Transgenic sugar beet event BV_CSM63713 and method for detecting and using the same

Abstract

A transgenic sugar beet event, Bv_CSM63713, is provided. Transgenic plant cells, plant parts, plants, seeds, agricultural and commodity products comprising event Bv_CSM63713 are also provided. Recombinant DNA molecules specific to event Bv_CSM63713, as well as methods of using and detecting Bv_CSM63713, are also provided. Sugar beet plants comprising event Bv_CSM63713 exhibit tolerance to benzoic acid-type auxins, such as dicamba, inhibitors of EPSPS, such as glyphosate, and inhibitors of glutamine synthetase, such as glufosinate.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 340,278, filed May 10, 2022, which is hereby incorporated by reference in its entirety.

[0002] Incorporation of Sequence Listing The sequence listing contained in the file named "MONS531WO_ST26.xml", which is 113 kilobytes (measured in MS - Windows), created on April 5, 2023, was submitted electronically with this specification and is hereby incorporated by reference in its entirety.

[0003] The present disclosure generally relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the present disclosure relates to compositions and methods for conferring herbicide tolerance to transgenic sugar beet plants. More specifically, recombinant DNA molecules of the sugar beet event Bv_CSM63713 are provided. Also provided are transgenic sugar beet plants, plant parts, seeds, cells, and agricultural products containing the sugar beet event Bv_CSM63713, as well as methods of using transgenic sugar beet plants, plant parts, seeds, cells, and agricultural products containing the sugar beet event Bv_CSM63713, methods of detecting the sugar beet event Bv_CSM63713, and methods of controlling weeds. Transgenic sugar beet plants, plant parts, seeds, and cells containing the sugar beet event Bv_CSM63713 exhibit tolerance to benzoic acid - type auxins, such as dicamba, inhibitors of 5 - enolpyruvylshikimate - 3 - phosphate synthase (EPSPS), such as glyphosate, and inhibitors of glutamine synthetase, such as glufosinate.

Background Art

[0004] Sugar beet (Beta vulgaris) is an important commercial crop in many countries, and the important role of herbicides in weed control in crop production is well established. Weeds compete with crops for space, nutrients, water, and light and can contaminate the harvest, so weed control is essential for obtaining good crop yields. Biotechnological methods have been shown to be useful for the production of transgenic sugar beets that are resistant to specific herbicides through the expression of heterologous genes (transgenes). Transgenic herbicide resistance enables the use of herbicides in the crop growing environment without phytotoxicity or with minimal phytotoxicity (e.g., less than about 10% phytotoxicity). Transgenic traits in sugar beet have been used to confer resistance to glyphosate and are widely used for weed control in the commercial production of sugar beet. Transgenic glyphosate resistance is generated by inserting into the sugar beet genome the ability to produce a variant of the enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS), which is the target of glyphosate. This variant, which is glyphosate resistant, is CP4-EPSPS from the CP4 strain of the genus Agrobacterium.

[0005] Herbicide tolerance traits may be used alone or in combination with other traits, such as tolerance to another herbicide. Combinations of herbicide tolerance traits are desirable to provide options for weed control that increase producer flexibility and allow the use of multiple modes of action of herbicides to control difficult weeds. Combining multiple desired traits within a genome can be achieved by crossing between two parents each having a desired trait and identifying progeny plants having the desired combination of traits, or by re-transforming a transgenic plant containing one or more desired trait(s) with one or more genes related to a further desired trait via random integration or targeted integration of the one or more genes related to the further desired trait. As another method, combining multiple desired traits can be achieved by inserting multiple genes as a single DNA molecule into one location, or locus, within the genome. Combining multiple herbicide tolerance traits at a single locus in sugar beet provides a useful tool in weed control that is much more easily and inexpensively maintained in the breeding process to form hybrids with subsequent diverse elite germplasm pools.

[0006] The expression of a transgene, and thus its effectiveness, in a transgenic plant, part, seed, or cell can be affected by many factors, such as the regulatory elements used in the transgene expression cassette, the combination and / or interaction of those regulatory elements, the chromosomal location of the transgene insertion site, the chromatin structure of the genome at or near the transgene insertion site, and the presence or proximity of any endogenous cis- and / or trans-regulatory elements or genes near the transgene insertion site. Furthermore, the productivity of the trait in a transgenic plant becomes even more complex when the transgenic insert contains multiple expression cassettes, each having different transgenes conferring different traits. These differences or factors can result in variations in the level of transgene expression, or the spatial or temporal pattern of transgene expression, between different transgenic insertion events of the same expression cassette. Additionally, different transgenic events can vary in terms of the molecular nature of the event. For example, a transgenic event may contain two or more copies of the transgene insertion at one or more chromosomal locations, the transgenic insertion may be of a truncated form compared to the intended insertion, may contain vector backbone sequences, the transgene may be inserted into an endogenous gene, or may be within a repetitive region. Such characteristics can lead to undesirable results, such as gene silencing, changes in the pattern and / or expression of the transgene, changes in the pattern and / or expression of the endogenous gene. There may also be undesirable phenotypic or agronomic differences between various events.

[0007] Even in the case of targeted array insertions, variability in the level of transgene expression between independent but genetically identical targeted array insertion (TSI) events has been observed in a subset of transgenic events (Verkest et al., 2019). This variability and silencing of expression occur independently of the transgene sequence and may be due to DNA methylation, which has led to different mechanisms of DNA methylation. The fact that significant variability in transgene expression was observed in a subset of clean TSI events indicates that selection is still required, as in the case of random integration events, to identify TSI events in which the gene of interest is stably expressed over generations, even when the integration event is targeted.

