미 농무부에 따르면, 몬산토사의 GM 밀은 이번에 발견된 Oregon주를 포함하여
미국 내 15개 주의 100군데 이상에서 시험재배를 실시했다고 합니다.
“The Agriculture Department said that during that seven-year period,
it authorized more than 100 field tests with the same
glyphosate-resistant wheat variety. Tests were conducted in in 15
states, including Oregon.”
(http://phys.org/news/2013-05-usda-non-approved-wheat-oregon-field.html)

로이터통신 보도에도 몬산토가 최소한 16개주 279군데에서 GM 밀 야외시험을
실시했으며, 그 면적이 4천 에이커가 넘는다고 합니다.
Government records show Monsanto conducted at least 279 field tests of
herbicide-resistant wheat on over 4,000 acres in at least 16 states
from 1994 until the company abandoned its field testing of wheat in
2004.
(http://www.reuters.com/article/2013/05/31/us-wheat-control-idUSBRE94U06H20130531)

이번에 몬산토사의 GM밀이 발견된 Oregon주의 농장은 GM 밀을 시험재배한 적이
없기 때문에 해당 농장주가 몬산토사를 상대로 소송을 제기했다고 하구요… 그리고
AP통신에  따르면… 몬산토사의 GM 밀이 시험재배된 16개주의 명단은 아래와 같습니다.
Arizona, California, Colorado, Florida, Hawaii, Idaho, Illinois,
Kansas, Minnesota, Montana, Nebraska, North Dakota, Oregon, South
Dakota, Washington and Wyoming.
(http://www.spokesman.com/stories/2013/jun/04/kansas-farmer-sues-monsanto-over-gmo-wheat/)


[PDF]

Evaluation of Broiler Performance When Fed Roundup-Ready …

www.poultryscience.org/ps/paperpdfs/04/​p0481325.pdf
 (첨부파일)
=================
GM Crop Database

http://www.cera-gmc.org/?action=gm_crop_database&mode=ShowProd&data=MON71800&frmat=SHORT

 

Database Product Description

 

MON71800

Host Organism

Triticum aestivum L. (Wheat)

 

Trait

Glyphosate herbicide tolerance.

 

Trait Introduction

Agrobacterium tumefaciens-mediated plant transformation.

 

Proposed Use

Production of T. aestivum for human food and livestock feed. This material will not be grown outside the normal production area for wheat.

 

Company Information

Monsanto Company
Chesterfield Village Research Center (MO)
700 Chesterfield Parkway North
St. Louis
MO  USA

 

 

Summary of Regulatory Approvals

 

Country	Environment	Food and/or Feed	Food	Feed	Marketing
Colombia			2004
United States		2004

Click on the country name for country-specific contact and regulatory information.

Abstract

 

Commercial wheat is comprised mainly of two species: common, or bread wheat (T. aestivum L.) and durum wheat (T. durum Desf.). Bread wheat is classified into several types, based on vernalisation requirement (winter and spring types) and kernel hardness. The hard types of bread wheat are high in protein, especially gliadins and glutenins. The high levels of these protein fractions in the flour impart elasticity to bread dough and allow it to expand during leavening and baking. Soft wheats are low in protein, and have low levels of gliadin and glutenin. These wheats are milled into flour for use in bakery products such as cakes, pastries, and unleavened breads. Durum wheat produces very hard, almost vitreous kernels due to its high protein content. This wheat is milled into semolina for the production of pasta and couscous.

Harvested wheat consists of a naked kernel, unlike other cereals such as rice, barley or oats that retain their hull (i.e., the palea and lemma) after harvest. The wheat kernel is loosely enclosed within the palea and lemma of each spikelet; these are eliminated as chaff during threshing. The wheat kernel is milled into white flour by removing the bran, aleurone layers and the germ prior to grinding; whole-wheat flour retains these fractions. By-products of wheat milling include: bran, germ, shorts and middlings. Some of these by-products are used as human food (i.e., bran, germ), and others, as livestock feed. Grain that does not meet the grade for food use can be used as animal feed, mainly for poultry and swine, but also for cattle. Wheat can also be fed as forage, either as pasture prior to stem elongation, or as ensilage. Wheat is also used in the brewing and distilling industries.

Weeds are a major production problem in wheat cultivation. Weeds compete for light, water and nutrients, and can also cause lodging and problems with harvesting. The seeds of several weed species are almost impossible to clean out of harvested wheat (e.g., Avena fatua L. wild oats), causing loss of quality and downgrading of the crop. Weeds can be managed using a combination of cultural practices (e.g., seed bed preparation, use of clean [certified] seed, narrow row spacing, fertilizer banding), integrated weed management (e.g., weed scouting, economic thresholds) and the use of herbicides. Depending on the weed species present, herbicides can be applied before the crop emerges (e.g., amitrole, glyphosate, trifluralin), or after (e.g., 2-4D, bromoxynil, dicamba, fenoxaprop-p-ethyl, MCPA, metsulfuron methyl). The build-up of weed populations can be stemmed by applying herbicides on summer-fallowed fields, and by practicing crop rotation, which allows the use of different herbicides. Rotating among herbicide groups also prevents the development of herbicide-resistant biotypes.

Roundup Ready® wheat (MON 71800) was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in spring wheat production. This genetically engineered spring wheat contains a novel form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that allows MON 71800 to survive an otherwise lethal application of glyphosate. The EPSPS gene introduced into MON 71800 was isolated from a strain of the common soil bacterium Agrobacterium tumefaciens strain CP4, and the novel form of the EPSPS enzyme produced by this gene is tolerant to glyphosate.

