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SPECIAL TOPIC 5 724 T A deficiencies in the developing world—has been the target on ahumanitariannonprofit basistohelpalleviatevitamin that containsthevitaminA precursorandwasdeveloped eties (STFigure5.1). EvenGoldenRice—a varietyofrice times resorttoviolencestoptheintroductionofGMvari the technology,opposeGMfooddevelopment,andsome - human populationandclimatechange—othersquestion ing —to helpaddressmalnutritioninaworldwithgrow - a globalvalueof$15billionforGMseeds. crops are planted on 170 million hectares of arable land, with ferent GMcropvarietieshadbeencreated.Worldwide, erance toagriculturalherbicides.By2012,morethan200dif - resistance toinsects,andsoybeanssugarbeetswithtol- taining thetropicaloillaurate,cornandcottonplantswith and zucchiniwithresistancetovirusinfection,canolacon- toes. Soonafterward,otherGMfoodsweredeveloped:papaya tomato thatstayedfirmandripelongerthannon-GMtoma- approved forsalein1994,wastheFlavrSavrtomato—a fied (GM)foodvarieties.Thefirstone, ated alargenumberofgeneticallymodi- flavor andnutrition. reduced pesticideuse,andenhanced agriculture, withincreasedproductivity, ised anexcitingnewphaseinscientific engineering ofanimalsandplantsprom- ing thesegenesintoorganisms.Genetic that conferdesirabletraits,thenintroduc- way—by identifyingandcloninggenes inamorepreciseandrapid could modifyagriculturallysignificant and 1980s,scientistsrealizedthatthey DNA technologiesemergedinthe1970s process isaslowone.Whenrecombinant through selective breeding, the and supportourcomplexcivilizations. evolved varieties that now feedourgrowing and breedingthemtoestablishthephenotype,wehave with naturallyoccurringormutagen-inducedvariations Although many people see great potential for GM Beginning inthe1990s,scientistscre- Although wehavehadtremendoussuccessshuffling FOR with desirablegenetictraits.Byselectingorganisms breeding techniquestocreateplantsandanimals hroughout theages,humanshaveusedselective

REVIEW “ productivity, reduced enhanced flavor and Special Topic G pesticide use, and pesticide use,and phase in scientific phase inscientific plants promised plants promised enetic engineering engineering enetic an exciting ne of and of animalsand with increased increased with Genetically Modified Foods Genetically Modified agriculture, agriculture, nutrition.” - ST Figure5–1 ST Western corporatecontroloflocalfoodcrops. to threat tohumanhealthandbiodiversitywouldlead a rice plants.TheattackersarguedthatGoldenRicewas minutes, theyhaduprootedandtrampledmostoftheGM Golden RiceintheBicolregionofPhilippines.Within15 ers brokethroughsecurityfencessurroundingafieldtrialof opposition and violence. On August8, 2013, 400 protest- of International. International. companyPioneerHi-Bred bythe USbiotech being tested ofactivistsopposed toGMcropsdestroyedplants hundreds InJuly2004, France. maizeinsouthwestern genetically-modified Opposition toGMfoodsisnotuniqueGoldenRice. w w s inModernGene

and examinethepromisesproblems will introduce the behind GM foods foods? InthisSpecialTopicchapter,we the debate. tion, andmisinformation—onbothsidesof bole andexaggeratedrhetoric,informa- foods. ThetopicofGMfoodattractshyper- extol thealmostlimitlessvirtuesofGM some scientistsandcommercialinterests safety andenvironmentalconcerns,while embrace thetechnologies.Opponentscite right bansonallGMfoods,whereasothers in 52countries.Somecountrieshaveout- marched againstGMfoodsinralliesheld In 2013,approximatelytwomillionpeople Anti-GM protesters att Anti-GM protesters So, what are the truths about GM ONLY acking a field of acking afield t ic s 5 10/06/14 5:38 PM ST 5 What Are GM Foods? 725

of the new technologies. We will look at some of the con- ST Table 5.1 Some GM Crops Approved for , Feed, or troversies and present information to help us evaluate the Cultivation in the United States* complex questions that surround this topic. Number of Crop Varieties GM Characteristics Soybeans 19 Tolerance to glyphosate Tolerance to herbicide What Are GM Foods? Reduced saturated Enhanced oleic acid GM foods are derived from genetically modified orga- Enhanced omega-3 fatty acid nisms (GMOs), specifically plants and animals of agricul- Maize 68 Tolerance to glyphosate herbicide tural importance. GMOs are defined as organisms whose Tolerance to glufosinate herbicide have been altered in ways that do not occur natu- Bt resistance rally. Although the definition of GMOs includes organisms Enhanced ethanol production that have been genetically modified by selective breeding, the Cotton 30 Tolerance to glyphosate herbicide most commonly used definition refers to organisms modi- Bt insect resistance fied through or recombinant DNA tech- Potatoes 28 Bt insect resistance nologies. Genetic engineering allows one or more genes to be Canola 23 Tolerance to glyphosate herbicide cloned and transferred from one to another—either Tolerance to glufosinate herbicide between individuals of the same species or between those of Enhanced lauric acid unrelated species. It also allows an organism’s endogenous Papaya 4 Resistance to papaya ringspot virus genes to be altered in ways that lead to enhanced or reduced ONLY beets 3 Tolerance to glyphosate herbicide expression levels. When genes are transferred between unre- Rice 3 Tolerance to glufosinate herbicide