[0008] Commercially useful multi-gene transgenic events require that each transgene in the transgenic insert be expressed in a manner necessary for the achievement of the trait, involving rigorous testing, evaluation, and selection. After the resistant trait is selected, individual expression cassettes are designed and tested in vitro and / or in planta, and the best expression cassette for each trait is selected. Such tests include testing different regulatory elements (e.g., promoters, introns, leaders, and 3’UTRs) and combinations of different regulatory elements for the desired spatial and temporal expression of the transgene, as well as examining whether the product (protein) of the transgene targets an intracellular compartment, such as the chloroplast. The expression cassettes selected for each trait are then combined into one construct, and the construct is tested to confirm that all of the expression cassettes function well and that each transgene is appropriately expressed. The selected combination of expression cassettes is then used for transformation to produce transgenic plants. From the perspective of where the transgene(s) can be inserted into the plant genome, Agrobacterium-mediated transformation using a T-DNA construct containing one or more transgene cassettes is mainly variable and random, so each transgenic event is unique, and the transgenic DNA is inserted randomly and uniquely at different plant genome positions. Thus, using the selected combination of expression cassettes, hundreds of unique multi-gene transgenic events are produced, each of which is the result of random insertion of foreign DNA at different plant genome positions.

[0009] For these reasons, the productivity of different transformation events from the same transformation construct can vary, and the identification of transformation events that confer the most beneficial traits or characteristics and have no other potential variants or concerns is necessary to select superior events for commercial use. Thus, it is necessary to produce and analyze a large number of individual transgenic events to select events with excellent commercial characteristics, which can be an important task involving analysis and selection among many different transformation events.

[0010] To establish multi-gene events for commercial use, extensive molecular characterization, greenhouse testing, and multi-year field testing with different germplasms across multiple locations and various conditions are required to enable the acquisition of broad agronomic, phenotypic, and molecular data. The data obtained are then analyzed and events suitable for commercial purposes are selected. After the commercial multi-gene events are identified as having the desired transgene expression, molecular characteristics, efficacy, and field productivity, they can be introgressed as a single locus with multiple herbicide tolerance traits into the genetic background of other sugarbeets using plant breeding methods. The resulting sugarbeet varieties contain the new traits in combination with other desired properties, such as natural traits, disease resistance traits, high-yield germplasms, and / or one or more other transgenic herbicide tolerance traits.

Summary of the Invention

[0011] Recombinant DNA molecules are provided herein. Examples of such recombinant DNA molecules include SEQ ID NO: 10, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, polynucleotides having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9, and sequences selected from the group consisting of perfect complements of any of the foregoing. In some embodiments, the recombinant DNA molecule is derived from a sugar beet plant, seed, plant part, plant cell, progeny plant, or commodity product comprising the Bv_CSM63713 event, and a representative sample of the seeds comprising the event has been deposited under ATCC Accession No. PTA-127098. In some embodiments, the recombinant DNA molecule is contained in a sugar beet plant, seed, plant part, plant cell, or progeny plant comprising the Bv_CSM63713 event, or a commodity product produced therefrom, and a representative sample of the seeds comprising the event has been deposited under ATCC Accession No. PTA-127098. The recombinant DNA molecule can be formed by inserting a heterologous nucleic acid molecule into the genomic DNA of a sugar beet plant or sugar beet cell. The recombinant DNA molecule can include an amplicon used for diagnosing the presence of the Bv_CSM63713 sugar beet event.

[0012] Provided is a DNA molecule that functions as a DNA probe. An example of such a DNA molecule is a DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that specifically hybridizes, under stringent hybridization conditions, to the DNA of the sugar beet event Bv_CSM63713 in a sample. Detecting the hybridization of the DNA molecule under stringent hybridization conditions is used for the diagnosis of the presence of the sugar beet event Bv_CSM63713 in the sample.

[0013] Similarly provided is a DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe specific for the detection of at least one of the following in a sample: the 5' junction sequence between the adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713, the 3' junction sequence between the transgenic insert of the sugar beet event Bv_CSM63713 and the adjacent sugar beet genomic DNA, SEQ ID NO: 9, and a fragment of SEQ ID NO: 9 comprising a continuous nucleotide of SEQ ID NO: 9 of sufficient length to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713.

[0014] The DNA probe may comprise SEQ ID NO: 36. Alternatively, the DNA molecule that functions as the DNA probe may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and the complement of any of the foregoing. The sample may be derived from a sugar beet plant, seed, plant part, plant cell, progeny plant, or commercial product.

[0015] Provided is a pair of DNA molecules. The pair of DNA molecules includes a first DNA molecule and a second DNA molecule. The first and second DNA molecules include a fragment of SEQ ID NO: 10 or its complement, and function as DNA primers when used together in an amplification reaction with a DNA containing the sugar beet event Bv_CSM63713, and produce an amplicon used for the diagnosis of the sugar beet event Bv_CSM63713 in a sample. For example, the first and second DNA molecules may include SEQ ID NO: 14 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 23, SEQ ID NO: 20 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 31 and SEQ ID NO: 26, SEQ ID NO: 33 and SEQ ID NO: 29, SEQ ID NO: 28 and SEQ ID NO: 29, or SEQ ID NO: 34 and SEQ ID NO: 35. The amplicon is a fragment of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, having a length of at least 10 nucleotides, and may include a nucleotide sequence selected from the group consisting of fragments containing nucleotides 1,000 - 1,001 or 12,722 - 12,723 of SEQ ID NO: 10.

[0016] Provided is a method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commercial products. In a first example of such a method, the method includes a) contacting the sample with any of the DNA molecules that function as a probe described herein, b) subjecting the sample and the DNA molecule that functions as a probe to stringent hybridization conditions, and c) detecting the hybridization of the DNA molecule that functions as a probe to the DNA molecule in the sample. The hybridization of the DNA molecule that functions as a probe to the DNA molecule in the sample is used for the diagnosis of the presence of the sugar beet event Bv_CSM63713 in the sample.

