Sugar beet line T120-7 was developed using recombinant DNA techniques to allow for the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Ignite®, Rely®, Liberty®, Harvest®, and Finale®) as a weed control option. This line was genetically engineered to express a new enzyme, phosphinothricin-N-acetyltransferase (PAT), derived from the common aerobic soil actinomycete, Streptomyces viridochromogenes.

Glufosinate is a short name for the ammonium salt, glufosinate-ammonium. It is a broad-spectrum contact herbicide and is used to control a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Glufosinate is a natural compound isolated from two species of Streptomyces fungi. It inhibits the activity of an enzyme, glutamine synthetase, which is necessary for the production of glutamine and for ammonia detoxification. The application of glufosinate leads to reduced glutamine and increased ammonia levels in the plant tissues. This causes photosynthesis to stop and the plant dies within a few days. Glufosinate also inhibits the same enzyme in animals. It is highly biodegradable, has no residual activity, and very low toxicity for humans and wild fauna. The PAT enzyme detoxifies phosphinothricin via acetylation into an inactive compound.

An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of line T120-7. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Event T120-7 was produced by Agrobacterium-mediated transformation of calli from the parent line R01 with plasmid vector pOCA18/Ac. The transfer-DNA (T-DNA) portion of the tumour inducing (Ti) bacterial plasmid was engineered to contain a modified form the PAT encoding gene as well as the nptII gene. In order to enhance plant expression of the protein, the nucleotide sequence of the pat gene was modified using site-directed mutagenesis to contain plant-preferred codons. These modifications did not result in changes to the predicted amino acid sequence of the PAT enzyme. Expression of NPTII was used as a selectable marker to screen for transformed plants during tissue culture regeneration and multiplication.

Associated with the pat gene were promoter sequences, as well as transcription termination and polyadenylation signal sequences, derived from the 35S transcript of cauliflower mosaic virus (CaMV). Expression of the NPTII encoding gene was regulated using promoter sequences from the A. tumefaciens nopaline synthase encoding gene (nos).

The Introduced DNA
Southern and PCR analyses of genomic DNA from event T120-7 indicated the presence of a single insertion of the T-DNA region, and no evidence of insertion of sequences outside the T-DNA borders.

Genetic Stability of the Introduced Trait
Stable insertion of the T-DNA was demonstrated by comparing the original transformant with four progenies produced either by self-pollination or crosses with nontransgenic sugar beet lines. From Southern blot analyses and trait segregation data, it was concluded that the trait was maintained over multiple generations and inherited in a Mendelian fashion.

Expressed Material
The expression levels of PAT enzyme were quantitated using enzyme linked immunosorbent assay (ELISA) and found to average 137 ng/g (fresh wt tissue; range 74-208 ng/g) in the roots, 966 ng/g (fresh wt tissue; range 732-1176 ng/g) in the tops, and undetectable in the pulp, molasses or refined sugar. The concentrations of NPTII averaged 20 ng/g (fresh wt tissue) in the roots, 44 ng/g (fresh wt tissue) in tops, and undetectable in molasses or refined sugar.

Field Testing
The transgenic sugar beet line T120-7 was field tested in the United States (1994, 1996, 1997) and in Canada, Western and Eastern Europe, and in the former Soviet Union. T120-7 was evaluated extensively and no differences were found in the agronomic characteristics, plant emergence and seedling vigour for line T120-7 compared to non-transformed counterpart beets and standard commercial sugar beet varieties growing in nearby fields.

Outcrossing
Sugar beet is an outcrossing, largely wind pollinated plant. It is normally a biennial, developing a large succulent root in the first year and a seed stalk the second. Certain conditions such as low temperatures after planting and longer day length may induce bolting and produce a seed stalk during the first growing season. Beet is also highly sensitive to frost and a poor competitor with other plants.

During the reproductive phase, large amounts of pollen are produced which can travel long distances. The genus Beta, including the wild relatives, is divided into four sections: Beta, Corolinnae, Procumbentes, and Nanae. Sugar beet hybridizes freely with all members of the section Beta and the resulting progeny are fertile but hybridization is unlikely with other members of the Chenopodiaceae family. Hybrids between sugar beet and members of the other three sections do not naturally occur without human intervention. Assuming proximity, synchrony of flowering and suitable conditions, B. vulgaris may freely hybridize with other varieties.

During production of T120-7, for purposes of than seed production, plants are harvested before the natural onset of the reproductive phase in the same manner that unmodified cultivars are grown. Since it is uncommon for sugar beets to bolt, except in fields or plots grown specifically for seed production, there is little opportunity for uncontrolled pollen flow due to adequate isolation distances enforced by seed certification agencies.

Specific concerns have been raised about potential outcrossing with subsp. macrocarpa, in the Imperial Valley, California. Isozyme studies indicated the introgression of genes from commercial sugar beets has occurred, although other reports show that gene flow between these two plant populations is not likely due to non-synchronous flowering periods, sterile F1 hybrids, and poor growth in F2 hybrids.

It was determined that the transgenic sugar beet line T120-7 was no more likely to become a weed than herbicide tolerant cultivars currently in use or that can be developed by traditional breeding techniques. T120-7 was unlikely to increase the weediness potential of any other cultivated plant or native wild species with which it may interbreed.