lated species, the resulting organism is called transgenic. SPECIAL TOPIC 5 Zucchini 2 Resistance to zucchini, watermelon, The term cisgenic is sometimes used to describe trans- squash and cucumber mosaic viruses fers within a species. In contrast, the term is a Alfalfa 2 Tolerance to glyphosate herbicide more general one, encompassing a wide range of methods that manipulate organisms or their components—such as isolating Plum 1 Resistance to plum pox virus or producing wine, cheese, or yogurt. Genetic modi- fication of plants or animals is one aspect of biotechnology. * Information from the International Service for the Acquisition of Agri-Biotech Applications, www.isaaa.org. In 2012, it was estimated that GM crops were grown in approximately 30 countries on 11 percent of the arable land on Earth. The majority of these GM crops (almost 90 GM food was approved for consumption, although percent) are grown in five countries—the United States, a GM salmon variety was nearing market approval in the Brazil, Argentina, Canada, and India. Of these five, the United States (Box 1). A number of agriculturally impor- United States accounts for approximately half of the acre- tant animals such as goats and sheep have been genetically age devoted to GM crops. According to the U.S. Depart- modified to produce pharmaceutical products in their milk. ment of Agriculture, 93 percent of soybeans and 88 percent The use of transgenic animals as bioreactors is discussed of maize grown in the United StatesREVIEW are from GM crops. In earlier in the text (see Chapter 22). the United States, more than 70 percent of processed foods contain ingredients derived from GM crops. Herbicide-Resistant GM Crops Soon after the release of the Flavr Savr tomato in the Weed infestations destroy about 10 percent of crops world- 1990s, agribusinesses devoted less to designing GM wide. To combat weeds, farmers often apply foods to appeal directly to consumers. Instead, the market before seeding a crop and between rows after the crops are shifted toward farmers, to provide crops that increased pro- growing. As the most efficient broad-spectrum herbicides ductivity. By 2012, approximately 200 different GM crop also kill crop plants, herbicide use may be difficult and lim- varieties were approved for use as food or livestock feed ited. Farmers also use tillage to control weeds; however, till- in the United States. However, only about two dozen are age damages structure and increases erosion. widelyFOR planted. These include varieties of soybeans, corn, Herbicide-tolerant varieties are the most widely planted sugar beets, cotton, canola, papaya, and squash. ST Table of GM crops, making up approximately 70 percent of all 5.1 lists some of the common GM food crops available for GM crops. The majority of these varieties contain a bac- planting in the United States. Of these GM crops, by far the terial gene that confers tolerance to the broad-spectrum most widely planted are varieties that are herbicide tolerant herbicide glyphosate—the active ingredient in commer- or insect resistant. At the time of writing this chapter, no cial herbicides such as Roundup®. Studies have shown that

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Box 1 The Tale of GM Salmon— Downstream Effects? Despite these assurances, envi- wild populations. A study published ronmental groups are planning to in 2013 shows that it is possible for fight the sale of GM salmon. Some the AquAdvantage salmon to breed grocery chains in the United States successfully with a close relative, the have banned GM fish, and legisla- brown trout.* In laboratory condi- t took 18 years and about $60 tors in several western U.S. states are tions, the hybrids grew more quickly million, but the first GM animal trying to block the approval of the than either the GM or non-GM varie- to be approved as human food— AquAdvantage salmon based on fears ties, and in closed stream-like systems, Ithe AquAdvantage salmon—may soon that the accidental release of these the hybrids outcompeted both paren- hit the U.S. market. fish could contaminate wild salmon tal fish varieties for food supplies. The AquAdvantage salmon is an populations with transgenes and dis- The authors point out that these Atlantic salmon that is genetically turb normal . results should be taken into account modified to grow twice as fast as its Supporters of GM fish point out during environmental assessments, non-GM cousins, reaching market- that the GM salmon are very unlikely although it is still not known whether able size in one and a half years rather to escape their facilities, and if any the hybrid salmon–trout variety than the usual three years. Scientists did escape, they would be poorly could successfully breed in the wild. at AquaBounty Technologies in adapted to wild conditions. Critics of If GM salmon could escape, breed, Massachusetts created the variety by the new GM salmon point out that and introduce transgenes into wild transforming an Atlantic salmon with the technique used to create sterile populations, there could be unknown a single gene encoding the Chinook triploids (pressure-shocking the fertili- negative downstreamONLY effects on fish salmon growth hormone. The gene zed eggs) still allows a small percent- ecosystems. was cloned downstream of the anti- age of fertile diploids to remain in *Oke, K.B., et al. 2013. Hybridization between freeze gene promoter from an the stock. They state that even a few genetically modified Atlantic salmon and wild eel. This promoter stimulates growth fertile fish, if they escaped into the brown trout reveals novel ecological interactions. hormone synthesis in the winter, a wild, could have long-term effects on Proc. R. Soc. B. 280 (1763): 20131047. time when the fish’s own growth hor- mone gene is not expressed. The rapid growth of the GM salmon allows fish farmers to double their productivity. AquaBounty intends to sell GM fish eggs to two facilities—one in the

SPECIAL TOPIC 5 SPECIAL TOPIC United States and one in Panama— that will raise the salmon and market them. To ensure that the fish will not escape the facilities, the company promises to sell only fertilized eggs that are female, triploid, and sterile. The facilities are to be approved only if the tanks are located inland and have sufficient filters to ensure that The AquAdvantage salmon grows twice as fast as a non-GM eggs and small fish cannot escape.REVIEWAtlantic salmon, reaching market size in half the time.

glyphosate is effective at low concentrations, is degraded effectiveness of glyphosate-tolerant crops. (This and other rapidly in soil and , and is not toxic to humans. concerns about herbicide-tolerant GM plants are described Farmers who plant glyphosate-tolerant crops can treat later in this chapter.) One method used to engineer a their fields with glyphosate, even while the GM crop is glyphosate-tolerant plant is described in the next section. growing. This approach is more efficient and economical than mechanical weeding and reduces soil damage caused Insect-Resistant GM Crops by repeatedFOR tillage. It is suggested that there is less environ- The second most prevalent GM modifications are those that mental impact when using glyphosate, compared with hav- make plants resistant to agricultural pests. Insect damage is ing to apply higher levels of other, more toxic, herbicides. one of the most serious threats to worldwide food produc- Recently, evidence suggests that some weeds may be tion. Farmers combat insect pests using crop rotation and developing resistance to glyphosate, thereby reducing the predatory organisms, as well as applying insecticides.