[0017] Provide another method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commercial products. The method includes a) contacting the sample with a pair of any DNA molecules described herein, b) performing an amplification reaction sufficient to produce a DNA amplicon, and c) detecting the presence of the DNA amplicon. The DNA amplicon includes at least one of the following: the 5' junction sequence between the adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713, the 3' junction sequence between the adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713, SEQ ID NO: 9, and a fragment of SEQ ID NO: 9 having a length sufficient to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713. The presence of the DNA amplicon indicates the presence of the sugar beet event Bv_CSM63713 in the sample. The DNA amplicon can be at least 10 nucleotides in length, at least 11 nucleotides in length, at least 12 nucleotides in length, at least 13 nucleotides in length, at least 14 nucleotides in length, at least 15 nucleotides in length, at least 16 nucleotides in length, at least 17 nucleotides in length, at least 18 nucleotides in length, at least 19 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 30 nucleotides in length, at least 35 nucleotides in length, at least 40 nucleotides in length, at least 45 nucleotides in length, at least 50 nucleotides in length, at least 60 nucleotides in length, at least 70 nucleotides in length, at least 80 nucleotides in length, at least 90 nucleotides in length, or at least 100 nucleotides in length.The DNA amplicon may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, and SEQ ID NO: 1, and fragments of any of SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, and SEQ ID NO: 1, which are at least 10 nucleotides in length and comprise a fragment containing nucleotides 1,000 - 1,001 or 12,722 - 12,723 of SEQ ID NO: 10.

[0018] Another method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample of DNA derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commodity products is provided. The method comprises a) contacting the sample with any of the DNA molecules that function as probes described herein, and b) performing a sequencing reaction to produce a target sequence. The target sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, perfect complements thereof, and fragments of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10, which are at least 10 nucleotides in length and comprise a fragment containing nucleotides 1,000 - 1,001 or 12,722 - 12,723 of SEQ ID NO: 10.

[0019] A further method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commodity products is provided. The method comprises a) contacting the sample with at least one antibody specific for at least one protein encoded by the sugar beet event Bv_CSM63713, and b) detecting binding of the antibody to the protein in the sample. Binding of the antibody to the protein indicates the presence of the sugar beet event Bv_CSM63713 in the sample.

[0020] Provided is a DNA detection kit for detecting the presence of the sugar beet event Bv_CSM63713 in a sample. An example of such a DNA detection kit is a kit containing any of the pairs of DNA primers described herein. Another example of a DNA detection kit is a kit containing any of the DNA molecules that function as probes described herein.

[0021] Similarly provided is a protein detection kit for detecting the presence of the sugar beet event Bv_CSM63713 in a sample. An example of such a kit is a kit containing at least one antibody specific to at least one protein encoded by the sugar beet event Bv_CSM63713. Detecting the binding of at least one antibody to at least one protein encoded by the sugar beet event Bv_CSM63713 in a sample is used for diagnosing the presence of the sugar beet event Bv_CSM63713 in the sample.

[0022] Provided are sugar beet seeds, plants, plant parts, or plant cells. The sugar beet seeds, plants, plant parts, or plant cells comprise a recombinant DNA molecule comprising a polynucleotide having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9, and a sequence selected from the group consisting of the full complements of any of the foregoing. The sugar beet seeds, plants, plant parts, or plant cells can express at least one herbicide tolerance gene selected from the group consisting of phosphinothricin N-acetyltransferase (PAT), dicamba monooxygenase (DMO), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and any combination thereof. The sugar beet seeds, plants, plant parts, or plant cells can be resistant to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof. The sugar beet plant, plant seed, plant part, or plant cell can comprise the sugar beet event Bv_CSM63713, and a representative sample of seeds containing the event has been deposited under ATCC accession number PTA-127098. The sugar beet plant, plant seed, plant part, or plant cell can further be defined as a progeny plant of any generation of a sugar beet plant comprising the sugar beet event Bv_CSM63713, or a sugar beet plant part, plant seed, or plant cell derived therefrom.

[0023] Further provided are sugar beet plants, plant parts, plant seeds, and plant cells. The sugar beet plants, plant parts, plant seeds, or plant cells contain the sugar beet event Bv_CSM63713, and a representative sample of seeds containing the sugar beet event Bv_CSM63713 has been deposited under ATCC accession number PTA-127098.

[0024] Any of the sugar beet plant parts described herein may include roots, beets, microspores, pollen, anthers, ovules, ovaries, flowers, embryos, stems, leaves, protoplasts, or callus.

[0025] A method for controlling or preventing weeds in an area is provided. An example of such a method includes planting sugar beet containing the event Bv_CSM63713 in the area, and applying an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof, whereby weeds in the area are controlled and there is no or less than about 10% phytotoxicity to the sugar beet. Applying the effective amount of the at least one herbicide may include applying at least two or more herbicides selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof throughout the growth period. The effective amount of dicamba may be from about 0.5 lb ae / acre to about 2 lb ae / acre of dicamba throughout the growth period. The effective amount of glufosinate may be from about 0.4 lb ai / acre to about 2.16 lb ai / acre of glufosinate throughout the growth period. The effective amount of glyphosate may be from about 0.75 lb ae / acre to about 2.25 lb ae / acre of glyphosate throughout the growth period.

[0026] Provided is a method for controlling volunteer sugar beets including the sugar beet event Bv_CSM63713 in a region. An example of such a method includes applying a herbicidally effective amount of at least one herbicide other than dicamba, glyphosate, or glufosinate, wherein application of the herbicide prevents the growth of sugar beets including the sugar beet event Bv_CSM63713. The herbicide other than dicamba, glyphosate, or glufosinate may be selected from the group consisting of paraquat, clethodim, clopyralid, desmedipham, triflusulfuron, 2,4-dichlorophenoxyacetic acid (2,4-D), and acetolactate synthase (ALS) inhibitors such as sulfonylurea (SU), imidazolinone, triazolopyrimidine, pyrimidinyl oxybenzoate, and sulfonylaminocarbonyltriazolinone.

[0027] Provided is a method for obtaining sugar beet plants or seeds of sugar beet plants that are resistant to glyphosate, dicamba, glufosinate, or any combination thereof. In one example of such a method, the method comprises: a) obtaining a population of progeny seeds or plants grown therefrom, at least one of which comprises the sugar beet event Bv_CSM63713; and b) identifying at least an initial progeny seed or plant grown therefrom that comprises the sugar beet event Bv_CSM63713. Identifying a progeny seed or plant that comprises the sugar beet event Bv_CSM63713 can comprise: a) growing the progeny seed or plant to produce a progeny plant; b) treating the progeny plant with an effective amount of at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate, and any combination thereof; and c) selecting a progeny plant that is resistant to at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate, and any combination thereof. Alternatively or additionally, identifying a progeny seed or plant that comprises the sugar beet event Bv_CSM63713 can comprise detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from the progeny seed or plant. Alternatively or additionally, identifying a progeny seed or plant that comprises the sugar beet event Bv_CSM63713 can comprise detecting the presence of at least one protein encoded by the sugar beet event Bv_CSM63713 in a sample derived from the progeny seed or plant.