The EPSPS enzyme is part of the shikimate pathway, an important biochemical pathway in plants involved in the production of aromatic amino acids and other aromatic compounds. When conventional plants are treated with glyphosate, the plants cannot produce the aromatic amino acids needed for growth and survival. EPSPS is present in all plants, bacteria, and fungi. It is not present in animals, since these organisms are unable to synthesize their own aromatic amino acids. Because the aromatic amino acid pathway is not present in mammals, birds, or aquatic life forms, glyphosate has little, if any, toxicity for these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources. MON 71800 was developed by introducing two CP4 EPSPS genes into the spring wheat variety ‘Bobwhite’ using Agrobacterium-mediated transformation.

The food and livestock safety of MON 71800 wheat was based on the safety assessment of the CP4 EPSPS protein and the level of expression of the protein in the grain. The CP4 EPSPS proteins constitutes a small amount of the total protein in MON 71800 so there is little dietary exposure. The lack of toxicity or allergenicity of CP4 EPSPS was demonstrated from the results of laboratory and safety studies. The nutritional equivalence and wholesomeness of MON 71800 wheat compared to conventional wheat was demonstrated by the analysis of key nutrients in the grain including proximates (e.g., crude protein, crude fat, crude fibre, ash, moisture), total dietary fibre, sugars, starch, amino acid and fatty acid composition, B vitamins and vitamin E, minerals, as well the composition in the anti-nutrient phytic acid.
==============

GM Crop Database

http://cera-gmc.org/index.php?action=gm_crop_database&mode=ShowProd&data=MON71800&frmat=LONG

 

Database Product Description

 

MON71800

Host Organism

Triticum aestivum L. (Wheat)

 

Trait

Glyphosate herbicide tolerance.

 

Trait Introduction

Agrobacterium tumefaciens-mediated plant transformation.

 

Proposed Use

Production of T. aestivum for human food and livestock feed. This material will not be grown outside the normal production area for wheat.

 

Company Information

Monsanto Company
Chesterfield Village Research Center (MO)
700 Chesterfield Parkway North
St. Louis
MO  USA

 

 

Summary of Regulatory Approvals

 

Country	Environment	Food and/or Feed	Food	Feed	Marketing
Colombia			2004
United States		2004

Click on the country name for country-specific contact and regulatory information.

Introduction

 

The spring wheat variety MON 71800 (Roundup Ready wheat) was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup? as a weed control option in spring wheat. This genetically engineered wheat variety contains a glyphosate-tolerant form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the soil bacterium Agrobacterium tumefaciens strain CP4. The novel form of this enzyme is termed hereafter CP4 EPSPS.

The EPSPS enzyme is part of the shikimate pathway that is involved in the production of aromatic amino acids and other aromatic compounds in plants (Steinrucken and Amrhein, 1980). When conventional plants are treated with glyphosate, the herbicide binds to EPSPS, thereby preventing the synthesis of aromatic amino acids needed for plant growth. The CP4 EPSPS enzyme in MON 71800 spring wheat has a reduced affinity for glyphosate; its enzymatic activity is therefore not hindered by the herbicide.

EPSPS is present in all plants, bacteria, fungi, but not in animals, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic pathway is not present in mammalian, avian or aquatic life forms, glyphosate has little if any toxicity for these organisms (U.S. EPA, 1993; WHO, 1994; Williams et al. 2000). The EPSPS enzyme is normally present in food derived from plant and microbial sources.

MON 71800 was developed by introducing the CP4 EPSPS coding sequences into the spring wheat variety ?Bobwhite? using Agrobacterium-mediated transformation.

General Description

 

Monsanto used Bobwhite spring wheat (designated as MON 71900) as the parental variety to produce Roundup Ready® wheat event MON 71800. Event MON 71800 was developed through Agrobacterium-mediated transformation using the double border, binary vector PVTXGT10. The vector consists of the T-DNA segment intended for transformation, as well as the backbone DNA which is not expected to be incorporated into the transformed wheat.

The T-DNA contains two cp4 epsps gene cassettes and the left and right T-DNA border sequences. The segment is approximately 7.0 Kb in size. The cp4 epsps coding sequence in both cassettes is derived from Agrobacterium sp. strain CP4 and begins with a leader chloroplast transit peptide coding sequence (ArabTP) derived from the Arabidopsis thaliana epsps gene. The ArabTP transit peptide directs the CP4 EPSPS protein expressed in event MON 71800 to chloroplasts. The ArabTP-cp4 epsps fusion coding sequence in each cassette is followed by the 3′ non-translated region of the nopaline synthase gene (nos 3′) from Agrobacterium tumefaciens that provides the transcriptional termination signal. The upstream regulatory sequence of the first cassette consists of the 5′ region of the rice actin1 gene (Pract1- ract1I) which contains the promoter, transcription start site and the first intron. The upstream regulatory sequence of the second cassette consists of an enhanced 35S promoter from the cauliflower mosaic virus (CMV) (P-e35S) and the intron of the corn heat shock protein gene (hsp70I). The two different promoters were used to drive expression of the gene product in both vegetative and reproductive tissues.