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The most widely used GM insect-resistant crops are synthesize beta-carotene (the precursor to A) in the Bt crops. Bacillus thuringiensis (Bt) is group of soil- the rice grain endosperm. dwelling bacterial strains that produce crystal (Cry) pro- deficiency is a serious problem in teins that are toxic to certain species of . These Cry more than 60 countries, particularly countries in Asia and are encoded by the bacterial cry genes and form Africa. The World Health Organization estimates that crystal structures during sporulation. The Cry proteins are 190 million of the world’s children and 19 million preg- toxic to Lepidoptera (moths and butterflies), Diptera (mos- nant women are vitamin A deficient. Between 250,000 and quitoes and flies), Coleoptera (beetles), and Hymenoptera 500,000 children with vitamin A deficiencies become blind (wasps and ants). Insects must ingest the bacterial spores or each year, and half of these will die within a year of los- Cry proteins in order for the toxins to act. Within the high ing their sight. As vitamin A is also necessary for immune pH of the insect gut, the crystals dissolve and are cleaved by system , deficiencies lead to increases in many insect protease enzymes. The Cry proteins bind to recep- other conditions, including diarrhea and virus infections. tors on the gut wall, leading to breakdown of the gut mem- The most seriously affected people live in the poorest coun- branes and death of the insect. tries and have a basic starch-centered , often mainly Each insect species has specific types of gut receptors rice. Vitamin A is normally found in dairy products and that will match only a few types of Bt Cry toxins. As there can be synthesized in the body from beta-carotene found are more than 200 different Cry proteins, it is possible to in orange-colored and and in green leafy select a Bt strain that will be specific to one pest type. vegetables. Bt spores have been used for decades as insecticides in Several approaches are being taken to alleviate the vita- both conventional and organic gardening, usually applied min A deficiency status ONLYof people in developing countries. in liquid sprays. Sunlight and soil rapidly break down the These include supplying high-dose vitamin A supplements Bt insecticides, which have not shown any adverse effects and growing fresh fruits and vegetables in home gardens. on groundwater, mammals, fish, or birds. Toxicity tests on These initiatives have had partial success, but the expense SPECIAL TOPIC 5 humans and animals have shown that Bt causes few nega- of delivering education and supplementation has impeded tive effects. the effectiveness of these programs. To create Bt crops, scientists introduce one or more In the 1990s, scientists began to apply recombinant cloned cry genes into plant cells using methods described DNA technology to help solve vitamin A deficiencies in in the next section. The GM crop plants will then manu- people with rice-based diets. Although the rice plant natu- facture their own Bt Cry proteins, which will kill the target rally produces beta-carotene in its , it does not pro- pest species when it eats the plant tissues. duce it in the rice grain endosperm, which is the edible part Although Bt crops have been successful in reducing of the rice. The beta-carotene precursor, geranylgeranyl- crop damage, increasing yields, and reducing the amounts diphosphate, is present in the endosperm, but the enzymes of insecticidal sprays used in agriculture, they are also con- that convert it to beta-carotene are not synthesized troversial. Early studies suggested that Bt crops harmed (ST Figure 5–2). Monarch butterfly populations, although more recent stud- In the first version of Golden Rice, scientists intro- ies have drawn opposite conclusions (Box 2). Other con- duced the genes phytoene synthase (psy) cloned from the cerns still exist and these will be discussed in subsequent daffodil plant and carotene desaturase (crtI) cloned from sections of this chapter. REVIEWthe bacterium Erwinia uredovora into rice plants. The bac- terial crtI gene was chosen because the encoded GM Crops for Direct Consumption by this gene can perform the functions of two of the miss- To date, most GM crops have been designed to help ing rice enzymes, thereby simplifying the transforma- farmers increase yields. Also, most GM food crops are not tion process. The resulting plant produced rice grains that consumed directly by humans, but are used as animal feed were a yellow color due to the presence of beta-carotene or as sources of processed food ingredients such as oils, (ST Figure 5–3). This strain synthesized modest levels starches, syrups, and . For example, 98 percent of the of beta-carotene—but only enough to potentially supply U.S. soybean crop is used as livestock feed. The remainder is 15–20 percent of the recommended daily allowance of vita- processed into a variety of food ingredients, such as , min A. In the second version of the GM plant, called Golden texturedFOR soy proteins, soybean oil, and soy flours. However, Rice 2, the daffodil psy gene was replaced with the psy gene a few GM foods have been developed for direct consump- from maize. Golden Rice 2 produced beta-carotene levels tion. Examples are rice, squash, and papaya (Box 3). that were more than 20-fold greater than those in Golden One of the most famous and controversial examples Rice. In the next section we describe the methods used to of GM foods is Golden Rice—a rice variety designed to create Golden Rice 2.

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Box 2 The Monarch Butterfly Story whereas survival within Bt cornfields human development, and changes in was 80 to 93 percent. agricultural practices. Tillage, mow- Although these studies established ing, and herbicide use have destroyed a low risk for Monarch butterflies, a millions of acres of milkweed . new twist in the tale may be emerging. Along with the use of conventional n 1999, three years after the Since 1993, the numbers of Monarch herbicides that kill weeds, the grow- introduction of Bt corn in the butterflies wintering in Mexico has ing of glyphosate-tolerant corn and United States, scientists pub- dropped dramatically. Numbers have soybean crops has resulted in efficient Ilished a report that ignited the anti- dropped significantly each year since suppression of weeds in fields that GM movement and triggered years 2007—including a 30 percent drop used to contain small numbers of of intensive research. The scientists in 2011–2012 and a 59 percent drop milkweed plants. By affecting milk- had conducted a laboratory assay in 2012–2013. The reasons for these weeds, herbicide-tolerant GM crops in which they fed milkweed leaves, declines appear to be complex. In may be contributing to the serious coated with pollen from either non- Mexico, butterfly reserves have suf- decline in Monarch butterfly popula- GM or Bt corn, to Monarch butterfly fered from logging, water diversion, tions. Scientists are proposing a pro- larvae. They concluded that Bt corn and drought. In the United States and gram to plant milkweed plants along pollen reduced larval survival by Canada, where Monarch butterflies north–south highways from Texas to approximately 50 percent. Concerned lay their eggs each year, milkweed Canada, to provide food for Monarch about the possibility of unintended habitat has suffered from drought, butterfly larvae. harm to nontarget organisms, the ONLY U.S. Department of Agriculture commissioned scientists from the United States and Canada to provide detailed follow-up research. In 2001, these studies culminated in a series of five published papers that examined Monarch butterfly and ecol- ogy and how these may be affected by different types of Bt corn and Bt proteins—in both laboratory and field conditions.

