GM Crop Database

Database Product Description

H7-1 (KM-ØØØ71-4)
Host Organism
Beta vulgaris (Sugar Beet)
Trait
Herbicide tolerant, glyphosate.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2005
Canada 2005 2005 2005
Colombia 2005 2010
European Union 2007 2007
Japan 2003 2007 2007
Korea 2006
Mexico 2006 2006
New Zealand 2005
Philippines 2005 2005
Russia 2006
Singapore 2014
Taiwan 2015
United States 2004 2004 2005

Introduction Expand

Sugar beet line H7-1 was developed using recombinant DNA techniques to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in sugar beet crops. The novel plants express an herbicide tolerant form of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) derived from the common soil bacterium, Agrobacterium tumefaciens strain CP4. Glyphosate specifically binds to and inactivates EPSPS, which is involved in the synthesis of the aromatic amino acids, tyrosine, phenylalanine and tryptophan (shikimate biochemical pathway). EPSPS is present in all plants, bacteria, and fungi but not in animals, which must obtain these essential amino acids from their diet. 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. The EPSPS enzyme is normally present in food derived from plant and microbial sources.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT figwort mosaic virus (FMV) 35S chloroplast transit peptide from A. thaliana P. sativum (pea) ribulose-1,5-bisphosphate carboxylase small subunit non-translated region 1 functional Native

Characteristics of Beta vulgaris (Sugar Beet) Expand

Center of Origin Reproduction Toxins Allergenicity

Beta is considered an Old World genus basically confined to the Mediterranean Basin and Middle East.

Sugar beet is an outcrossing, largely wind pollinated, plant. During the reproductive phase, large amounts of pollen are produced which can travel long distances. Sugar beet hybridizes freely with all members of the section Beta (within genus Beta).

Saponins (triterpenoid glycosides) are the only known toxicants found in sugar beet and are actively eliminated in sugar processing.

There are rare reports of allergic reactions in sugar beet field workers, associated with exposure to the seeds of the plant.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Agrobacterium tumefaciens strain CP4 CP4 epsps

Agrobacterium tumefaciens is a common soil bacterium that is responsible for causing crown gall disease in susceptible plants. There have been no reports of adverse effects on humans or animals.

Modification Method Expand

Sugar beet line H7-1 was produced by Agrobacterium-mediated transformation of the sugar beet line 3S0057 with the binary plasmid vector PV-BVGT08. The T-DNA region of the plasmid vector contained sequences encoding CP4 EPSPS from Agrobacterium tumifaciens strain CP4. Associated with the CP4 EPSPS encoding gene were modified promoter sequences from the 35S transcript of figwort mosaic virus (P-FMV), sequences encoding the chloroplast transit peptide (ctp2) from the Arabidopsis thaliana EPSPS gene, and transcription termination and polyadenylation signal sequences from the ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) E9 encoding gene from pea (Pisum sativum). Right border and left border sequences of the T-DNA region of the plasmid vector were derived from Agrobacterium tumefaciens. The plasmid also contained a vegetative and bacterial origin of replication for maintenance and replication of the plasmid in both A. tumefaciens and E. coli. The aad gene, encoding the Tn7 AAD 3’ adenyltransferase that provides resistance to spectinomycin and streptomycin, was also present in the plasmid, as well as rop, a sequence that helps to maintain and control the copy number of the plasmid in E. coli.

The plasmid vector PV-BVGT08 did not contain marker gene sequences. The selection of successfully transformed cells was achieved by applying glyphosate and screening for tolerant cells.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analyses of the genomic DNA from H7-1 confirmed the existence of a single intact copy of the CP4 EPSPS expression cassette, including the promoter, terminator, and chloroplast transit peptide sequences. The analyses also confirmed that none of the plasmid backbone sequences were integrated into the genome of H7-1. Further analyses demonstrated the nearly complete integration of the Left Border sequence and the omission of the Right Border sequence.

Genetic Stability of the Trait

Southern blot analyses were conducted on genomic DNA from the F2, F3 and F4 generations of the original transformant. The results demonstrated the stable integration of the CP4 EPSPS expression cassette at a single locus in all generations. The results of Mendelian segregation analysis confirmed that the glyphosate-tolerance trait is inherited as a dominant trait.