Reference: U.S. Food and Drug Administration

Summary of Introduced Genetic Elements

 

Code	Name	Type	Promoter, other	Terminator	Copies	Form
CP4 epsps	5-enolpyruvyl shikimate-3-phosphate synthase  (Agrobacterium tumefaciens CP4)	HT	enhanced CaMV 35S chloroplast transit peptide from A. thaliana EPSPS gene (CTP2)	A. tumefaciens nopaline synthase (nos) 3′-untranslated region
CP4 epsps	5-enolpyruvyl shikimate-3-phosphate synthase  (Agrobacterium tumefaciens CP4)	HT	rice actin I promoter and intron sequences chloroplast transit peptide from A. thaliana EPSPS gene (CTP2)	A. tumefaciens nopaline synthase (nos) 3′-untranslated region

Characteristics of Triticum aestivum (Wheat)

 

Center of Origin	Reproduction	Toxins	Allergenicity
Asia Minor, Tigris-Euphrates drainage basin of the Middle East, as well as the regions of southern Caucasus and Crimea.	Primarily self-pollinated (autogamous). Some outcrossing by wind-pollination of less than 10%. Seed does not display dormancy.	Phytic acid, trypsin inhibitor, lectins. Gliadins responsible for celiac enteropathy.	Glutenins and gliadins (e.g., the IgE-inducing alpha-gliadin).

Donor Organism Characteristics

 

Latin Name	Gene	Pathogenicity
Agrobacterium tumefaciens strain CP4	CP4 EPSPS	A. tumefaciens is a common soil bacterium that is responsible for causing crown gall disease in susceptible plants. There have been no reports of adverse affects on humans or animals.

Modification Method

 

The wheat line MON 71800 was produced by Agrobacterium-mediated transformation of plant cells from ?Bobwhite? spring wheat. The plasmid vector PV-TXGT10 used for the transformation contained two cp4 epsps gene cassettes coding for glyphosate tolerance. Each gene cassette consisted of chloroplast transit peptide coding sequences from the Arabidopsis thaliana epsps gene (ArabTP) associated with the sequences of the cp4 epsps gene. Two different promoters were used to regulate the expression of each cp4 epsps gene: 1) the enhanced 35S promoter from the cauliflower mosaic virus and 2), the promoter, transcription start site, and first intron of the 5? region of the rice actin1 gene. Terminator sequences in each gene casette consisted of the 3? non-translated region of the nopaline synthase gene (nos 3?). The PV-TXGT10 vector backbone contained the origin of replication sequences ori-V and ori-322/rop. The vector backbone also contained the aad gene, which codes for streptomycin adenyltransferase, to allow the selection of bacteria containing the PV-TXGT10 vector.

Characteristics of the Modification

 

The Introduced DNA
Southern blot analysis and Polymerase Chain Reaction (PCR) amplification of the genomic DNA of MON 71800 wheat demonstrated one site of integration of a single copy of the T-DNA insert of PV-TXGT10. Southern blot analysis also confirmed the insertion of one intact copy of each cp4 epsps gene cassette, including the promoter, terminator and chlorophyll transit peptide sequences. None of the vector backbone sequences were integrated into the genome of MON 71800 wheat.

Genetic Stability of the Introduced Trait
The stability of the inserted DNA was evaluated, across several generations of wheat plants, using Southern blot analysis. The plants tested were progeny from several generations of self-fertilization, as well as from crosses with commercial varieties. The results of the genomic DNA blot analysis confirmed the stable inheritance of the inserted cp4 epsps gene cassettes. The stability of the introduced trait was also demonstrated after 18 generations of selfing of the original homozygous glyphosate-resistant plants. These generations showed no decrease in tolerance to glyphosate.

Mendelian segregation studies for the inheritance of the glyphosate tolerant trait were conducted with heterozygous first generation plants: these were selfed and the progeny were sprayed with glyphosate. The resulting 3:1 ratio of tolerant to sensitive plants was statistically significant and confirmed the inheritance of a single insertion site of the glyphosate tolerant trait.

Expressed material
In plant cells, the EPSPS enzyme is transported to the chloroplast by a transit peptide, which then cleaves from the enzyme. The introduced gene sequences in MON 71800 included a gene for a chloroplast transit peptide from Arabidopsis thaliana. Thorough analyses were conducted to investigate whether the same mechanism of binding, transporting, and cleaving of the chloroplast transit peptide to the CP4 EPSPS enzyme exists in MON 71800. The analyses revealed two forms of CP4 EPSPS expressed in MON 71800: one in which the chloroplast transit peptide has completely cleaved from the full length enzyme, and the other in which only part of the transit peptide is bound to the enzyme. Further analysis of Western blots showed that of the total amount of CP4 EPSPS expressed in MON 71800, 80% is in the form where the transit peptide is fully cleaved and 20%, where part of the transit peptide is still bound to the enzyme.

An enzyme-linked immunosorbent assay (ELISA) analysis was used to quantify the levels of the CP4 EPSPS proteins in forage and grain from MON 71800. The mean levels of both CP4 EPSPS proteins, on a fresh weight basis, were 106 μg/g in forage and 13 μg/g in grain. Assuming 14.5% moisture in stored grain, the concentration of both CP4 EPSPS proteins in the grain is approximately 0.0015%, on a dry matter basis.

Food and/or Feed Safety Considerations

 

Dietary exposure
The genetic modification of MON 71800 spring wheat will not result in any change in the consumption pattern of wheat and wheat-based products. MON 71800 is expected to be used in similar applications as other spring wheat cultivars by the food industry. MON 71800 did not express any novel compositional characteristics, as confirmed by the similarity in composition of the modified line to its parental counterpart, and other conventional spring wheat varieties. Furthermore, the availability of many spring wheat cultivars for cultivation, and the normal variation in wheat composition due to differences in grade and growing conditions, result in a wide variation in the composition of conventional wheat grain. Consequently, the dietary exposure of consumers in the United States to MON 71800 is anticipated to be the same as for other varieties of commercially available spring wheat.