SPECIAL TOPIC 5 SPECIAL TOPIC Data from these studies explained and contradicted the initial study. First, the authors of the original 1999 study had fed larvae on pol- len contaminated with ground-up corn anthers, which contain 100-fold higher levels of the Cry1Ab protein than found in pollen. In the field, larvae do not eat anthers or other parts of corn plants, as larvae feedREVIEW exclusively on milkweed leaves. Sec- ond, in laboratory tests, larvae were found to be sensitive to Cry1Ab and Cry1Ac proteins, but not to other Cry proteins. Third, the levels of Cry1Ab and Cry1Ac proteins present in Bt cornfields had little, if any, effects on Monarch butterfly larvae. The studies also examined the survival of larvae on Bt cornfields compared with non- Bt cornfields that are sprayed with a FORpyrethroid insecticide used to control cornfield pests. Larval survival on milkweed within pesticide-treated fields was between 0 and 10 percent, Monarch butterfly larvae feed exclusively on milkweed.

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Box 3 The Success of Hawaiian Within three years, papaya harvests in is genetically modified. GM papaya GM Papaya Hawaii doubled and consumer accep- is approved for sale in the United tance was positive. Virus-resistant GM States, Canada, and Japan. papaya is credited with saving the Since the development of GM Hawaiian papaya industry. papaya in Hawaii, efforts to develop n the mid-1990s, the papaya An interesting side-effect of the similar varieties in other parts of the ringspot virus (PRSV) spread rap- presence of GM papaya in Hawaii world have stalled because of increas- idly throughout Hawaii’s papaya was the recovery of non-GM and ing public resistance to GM foods. Ifields and threatened to destroy the organically grown papaya. Because Since 2010, thousands of GM industry within a few years. To try PRSV levels declined due to the pres- papaya trees in Hawaii have been cut to stop the destruction of Hawaiian ence of virus-resistant fields and the down and destroyed by anonymous papaya, a team of scientists from the abandoning of infected fields, some attackers. Efforts to introduce GM University of Hawaii, the USDA Agri- growers can now produce non-GM papaya in Thailand have failed, and cultural Research Center in Hawaii, papaya, albeit on a smaller scale than the government recently banned GM and the Upjohn Company cloned before the virus spread throughout foods. Japan has approved the sale of the coat protein gene of PRSV and Hawaii. At the present time, more GM papaya, but only if it is labeled as introduced it into cultured papaya than 70 percent of Hawaiian papaya genetically modified. cells using biolistic transformation. The goal was to create PRSV resis- (a) (b) tance using a mechanism known as ONLY pathogen-derived resistance. The presence of virus coat proteins within

the plant is thought to interfere with S the disassembly and movement of an P E

infecting virus, slowing or preventing CI

infection. Researchers tested resis- AL tance to PRSV in the transformed

papaya plants and developed two TO GM varieties—SunUp and Rainbow. PIC 5 SunUp was homozygous for the PRSV coat protein gene, and Rainbow was an F1 hybrid of SunUp and a non-GM variety Kapoho. After three years of field testing and two years of moving through federal regulatory processes, GM papaya was approved for use. Seeds were given for free to farmers A test field of GM papaya. The “Rainbow” variety, growing in who immediately planted them to the center of the field, is unaffected by PRSV. The “Sunrise” non- replace their virus-devastatedREVIEW fields. GM variety surrounds the GM papaya and is severely infected. Clinical trials have shown that the beta-carotene in Despite the promise of Golden Rice 2, controversies Golden Rice 2 is efficiently converted into vitamin A in remain. Critics of GM foods suggest that Golden Rice could humans and that about 150 grams of uncooked Golden make farmers too dependent on one type of food or might Rice 2 (which is close to the normal daily rice consumption have long-term health or environmental effects. These and of children aged 4–8 years) would supply all of the child- other controversies surrounding GM foods are discussed in hood daily requirement for vitamin A. subsequent sections of this chapter. At the present time, Golden Rice 2 is undergoing field, biosafety, and efficacy testing in preparation for approval byFOR government regulators in Bangladesh and the Philip- pines. If Golden Rice 2 proves useful in alleviating vitamin Methods Used to Create GM Plants A deficiencies and is approved for use, seed will be made available at the same price as non-GM seed and farmers Most GM plants are created using one of two approaches: will be allowed to keep and replant seed from their own the biolistic method or tumefaciens- crops. mediated transformation technology. Both methods

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Geranylgeranyl- diphosphate

psy gene Phytoene synthase from maize (psy gene)

Phytoene

Phytoene desaturase (pds gene)

crtI gene from Zeta-carotene bacteria z-carotene desaturase (zds gene)

Lycopene

Lycopene b-cyclase (lcy gene) ONLY Beta-carotene

ST Figure 5–2 Beta-carotene pathway in Golden Rice 2. Rice plant enzymes and genes involved in beta-carotene synthesis are shown on the right. The enzymes that are not expressed in rice endosperm are indicated with an “X.” The genes inserted into Golden Rice 2 are shown on the left.

target plant cells that are growing in vitro. Scientists can ST Figure 5–3 Non-GM and Golden Rice 2. Golden Rice 2 SPECIAL TOPIC 5 SPECIAL TOPIC generate plant cultures from various types of plant contains high levels of beta-carotene, giving the rice endosperm tissues, and these cultured cells will grow in either liquid a yellow color. The intensity of the color reflects the amount of beta-carotene in the endosperm. cultures or on the surface of solid growth media. When grown in the presence of specific and hormones, these cultured cells will form clumps of cells called calluses, which, when transferred to other types of media, will form These characteristics are conferred by a 200-kb tumor- . When the rooted plantlets are mature, they are trans- inducing plasmid called a . After infection with ferred to soil medium in greenhousesREVIEW where they develop Agrobacterium, the Ti plasmid integrates a segment of its into normal plants. DNA known as transfer DNA (T-DNA) into random loca- The biolistic method is a physical method of introduc- tions within the plant . (ST Figure 5–4). To use the ing DNA into cells. Particles of heavy metals such as gold Ti plasmid as a transformation vector, scientists remove are coated with the DNA that will transform the cells; these the T-DNA segment and replace it with cloned DNA of the particles are then fired at high speed into plant cells in vitro, genes to be introduced into the plant cells. using a device called a gene gun. Cells that survive the In order to have the newly introduced gene expressed bombardment may take up the DNA-coated particles, and in the plant, the gene must be cloned next to an appropri- the DNA may migrate into the nucleus and integrate ate promoter sequence that will direct transcription in the into a plant chromosome. Plants that grow from the bom- required plant tissue. For example, the beta-carotene path- bardedFOR cells are then selected for the desired phenotype. way genes introduced into Golden Rice were cloned next Although biolistic methods are successful for a wide to a promoter that directs transcription of the genes in the range of plant types, a much improved transformation rate rice endosperm. In addition, the transformed gene requires is achieved using Agrobacterium-mediated technology. Agro- appropriate transcription termination signals and signal bacterium tumefaciens (also called Rhizobium radiobacter) sequences that allow insertion of the encoded protein into is a soil microbe that can infect plant cells and cause tumors. the correct cell compartment.

