuge depends relatives form in 12 of the 13 most com- restricted because of the presence of on consistent, appropriate implementa- mon crops worldwide in some part of related wild species. There is a need for tion and monitoring. Grower compliance their agricultural range, and this process similar regulation globally, particularly in to a resistance management strategy is has been implicated in enhanced weedi- centers of crop origin, to insure that Bt essential to delaying the development ness about half of the time. crops are not grown in areas where of resistance. Similarly, proper application of Bt sprays would help to defer the development of resistance resulting from overuse, but presently only Bt TABLE 2. crops have mandated resistance management strategies. BARRIERS TO GENE FLOW THROUGH POLLEN EXAMPLE/EXPLANATION

New Bt crops that express multiple ICPs PHYSICAL BARRIERS with different modes of action are PLACEMENT OF BARRIERS TO POLLEN SPREAD ...... PLANT NON-ENGINEERED BORDERS currently being developed in a ‘gene stacking’ strategy. This technique should RESTRICT LOCATION OR TIMING limit selection for resistance in target OF CROP PLANTING ...... PROHIBIT USE NEAR WILD RELATIVES and secondary pest species because the CONTAINMENT OF probability of multiple, rare resistance ENGINEERED CROPS ...... GROW CROPS IN GREENHOUSES mechanisms arising simultaneously is astronomically low. PHYSIOLOGICAL BARRIERS HARVEST ENGINEERED PLANTS Gene Flow to BEFORE FLOWERING ...... ONIONS, SUGAR BEETS ENGINEER TRAITS INTO PLANTS Other Organisms THAT SELF-POLLINATE ...... POTATOES, TOMATOES Although it is theoretically possible for ENGINEER TRAITS INTO PLANTS microorganisms to incorporate DNA THAT ARE STERILE ...... BANANAS from transgenic plants, this has only MAKE THE ENGINEERED been accomplished under specifically PLANTS STERILE ...... “TERMINATOR TECHNOLOGY” optimized laboratory conditions and at a PERFORM GENETIC MODIFICATIONS ON PLASTIDS ...... PLASTIDS ARE NOT IN POLLEN

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potential outcrossing with wild or has been shown that farm workers do weedy relatives could introduce traits not develop respiratory, cutaneous, or that might disturb biodiversity of culti- In some cases, Bt crops can improve eye diseases from exposure to large vated crop species. Any gene flow that food safety. For example, approximately amounts of Bt in sprays. They do makes a weed more competitive in the 59% of corn grain globally is contami- develop antibodies to ICPs, but in no wild will not be readily reversed in the nated with fumonisins, fungal toxins case has the presence of these antibod- way that use of some short-lived envi- produced by the genus Fusarium that ies been linked to acute or chronic ronmentally damaging chemical can be can cause liver and kidney damage in disease. Thus, the use of Bt as a biologi- halted. There are a wide variety of many species, and are probable human cal pesticide has proven to be methods to limit gene flow between carcinogens. These fungal pathogens remarkably safe for vertebrates, particu- related plant species (Table 2). enter the plant through wounds caused larly in comparison with synthetic by boring insects that are among the chemical insecticides. The health bene- Human Health most important insect pests of corn fits to farm workers are clearly Considerations worldwide. Bt corn is protected against illustrated in a recent report indicating Issues have been raised regarding con- damage from corn borers and consis- that farmers in China currently growing sumer and worker safety of Bt use, tently has 90% less fumonisin than Bt cotton applied 80% less toxic insecti- whether applied as a foliar spray or conventional plants. Thus, protection cides than those growing conventional expressed in plants. Food safety issues against insect damage and subsequent cotton varieties. The farmers growing Bt involve the potential toxicity and aller- fungal infection may have important cotton also reported more than four-fold genicity of proteins, changes in the health implications for consumers and fewer instances of symptoms, such as nutritional composition of plants, and farm animals exposed to fumonisins in headache, nausea, skin pain, or diges- the safety of the antibiotic resistance their diet. tive problems from applying pesticides. marker genes used during the process Bt cotton is the GE crop most frequently of genetic engineering. These issues are Bt sprays and transgenic crops have not planted by small farmers worldwide. reviewed in a recent joint FAO/WHO had any known significant harmful Small farmers in developing countries publication. Bt proteins in all Bt plants effects on vertebrates, including mam- are at particular risk of exposure to pes- and sprays registered for food consump- mals and human beings. There have ticides because they often apply tion break down rapidly in simulated been only a few reports of Bt bacteria pesticides by hand, without safety pre- digestive systems, do not resemble any being isolated from humans with cautions, such as respirators, goggles, known food allergen or protein toxin and wounds or infections, and in these rare and gloves. have no oral toxicity, even when admin- cases there is no evidence that Bt was istered in high doses. the cause of any lasting injury. It also For the Future Improving our knowledge While transgenic crops are being The StarLink® Story adopted at unprecedented rates in Like other varieties of Bt corn, StarLink® produces an ICP that confers large- and small-scale farming systems resistance to important insect pests, such as the European corn borer. worldwide, the technology remains con- However, StarLink® is the only Bt corn variety producing a particular ICP troversial. Debate has arisen on known as Cry9C. At the time of its commercial launch, StarLink® had implications ranging from user, con- been approved for use in animal feed only and not in foods for human sumer, and environmental safety to consumption. In September of 2000 StarLink® corn was detected in Taco global economics and world hunger. Bell tortillas, prompting a rapid response that resulted in the voluntary Many of the short- and long-term impli- recall of all StarLink® corn food products. The Scientific Advisory Panel of cations have been predicted based on the EPA subsequently reevaluated the latest information available and past and ongoing experience, but much concluded that while no known health effects were associated with remains to be learned because the tech- StarLink®, it did not meet the most stringent standards to rule out any nology is new. Continued collaboration is possibility of allergenicity. The product is no longer marketed and regis- therefore needed among scientists in tration has been withdrawn. The EPA estimates that StarLink® corn academia, government, environmental will be essentially eliminated from US grain supplies in 2002 groups, and industry to critically define, (http://www.epa.gov/oppbppd1/biopesticides/index.htm).