Nutritional and Compositional Data
The nutritional components of MON 71800 grain and forage were determined analytically and compared to those of the parental line ‘Bobwhite’ and several commercial varieties grown at five locations in the United States and Canada. For the grain, these components included proximates (crude protein, crude fat, ash, moisture, total carbohydrates), total dietary fibre, sugars, starch, amino acids, fatty acids, B vitamins, vitamin E and minerals. Forage samples were analyzed for proximates, acid detergent fibre, neutral detergent fibre, calcium and phosphorus. At some of the locations, for both the grain and the forage, there were statistically significant differences in the levels of certain components between MON 71800 and its parental line. These differences within locations could be attributed to environmental effects rather than to any unintended effect of the genetic modification in MON 71800. However, the combined data from all test locations demonstrated that the nutritional composition of MON 71800 grain and forage was comparable to that of the parental line ‘Bobwhite,’ and other commercial spring wheat varieties.

Phytic acid occurs naturally in wheat and other cereals. It is indigestible by humans and non-ruminant livestock, and inhibits the absorption of iron and other minerals. Grain samples of MON 71800, the parental line ‘Bobwhite,’ other commercial wheat varieties were analyzed to determine levels of phytic acid. The concentration of phytic acid in MON 71800 was comparable to that in the parental line, and was within the range of values determined for the commercial varieties and those found in the literature.

Toxicity and Allergenicity
The potential for toxicity and allergenicity of MON 71800 wheat was investigated using the following data and information: results from the determination of amino acid sequence similarity between the CP4 EPSPS proteins and known toxins and allergens; analysis for possible glycosylation of the CP4 EPSPS proteins; analysis of the stability the novel proteins in simulated gastric fluids; results from an acute oral toxicity study in mice using the CP4 EPSPS proteins; and, information on the safety of the cp4 epsps gene donor, A. tumefaciens strain CP4. The potential for increased allergenicity of the grain from MON 71800 was investigated, specifically with regard to endogenous wheat allergens that induce an IgE reaction in susceptible humans. Various immunilogical assays, using sera from humans with an IgE-mediated wheat allergy, were performed with extracts from MON 71800, the parental line, and several other commercial wheat varieties. The possibility that the genetic modification would have also altered the levels of gliadin, proteins that cause celiac enteropathy in susceptible persons, was also investigated. Gliadin levels were measured, and gluten levels calculated in MON 71800, its parental line, and other commercial varieties.

The CP4 EPSPS proteins in MON 71800 showed no amino acid sequence similarity with known toxins and allergens and neither of the two forms of the protein are glycosylated. Both forms of the CP4 EPSPS protein were rapidly digested under simulated gastric fluid conditions, and the enzyme activity of each protein was substantially diminished within the same period as for the digestion. The results of the acute oral toxicity study on mice, at the highest administered dose, showed no adverse effects of the CP4 EPSPS proteins. Gluten levels were not significantly altered by the genetic modification of MON 71800, neither were the endogenous allergens, as demonstrated by the results of the immunological studies.

Both forms of the CP4 EPSPS protein were expressed at very low levels in MON 71800 grain and forage. This fact, along with negative results of the various safety studies, sequence homology investigations, and immunological assays led to the conclusion that MON 71800 spring wheat did not demonstrate any potential for toxicity and novel allergenicity, nor any altered endogenous allergenicity, compared to conventional spring wheat varieties.

Links to Further Information

 

Food Standards Australia New Zealand[PDF Size: 296073 bytes]

Initial Assessment Report: Application A524 – Food derived from herbicide – tolerant wheat MON 71800

U.S. Food and Drug Administration[PDF Size: 164547 bytes]

Biotechnology Consultation Note to the File BNF No. 000080

References

 

Steinrucken, H.C. & Amrhein, N. (1980). The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase. Biochemical and Biophysical Research Communications, 94, 1207-1212.

U.S. EPA. (1993). Reregistration Eligibility Decision (RED): Glyphosate. Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, D.C.

WHO. (1994). Glyphosate. World Health Organization (WHO), International Programme of Chemical Safety (IPCS), Geneva. Environmental Health Criteria No. 159.

Williams, G.M., Kroes, R. & Munro, I.C. (2000). Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regulatory Toxicology and Pharmacology 31, 117-165.


================

Biotechnology Consultation Note to the File BNF No. 000080

Date: July 22, 2004

http://www.fda.gov/Food/FoodScienceResearch/Biotechnology/Submissions/ucm155777.htm

Subject: Monsanto Roundup Ready® Wheat Event MON 71800

Keywords:   Wheat, Roundup Ready®, Triticum aestivum, Glyphosate (N-phosphonomethyl-glycine), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), cp4 epsps gene, Agrobacterium sp. CP4 strain, Herbicide-Tolerant, Glyphosate-Tolerant, event MON 71800, Bobwhite

1. Introduction

In a submission dated June 28, 2002, Monsanto provided information to support the safety and nutritional assessment of their glyphosate-tolerant (Roundup Ready®) wheat (Triticum aestivum) containing a transformation event designated MON 71800. The company provided additional information in a submission dated April 25, 2003. Monsanto concluded that their Roundup Ready® wheat event MON 71800 and the foods and feed derived from it are as safe and nutritious as current commercial varieties of wheat and the comparable foods and feed derived from them. Monsanto has previously completed consultations for other Roundup Ready® crops which are also tolerant to glyphosate. These other crops include bioengineered soybean, canola, corn, and cotton (BNFs 01, 20, 71, and 86, respectively). In a letter to FDA dated June 9, 2004, Monsanto states that the firm is deferring all further commercial development efforts to introduce Roundup Ready® wheat until such time that other wheat biotechnology traits are introduced. In its letter, Monsanto requests that FDA complete the consultation process for Roundup Ready® wheat event MON 71800.