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Cytokinin survive on synthetic culture medium that contains only mannose as a source. Cells that are cotransformed with the pmi gene under control of an appropriate pro- T-DNA Region moter and the gene of interest can be positively selected by Left Border Right Border growing the plant cells on a mannose-containing medium. This type of positive selection was used to create Golden Rice 2. Studies have shown that purified PMI protein is Ti Plasmid easily digested, nonallergenic, and nontoxic in mouse oral Opine toxicity tests. A variation in positive selection involves use of a marker gene whose expression results in a visible phe- notype, such as deposition of a colored pigment. Virulence The following descriptions illustrate the methods used Origin of Region Replication to engineer two GM crops: Roundup-Ready soybeans and (ORI) Golden Rice 2. ST Figure 5–4 Structure of the Ti plasmid. The 250-kb Roundup-Ready® Soybeans Ti plasmid from Agrobacterium tumefaciens inserts the T-DNA portion of the plasmid into the host cell’s nuclear genome The Roundup-Ready soybean GM variety received mar- and induces tumors. Genes within the virulence region code ket approval in the United States in 1996. It is a GM plant for enzymes responsible for transfer of T-DNA into the plant with resistance to the herbicide glyphosate, the active genome. The T-DNA region contains auxin and genes ingredient in Roundup, a commercially available broad- that encode hormones responsible for cell growth and tumor ONLY formation. The opine genes encode compounds used as energy spectrum herbicide. Glyphosate interferes with the enzyme sources for the bacterium. The T-DNA region of the Ti plasmid 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), is replaced with the gene of interest when the plasmid is used as which is present in all plants and is necessary for plant syn- S P

a transformation vector. thesis of the aromatic amino acids , , E CI and . EPSPS is not present in mammals, which AL obtain aromatic amino acids from their diets. Selectable Markers To produce a glyphosate-resistant soybean plant, TO The rates at which T-DNA successfully integrates into the researchers cloned an epsps gene from the Agrobacterium PIC 5 plant genome and becomes appropriately expressed are strain CP4. This gene encodes an EPSPS enzyme that is low. Often, only one cell in 1000 or more will be successfully resistant to glyphosate. They then cloned the CP4 epsps transformed. Before growing cultured plant cells into mature gene downstream of a constitutively expressed promoter plants to test their phenotypes, it is important to eliminate from the cytomegalovirus to allow gene expression in all the background of nontransformed cells. This can be done plant tissues. In addition, a short peptide known as a chlo- using either positive or negative selection techniques. roplast transit peptide (in this case from petunias) was An example of negative selection involves use of a cloned onto the 5¿ end of the epsps gene-coding sequence. This marker gene such as the hygromycin-resistance gene. allowed newly synthesized EPSPS protein to be inserted into This gene, together with an appropriate promoter, can be the soybean chloroplast (ST Figure 5–5). The final plasmid introduced into plant cells alongREVIEW with the gene of interest. contained two CP4 epsps genes and, for the initial experi- The cells are then incubated in culture medium containing ments, a beta-glucuronidase (GUS) gene from E. coli. The hygromycin—an antibiotic that also inhibits the growth of eukaryotic cells. Only cells that express the hygromycin- pV-GMGT04 pV-GMGT04 resistance gene will survive. It is then necessary to verify E35S ctp4 CP4 epsps nos that the resistant cells also express the cotransformed gene. This is often done by techniques such as PCR amplification ST Figure 5–5 Portion of plasmid pV-GMGT04 used to cre- using gene-specific primers. Plants that express the gene of ate Roundup-Ready soybeans. A 1365-bp fragment encoding interest are then tested for other characteristics, including the EPSPS enzyme from Agrobacterium CP4 was cloned down- the phenotype conferred by the introduced gene of interest. stream from the cauliflower mosaic virus E35S promoter and the An example of positive selection involves the use of petunia chloroplast transit peptide signal sequence (ctp4). CTP4 FOR signal sequences direct the EPSPS protein into chloroplasts, a selectable marker gene such as that encoding phospho- where aromatic amino acids are synthesized. The CP4 epsps cod- mannose isomerase (PMI). This enzyme is common in ing region was cloned upstream of the nopaline synthase (nos) animals but is not found in most plants. It catalyzes the transcription termination and polyadenylation sequences. The interconversion of mannose 6-phosphate and fructose CP4 epsps sequences encode a 455-amino-acid 46-kDa ESPSP 6-phosphate. Plant cells that express the pmi gene can protein.

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GUS gene acted as a positive marker, as cells that expressed Glu crtI nos Glu psy nos Ubi1 pmi nos the plasmid after transformation could be detected by the