Expressed material

Levels of CP4 EPSPS were quantified in samples of H7-1 processed roots (brei) and leaves (tops) from 6 field trials conducted in Europe. Quantification was achieved using a double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) technique, and results were expressed on a fresh weight basis. The mean level of CP4 EPSPS in leaves was 161 µg/g (range: 112 – 201 µg/g), and 181 µg/g (145-202 µg/g) in processed roots.

Sugar beet intended for human consumption is generally converted directly to refined white sugar through extensive purification processes. The levels of CP4 EPSPS protein introduced into H7-1 sugar beet were not measured in refined sugar. It was assumed that since refined sugar does not contain detectable levels of protein, there would be virtually no human exposure to CP4 EPSPS. Consequently, there is no anticipated human exposure to this protein as a result of consumption of refined sugar derived from H7-1 sugar beet.

Environmental Safety Considerations Expand

Field Testing

H7-1 was field tested in the United States from 1998 to 2002, and in Europe (France and Germany) in 1998-99. In addition to evaluation in different environments, H7-1 was also evaluated in different genetic backgrounds that represented regionally adapted cultivars. Field trial evaluations included agronomic performance, disease and insect resistance, leaf morphology, seed germination and dormancy, bolting, flowering onset and seed harvest date. In most field trials, H7-1 was compared to a control that represented a nearly equivalent genotype. No significant differences were observed in any of these characteristics between H7-1 and the non-transgenic controls.

In only 6 out of 98 trials conducted in the United States were significant differences in disease and insect susceptibilities observed between H7-1 and the control and conventional cultivars. No differences were observed in the European trials. Some of these differences represented increased susceptibility, while others represented a decrease compared to the controls. Since there were no trends associated with these differences for H7-1, these were likely due to interactions between environment and genotype, rather than with the novel protein in H7-1.

Outcrossing

Sugar beet is an outcrossing species, and is mainly wind pollinated. It is a biennial, developing a rosette of leaves and a large taproot in the first year, and bolting (i.e., developing an inflorescence bearing stem) in the second year. Bolting is induced only after the plant has undergone vernalization at the end of the first season. In exceptional circumstances, bolting may be induced in the first year by low temperatures and longer daylength (e.g., by growing a cultivar outside of its adapted area). Large amounts of pollen are produced during the reproductive phase; this pollen can be wind-borne over long distances.

The genus Beta is comprised of four sections: i) Beta, to which sugar beet belongs; ii) Corollinae; iii) Nanae; and iv) Procumbentes. All species within the Beta section (B. vulgaris ssp. maritima, B. macrocarpa) are sexually cross-compatible and produce fertile progeny. Hybrids between sugar beet and members of the other sections do not occur naturally, and cross-breeding with other members of the Chenopodiaceae is unlikely. Assuming proximity, synchronicity of flowering, and suitable conditions sugar beet may freely hybridize with the botanical varieties within the vulgaris subspecies.

Wild relatives that are sexually compatible with sugar beet exist in parts of Europe, Asia, and Central and South America. Hybrids resulting from crosses between cultivated sugar beets and B. macrocarpa, and those with B. vulgaris ssp. maritima have been reported in Europe. The potential therefore exists for introgression of the glyphosate tolerance trait from H7-1 into these wild relatives. In North America, concerns have been raised about potential outcrossing with populations of B. macrocarpa in the Imperial Valley of California. Studies have indicated the introgression of genes from commercial sugar beets, although other reports show that gene flow between these two populations is not likely due to non-synchronous flowering and the production of sterile F1 hybrids. Sugar beet plants have escaped from past commercial cultivation in the San Francisco Bay area and persist to this day. However, introgression of the glyphosate tolerance trait into these plants is highly unlikely as sugar beets are no longer in commercial production in that area.

As with conventional sugar beet cultivars, H7-1 plants are harvested prior to the initiation of the bolting phase. Since sugar beets are usually not allowed to bolt, except in fields or plots grown for seed production, there is little opportunity for uncontrolled pollen flow. Sugar beets grown for seed production are also spatially isolated from other sugar beet fields to minimize pollen flow. Since the cultivation practices for H7-1 sugar beet are identical to those for conventional sugar beet varieties, the potential for outcrossing would only exist when H7-1 is grown for seed production.

Glyphosate-tolerant hybrids resulting from the introgression of the transgene from H7-1 to commercial sugar beet cultivars, or compatible wild relatives, would possess no other competitive advantage than herbicide tolerance. This advantage would only be expressed in managed ecosystems, such as cultivated fields. Glyphosate-tolerant hybrids could be managed with conventional weed control practices, including herbicides other than glyphosate.