[0028] A method for determining the zygosity of a sugar beet plant, plant part, plant seed, or plant cell containing sugar beet event Bv_CSM63713 is provided. One example of such a method includes: a) contacting a sample containing DNA from the sugar beet plant, plant part, plant seed, or plant cell with a primer set capable of producing a first amplicon used to diagnose the presence of sugar beet event Bv_CSM63713 and a second amplicon used to diagnose wild-type sugar beet genomic DNA not containing sugar beet event Bv_CSM63713; b) performing a nucleic acid amplification reaction; and c) detecting the first amplicon and the second amplicon. The presence of both amplicons indicates that the sample is heterozygous for sugar beet event Bv_CSM63713, and the presence of only the first amplicon indicates that the sample is homozygous for sugar beet event Bv_CSM63713. Specific examples of primer sets that may be used are primer sets comprising SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:29.

[0029] Provide another method for determining the zygosity of sugar beet plants, plant parts, plant seeds, or plant cells containing the sugar beet event Bv_CSM63713. The method comprises: a) contacting a sample containing DNA derived from the sugar beet plant, plant part, plant seed, or plant cell with a probe set comprising at least a first probe that specifically hybridizes to the sugar beet event Bv_CSM63713 and at least a second probe that specifically hybridizes to the sugar beet genomic DNA disrupted by the insertion of the foreign DNA of the sugar beet event Bv_CSM63713 but does not hybridize to the sugar beet event Bv_CSM63713; and b) hybridizing the probe set with the sample under stringent hybridization conditions. Detecting hybridization of only the first probe under the hybridization conditions is used for diagnosing sugar beet plants, plant parts, seeds, or plant cells that are homozygous for the sugar beet event Bv_CSM63713. Detecting hybridization of both the first probe and the second probe under the hybridization conditions is used for diagnosing sugar beet plants, plant parts, seeds, or plant cells that are heterozygous for the sugar beet event Bv_CSM63713.

[0030] Provided is a DNA construct. An example of such a DNA construct includes a first expression cassette, a second expression cassette, and a third expression cassette. The first expression cassette comprises, in operable linkage, i) a chlorophyll A-B binding protein (Cab1) promoter and leader from Arabidopsis thaliana, ii) a phosphinothricin N-acetyltransferase (PAT) coding sequence, and iii) a 3’UTR of a small heat shock protein (Hsp20) from Medicago truncatula. The second expression cassette comprises, in operable linkage, i) a ubiquitin (Ubq1) promoter, leader, and intron from Cucumis melo, ii) a coding sequence for a chloroplast transit peptide of ribulose bisphosphate carboxylase small subunit (RbcS) from Pisum sativum, iii) a dicamba monooxygenase coding sequence (DMO), and iv) a 3’UTR of a putative protein from Medicago truncatula. The third expression cassette comprises, in operable linkage, i) an inclusion body matrix protein enhancer from Dahlia mosaic virus, ii) a promoter, leader, and intron of S-adenosyl-L-methionine synthetase (SAMS2) from Cucumis melo, iii) a coding sequence for a chloroplast transit peptide of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Arabidopsis thaliana, iv) a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) coding sequence, and v) a 3’UTR of a hypothetical protein from Medicago truncatula. For example, the DNA construct may comprise SEQ ID NO: 9.

[0031] Also provided are sugar beet plants, plant seeds, plant parts, or plant cells comprising any of the DNA constructs described herein.

[0032] Provided is a method for improving tolerance to at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate, and any combination thereof in sugar beet plants. The method includes a) inserting any of the DNA constructs described herein into the genome of sugar beet cells, b) generating sugar beet plants from the sugar beet cells, and c) selecting sugar beet plants containing the DNA construct. The selecting may include treating the sugar beet cells or plants with an effective amount of at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate, and any combination thereof.

[0033] Also provided is a sugar beet plant, plant seed, plant part, or plant cell that is tolerant to herbicides having the mode of action of three different herbicides at a single genomic locus. The sugar beet plant, plant seed, plant part, or plant cell may contain any of the DNA constructs described herein.

[0034] Provided are sugar beet seeds, plants, plant parts, or plant cells that are tolerant to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof. The sugar beet seeds, plants, plant parts, or plant cells contain any of the DNA constructs provided herein.

[0035] Any of the sugar beet seeds, plants, plant parts, or cells can be obtained by any of the methods for improving tolerance to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof in the sugar beet plants provided herein.

[0036] Provided is a method for producing a progeny sugar beet plant comprising the sugar beet event Bv_CSM63713. The method comprises: a) selfing or crossing a first sugar beet plant comprising the sugar beet event Bv_CSM63713 with itself or with a second sugar beet plant; b) collecting one or more seeds produced from the cross; c) growing the one or more seeds to produce one or more progeny plants; and d) selecting at least an initial progeny plant or seed comprising the sugar beet event Bv_CSM63713. Also provided are inbred and hybrid sugar beet plants and seeds comprising the sugar beet event Bv_CSM63713 produced by the method.

[0037] Also provided are non-viable sugar beet plant material and non-regenerable sugar beet plant material. The material can comprise any of the recombinant DNA molecules or DNA constructs described herein.

[0038] Also provided is non-viable sugar beet plant material or non-regenerable sugar beet plant material comprising the sugar beet event Bv_CSM63713, and a representative sample of seeds comprising the sugar beet event Bv_CSM63713 has been deposited under ATCC accession number PTA-127098.

