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Biotechnology Consultation
Note to the File
BNF No. 000090
Date: August 7, 2004
Subject: Glyphosate-tolerant sugar beet event H7-1
Keywords: sugar beet, Beta vulgaris, glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Agrobacterium sp. strain CP4, Roundup®, herbicide tolerant
In a submission dated April 16, 2003, Monsanto Company (Monsanto) and KWS SAAT AG (KWS) (jointly the notifiers) submitted a safety and nutritional assessment of Roundup Ready® (glyphosate-tolerant) sugar beet event H7-1. The notifiers provided additional information in support of this safety and nutritional assessment on December 4, 2003. The notifiers concluded that food and feed derived from glyphosate-tolerant sugar beet event H7-1 are as safe and nutritious as food and feed derived from conventional varieties currently being marketed.
This current submission is in addition to a previously completed consultation for glyphosate-tolerant sugar beet event 77 in BNF 000056. Monsanto has previously completed consultations for other Roundup Ready® crops which are also tolerant to glyphosate. These other crops include bioengineered soybean (BNF 000001), canola (BNF 000020, BNF 000077), corn (BNF 000035, BNF 000051, BNF 000071), and cotton (BNF 000026).
The intended effect of the genetic modification is to confer tolerance to the herbicide glyphosate [N-phosphonomethyl glycine], which is the active ingredient in Roundup®. Resistance is conferred by the introduction of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) derived from Agrobacterium sp. strain CP4 into sugar beet. Glyphosate functions as a herbicide by inhibiting the endogenous EPSPS enzyme, which catalyzes a reaction necessary for the synthesis of aromatic amino acids. The EPSPS enzyme derived from Agrobacterium sp. strain CP4 that is introduced by the H7-1 transformation event has reduced affinity for glyphosate as compared with the native plant EPSPS enzyme. Therefore, the presence of the new enzyme in plant tissues prevents deleterious effects of glyphosate on aromatic amino acid synthesis in this transgenic sugar beet variety.
The parental variety is a KWS proprietary multi-germ sugar beet line known as 3S0057.
The notifiers constructed the double border, binary plasmid vector PV-BVGT08 for the transformation of sugar beet line 3S0057. The plasmid contains a single copy of the EPSPS expression cassette. The T-DNA region of PV-BVGT08 contains the following elements intended to be incorporated into the target sugar beet genome during the Agrobacterium-mediated transformation process.
| Name | Description |
|---|---|
| RB | Right Border Sequence derived from Agrobacterium tumefaciens |
| P-FMV | Modified figwort mosaic virus 35S promoter |
| ctp2 | Chloroplast transit peptide sequence from the EPSPS gene from Arabidopsis thaliana |
| cp4 epsps | 5-enolpyruvylshikimate-3-phosphate synthase enzyme (EPSPS) gene derived from Agrobacterium sp. strain CP4 |
| E9 3' | Polyadenylation sequence from the Pisum sativum rbcS E9 gene |
| LB | Left Border Sequence derived from Agrobacterium tumefaciens. |
Plasmid PV-BVGT08 also contains several genes necessary for maintenance and selection of the plasmid in the intermediate host Escherichia coli that are not ultimately incorporated into the plant genome. Plasmid PV-BVGT08 contains both a vegetative and bacterial origin of replication that allow replication of the plasmid in both A. tumefaciens and E. coli. The plasmid contains the aad gene encoding the Tn7 AAD 3' adenyltransferase that provides resistance to spectinomycin and streptomycin. The plasmid also contains a sequence, known as rop, which represses the formation of RNA primer allowing maintenance and copy number control of the plasmid in E. coli.
The notifiers report that KWS sugar beet line 3S0057 was transformed with the T-DNA region of the plasmid vector PV-BVGT08, resulting in the introduction of a single copy of the EPSPS expression cassette into the sugar beet genome. The notifiers confirmed the size of the insert as approximately 3.4 kilobase pairs. Transformed cells were selected by application of glyphosate.
The notifiers used Southern blot analysis to characterize the DNA introduced in event H7-1. Genomic DNA from event H7-1 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, the notifiers conclude that event H7-1 contains a single copy of the EPSPS expression cassette. Components of the cassette (P-FMV, ctp2, cp4 epsps, E9 3') are intact. The notifiers also conclude that event H7-1 does not contain plasmid backbone sequences.
The notifiers used an inverse-PCR technique to identify the 5' and the 3' genomic DNA sequences that flank the integrated insert in the transformation event H7-1. This analysis showed that the integrated cassette contained a nearly complete Left Border sequence and was devoid of the Right Border sequence.