2. Intended Effect

The intended effect of the genetic modification is to confer tolerance to the herbicidal compound glyphosate (N-phosphonomethyl-glycine) which is the active ingredient in Roundup® agricultural herbicides. In glyphosate-sensitive plants, glyphosate binds to the plant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and prevents the synthesis of aromatic amino acids that are necessary for plant growth. Roundup Ready® wheat MON 71800 contains the epsps gene from Agrobacterium sp. strain CP4 (cp4 epsps gene) encoding the CP4 EPSPS enzyme. This enzyme has a reduced affinity for glyphosate when compared to the native plant EPSPS enzyme. As a result, wheat plants expressing the CP4 EPSPS enzyme are tolerant to glyphosate and survive spraying with Roundup®

3. Development of Roundup Ready® Wheat Event MON 71800

3.1. The Parent Plant Wheat

Monsanto describes the history and biology of wheat. The scientific name for common bread wheat is Triticum aestivum L. Wheat belongs to the order Poales (Glumiflorae), family Poaceae (Graminae), tribe Triticeae, genus Triticum. T. aestivum is hexaploid with a total of 42 chromosomes. Modern wheat cultivars are either tetraploid (durum) or hexaploid (common and club types). Wheat is predominantly self-pollinating.

3.2.  Genetic Modifications and Characterization of the Introduced DNA

Monsanto used Bobwhite spring wheat (designated as MON 71900) as the parental variety to produce Roundup Ready® wheat event MON 71800. Event MON 71800 was developed through Agrobacterium-mediated transformation using the double border, binary vector PV-TXGT10. The vector consists of the T-DNA segment intended for transformation, as well as the backbone DNA which is not expected to be incorporated into the transformed wheat.

The T-DNA contains two cp4 epsps gene cassettes and the left and right T-DNA border sequences. The segment is approximately 7.0 Kb in size. The cp4 epsps coding sequence in both cassettes is derived from Agrobacterium sp. strain CP4 and begins with a leader chloroplast transit peptide coding sequence (ArabTP) derived from the Arabidopsis thaliana epsps gene. The ArabTP transit peptide directs the CP4 EPSPS protein expressed in event MON 71800 to chloroplasts. The ArabTP-cp4 epsps fusion coding sequence in each cassette is followed by the 3′ non-translated region of the nopaline synthase gene (nos 3′) from Agrobacterium tumefaciens that provides the transcriptional termination signal. The upstream regulatory sequence of the first cassette consists of the 5′ region of the rice actin1 gene (P-ract1-ract1I) which contains the promoter, transcription start site and the first intron. The upstream regulatory sequence of the second cassette consists of an enhanced 35S promoter from the cauliflower mosaic virus (CMV) (P-e35S) and the intron of the corn heat shock protein gene (hsp70I). The two different promoters were used to drive expression of the gene product in both vegetative and reproductive tissues.

The vector backbone contains the origin of DNA replication ori-V that allows maintenance of PV-TXGT10 in Agrobacterium as well as the origin of replication ori-322/rop that allows the replication of PV-TXGT10 in the intermediate host E. coli. The vector backbone also contains the aad gene encoding the selectable marker enzyme streptomycin adenyltransferase that allows selection of bacteria containing PV-TXGT10.

Monsanto used Southern blot analysis to characterize the DNA introduced in the transformation event MON 71800. Genomic DNA isolated from event MON 71800 and the parent line MON 71900 was digested with restriction enzymes and subjected to Southern blot analysis using radiolabeled DNA probes corresponding to different segments of the T-DNA and plasmid backbone. Based on this analysis, Monsanto concludes that: 1) event MON 71800 contains a single T-DNA insert of the expected size (approximately 7.0 Kb) comprising one intact copy of each cp4 epsps expression cassette; 2) event MON 71800 does not contain the plasmid backbone sequences, including ori-V, ori-322/rop, and aad coding sequence; and 3) all genetic elements present in the expression cassettes before transformation are also present in event MON 71800.

Monsanto performed polymerase chain reaction (PCR) on event MON 71800 genomic DNA to verify the presence of the unique T-DNA insert-to-plant junction sequences. This analysis yielded PCR products of the expected size, thereby confirming the presence of these unique sequences in event MON 71800.

3.3. Stability of the Introduced DNA

Monsanto describes the experiments it conducted to evaluate the stability of the DNA insert. Monsanto used Southern blot analysis to test the insert stability across several generations of wheat plants containing event MON 71800. The tested plants included R2, R3, R4, and R5 progeny derived from the initial MON 71800 transformant by self-fertilization and three pre-commercial wheat varieties (Westbred 926 BC4F1, HJ-98 BC4F1, and BW251 BC5F4) containing event MON 71800 introduced by traditional breeding. These seed materials represent the 2nd, 3rd, 4th, 5th, 8th, 8th, and 12th generations, respectively. The non-transgenic wheat lines MON 71900, Westbred 926, HJ-98, and BW251 were used as control lines. Genomic DNA isolated from the seeds of all of these lines was digested with the restriction enzyme Bam HI and probed with the cp4 epsps coding sequence. The DNA from the non-transgenic control lines did not contain detectable hybridization bands. The digested transformation plasmid used as a positive control produced two bands of the expected length (3.7 and 8.6 Kb). The DNA from the test lines containing event MON 71800 produced two DNA bands – the expected 3.7 Kb band (which represents the internal segment of the insert) and a 9.0 Kb fragment (which represents a border segment containing the 3′ end of the insert and a portion of the flanking wheat DNA sequence). Monsanto concludes that these experiments demonstrate the stability of the inserted DNA spanning six different seed generations.