Weediness Potential

Sugar beet plants are not a serious weed, although sugar beets have escaped cultivation and their progeny have persisted in the environment for many years. Sugar beets may also volunteer in fields the year following cultivation. Volunteers may also arise from the presence of wild beet, the bolting of beet plants, the development of groundkeepers which arise initially form vegetative growth of beet crowns or tops left after harvest or the germination of seed (which may be dormant in soil for up to 10 years). Volunteer plants may be controlled by mechanical means or by the use of herbicides registered for use on sugar beet volunteers.

Field trials were conducted to address concerns that H7-1 sugar beet might become weedier, compared to conventional sugar beet cultivars. H7-1 and its control lines were evaluated for plant growth and reproductive characteristics related to persistence and competitiveness. Results from these trials revealed no significant differences between H7-1 and the control lines, for characteristics such as plant vigour, bolting, seedling emergence, seed germination, and seed dormancy. Based on these results, H7-1 is not expected to become weedier, nor is it expected to become invasive of natural habitats, compared to conventional sugar beet cultivars.

Secondary and Non-target Adverse Effects

The herbicide-tolerance trait in H7-1 sugar beet is conferred by the novel protein CP4 EPSPS, an enzyme originally isolated from the soil bacterium Agrobacterium tumifaciens strain CP4. The novel form of this enzyme is similar to that naturally found in plants, bacteria and fungi. The lack of toxicity of CP4 EPSPS has been substantiated in previous regulatory approvals of glyphosate-tolerant plants, and its high substrate specificity makes it unlikely to metabolize any other endogenous substances to produce toxic compounds. The cultivation of H7-1 sugar beet would therefore not be expected to result in deleterious effects on non-target and beneficial organisms that would be attributed to the novel protein. This was also supported by field trial observations that revealed no negative effects from the cultivation of H7-1 on honeybees and earthworms, compared to conventional sugar beet cultivars.

Results from field trials to evaluate disease and insect susceptibilities demonstrated generally that H7-1 was not more susceptible than conventional sugar beet cultivars. H7-1 would therefore not be expected to become a host for plant pathogens, or insect pest, any more than conventional sugar beet cultivars.

Impact on Biodiversity

H7-1 sugar beet did not exhibit any novel characteristics that would increase its weediness, or cause it to become an invasive species, compared to conventional sugar beet. The expression of the novel protein (CP4 EPSPS) in H7-1 has not been observed to negatively impact non-target or beneficial organisms. While the potential for gene transfer from H7-1 to cultivated sugar beet is possible, the transfer of the herbicide tolerance trait to wild relatives in the United States is insignificant. However, this is not the case in other areas, such as Europe, where wild relatives of sugar beet exist. The introgression of the glyphosate tolerance trait into wild relatives such as B. vulgaris ssp. maritima (sea beet) is therefore possible.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Sugar beet intended for human consumption is generally converted directly to refined white sugar through extensive purification processes. The levels of CP4 EPSPS protein introduced into H7-1 sugar beet were not measured in refined sugar. CP4 EPSPS was expressed at very low levels in the roots, and since refined sugar does not contain detectable levels of protein, there would be virtually no human exposure to CP4 EPSPS. Consequently, there is no anticipated human exposure to this protein as a result of consumption of refined sugar derived from H7-1 sugar beet.

Nutritional and Compositional Data

Detailed analyses conducted over multiple years and environments were carried out to establish the nutritional composition of H7-1 sugar beet. Analyses included crude ash, crude fibre, crude fat, crude protein, carbohydrate (by calculation), dry matter, acid detergent fibre and neutral detergent fibre in both tops and processed roots (brei). Additional quality components were measured in brei including, invert sugar content, polarization (% sucrose), sodium, potassium and alpha-amino nitrogen. No biologically meaningful differences in any compositional and quality parameters were identified between H7-1 sugar beet and the non-transgenic controls. It was concluded that H7-1 sugar beet is equivalent to other commercially available sugar beet with respect to composition and nutritional quality.
The presence of naturally occurring toxins and allergens in glyphosate-tolerant sugar beet line H7-1 was investigated. Saponins are the only known toxicants found in sugar beet and are actively eliminated in sugar processing. These are also considered antinutrients; feed intake is reduced due to the bitter taste imparted by these compounds. Saponins were quantified in tops and root and compared to the non-transgenic control. Levels of saponin in H7-1 did not differ significantly from those of the control lines, and were within the range observed for conventional sugar beet cultivars.