To examine the inheritance of the glyphosate tolerance trait, the notifiers conducted numerous crosses using conventional breeding techniques resulting in 27 breeding experiments over four generations. These studies indicate that the introduced trait (glyphosate tolerance) was stably inherited through generations and segregated according to the Mendelian inheritance pattern.
To ascertain whether the integrated DNA in event H7-1 was stably incorporated into the genome, the notifiers performed Southern blot analyses on genomic DNA from generational descendants (F2, F3, F4) of the original transformant. The notifiers state that these analyses demonstrate that the inserted DNA, containing the full-length EPSPS expression cassette, was stably integrated at a single locus in all progeny.
The CP4 5-enolpyruvylshikimate-3-phosphate synthase expressed in sugar beet event H7-1 is a 47.6 kDa protein consisting of a single polypeptide of 455 amino acids. Native plant EPSPS enzymes are involved in the biosynthesis of aromatic amino acids that are necessary for growth and development of the plant. The CP4 EPSPS protein is similar in structure and function to native plant EPSPS enzymes, but has a much reduced affinity for glyphosate. Western blot analysis shows that CP4 EPSPS protein in event H7-1 leaf tissue and E. coli produced CP4 EPSPS protein are equivalent in molecular weight and are recognized by antibodies specific for the CP4 EPSPS protein.
Field trials were conducted at six locations in Europe in sugar beet producing regions. The event H7-1 sugar beets were treated with Roundup® herbicide. Samples of both brei (processed roots) and tops (leaves) were collected from each location and analyzed for their concentration of CP4 EPSPS by a double-antibody sandwich ELISA using a mouse monoclonal anti-CP4 EPSPS antibody as the capture antibody and a goat polyclonal anti-CP4 EPSPS conjugated to horseradish peroxidase (HRP) as the detection antibody. The mean level of the CP4 EPSPS protein in top tissue was 161 μg/g with a range of 112-201 μg/g (fresh weight). The mean level of the CP4 EPSPS protein in brei was 181 μg/g with a range of 145-202 μg/g (fresh weight).
The notifiers conclude that the concentration of CP4 EPSPS protein in sugar beet H7-1 tissues is very low, and cite a publication documenting that refined sugar produced from sugar beets does not contain detectable levels of protein. Therefore, the notifiers have not calculated human exposure to CP4 EPSPS. The notifiers also describe the history of safe use of CP4 EPSPS expressed in other crops and the similarity between native plant EPSPS enzymes and CP4 EPSPS.
The notifiers describe an acute toxicity study conducted in mice where the mice were dosed by gavage with up to 572 mg/kg of CP4 EPSPS. The notifiers report that no adverse events were observed at any dose level.
The notifiers compared the amino acid sequence of CP4 EPSPS to protein sequences in the ALLPEPTIDES database using the FASTA algorithm. The notifiers report that no biologically relevant sequence similarities between CP4 EPSPS protein and known toxins were observed.
The notifiers describe their assessment of the allergenic potential of CP4 EPSPS protein. The notifiers state that the CP4 EPSPS protein was derived from Agrobacterium sp. strain CP4 and that there have been no reports of allergy to Agrobacterium species. The notifiers searched the ALLERGEN3 database (compiled by Monsanto) containing sequences of known allergens and gliadin1 for amino acid sequence homology to the CP4 EPSPS protein. The notifiers conclude that there was no immunologically significant amino acid sequence homology between the CP4 EPSPS protein and amino acid sequences of allergens in the ALLERGEN3 database.
The notifiers discuss two studies performed to assess the in vitro digestibility of CP4 EPSPS. In both studies, the CP4 EPSPS protein expressed in E. coli was used. In the first study, the CP4 EPSPS protein was exposed to simulated gastric and intestinal fluids that were prepared according to the U.S. Pharmacopoeia (1990). The degradation of the protein was assessed by western blot analysis. The half-life of the CP4 EPSPS protein was reported to be less than 15 seconds in the gastric fluid and less than ten minutes in the intestinal fluid (Harrison, et al., 1996).
In the second study, the in vitro digestibility of CP4 EPSPS was assessed in simulated gastric fluid (SGF) that contained a new formulation of the peptic enzyme pepsin. The degradation of CP4 EPSPS in SGF was assessed by the SDS-PAGE colloidal blue gel staining method, western blot analysis, and EPSPS enzymatic activity assay. The notifiers report that SDS-PAGE colloidal blue staining indicated that 98% of the CP4-EPSPS protein was digested within 15 seconds, with no degenerative bands observed. Western blot analysis indicated that greater than 95% of the CP4-EPSPS protein was digested within 15 seconds, and the enzymatic activity assay indicated that the EPSPS activity was reduced to less than 10% of the initial activity within 15 seconds of exposure to SGF.