presence of a blue precipitate. The final cell line chosen for ST Figure 5–7 T-DNA region of T1 plasmid pSYN12424. production of Roundup-Ready soybeans did not contain The Ti plasmid used to create Golden Rice 2 contained the caro- the GUS gene. tene desaturase (crtI) gene cloned from bacteria, the phytoene The plasmids were introduced into cultured soybean synthase (psy) gene cloned from maize, and the phosphoman- cells using biolistic bombardment. Afterward, cells were nose isomerase (pmi) gene cloned from E. coli. The glutelin (Glu) gene promoter directs transcription in rice endosperm, and the treated with glyphosate to eliminate any nontransformed polyubiquitin (Ubi1) promoter directs transcription in all tissues. cells. (ST Figure 5–6). The resulting calluses were grown Transcription termination signals were provided by the nopaline into plants, which were then field tested for glyphosate synthase (nos) gene 3¿ region. resistance and a large number of other parameters, includ- ing composition, toxicity, and allergenicity. the nos gene terminator region (nos). The Glu promoter Golden Rice 2 directs transcription of the specifically in the To create Golden Rice 2, scientists cloned three genes into rice endosperm. The nos terminator was cloned from the the T-DNA region of a Ti plasmid. The Ti plasmid, called Agrobacterium tumefaciens nopaline synthase gene and pSYN12424, is shown in ST Figure 5–7. The first gene was supplies the transcription termination and polyadenyl- the carotene desaturase (crtI) gene from Erwinia uredovora, ation sequences required at the 3¿ end of plant genes. The fused between the rice glutelin gene promoter (Glu) and second gene was the phytoene synthase (psy) gene cloned from maize. The maize psy geneONLY has approximately 90 per- cent sequence similarity to the rice psy gene and is involved in carotenoid synthesis in maize endosperm. This gene was also fused to the Glu promoter and the nos terminator Load pV-GMGT04 plasmids sequences in order to obtain proper transcription initiation into gene gun and termination in rice endosperm. The third gene was the selectable marker gene, phosphomannose isomerase (pmi), cloned from E. coli. In the Golden Rice 2 Ti plasmid, the pmi gene was fused to the maize polyubiquitin gene pro- moter (Ubi1) and the nos terminator sequences. The Ubi1 Bombard cells promoter is a constitutive promoter, directing transcrip- SPECIAL TOPIC 5 SPECIAL TOPIC with plasmids tion of the pmi gene in all plant tissues. To introduce the pSYN12424 plasmid into rice cells, researchers established embryonic rice cell cultures and infected them with Agrobacterium tumefaciens that con- Select transformed cells with glyphosate tained pSYN12424 (ST Figure 5–8). The cells were then placed under selection, using culture medium containing only mannose as a carbon source. Surviving cells express- REVIEWing the pmi gene were then stimulated to form calluses that were grown into plants. To confirm that all three genes were present in the transformed rice plants, samples were taken Grow surviving cells into plantlets and analyzed by the polymerase chain reaction (PCR) using gene-specific primers. Plants that contained one integrat- ed copy of the transgenic construct and synthesized beta- carotene in their seeds were selected for further testing.

GM Foods Controversies ST FigureFOR 5–6 Method for creating Roundup-Ready soy- beans. Plasmids were loaded into the gene gun and fired at high GM foods may be the most contentious of all products of pressure into cells growing in tissue cultures. Cells were grown in the presence of glyphosate to select those that had integrated modern biotechnology. Advocates of GM foods state that and expressed the epsps gene. Surviving cells were stimulated to the technologies have increased farm productivity, reduced form calluses and to grow into plantlets. pesticide use, preserved , and have the potential to feed

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created for different purposes and are used in ways that are Ti plasmid pSYN12424 both planned and unplanned. Each construction is unique and therefore needs to be assessed separately. Introduce Ti plasmid into A. tumifaciens We will now examine two of the main GM foods con- troversies: those involving human health and safety, and Agrobacterium environmental effects. tumifaciens Health and Safety Infect cultured cells GM food advocates often state that there is no evidence that GM foods currently on the market have any adverse health effects, either from the presence of toxins or from potential allergens. These conclusions are based on two observations. Select by growing on mannose medium First, humans have consumed several types of GM foods for more than 20 years now, and no reliable reports of adverse effects have emerged. Second, the vast majority of toxicity tests in animals, which are required by government regula- Grow into calluses and plants tors prior to approval, have shown no negative effects. A few negative studies have been published, but these have been criticized as poorly executed or nonreproducible. Critics of GM foodsONLY counter the first observation in several ways. First, as described previously, few GM foods are eaten directly by consumers. Instead, most are used as

Select plants for high livestock feed, and the remainder form the basis of purified SPECIAL TOPIC 5 endosperm color (beta-carotene) food ingredients. Although no adverse effects of GM foods in livestock have been detected, the processing of many food ingredients removes most, if not all, plant proteins and DNA. Hence, of GM food-derived ingre- dients may not be a sufficient test for health and safety. - - + + + Second, GM foods critics argue that there have been few

ST Figure 5–8 Method for creating Golden Rice 2. Rice plant human clinical trials to directly examine the health effects cells were transformed by pSYN12424 and selected on man- of most GM foods. One notable exception is Golden Rice nose-containing medium, as described in the text. Plants that 2, which has undergone two small clinical trials. They also produced high levels of beta-carotene in rice grain endosperm say that the toxicity studies that have been completed are (+ +), based on the intensity of the grain’s yellow color, were performed in animals—primarily rats and mice—and most selected for further analysis. of these are short-term toxicity studies. Supporters of GM foods answer these criticisms with growing human populations. Critics claim that GM foods several other arguments. The first argument is that short- are unsafe for both humans andREVIEW the environment; accord- term toxicity studies in animals are well-established meth- ingly, they are applying pressure on regulatory agencies to ods for detecting toxins and allergens. The regulatory pro- ban or severely limit the extent of GM food use. These cam- cesses required prior to approval of any GM food demand paigns have affected regulators and politicians, resulting in data from animal toxicity studies. If any negative effects are a patchwork of regulations throughout the world. Often the detected, approval is not given. Supporters also note that debates surrounding GM foods are highly polarized and several dozen long-term toxicity studies have been pub- emotional, with both sides in the debate exaggerating their lished that deal with GM crops such as glyphosate-resistant points of view and selectively presenting the data. So, what soybeans and Bt corn, and none of these has shown long- are the truths behind these controversies? term negative effects on test animals. A few studies that One point that is important to make as we try to answer report negative long-term effects have been criticized as thisFOR question is that it is not possible to make general state- poorly designed and unreliable. GM food advocates note ments about all “GM foods.” Each GM crop or organism that human clinical trials are not required for any other contains different genes from different sources, attached food derived from other genetic modification methods such to different expression sequences, accompanied by differ- as selective breeding. During standard breeding of plants ent marker or selection genes, inserted into the genome and animals, genomes may be mutagenized with radia- in different ways and in different locations. GM foods are tion or chemicals to enhance the possibilities of obtaining