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assess, and compare the risks and benefits of Bt sprays and crops relative to each other and to conventional pest control strategies. Scientific analyses should monitor actual on-farm practices, include conventional laboratory and field studies, and cover environmental impacts, effects on nontarget organisms and consumer safety. A thorough and objective evaluation of known and unre- solved safety issues is essential to appropriate regulation of transgenic crops in relation to the security, sustain- ability, and economics of food production both on a regional scale and globally. The eventual worldwide acceptance of genetic engineering will depend not only on safety but also diverse cultural and societal norms.

With regard to Bt plants and formulations, many fundamental questions still are not required for foliar sprays of Bt or ble impacts on important soil organisms. require answers. For example, what is other insecticides With increasing Additional information also is needed on the baseline natural abundance and dis- deployment of Bt crops, it is important to the frequency with which pollen from Bt tribution of B. thuringiensis in the develop more sophisticated and reliable crops may fertilize other plants. In many environment? When we breathe, how resistance monitoring and management cases, long-term environmental impact many Bt spores do we inhale? How strategies if sustained benefits are to be studies were not possible before Bt many spores and crystals are consumed gained from Bt technology. The potential crops were commercialized because of when we eat uncooked vegetables or for resistance development in target and the scale and duration over which moni- organically grown crops sprayed with nontarget organisms needs to be better toring is required. Agricultural systems Bt? How does this compare with the understood. What are the frequencies of are disruptive to the environment, so it amounts of ICPs ingested with Bt crops, resistance alleles and what are the best is imperative that these effects be and what is the relevance to consumer ways to detect them? Are they domi- clearly distinguished from those specifi- health? What factors influence the nant, recessive, or polygenic? What are cally associated with Bt formulations stability of insecticidal crystals and the mechanisms of action of ICPs at the and genetically engineered plants. proteins introduced into soil in the molecular level and how do they influ- form of sprays and plant exudates and ence target insect specificity and Research should continue to generate residues, and under what conditions resistance? Do ICPs delivered in sprays and evaluate Bt engineered crops that might persistence have biologically and plants differ in their effects will benefit developing nations, such as significant consequences? on specificity and resistance? Will the work being done by the International increased deployment of Bt crops influ- Rice Research Institute. Important con- Resistance of a pest to any insecticide is ence the rate of emergence of insect tributions will also come from other a serious concern. The U.S. Environ- populations resistant to Bt sprays? international centers of the Consultative mental Protection Agency requires Group on International Agricultural specific insect resistance management The need remains for a wide range of Research that focus on wheat, corn, and programs for Bt corn, cotton, and pota- field studies to enable rigorous quantita- potatoes. This research is enhanced by toes, which include refugerequirements, tive assessments of the direct and collaborations among scientists in acad- annual resistance monitoring, and reme- indirect effects of Bt sprays and crops emic, government, and international dial action programs (http://www.epa. on natural enemies and non-target research foundation laboratories in both gov/pesticides/biopesticides/otherdocs/b organisms, especially as compared with t_brad2/4%20irm.pdf). These programs treatments that include standard pesticide controls. Longer-term and more complex studies should evaluate possi-