3.4 Inheritance of the Roundup Ready® Trait

Monsanto tested the inheritance of the Roundup Ready® trait as follows: the original event MON 71800 R0 plant was selected on a glyphosate medium. Initial R1 plants were sprayed with Roundup® herbicide and evaluated for their resistance and sensitivity. Homozygous glyphosate-resistant R1 plants were identified by a PCR-based homozygosity assay and confirmed by spray testing their R2 progeny with Roundup® herbicide. One of the homozygous glyphosate-resistant plants from the R1 generation was used to increase the number of generations to R18 by self-pollination. These generations showed no decrease in vegetative or reproductive tolerance and no sensitivity to Roundup® confirming the stability of the Roundup Ready®trait.

Monsanto also described a Mendelian segregation study for the Roundup Ready® trait in event MON 71800. Monsanto selected heterozygous R1 plants and self pollinated them to produce progeny. The R2 plants were sprayed with Roundup® herbicide and scored for their resistance or sensitivity to the herbicide. The ratio of resistant to sensitive plants was found to be in conformance with the expected 3:1 ratio by the chi square test. Monsanto concludes that this result is consistent with a single insertion site of the Roundup Ready® trait in the wheat genome.

4. Expressed Proteins: the CP4 EPSPS Enzymes

4.1. Identity, Function and Characterization

Monsanto notes that the CP4 EPSPS enzyme is structurally and functionally similar to native plant EPSPS enzymes, which are involved in the biosynthesis of aromatic amino acids that are necessary for growth and development of the plant.

In order for EPSPS to function in plants, it must be transported to the chloroplast. In event MON 71800, the A. thaliana chloroplast transit peptide coding sequence was joined to the cp4 epsps coding sequence so that a fusion protein of CP4 EPSPS (47.6 kilodaltons (kDa), 455 amino acids) and the A. thaliana chloroplast transit peptide (1.6 kDa, 76 amino acids) would be expressed. The transit peptide directs the protein to the chloroplast. Typically, transit peptides are completely cleaved from the protein following delivery to the chloroplast. However, there are examples in the literature of alternatively processed forms where the transit peptide is only partially cleaved.

Monsanto isolated and purified CP4 EPSPS expressed in the grain of event MON 71800 and used the following techniques for characterization: sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); western blot analysis; glycosylation analysis; enzymatic assay; N-terminal amino acid sequence analysis; and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Monsanto identified two forms of the CP4 EPSPS protein in the grain of event MON 71800, i.e., a “mature form” and an “alternatively processed form”. The mature form is the full length CP4 EPSPS, where the transit peptide was fully cleaved; the alternatively processed form is the full length CP4 EPSPS protein plus seven amino acids from the transit peptide. Monsanto used the mature form of CP4 EPSPS produced in E. coli for comparison in the characterization analyses.

Based on quantitative image analysis of western blots, Monsanto reports that the relative percentages of the CP4 EPSPS proteins produced in event MON 71800 are 80% mature form and 20% alternatively processed form. Glycosylation analysis (ECL glycoprotein detection system) indicated that neither form of the protein is glycosylated. In addition, Monsanto concludes from enzyme activity assays that the average specific activity of both plant-produced CP4 EPSPS proteins is comparable to the specific activity of the protein produced in E. coli.

4.2. Expression Level and Human Exposure

Monsanto used a direct double antibody sandwich enzyme-linked immunosorbent assay (ELISA) analysis to estimate the levels of the CP4 EPSPS proteins in forage and grain tissues collected from event MON 71800. The forage and grain tissues from the parental line MON 71900 were used as control samples. Monsanto provides the results of the assay which show that the average level of CP4 EPSPS proteins are 106 μg/g in forage and 13 μg/g in grain on a fresh weight basis. Monsanto estimates a per capita exposure to the CP4 EPSPS proteins to be 0.039 mg/kg body weight/day assuming no loss due to food processing.

4.3. Presence in Food Crops

Monsanto discusses the similarity of the CP4 EPSPS proteins present in event MON 71800 to native EPSPS proteins that occur in plants and microorganisms and to CP4 EPSPS proteins present in bioengineered crops. Monsanto states that the CP4 EPSPS proteins expressed in event MON 71800 are similar to naturally occurring EPSPS proteins present in a variety of food and feed sources, such as, soybean, corn, and Baker’s yeast. Monsanto also states that the mature CP4 EPSPS protein expressed in event MON 71800 grain has been consumed by humans and animals since 1996, through the consumption of Roundup Ready® crops, such as soybean, corn and canola. The mature protein is identical to or shares greater than 99% amino acid sequence identity with the CP4 EPSPS proteins produced in these food crops. The alternatively processed form of CP4 EPSPS shares greater than 98% amino acid sequence identity with the CP4 EPSPS protein produced in other Roundup Ready® food crops.

4.4. Assessment of Potential Allergenicity

4.4.1. Donor

Agrobacterium species, the source of the CP4 EPSPS gene, are not known to be allergenic.

4.4.2. Amino Acid Sequence Homology

Monsanto searched the ALLPEPTIDES database for amino acid sequence homology to the CP4 EPSPS proteins found in event MON 71800 using the FASTA algorithm. The ALLPEPTIDES database is comprised of publically available protein sequences in SwissProt version 39+, TrEMBL (updated weekly), and GenPept version 124. Monsanto concluded that the CP4 EPSPS proteins do not demonstrate sequence similarity to proteins known to pose human health risks.

In addition, Monsanto screened all overlapping peptides of eight or more contiguous amino acids of the CP4 EPSPS proteins against the ALLERGEN3 database using a pairwise comparison algorithm. ALLERGEN3 is an allergen and gliadin protein sequence database compiled by Monsanto. Neither of the two forms of CP4 EPSPS contained sequences of eight or more contiguous amino acids identical to those from proteins in the allergen database.