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a desired phenotype. This type of manipulation has the cases, the presence of these weeds is affecting crop yields. potential to introduce into genes other than the One reason for the rapid rise of resistant weeds is that ones that are directly selected. Also, plants and animals farmers have abandoned other weed-management prac- naturally exchange and shuffle DNA in ways that cannot tices in favor of using a single broad-spectrum herbicide. be anticipated. These include interspecies DNA transfers, This strong selection pressure has brought the rapid evo- transposon integrations, and chromosome modifications. lution of weed species bearing gene variants that confer These events may result in unintended changes to the phys- herbicide resistance. In response, biotechnology compa- iology of organisms—changes that could potentially be as nies are developing new GM crops with tolerance to mul- great as those arising in GM foods. tiple herbicides. However, scientists argue that weeds will also develop resistance to the use of multiple herbicides, Environmental Effects unless farmers vary their weed management practices Critics of GM foods point out that GMOs that are released and incorporate tillage, rotation, and other herbicides into the environment have both documented and potential along with using the GM crop. Scientists point out that consequences for the environment—and hence may indi- herbicide resistance is not limited to the use of GM crops. rectly affect human health and safety. GM food advocates Weed populations will evolve resistance to any herbicide argue that these potential environmental consequences can used to control them, and the speed of will be be identified and managed. Here, we will describe two dif- affected by the extent to which the herbicide is used. ferent aspects of GM foods as they may affect the natural Since 1996, more than eight different species of environment and agriculture. insect pests have evolved some level of resistance to Bt insecticidal proteins. For example, in 2011 scientists 1. Emerging herbicide and insecticide resistance. Many ONLY reported the first cases of resistance of the western published studies report that the planting of herbicide- corn rootworm to Bt maize expressing the cry3Bb1 tolerant and insect-resistant GM crops has reduced gene, in maize fields in Iowa. In 2010, scientists from the quantities of herbicides and insecticides that are Monsanto detected large numbers of pink bollworms broadly applied to agricultural crops. As a result, the with resistance to the toxin expressed from the cry- effects of GM crops on the environment have been 1Ac gene in one variety of Bt cotton. In order to slow assumed to be positive. However, these positive effects down the development of Bt resistance, several strate- may be transient, as herbicide and insecticide resis- gies are being followed. The first is to develop varieties of tance is beginning to emerge. (ST Figure 5–9). GM crops that express two Bt toxins simultaneously. Since glyphosate-tolerant crops were introduced in Several of these varieties are already on the market SPECIAL TOPIC 5 SPECIAL TOPIC the mid-1990s, more than 24 glyphosate-resistant weed and are replacing varieties that express only one Bt cry species have appeared in the United States. Resistant gene. The second strategy involves the use of “refuges” weeds have been found in 18 other countries, and in some surrounding fields that grow Bt crops. These refuges contain non-GM crops. Insect pests grow easily with- in the refuges, which place no evolutionary pressure on the insects for resistance to Bt toxins. The idea is for these nonselected insects to mate with any resis- REVIEWtant insects that appear in the Bt crop region of the field. The resulting hybrid offspring will be heterozy- gous for any resistance gene variant. As long as the resistance gene variant is recessive, the hybrids will be killed by eating the Bt crop. In fields that use refuges and plant GM crops containing two Bt genes, resis- tance to Bt toxins has been delayed or is absent. As with emerging herbicide resistance, farmers are also encouraged to combine the use of Bt crops with con- ventional pest control methods. FOR 2. The spread of GM crops into non-GM crops. There have been several documented cases of GM crop plants ap-

ST Figure 5–9 Herbicide-resistant weeds. Water hemp pearing in uncultivated areas in the United States, Cana- weeds, resistant to glyphosate herbicide, growing in a field of da, Australia, Japan, and Europe. For example, GM sugar Roundup-Ready soybeans. beet plants have been found growing in commercial top

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soils. GM canola plants have been found growing in would, however, make it more difficult to eradicate ditches and along roadways, railway tracks, and in fill the plants. This is illustrated in a case of escaped GM soils, far from the fields in which they were grown. A bentgrass in Oregon, where it has been difficult to get 2011 study1 found “feral” GM canola plants growing in rid of the plants because it is no longer possible to use 288 of 634 sample sites along roadways in North Dakota. the relatively safe herbicide glyphosate. The potential Of these plants, 41 percent contained the CP4 EPSPS for environmental damage may be greater if the GM protein (conferring glyphosate resistance), and 39 per- transgenes did confer an advantage—such as insect cent contained the PAT protein (conferring resistance to resistance or tolerance to drought or flooding. the herbicide glufosinate). In addition, two of the plants In an attempt to limit the spread of transgenes from (0.7 percent of the sample) expressed both proteins (re- GM crops to non-GM crops, regulators are consider- sistant to both herbicides). GM plants that express both ing a requirement to separate the crops so that pollen proteins have not been created by genetic modification would be less likely to travel between them. Each crop and were assumed to have arisen by cross-fertilization of plant would require different isolation distances to take the other two GM crops. The researchers who conducted into account the dynamics of pollen spreading. Several this survey were not surprised to find GM canola along other methods are being considered. For example, one transportation routes, as seeds are often spilled during proposal is to make all GM plants sterile using RNAi shipping. More surprising was the extent of the distribu- technology. Another is to introduce the transgenes into tion and the presence of hybridized GM canola plants. chloroplasts. As chloroplasts are inherited maternally, One of the major concerns about the escape of GM their genomes would not be transferred via pollen. All of crop plants from cultivation is the possibility of out- these containment methodsONLY are in development stages crossing or —the transfer of transgenes from and may take years to reach the market. GM crops into sexually compatible non-GM crops or

wild plants, conferring undesired phenotypes to the SPECIAL TOPIC 5 other plants. Gene flow between GM crops and adja- cent non-GM crops is of particular concern for farm- The Future of GM Foods ers who want to market their crops as “GM-free” or “organic” and for farmers who grow seed for planting. Over the last 20 years, GM foods have revealed both prom- Gene flow of GM transgenes has been document- ise and problems. GM advocates are confident that the next ed in GM and non-GM canola as well as sugar beets, generation of GM foods will show even more promising and in experiments using rice, wheat, and maize. GM prospects—and may also address many of the problems. critics often refer to controversial studies about GM Research is continuing on ways to fortify staple crops outcrossing in Oaxaca, Mexico. In the first study in with nutrients to address diet problems in poor countries. 2001, it was reported that the local maize crops con- For example, Australian scientists are adding genes to tained transgenes from Monsanto’s Roundup-Ready bananas that will not only provide resistance to Panama and Bt insect-resistant maize. As GM crops were not —a serious fungal disease that can destroy crops— approved for use in Mexico, it was thought that the but also increase the levels of beta-carotene and other transgenes came from maize that had been imported nutrients, including iron. Other GM crops in the pipeline from the United States as aREVIEW foodstuff, and then had include plants engineered to resist drought, high salinity, been planted by farmers who were not aware that the starvation, and low temperatures. seeds were transgenic. Over the next ten years, subse- Scientists hope that new genome information and quent studies reported mixed results. In some studies, more precise technologies will allow them to accurately the transgenes were not detected, and in others, the edit a plant’s endogenous genes—decreasing, increasing, or same transgenes were detected. There is still no con- eliminating expression of one or more of the plant’s genes sensus about whether gene flow has occurred between in order to create a desirable phenotype. These approaches the GM and non-GM maize in Mexico. avoid the use of transgenes and address some of the con- It is thought that the presence of glyphosate- cerns about GM foods. The current techniques that resistant transgenes in wild plant populations is not researchers use to introduce genes into plant cells result in FORlikely to be an environmental risk and would confer random insertions into the genome. New techniques are no positive fitness benefits to the hybrids. The pres- being devised that will allow genes to be inserted into pre- ence of glyphosate-resistant genes in wild populations cise locations in the genome, avoiding some of the poten- tial unknown effects of disrupting a plant’s normal genome 1Schafter, M.G. et al. 2011. PLoS One 6:e25736. with random integrations.