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developing and developed countries. All varieties of ICPs and other types of phases of such projects—from design insecticides of microbial origin. to field-testing and consideration of environmental issues—benefit from tems. Expression also can be engi- Communication these collaborations. neered very specifically in the bacteria Colloquium participants believe the sci- used in sprays by manipulating genetic entific community could do a great deal Technology elements that aid in synthesis of ICPs. more to encourage the expansion of Gene expression technology will play a government and university programs critical role in the improvement of Bt Protein engineering has the potential to aimed at communicating to the general products. ICP genes in transgenic plants make a significant contribution to Bt public the risks and benefits of using Bt can be genetically engineered for opti- applications and insect control. For insecticides and Bt crops. Scientists can mal efficacy and risk management. example, based on the current under- work with their professional societies to Refined engineering of genes may soon standing of the structure and mode of present a balanced view of the risks and enable tissue-specific or inducible action of ICPs, researchers have engi- benefits of these new technologies. This expression such that ICP proteins are neered proteins with increased toxicity can be done through white paper synthesized only at sites in plants to target insects. In the future, the reports like this one, forums at scientific where insects are feeding. same techniques can be used to nar- meetings, articles in newsletters, and row the host range, reducing the being available to members of the press For crops such as corn and cotton, in impact on non-target insects or biologi- and the public. This report is available on which the pollen does not inherit plas- cal control agents by increasing the the web site of the American Society for tids, plastid transformation systems specificity of pesticidal proteins. Microbiology (http://www.asmusa.org/ would prevent the dissemination of Bt Genomic information of insects may acasrc/aca1.htm) transgenes through pollen. This also pro- suggest additional ways to improve the vides the opportunity to introduce efficacy and specificity with which ICPs It is essential to foster public under- unmodified bacterial genes instead of interact with their molecular targets. To standing that long-term environmental synthetic genes and high protein yields augment the arsenal of insecticidal pro- effects of Bt crops and products gener- characteristic of plastid expression sys- teins, searches should continue for new ally cannot be studied until after the products have been commercialized. Prior to the registration of the first Bt crop in 1995, the EPA evaluated studies of potential effects of Bt endotoxins in the environment. Based on their find- ings, they approved the use of Bt crops but realized that additional knowledge was needed over the longer term to assess the risks and benefits of this technology. Environmental effects cannot be conclusively studied in the laboratory; they must be studied in the field. This post-release analysis is essentially what is done to assess the environmental effects of traditional plant breeding and agricultural practices. The public should be reassured that the use and monitoring of transgenic crops con- tinues to be more rigorously regulated by governmental agencies than any other agricultural technology.

The positive aspects and the risks of agricultural biotechnology should be

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clearly communicated to the public and compared with the benefits and risks of alternative technologies. Evidence col- Recommendations lected to date indicates that genetic engineering of crops is at least as safe Research Communication as conventional means of generating ■ Develop and maintain integrated ■ Professional societies should help new crops. Many of the benefits of Bt international databases on Bacillus coordinate the dialogue between the crops, such as decreased production thuringiensis and ICPs. international scientific community, costs, reduced environmental impact, the press, and the public. and lowered levels of contaminants in ■ Continue support for culture the food supply, are not easily recog- collections of Bacillus thuringiensis ■ Scientists should participate in exist- nized by the consumer. Alternative isolates and strains of related ing statewide outreach programs scenarios to the use of Bt crops should species. (such as cooperative extension be presented, and the local, national, services) that can disseminate infor- and international consequences of these ■ Continue investigations into the mation about the technology to the scenarios should be considered in rela- persistence of ICPs and possible public and farming communities. tion to food security, human health, and long-term effects on nontarget the environment. organisms and the environment. ■ A pamphlet for the public should be developed presenting objective infor- Enhanced communication should be ■ Evaluate and implement improved mation about Bt technologies. strongly encouraged among university resistance management strategies. and government researchers assessing ■ A web site on Bt should be devel- the environmental effects of Bt crops ■ Evaluate gene flow to relatives of oped as an educational tool for the worldwide. This is particularly important crops and improve strategies to public. because Bt crops are grown in a variety manage it through cultural practices of agroecosystems, the same insects and engineered properties including ■ An international database should are often a problem in different coun- the incorporation of Bt genes into be developed and maintained to tries, and both insects and weedy the plastid genomes of engineered centralize information concerning species frequently span national bor- crop plants. risk-benefit assessment issues asso- ders. An international clearinghouse and ciated with the global adoption of database would facilitate information new technologies in agriculture. exchange pertaining to risks of emergence of resistance, nontarget effects, and potential gene flow to weeds and crops. In addition to its value to the scientific community, this centralized, science-based resource will help to build public confidence in genetic engineering by documenting the activities of ongoing research and monitoring programs.

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Cao, J, AM Shelton and ED Earle. 2001. Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter. Molecular Breeding 8:207-216.

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