4.4.3. Stability in Simulated Digestive Fluid

Monsanto examined the in vitro stability of both forms of CP4 EPSPS in simulated gastric fluids (SGF) prepared according to U.S. Pharmacopeia (1990). Stability was assessed by colloidal blue staining of SDS-PAGE gels, western blot analysis, and EPSPS enzyme activity assay.

In experiments performed with the purified E. coli-produced mature form of CP4 EPSPS, 95-98% of the protein was digested in SGF within 15 seconds, with no detectable degradation products present. The enzymatic activity of this form of CP4 EPSPS decreased by greater than 90% following SGF treatment for 15 seconds. Monsanto also refers to previous experiments which show that purified E. coli-produced mature form of CP4 EPSPS was digested in less than ten minutes in simulated intestinal fluid (SIF) (Harrison et al., 1996).

In experiments performed with the purified E. coli-produced alternatively processed form of CP4 EPSPS, 98% of the protein was digested in SGF within 15 seconds. The enzymatic activity of this form of CP4 EPSPS was lost following SGF treatment for 15 seconds. In addition, purified E. coli-produced alternatively processed form of CP4 EPSPS was degraded within four to eight hours of SIF treatment.

Monsanto performed additional experiments examining the in vitro stability of the CP4 EPSPS proteins produced in event MON 71800 grain in SGF. These experiments were performed to assess the stability of the CP4 EPSPS proteins within the matrix of other wheat proteins. The results indicated that these proteins were rapidly digested within a matrix of wheat grain proteins, with greater than 95% of CP4 EPSPS digested within 15 seconds.

Finally, Monsanto calculated that the amount of CP4 EPSPS proteins present in event MON 71800 grain is very low and represents only a small portion of the total protein. From these data and information, Monsanto concluded that the CP4 EPSPS proteins in event MON 71800 do not pose a significant allergenic risk.

4.5. Assessment of Potential Toxicity

Monsanto provided information about Agrobacterium sp. strain CP4, the donor of the cp4 epsps gene. Agrobacterium species are non-pathogenic and non-toxigenic. The safety of the Agrobacterium sp. strain CP4 has been previously evaluated during Monsanto’s consultations with FDA on other Roundup Ready&174; crops.

In two separate studies, Monsanto assessed the acute oral toxicity of the CP4 EPSPS proteins in mice. They used both the mature and alternatively processed CP4 EPSPS proteins, each expressed in E. coli. Each protein was administered by oral gavage. The highest doses tested were 572 mg/kg body weight for the mature protein and 1028 mg/kg body weight for the alternatively processed protein. Monsanto reports that no acute toxicity resulted from the oral administration of either protein in male or female mice at any of the doses administered in the experiments.

Monsanto compared the amino acid sequences of the CP4 EPSPS proteins expressed in the grain of event MON 71800 to the amino acid sequences of proteins (including toxins) available in the database ALLPEPTIDES using FASTA algorithm. Monsanto concluded that its search showed no relevant similarities between the CP4 EPSPS proteins present in event MON 71800 and proteins that are known to cause adverse health effects in humans or animals.

5. Food and Feed Uses of Wheat

Monsanto describes historical and current uses of wheat in food and animal feed. Wheat has been cultivated for use in food for several millennia. In the U.S., wheat is one of the most abundant crops in terms of planted acreage. Wheat grain contains approximately 60% carbohydrate, 10-16% protein, 2% fat, and 13% water. It is mainly used for the production of flour which is used in baked goods or other flour-based foods. U.S. consumers currently consume approximately 143 pounds of wheat flour per capita per year.

The use of wheat as animal feed is minor when compared to its use as food. Wheat grain is used as feed for poultry, swine, and cattle. It is estimated that on average 13 percent of total U.S. wheat grain has been utilized for animal feed in recent years. Wheat forage is also used as an animal feed source and is used for grazing cattle over the winter months in certain regions of the U.S.

Monsanto is not aware of any food or feed uses of standard spring wheat varieties that are not also applicable to spring wheat varieties containing event MON 71800.

6. Compositional Analysis of Roundup Ready® Wheat

Monsanto compared MON 71800 wheat to the parent line (Bobwhite MON 71900) and to commercially available non-transgenic spring wheat varieties.

6.1 Overview of the Approach to Compositional Analysis

To assess whether Roundup Ready® wheat event MON 71800 is as safe and nutritious as wheat varieties currently consumed, Monsanto conducted compositional analyses of grain and forage from event MON 71800, the non-transgenic parent line MON 71900 (Bobwhite), and several commercial wheat varieties. To conduct these compositional analyses, Monsanto collected both grain and forage samples from field trials and purchased commercially available grain produced from certified seed.

Monsanto planted event MON 71800, the parental line MON 71900, and 18 different commercial lines at five North American sites (three sites in the U.S. and two sites in Canada). Monsanto considered that these five sites provided a variety of environmental conditions representative of regions where Roundup Ready® wheat can be grown as a commercial product. Monsanto analyzed 76 components in grain collected from event MON 71800, the parental line MON 71900, the 18 different commercial spring wheat lines that Monsanto grew as part of its field trials, and grain from 4 spring wheat varieties purchased from certified seed producers. Monsanto also analyzed 9 components in forage tissues collected from event MON 71800, the parental line MON 71900, and the 18 different commercial spring wheat lines that Monsanto grew as part of its field trials.