[0039] Also provided are commodity products. Examples of such commodity products are commodity products comprising any of the recombinant DNA molecules or DNA constructs described herein. The commodity products can be produced from transgenic sugar beet plants, plant parts, plant seeds, or plant cells comprising the sugar beet event Bv_CSM63713. The commodity products can include, for example, whole seeds or processed seeds, non-growing seeds, processed plant parts, processed plant tissues, dried plant tissues, dried plant parts, frozen plant tissues, frozen plant parts, plant parts processed for animal feed, fibers, pulp, pulp pellets, pulp fragments, tailings, squeezed juice, syrup, molasses, extracts, raffinates, betaine, separator molasses soluble matter (SMS), or any other human food, growing seeds, growing plant parts (e.g., roots and leaves), or growing plant cells.

[0040] Provided is a method for producing a commercial product. The method includes: a) obtaining a transgenic sugar beet plant, plant part, or plant seed comprising the sugar beet event Bv_CSM63713; and b) producing a commercial product from the transgenic sugar beet plant, plant part, or plant seed.

[0041] Provided is a method for controlling, preventing, or reducing the occurrence of herbicide-resistant weeds. The method includes cultivating a sugar beet plant comprising a transgene conferring resistance to a herbicide having three different herbicide modes of action at a single genomic locus in a crop growth environment. The three different herbicide modes of action can be selected from the group consisting of inhibition of glutamine synthetase, benzoic acid-type auxin, and inhibition of EPSPS.

[0042] Also provided is a method for controlling, preventing, or reducing the occurrence of herbicide-resistant weeds. The method includes: a) cultivating a sugar beet plant comprising any of the DNA constructs described herein for conferring resistance to a herbicide having three different herbicide modes of action at a single genomic locus in a crop growth environment; and b) applying at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof to the crop growth environment, wherein the sugar beet plant is resistant to the at least one herbicide.

[0043] Provided is a method for reducing the number of genetic loci for sugar beet breeding by inserting a transgene at a single genomic locus for resistance to three different classes of herbicides. The transgene can be inserted as a single molecularly linked transgenic insert. The single molecularly linked transgenic insert can confer commercial-level resistance to at least one herbicide for each herbicide mode of action.

[0044] Further provided are sugar beet plants, plant cells, plant parts, and plant seeds. The sugar beet plants, plant cells, plant parts, and plant seeds contain a recombinant DNA construct integrated into chromosome 4. The recombinant DNA construct confers resistance to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof. The recombinant DNA construct is integrated at the position of the chromosome where SEQ ID NO: 11 and SEQ ID NO: 12 are located side by side.

Brief Description of the Drawings

[0045]

Figure 1

Figure 2

[0046] Brief Description of the Sequences SEQ ID NO: 1 is a 30-nucleotide sequence representing the 5' junction region of sugar beet genomic DNA and the integrated transgene insert. SEQ ID NO: 1 corresponds to nucleotide positions 986-1015 of SEQ ID NO: 10.

[0047] SEQ ID NO: 2 is a 30-nucleotide sequence representing the 3' junction region of the integrated transgene insert and sugar beet genomic DNA. SEQ ID NO: 2 corresponds to nucleotide positions 12708-12737 of SEQ ID NO: 10.

[0048] SEQ ID NO: 3 is a 60-nucleotide sequence representing the 5' junction region of sugar beet genomic DNA and the integrated transgene insert. SEQ ID NO: 3 corresponds to nucleotide positions 971-1030 of SEQ ID NO: 10.

[0049] SEQ ID NO: 4 is a 60-nucleotide sequence representing the 3' junction region of the integrated transgene insert and sugar beet genomic DNA. SEQ ID NO: 4 corresponds to nucleotide positions 12693-12752 of SEQ ID NO: 10.

[0050] Sequence number 5 is a 100-nucleotide sequence representing the 5'-junction region of the sugar beet genomic DNA and the integrated transgene insert. Sequence number 5 corresponds to nucleotide positions 951 to 1050 of sequence number 10.

[0051] Sequence number 6 is a 100-nucleotide sequence representing the 3'-junction region of the integrated transgene insert and the sugar beet genomic DNA. Sequence number 6 corresponds to nucleotide positions 12673 to 12772 of sequence number 10.

[0052] Sequence number 7 is a 1050-nucleotide sequence representing the 5'-genomic flanking region of the sugar beet genomic DNA and the 50-nucleotide integrated transgene insert. Sequence number 7 corresponds to nucleotide positions 1 to 1050 of sequence number 10.

[0053] Sequence number 8 is a 1050-nucleotide sequence representing the 50-nucleotide of the integrated transgene insert and the 3'-genomic flanking region of the sugar beet genomic DNA. Sequence number 8 corresponds to nucleotide positions 12673 to 13722 of sequence number 10.

[0054] Sequence number 9 is an 11722-nucleotide sequence corresponding to the transgene insert of the sugar beet event Bv_CSM63713. Sequence number 9 corresponds to nucleotide positions 1001 to 12722 of sequence number 10.

[0055] Sequence number 10 is a 13722-nucleotide sequence corresponding to the contig nucleotide sequence of the 5'-sugar beet genomic DNA sequence (sequence number 11), the transgene insert in event Bv_CSM63713 (sequence number 9), and the 3'-sugar beet genomic DNA sequence (sequence number 12).

[0056] Sequence number 11 is a 1000-nucleotide sequence representing the 5'-flanking sugar beet genomic DNA up to the transgene insert (sequence number 9). Sequence number 11 corresponds to nucleotide positions 1 to 1000 of sequence number 10.

[0057] Sequence number 12 is a 1000-nucleotide sequence representing the 3'-adjacent sugar beet genomic DNA after the transgene insert (sequence number 9). Sequence number 12 corresponds to nucleotide positions 12723 to 13722 of sequence number 10.

[0058] Sequence number 13 is a 2007-nucleotide sequence representing the wild-type sugar beet genomic DNA at the position where the transgenic sequence was inserted into event Bv_CSM63713. As shown in Figure 1, integration of the transgenic insert (sequence number 9) into the sugar beet genome resulted in a 7-nucleotide deletion (ACCTCGC) in the genomic sequence of sugar beet event Bv_CSM63713.