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Researchers are also devising more creative ways to protect plants from insects and . One intriguing project involves introducing into wheat a gene that encodes a pheromone that acts as a chemical alarm signal to aphids. If successful, this approach could protect the wheat plants from aphids without using toxins. Another project involves cassava, which is a staple crop for many Africans and is afflicted by two viral diseases—cassava mosaic virus and brown streak virus—that stunt growth and cause rot (ST Figure 5–10). Although some varieties of cassava are resistant to these viruses, the cycle of cassava is so long that it would be difficult to introduce resistance into other varieties using conventional breeding techniques. Scientists plan to transform plants with genes from resistant cassava. ST Figure 5–10 Cassava tubers infected with brown streak This type of cisgenic gene transfer is more comparable to virus. Cassava, a major food crop in sub-Saharan Africa, is traditional breeding than transgenic techniques. threatened by infection with the mosaic virus, which stunts plant In the future, GM foods will likely include additional growth, and the brown streak virus, which causes root rot. GM animals. As described in Box 1, a transgenic Atlantic salmon variety is likely to receive marketing approval in the the incidence of avian influenza in poultry production, but near future. In another project, scientists have introduced also reduce the transmissibility ONLYof avian influenza viruses to a DNA sequence into chickens that protects the birds from humans. spreading avian influenza. The sequence encodes a hairpin Although these and other GM foods show promise for RNA with similarity to a normal viral RNA that increasing agricultural productivity and decreasing disease, binds to the viral polymerase. The presence of the hairpin the political pressure from anti-GM critics remains a pow- RNA inhibits the activity of the viral polymerase and inter- erful force. An understanding of the science behind these feres with viral propagation. If this strategy proves useful in technologies will help us all to evaluate the future of GM vivo, the use of these GM chickens would not only reduce foods. SPECIAL TOPIC 5 SPECIAL TOPIC Selected Readings and Resources

Journal Articles Schafer, M.G., et al. 2011. The establishment of genetically engi- neered canola populations in the US. PLoS One 6(10): e25736. Butler, D. 2012. Hyped GM maize study faces growing scrutiny. Whitty, C.J.M., et al. 2013. Africa and Asia need a rational debate 490: 158. on GM crops. Nature 497: 31–33. Cressey, D. 2013. A new breed. Nature 497: 27–29. Domingo, J.L. 2007. Toxicity studies of genetically modified REVIEWWeb Sites plants: A review of the published literature. Crit. Rev. Food Sci. Nutrition 47: 721–733. GMO Compass: EU supported GMO database. http://www. Enserink, M. 2008. Tough lessons from Golden Rice. Science gmo-compass.org. 320: 468–471. Adoption of Genetically Engineered Crops in the US. U.S. Gassmann, A.J., et al. 2011. Field-evolved resistance to Bt maize Department of Agriculture, Economic Research Service. http:// by western corn rootworm. PLoS One 6(7): e22629. www.ers.usda.gov/data-products/adoption-of-genetically-engi- Gilbert, N. 2013. A hard look at GM crops. Nature 497: 21–26. neered-crops-in-the-us.aspx#.UdmLqW3fJyI Gonsalves, D. 2004. Transgenic papaya in Hawaii and beyond. GM Approval Databases and Information. International Service AgBioForum 7: 36–40. for the Acquisition of Agri-Biotech Applications. http://isaaa.org Oke, K.B., et al. 2013. Hybridization between genetically modi- Twenty questions on genetically modified foods. World Health fied Atlantic salmon and wild brown trout reveals novel ecological Organization. http://www.who.int/foodsafety/publications/ interactions.FOR Proc. R. Soc. B., 280: 20131047. biotech/20questions/en/ Paine, J.A., et al. 2005. Improving the nutritional value of Golden Golden Rice. The International Rice Research Institute. http:// Rice through increased pro-vitamin A content. Nature Biotech. irri.org/golden-rice 23(4): 482–487.

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What measures have been taken to alleviate vitamin A defi- to alleviate vitamin have been taken What measures have To date, how successful developing countries? ciencies in been? these strategies and how was it created? What is Golden Rice 2, can be transformed using biolistic Describe how plants method compare with Agrobacterium methods. How does this tumefaciens-mediated transformation? negative selection techniques contribute How do positive and GM crops? to the development of soybean variety was devel- Describe how the Roundup-Ready used to transform the soybean plants. oped, and what genes were be may they that foods is to GM objections major the of One disagree, and why? harmful to human health. Do you agree or

6. 7. 8. 9. 3. 10.

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iscussion Questions D and approval, the development, regulating laws are the What use of GM foods in your region and nationally? should be Do you think that foods containing GM ingredients and disadvan- labeled as such? What would be the advantages tages to such a strategy? FOR How does glyphosate , and how has it been used with GM How does glyphosate yields? crops to increase agricultural by which the Cry proteins from Describe the mechanisms Bacillus thuringiensis act as insecticides. How do genetically modified organisms compare with - compare with modified organisms How do genetically through selective breeding? isms created as transgenic or cisgenic? GM crops be considered Can current Why? 200 GM crop varieties that have been Of the approximately are widely used. What are these varieties, developed, only a few they? and how prevalent are Review Questions Review

1. 2. 4. 5. 1. 2. 3.

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