Monsanto used standard analytical methods or other suitable methods for compositional analyses and provides references and descriptions for all analytical methods used. Monsanto provided the analytical results for the five test sites individually, and for all the sites combined. Monsanto reported the level of each analyzed component (mean and range) in the transgenic line MON 71800 and non-transgenic control line MON 71900; the difference between the level of each component (mean and range) obtained for line MON 71800 and line MON 71900; the range of levels obtained for 22 commercial lines; and the historical range based on values in the literature.

Monsanto subjected all of the compositional data obtained for Roundup Ready® wheat event MON 71800 and the parental control line MON 71900 to statistical analysis using a mixed model analysis of variance. Monsanto conducted the statistical analysis on data from each of the five replicated trials, as well as on data combined from all five trials.

6.2 Grain

Monsanto determined the levels of the following components of wheat grain:

• Proximates: protein, fat, carbohydrate (by calculation), ash and moisture
  
• Sugars: arabinose, fructose, galactose, glucose, maltose, raffinose, sucrose and xylose
  
• Total dietary fiber (TDF)
  
• Amino acid composition
  
• Fatty acid composition (16:0 palmitic, 16:1 palmitoleic, 18:0 stearic, 18:1 oleic, 18:2 linoleic, 18:3 linolenic, 20:0 arachidic, and 20:1 eicosenoic)
  
• B vitamins: niacin, riboflavin (vitamin B₂), thiamin (B₁), and vitamin B₆
  
• Vitamin E
  
• Minerals: cadmium, calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, and zinc
  
• Starch

Monsanto also analyzed wheat grain for 14 other fatty acids, sodium, and the sugars mannose and stachyose; however, these substances were not detected in any samples tested (i.e., either from line 71800, line 71900, or commercial varieties).

Based on the combined statistical analysis of data from all test sites, Monsanto did not identify any statistically significant differences in grain composition between the event MON 71800 line and the parental MON 71900 line. Monsanto did identify some statistically significant differences (p<0.05) in composition between MON 71800 and MON 71900 at individual sites. In total, Monsanto found small differences in the levels of 16 components. However, these differences were found at only one or two test sites, and were not consistent over all trials. Monsanto also reported that for grain, all measured parameters fell within the range detected by Monsanto for the commercial varieties. The values also fell within the range found in the literature, except for the two amino acids, serine and valine; however, there were no differences in the levels of these amino acids when comparing MON 71800, MON 71900, or the commercial wheat varieties. Monsanto concludes that grain from event MON 71800 is compositionally equivalent to that of the non-transgenic parental control wheat and other wheat varieties grown commercially.

6.3 Forage

Monsanto determined the levels of the following components of wheat forage:

• Proximates: protein, fat, carbohydrate (by calculation), ash and moisture
  
• Fiber: acid detergent fiber, neutral detergent fiber
  
• Minerals: calcium and phosphorus

Based on the combined statistical analyses from individual test sites, Monsanto identified no statistically significant differences (p<0.05) in composition between forage samples from MON 71800 and MON 71900. In the individual tests, Monsanto found significant (p<0.05) differences in the levels of five components from two out of five test sites. However, the differences were small and were not consistent over all locations as indicated by the combined analysis. In addition, the range of values for those components fell within the interval of values determined for the commercial varieties and within values reported in the literature.

Monsanto concludes that the forage from event MON 71800 is compositionally equivalent to that of the non-transgenic parental control wheat and other wheat varieties grown commercially.

6.4 Antinutrients

Phytic acid is an antinutrient that occurs naturally in wheat. Monsanto measured phytic acid in the grain of all test and control lines. The mean level of phytic acid in event MON 71800 was comparable to that of the parental line MON 71900 and fell within the range established for the 22 commercial lines and within the historical range for wheat based on values in the literature.

6.5 Endogenous Allergens

Because wheat is known to cause allergic reactions in sensitive individuals, Monsanto performed an evaluation to assess whether the transformation process may have increased the overall allergenicity of wheat grain from event MON 71800. Monsanto conducted IgE-inhibition ELISAs and IgE-immunoblot analysis with extracts from event MON 71800, parental MON 71900, and seven other commercial varieties of wheat. Sera used in these assays were obtained from ten human subjects with IgE-mediated allergic response to wheat consumption as evidenced by clinical history and response in single or double blind oral food challenge. From these experiments, Monsanto concluded that the transformation process did not significantly alter endogenous allergens in event MON 71800, because IgE binding properties were similar to those of other commercial wheat varieties.

6.6 Gluten

Celiac disease (gluten-sensitive enteropathy) is caused by a specific immune response to antigens present in gluten in susceptible individuals. Gluten refers to a mixture of glutenin and gliadin proteins present in wheat, barley and rye. Monsanto measured gliadin levels and calculated gluten levels in event MON 71800, parental MON 71900, and 22 other commercial wheat varieties. Gliadin and gluten levels in event MON 71800 are comparable to those of the parental and other commercial varieties tested.

6.7 Summary of Compositional Analysis

Monsanto concludes that the results of its compositional analysis established that the levels of nutrients and other components of the grain and forage derived from event MON 71800 fall within the ranges found for commercially available wheat varieties. Monsanto considered that the few minor differences in the levels of certain components seen at individual test sites are unlikely to be meaningful, and that grain and forage from event MON 71800 are compositionally equivalent to that of the non-transgenic parental control wheat and other wheat varieties grown commercially.

7. Conclusions

Monsanto has concluded that Roundup Ready® wheat event MON 71800 is not materially different in composition, safety, or any other relevant parameter from wheat now grown, marketed, and consumed. At this time, based on Monsanto’s data and information, the agency considers Monsanto’s consultation on Roundup Ready® event MON 71800 wheat to be complete.

Jason Dietz