[0059] Sequence number 14 is a 47-nucleotide sequence corresponding to a forward primer called txht024d01-X and is used to identify the DNA of sugar beet event Bv_CSM63713 in a sample. The primer contains a 5'-21-nucleotide oligo sequence used in Kompetitive Allele-Specific PCR (KASP). Nucleotides 20 to 47 of sequence number 14 correspond to positions 972 to 999 of sequence number 10 (the oligo tail nucleotides 20 to 21 (CT) of the oligo are also aligned with sequence number 10 together with nucleotides 22 to 47).

[0060] Sequence number 15 is a 26-nucleotide sequence corresponding to the forward primer txht024d01-X described in sequence number 14 but does not contain the 21-nucleotide oligo sequence.

[0061] Sequence number 16 is a 41-nucleotide sequence corresponding to a reverse primer called sxcp4xxxs1-X used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of the CP4 EPSPS gene in the regenerated sugar beet events for event selection. Nucleotides 1 to 21 of sequence number 16 are an oligo nucleotide tail sequence.

[0062] Sequence number 17 is a 27-nucleotide sequence corresponding to the forward primer called sxcp4xxxs1-C, which is used in KASP to detect the presence of the EPSPS gene of CP4 in the regenerated sugar beet events for event selection.

[0063] Sequence number 18 is a 26-nucleotide sequence corresponding to the reverse primer called txht024d01-R, which is used to identify the DNA of the sugar beet event Bv_CSM63713 in the sample when combined with sequence number 14 or 15, and corresponds in the reverse direction to positions 1004 - 1029 of sequence number 10.

[0064] Sequence number 19 is a 48-nucleotide sequence corresponding to the forward primer called txht024d02-X, which is used to identify the DNA of the sugar beet event Bv_CSM63713 in the sample. The primer contains a 21-nucleotide oligo sequence at the 5'. Nucleotides 22 - 48 of sequence number 19 correspond to positions 12697 - 12723 of sequence number 10.

[0065] Sequence number 20 is a 27-nucleotide sequence corresponding to the forward primer txht024d02-X described in sequence number 19, but does not contain the 21-nucleotide oligo sequence.

[0066] Sequence number 21 is a 43-nucleotide sequence corresponding to the reverse primer called sxpatxxxs2-X, which is used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of the PAT gene in the regenerated sugar beet events for event selection. Nucleotides 1 - 21 of sequence number 21 are the oligo nucleotide tail sequence.

[0067] Array number 22 is a 25-nucleotide sequence corresponding to a forward primer called sxpatxxxs2-C, which is used in KASP to detect the presence of the PAT gene in the regenerated sugar beet events for event selection.

[0068] Array number 23 is a 29-nucleotide sequence corresponding to a reverse primer called txht024d02-R, which is used to identify the DNA of the sugar beet event Bv_CSM63713 in the sample when combined with array number 19 or 20, and corresponds in the reverse direction to positions 12727 - 12755 of array number 10.

[0069] Array number 24 is a 44-nucleotide sequence corresponding to a reverse primer called txht024s01-X, which is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample and hybridizes to the region of the sugar beet genome. The primer contains a 5'-21-nucleotide oligo sequence used in Kompetitive Allele-Specific PCR (KASP). Nucleotides 22 - 37 of array number 24 correspond in the reverse direction to positions 12723 - 12738 of array number 10. Nucleotides 38 - 44 of array number 24 correspond to the 7-nucleotide wild-type sugar beet genomic DNA sequence (ACCTCGC) deleted in the sugar beet event Bv_CSM63713 shown in array number 13, as shown in Figure 1.

[0070] Array number 25 is a 45-nucleotide sequence corresponding to a reverse primer called txht024s01-Y, which is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample. The primer contains a 5'-21-nucleotide oligo sequence used in Kompetitive Allele-Specific PCR (KASP). Nucleotides 21 - 45 of array number 25 correspond in the reverse direction to positions 1001 - 1025 of array number 10 (the nucleotide 21 (T) of the oligo tail is also aligned with array number 10 together with nucleotides 22 - 45).

[0071] Sequence number 26 is a 30-nucleotide sequence corresponding to the forward primer named txht024s01-R used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample. This corresponds to positions 957 to 986 of sequence number 10.

[0072] Sequence number 27 is a 42-nucleotide sequence corresponding to the forward primer named txht024s02-X used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample, and hybridizes to a region of the sugar beet genome. The primer contains a 5'-21-nucleotide oligotail sequence used for Kompetitive Allele-Specific PCR (KASP). Nucleotides 21 to 35 of sequence number 27 correspond to positions 986 to 1000 of sequence number 10. (Nucleotide 21 "T" of the oligotail also aligns with sequence number 10 together with nucleotides 22 to 35). Nucleotides 36 to 42 of sequence number 27 correspond to the wild-type sugar beet genomic DNA sequence (ACCTCGC) of 7 nucleotides deleted in the sugar beet event Bv_CSM63713 described in sequence number 13.

[0073] Sequence number 28 is a 48-nucleotide sequence corresponding to the forward primer named txht024s02-Y used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample. Nucleotides 22 to 48 of sequence number 28 correspond to positions 12696 to 12722 of sequence number 10, and nucleotides 1 to 21 of sequence number 28 are an oligonucleotide tail sequence.

[0074] Sequence number 29 is a 30-nucleotide sequence corresponding to the reverse primer named txht024s02-R used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample. This corresponds in the reverse direction to positions 12743 to 12772 of sequence number 10.

[0075] Array number 30 corresponds to array number 24, but is a 23-nucleotide sequence that does not contain the 21-nucleotide oligo sequence. This is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample and hybridizes to the region of the sugar beet genome.

[0076] Array number 31 corresponds to array number 25, but is a 24-nucleotide sequence that does not contain the 21-nucleotide oligo sequence. This is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample.

[0077] Array number 32 corresponds to array number 27, but is a 21-nucleotide sequence that does not contain the 21-nucleotide oligo tail. This is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample and hybridizes to the region of the sugar beet genome.