Toxicity and Allergenicity

The potential for toxicity and allergenicity of the CP4 EPSPS protein in H7-1 sugar beet was assessed. An amino acid sequence homology search, using the ALLPEPTIDES database, revealed no similarities between CP4 EPSPS and known toxins. A sequence homology search conducted using the ALLERGEN3 database also revealed no similarities between the novel protein and known allergens, including gliadin. The CP4 EPSPS protein was degraded within one minute of exposure to simulated gastric and intestinal fluids, and the enzymatic activity reduced to less than 10% of the initial activity within 15 seconds of exposure. No adverse effects were observed in an acute toxicity study where mice were fed (by gavage) doses of CP4 EPSPS up to 572 mg/kg.

The results of the digestibility and acute oral toxicity studies, sequence homology searches, and the very low expression levels of the novel protein in roots (1.44% of total protein) support the conclusion that the H7-1 is neither toxic nor allergenic, and is as safe as conventional sugar beet varieties.

Abstract Collapse

Sugar beet (Beta vulgaris L. ssp. vulgaris var. altissima) had a global harvest of 256 million metric tonnes in 2006. The major producers of sugar beet in 2004 were France, the United States, Germany, Russian Federation, Turkey, Ukraine and Poland. Sugar beet is a botanical variety of B. vulgaris ssp. vulgaris, as are other comestible and fodder beets, and is a member of the goosefoot (Chenopodiaceae) family. It is an important source of sugar (sucrose), accounting for approximately 40 percent of global production.

Sucrose is present in limited quantities in many plants, including various palms and the sugar maple, but sugar beet and sugarcane are the only commercially important sources. More than half of the world sugar supply is obtained from sugarcane, which is grown in tropical and subtropical climates. The rest is supplied by the sugar beet, which is grown in temperate countries. Sugar is manufactured from the roots of the sugar beet; the leaves and tops are removed after harvesting and used as livestock feed. The roots are cut into cossettes, or chips, from which the juice is extracted. The juice is processed to yield sugar and beet molasses, and the remaining pulp is used for domestic animal feed. Beet molasses is also fed to livestock; table molasses is not made from sugar beets because of difficulties in purification. The sugar (sucrose) that is produced from the sugar beet is identical to that derived from sugarcane. It is widely used as a sweetener for foods, in preserves, and in the manufacture of candies, baked goods, and soft and alcoholic beverages. Sugar is also used as the raw material for fermentation products such as ethyl alcohol, butyl alcohol, glycerin, citric acid, and levulinic acid. Sugar is an ingredient in some transparent soaps, and it can be converted to esters and ethers, some of which yield tough, insoluble, and infusible resins.

Effective weed management is critical to sugar beet production. Weeds cause significant losses in sugar beet yield and crop quality, and also cause harvesting problems. The low growth habit of the sugar beet plant renders it more susceptible to competition from weeds compared to other crops, such as corn or soybean. Many weed species grow taller than sugar beet and thus aggressively compete for light, water and nutrients. The economic threshold for weed control in sugar beet production is therefore quite low, given the high value of the crop, and the significant losses caused by the presence of weeds. Weeds control strategies include the use of herbicides alone, and herbicides in combination with hand weeding. Sugar beet herbicides can be applied prior to crop emergence (e.g., triallate, EPTC, ethofumesate), and post-emergence (e.g., ethofumesate, triflusulfuron, desmedipham, phenimedipham, sethoxydim).
H7-1 sugar beet was developed to allow for the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option. This genetically engineered sugar beet line contains a form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that allows it to survive the otherwise lethal application of glyphosate. The EPSPS gene that was introduced into H7-1 was isolated from the common soil bacterium Agrobacterium tumefaciens, strain CP4 and the form of 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, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic 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.

The sugar beet line H7-1 was field tested in the United States from 1998 to 2002, and in Europe (France and Germany) in 1998-99. H7-1 was evaluated extensively and no differences were found in agronomic performance, disease and insect resistance, leaf morphology, seed germination and dormancy, seedling and plant vigour, bolting, flowering onset, and seed harvest date when compared to conventional sugar beet cultivars. Field observations demonstrated that the cultivation of H7-1 did not negatively affect beneficial and non-target organisms, compared to that of conventional sugar beet.