Weediness
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. Occasionally, sugar beet volunteers may arise from the presence of wild beet, the bolting of fodder beet plants, the development of groundkeepers, which arise initially from vegetative growth of beet crowns or tops left after harvest or the germination of seed (which may be dormant in the soil for up to 10 years). Volunteer plants may be controlled by mechanical means or the use of registered herbicides that can be used on sugar beet volunteers.

Sugar beet plants have escaped from past commercial cultivation in the San Francisco Bay area and persist to this day. However, transgene movement via pollen to these plants is highly unlikely as sugar beets are no longer in commercial production in the Bay area. Other populations of sexually compatible plants are located in the Imperial Valley of California, but no wild populations exist outside of California.

The movement of the glufosinate tolerance trait from T120-7 to any other sexually compatible plant should not have a significant impact as the glufosinate tolerance would not confer any competitive advantage to these plants, especially if glufosinate is not applied to these plants. This would only occur in managed ecosystems where glufosinate is applied for broad-spectrum weed control, or in plant varieties developed to exhibit glufosinate tolerance and in which glufosinate is used to control weeds. As with glufosinate tolerant sugar beet volunteers, these individuals, should they arise, would be controlled using other available chemical means. Hybrids, if they developed, could potentially result in the loss of glufosinate as a tool to control these species. However, this can be avoided by the use of sound crop management practices including not using the same herbicide every year.

Secondary and Non-Target Adverse Effects
It was concluded that the genes inserted into the transgenic sugar beet line T120-7 would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms. Field observations of event T120-7 revealed no negative effects on nontarget organisms. The lack of known toxicity for the introduced PAT enzyme suggests no potential for deleterious effects on beneficial organisms such as bees and earthworms. The high specificity of the enzyme for its substrates makes it unlikely that the introduced enzyme would metabolize endogenous substrates to produce compounds toxic to beneficial organisms.

Impact on Biodiversity
Genetically engineered event T120-7 sugar beet is no more likely to become a weed than lines developed by traditional breeding techniques. It is unlikely to increase the weediness potential of any other cultivated plant or native wild species with which it may interbreed. It will not harm threatened and endangered species and non-target organisms. It was concluded that there was no potential impact of event T120-7 on biodiversity.

Dietary Exposure
Approximately 26% of the world sugar consumed and 47% of the sugar consumed in the United States is produced from sugar beets. Sugar beet is generally converted directly to refined white sugar (which is composed almost entirely of sucrose) through extensive purification processes. The PAT and NPTII proteins were expressed at low levels in the sugar beet roots and tops, and neither protein was detected in refined sugar or molasses derived from sugar beet T120-7, indicating no likelihood for exposure to these proteins from these products.

Sugar beet pulp is a by-product and is normally dried and pelleted for use as a livestock feed. In recent years, it has been purified and used as an additive, at less than 1%, in some specific foods such as food fibre for breakfast cereals etc. The PAT protein was not detected in the pulp and NPTII was detected at very low levels.

Nutritional Data
The transgenic T120-7 line was compared with nontransgenic sugar beets on the basis of numerous compositional components, including crude fat, crude protein, fibre, ash, carbohydrate, calories, calcium, magnesium, phosphorus, potassium, and sodium. With the exception of fibre content, no statistically significant differences were observed. The differences in fibre content were ascribed to environmental factors associated with different growing locations and conditions.

The process fractions, refined sugar, molasses and dried pulp, were assessed for the proximate variables: fatty acids, amino acids, minerals and sugar profiles. Analysis of the dried pulp fraction revealed differences for ash content and some amino acids. It was determined that these differences in nutrient content would not impact on animal nutrition, as dried pulp comprises only a portion, less than 25%, of the total diet for cattle feed use. The molasses fraction revealed differences in fat, protein, calcium, C10:0 and some amino acids. These differences were small and not expected to impact the nutritional content of animal feed. Analysis of the refined sugar fraction showed a difference in tryptophan levels. Importantly, there were no significant differences between transgenic and nontransgenic sugar beets in carbohydrate content, which is the major nutrient in refined sugar (98%).

Toxicity and Allergenicity
It was determined that the PAT enzyme has a very low potential for toxic or allergenic effects based on its physiochemical characteristics (e.g., rapid breakdown under mammalian digestive conditions using simulated gastric and intestinal fluids, and lack of heat stability), its low concentrations in plant tissues and thus food or livestock feed products derived from them, and the lack of amino acid sequence homology with any known protein toxins or allergens.

DD2002-39: Determination of the Safety of Aventis CropScience Canada Inc’s Glufosinate Ammonium Tolerant Sugar Beet (Beta vulgaris) Lines Derived from Event T120-7.

NOVEL FOOD INFORMATION: FOOD BIOTECHNOLOGY GLUFOSINATE AMMONIUM TOLERANT SUGAR BEET (EVENT T120-7)

AgrEvo USA Company Petition for Determination of Nonregulated Status: Glufosinate Tolerant Sugar Beet, Transformation Even T120-7

Memorandum to file concerning glufosinate herbicide tolerant sugar beet.

AgrEvo USA Company Petition 97-336-01p for Determination of Nonregulated Status for Transgenic Glufosinate Tolerant Sugar Beet Transformation Event T120-7

US Food and Drug Administration (1994). Secondary Food Additives Permitted in Food for Human Consumption; Food Additives Permitted in Feed and Drinking Water of Animals; Aminoglycoside 3'-Phosphotransferase II; Final Rule. Federal Register, 59:26700-26711.