[0078] Array number 33 corresponds to array number 28, but is a 27-nucleotide sequence that does not contain the 21-nucleotide oligo sequence. This is used for the zygosity assay of the DNA of the sugar beet event Bv_CSM63713 in the sample.

[0079] Array number 34 is a 17-nucleotide sequence corresponding to the forward primer called 2109_fwd1 and is used to identify the DNA of the sugar beet event Bv_CSM63713 in the sample. This corresponds to positions 12686 - 12702 of array number 10.

[0080] Array number 35 is a 24-nucleotide sequence corresponding to the reverse primer called 2109_rev1 and is used to identify the DNA of the sugar beet event Bv_CSM63713 in the sample, corresponding in the reverse direction to positions 12739 - 12762 of array number 10.

[0081] SEQ ID NO: 36 is a 16-nucleotide sequence corresponding to a probe called 2109_probe1, which is used to identify the DNA of the sugar beet Bv_63713 event in a sample and corresponds to positions 12710 to 12725 of SEQ ID NO: 10.

[0082] SEQ ID NO: 37 is a 40-nucleotide sequence corresponding to a forward primer called txdmoxxxxx-X, which is used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of the DMO gene in the regenerated sugar beet events for event selection. Nucleotides 1 to 21 of SEQ ID NO: 40 are oligonucleotide tail sequences.

[0083] SEQ ID NO: 38 is a 29-nucleotide sequence corresponding to a reverse primer called txdmoxxxxx-C, which is used in KASP to detect the presence of the DMO gene in the regenerated sugar beet events for event selection.

[0084] SEQ ID NO: 39 and 40 are 39- and 25-nucleotide sequences corresponding to primers called txmonlbxxx-X and txmonlbxxx-C, respectively, which are used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of the left border (LB) of the Agrobacterium T-DNA that is overread in the regenerated sugar beet events for event selection. Nucleotides 1 to 21 of each primer are oligonucleotide tail sequences.

[0085] SEQ ID NO: 41 and 42 are 40- and 28-nucleotide sequences corresponding to primers called txmonrbxxx-X and txmonrbxxx-C, respectively, which were used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of the right border (RB) of the Agrobacterium T-DNA that is overread in the regenerated sugar beet events for event selection. Nucleotides 1 to 21 of each primer are oligonucleotide tail sequences.

[0086] SEQ ID NOs: 43 and 44 are 42- and 23-nucleotide sequences corresponding to primers named txvir2dd04-X and txvir2dd04-C, respectively, which were used in Kompetitive Allele-Specific PCR (KASP) to detect the presence of Agrobacterium in the regenerated sugar beet events for event selection. Nucleotides 1 to 21 of each primer are oligonucleotide tail sequences.

[0087] SEQ ID NO: 45 is a 24-nucleotide sequence corresponding to a forward primer named GluA3-F for the amplification of glutamine synthetase, which serves as an internal control for sugar beet PCR.

[0088] SEQ ID NO: 46 is a 23-nucleotide sequence corresponding to a reverse primer named GluA3-R for the amplification of glutamine synthetase, which serves as an internal control for sugar beet PCR.

[0089] SEQ ID NO: 47 is a 27-nucleotide sequence corresponding to a probe named GluD1 for the amplification of glutamine synthetase, which serves as an internal control for sugar beet PCR.

[0090] SEQ ID NOs: 48 to 77 are nucleotide sequences of genetic elements in the transgenic insert of the sugar beet event Bv_CSM63713, which are further described in Table 15 below.

BEST MODE FOR CARRYING OUT THE INVENTION

[0091] The following definitions, explanations, and methods are provided to better define the present invention and to guide those skilled in the art in practicing the present invention. Unless otherwise noted, terms are to be understood according to their conventional usage by those skilled in the relevant art.

[0092] Herbicide tolerance is an important agronomic trait for effective weed control to maintain favorable crop growth conditions and crop yields, and is achieved by manipulating herbicide tolerance-introducing genes in crops using modern plant biotechnology techniques. The sugar beet event Bv_CSM61723 provides tolerance to three different herbicide chemistries via different modes of action for weed control and herbicide-tolerant weed management.

[0093] Using plant transformation technology, foreign DNA (also known as transgenic DNA) is randomly inserted into the chromosomes of the cell's genome to produce genetically engineered cells, also called "transgenic" or "recombinant" cells. Using this technology, many individual cells are transformed, each resulting in a unique transgenic event due to the random insertion of foreign DNA into the genome. Transgenic plants are then regenerated from the individual transgenic cells. As a result, all cells of the transgenic plant contain the transgenic event inserted as an integral part of its genome. This transgenic plant can then be used to produce progeny plants, each containing a unique transgenic event.

[0094] The sugar beet event Bv_CSM63713 is provided. Event Bv_CSM63713 was produced by Agrobacterium-mediated transformation of the shoot meristem of sugar beet, involving (i) transformation of thousands of sugar beet cells with four different DNA constructs containing three expression cassettes (each expression cassette was selected after being tested in combination with the other two expression cassettes following individual tests), (ii) regeneration of a population of transgenic plants, each containing a unique transgenic event, and (iii) rigorous multi-year event selection involving testing and analysis of the molecular characteristics, herbicide tolerance efficacy, and agronomic characteristics in various genetic backgrounds for hundreds of events through tens of thousands of plants. The sugar beet event Bv_CSM63713 was thus produced and selected as a unique and superior event useful for large-scale agronomic commercial purposes.

[0095] As used herein, "expression cassette", "cassette", or "transgene cassette" refers to a recombinant DNA molecule or sequence that includes a combination of different elements for expressing the RNA and / or protein encoded by the coding sequence of the transgene in a transformed plant cell or transformed plant containing the transgene. The "expression cassette", "cassette", or "transgene cassette" provided herein includes one or more regulatory element(s) operably linked to a coding sequence or a transcribable DNA sequence. Examples of such regulatory elements include a promoter, a leader, a 5' untranslated region (5'UTR), an intron, and / or a 3' untranslated region (3'UTR) region. The "expression cassette", "cassette", or "transgene cassette" is recombinant and heterologous with respect to the genome of the transformed plant cell. For the purposes of the present disclosure, such "expression cassette", "cassette", or "transgene cassette" is a recombinant DNA molecule or sequence that encodes a protein conferring resistance to at least one class of herbicides described herein. Table 15 provides a list of genetic elements included in three transgene cassettes in the transgenic insert (SEQ ID NO: 9) of the sugar beet event Bv_CSM63713.