Sugar beet is an outcrossing species, and is mainly wind pollinated. It is a biennial, developing a rosette of leaves and a large taproot in the first year, and bolting (i.e., forming an inflorescence bearing stem) in the second year. Bolting is induced only after the plant has undergone vernalisation at the end of the first season. Certain environmental conditions, such as low temperatures and longer daylength, may induce bolting in the first year. Large amounts of pollen are produced during the reproductive phase; this pollen can be wind-borne over long distances. Sugar beet is also very sensitive to frost, and is a poor competitor with other plants.

The genus Beta is comprised of four sections: i) Beta, to which sugar beet belongs; ii) Corollinae; iii) Nanae; and iv) Procumbentes. All species within the Beta section (e.g., B. vulgaris ssp. maritima, B. macrocarpa) are cross-compatible and produce fertile progeny. Hybrids between sugar beet and members of the other three sections do not occur naturally, and cross-breeding with other members of the Chenopodiaceae is unlikely. As with conventional sugar beet cultivars, H7-1 plants are harvested prior to the initiation of the bolting phase. Since sugar beets are usually not allowed to bolt, except in fields or plots grown for seed production, there is little opportunity for uncontrolled pollen flow. Sugar beets grown for seed production are also isolated from other sugar beet fields to minimize pollen flow. This is usually achieved by the use of isolation distances enforced by seed certification agencies.

The development of glyphosate-tolerant hybrids is possible due to the ability of sugar beet to outcross with related plants. However, the glyphosate tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environment routinely subjected to glyphosate applications. In the event that a glyphosate-tolerant hybrid survived, it could be controlled using mechanical and/or other available chemical means. H7-1 is considered unlikely to increase the weediness potential of any other cultivated plant or native wild species with which it may interbreed. H7-1 also does not possess growth and reproductive characteristics that would render it weedier than conventional sugar beet.

Sugar beet intended for human consumption is generally converted directly to refined white sugar through extensive purification processes. The levels of CP4 EPSPS protein introduced into H7-1 sugar beet were not measured in refined sugar. CP4 EPSPS was expressed at very low levels in the roots, and since refined sugar does not contain detectable levels of protein, there would be virtually no human exposure to CP4 EPSPS. Consequently, there is no anticipated human exposure to this protein as a result of consumption of refined sugar derived from H7-1 sugar beet.

Detailed analyses were carried out to establish the nutritional value of H7-1 sugar beet. The transgenic H7-1 line was compared with non-transgenic sugar beets in an analysis of numerous compositional components, including crude ash, crude fibre, crude protein, carbohydrate and dry matter in both tops and roots (processed). Additional quality components were measured in processed roots including, invert sugar content, polarization (% sucrose), sodium, potassium and alpha-amino nitrogen. No biologically meaningful differences in any compositional or quality parameters relevant to food were identified between non-transgenic, control sugar beet and sugar beet H7-1. It was concluded that glyphosate-tolerant sugar beet H7-1 is equivalent to other commercially available sugar beet varieties with respect to composition and nutritional quality.

The presence of naturally occurring toxins and allergens in glyphosate-tolerant sugar beet line H7-1 was investigated. Saponins are the only known toxicants found in sugar beet and are actively eliminated in sugar processing. These are also considered antinutrients since they impart a bitter taste to animal feed and reduce intake. Levels of saponin in H7-1 did not differ significantly from those of the control lines, and were within the range observed for conventional sugar beet cultivars.

The potential toxicity and allergenicity of the CP4 EPSPS protein was assessed. No significant homologies between the amino acid sequence of CP4 EPSPS and those of known toxins or allergens were detected. The protein did not display the characteristics usually associated with allergenic substances, and was readily degraded under conditions simulating the mammalian digestive environment. Acute oral gavage studies, in which mice were fed high doses of the CP4 EPSPS protein, did not reveal any toxic effects and provided further support to the conclusion that this protein, and thus the transgenic H7-1 sugar beet line, possessed little or no potential for toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office Colombian Agricultural Institute European Commission: Community Register of GM Food and Feed European Food Safety Authority Food Standards Australia New Zealand Health Canada Novel Foods Japanese Biosafety Clearing House, Ministry of Environment Philippines Department of Agriculture, Bureau of Plant Industry U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S. Food and Drug Administration

This record was last modified on Thursday, August 18, 2016