The notifiers further note that the CP4 EPSPS protein represents no more than 1.44% of the total protein in event H7-1 root samples on a dry weight basis and that sugar beet processing to refined sugar would remove all protein. The notifiers conclude that the CP4 EPSPS protein in event H7-1 is safe for human and animal consumption.
The main food use of sugar beet, Beta vulgaris, is for the extraction of sucrose from sugar beet root tissue through a process where the root tissue is extracted with water, followed by purification, evaporation, and separation of sucrose crystals by centrifugation. The final product is granular sugar. This process also yields sugar beet molasses and sugar beet pulp, which are often pelleted and used in animal feed. The leafy sugar beet "tops" are usually left in the field, but they may occasionally be fed to ruminant animals.
To assess whether glyphosate tolerant sugar beet event H7-1 is as safe and nutritious as conventional sugar beet varieties currently consumed, the notifiers compared the composition of the hybrid lines containing event H7-1, produced through conventional breeding, to the composition of the corresponding non-transgenic, segregant control lines derived from the breeding process and eight commercial sugar beet varieties.
The notifiers collected sugar beet samples for compositional analyses from field trials conducted in 1999 at five locations in Europe in traditional sugar beet production areas (France, Germany, Italy, Spain and the United Kingdom). At each location, the hybrid plants containing event H7-1, the non-transgenic control plants, and eight commercial varieties were grown. Event H7-1 plants were treated with Roundup® herbicide during the growing season.
The notifiers analyzed tops (leaves) and brei (processed roots) using standard analytical methods or other suitable methods. These methods are briefly described in the notice. The notifiers provide analytical results as means and ranges from replicated samples from all test sites combined. The notifiers conducted the statistical analysis on the mean values to detect statistically significant differences between event H7-1 and its non-transgenic control at p<0.05. The notifiers also provide the mean values and ranges for the commercial varieties as well as published values. In a separate trial, the notifiers measured acid detergent fiber (ADF) and neutral detergent fiber (NDF) in brei in H7-1 and 5 non-bioengineered varieties of sugar beets.
The notifiers analyzed the samples of sugar beet tops and brei for the following components:
For brei, the notifiers report detecting no statistically significant differences in the mean levels of proximate components and quality parameters between event H7-1 and non-transgenic segregant controls. The notifiers did report detecting statistically significant differences between event H7-1 and non-transgenic controls in the levels of two out of eighteen amino acids analyzed, with alanine level being statistically significantly higher and glutamic acid level being statistically significantly lower in event H7-1 than the corresponding levels in non-transgenic controls. However, the notifiers note that the mean values for these two amino acids in event H7-1 fall within the ranges reported for both the non-transgenic control and commercial varieties. Therefore, the notifiers do not deem these differences to be biologically significant. In a separate trial, the notifiers measured ADF and NDF in H7-1 and 5 commercial non-bioengineered varieties of sugar beets and found that the values obtained for H7-1 were within the range of values measured in the commercial varieties. The notifiers conclude that brei from event H7-1 is compositionally equivalent to that of the non-transgenic control and other commercial sugar beet varieties.
For the top tissue, the notifiers report finding a statistically significant difference (p<0.05) between event H7-1 and non-transgenic controls in the levels of dry matter and four out of eighteen amino acids measured. The level of alanine in event H7-1 was statistically significantly higher and the levels of histidine, phenylalanine, and tyrosine were statistically significantly lower than the corresponding levels in non-transgenic controls. However, the notifiers note that the mean levels of dry matter and the four amino acids fall within the ranges reported for both the non-transgenic control and commercial varieties. Therefore, the notifiers do not deem these differences to be biologically significant. The notifiers conclude that the top tissue from event H7-1 is compositionally equivalent to that of the non-transgenic control and commercial sugar beet varieties.
The notifiers analyzed the root and top samples from event H7-1, the non-transgenic control, and the reference commercial varieties for saponins. Saponins are triterpenoid glycosides that occur naturally in many plant foods such as legumes, potatoes, tea and sugar beet. Saponins have a bitter taste and can act as a deterrent to foraging. The notifiers report that the saponin levels in event H7-1 tissues were not statistically significantly different from the levels in non-transgenic controls and were comparable to the levels in the tissues of the commercial reference varieties.
The notifiers conclude that glyphosate tolerant sugar beet event H7-1 is not materially different in composition, safety, or other relevant parameters from sugar beet now grown, marketed, and consumed. At this time, based on the notifiers' data and information, the agency considers the notifiers' consultation on glyphosate tolerant sugar beet event H7-1 to be complete.
/s/
Richard E. Bonnette
(1)Gliadin is a protein found in wheat and other grass family grains. Exposure to gliadin may cause or exacerbate celiac disease (gluten-sensitive enteropathy), an immunologically-mediated disease, in genetically predisposed persons.