[0096] The act of inserting transgenic DNA into the genome of a sugar beet plant is accomplished by plant transformation methods known in the art and creates a new transgenic genomic DNA sequence known as a "transgenic event" or "event". The DNA sequence of the event includes the inserted foreign DNA (referred to as the "transgenic insert") and genomic DNA (referred to as "flanking DNA") that is adjacent to, or "flanks", the transgenic insert on either side of the insertion site. As used herein, the term "flanking" with respect to a transgenic event means that the plant genomic sequence(s) includes the transgenic event at the 5' and / or 3' end(s) of the insertion of the transgenic event in the genome of the transformed plant, plant part, plant tissue, or plant cell. Similarly, "flanking DNA" refers to a length of genomic DNA sequence that is adjacent to the transgenic DNA insertion in the genome of the transformation event at the 5' and / or 3' end(s) of the insertion. Thus, "5' flanking" means the sugar beet genomic DNA sequence that is adjacent to the transgenic DNA insertion and is upstream (or on the 5' end side) of it. For example, "5' flanking" can include the sugar beet genomic DNA sequence immediately upstream (5' end side) of the transgenic insert, or any sugar beet genomic DNA sequence upstream (5' end side) of the transgenic insert that is not immediately adjacent to the transgenic insert but is within about 5000 nucleotides, about 3000 nucleotides, or about 1000 nucleotides upstream of the transgenic insert. Similarly, "3' flanking" means the sugar beet genomic DNA sequence that is adjacent to the transgenic insert and is downstream (or on the 3' end side) of it.For example, "3' adjacent" refers to the sugar beet genomic DNA sequence downstream (3' end side) directly adjacent to the transgenic insertion, or any sugar beet genomic DNA sequence downstream (3' end side) of the transgenic insertion, which is not directly adjacent to the transgenic insertion but is within about 5000 nucleotides, about 3000 nucleotides, or about 1000 nucleotides downstream of the transgenic insertion. The DNA sequence of the event is unique and specific to the event and can be easily identified when compared to other DNA sequences, such as the sequences of other events or the sequences of untransformed sugar beet genomic DNA. The sugar beet event Bv_CSM63713 has a novel and unique DNA sequence provided as SEQ ID NO: 10, which includes a contiguous sequence comprising a 5' sugar beet genomic flanking sequence provided as SEQ ID NO: 11, a transgenic insert sequence provided as SEQ ID NO: 9, and a 3' sugar beet genomic flanking DNA sequence provided as SEQ ID NO: 12. Thus, the sugar beet event Bv_CSM63713 is an essential part of the chromosomes of transgenic sugar beet cells and plants containing the event and, therefore, is a DNA molecule that is fixed and can be transmitted to progeny cells and plants.

[0097] Also provided are the original transformed cells and plant progeny comprising the sugar beet event Bv_CSM63713. Such progeny can be produced by cell tissue culture, by selfing of sugar beet plants comprising the sugar beet event Bv_CSM63713, or by cross-pollination between a sugar beet plant comprising the sugar beet event Bv_CSM63713 and another plant, whether or not it comprises the event, or by any other method known in the art including any plant cell or tissue culture method, and the progeny comprise the sugar beet event Bv_CSM63713. Such other plants may be transgenic plants comprising the same or different event(s), or non-transgenic plants, for example, those derived from different varieties. The sugar beet event Bv_CSM63713 is passed on from the original parent to the progeny through each generation. A "transgenic plant" or "plant" is thus regenerated from a transformed plant cell and may be the original transformed plant comprising the transgenic DNA and event, or in the case of a progeny plant of the original transformed plant, which may be separated from the transformant on a generation-by-generation basis, and the transgenic DNA and event are retained in the same specific location and sequence relationship within the genome of the plant.

[0098] As used herein, the term "beet" refers to Beta vulgaris including all subspecies of Beta vulgaris, and further includes all plant species that can cross with Beta vulgaris. This includes Beta vulgaris subspecies adanensis, Beta vulgaris subspecies maritima, and Beta vulgaris subspecies vulgaris, Beta macrocarpa, and Beta patula. Within Beta vulgaris subspecies vulgaris, there are included 1) the Altissima group, for example, sugar beet, 2) the Cicla group, for example, spinach beet or chard, 3) the Flavescens group, for example, Swiss chard, 4) the Conditiva group, for example, beetroot or garden beet, and 5) the Crassa or fodder beet group, for example, mangelwurzel.

[0099] This disclosure describes the introduction of event Bv_CSM63713 into sugar beet, and thus the term "sugar beet event Bv_CSM63713" is used herein to refer to that event. However, one of ordinary skill in the art will understand that event Bv_CSM63713 can be readily introduced into other Beta vulgaris and plant species that can be crossed with Beta vulgaris by cross-pollination or outcrossing between a sugar beet plant containing the sugar beet event Bv_CSM63713 and another plant not containing the sugar beet event Bv_CSM63713, or by any other method known in the art. For example, to introduce event Bv_CSM63713 into fodder beet, a plant containing or comprising event Bv_CSM63713 is cross-pollinated with a fodder beet plant.This may be achieved or facilitated by human intervention. For example, collecting the pollen of a plant including event Bv_CSM63713 by hand, placing this pollen on the style or stigma of a forage beet plant that does not include event Bv_CSM63713, and event Bv_CSM63713 is inherited from the male parent including Bv_CSM63713 to the offspring of the forage beet, or removing, destroying, or covering the stamens or anthers of the forage beet plant by hand and / or by human actions (e.g., by manual intervention or by applying a chemical emasculant) to prevent natural self-pollination and effect cross-pollination by pollen from a plant including event Bv_CSM63713 for pollination to occur, or placing in a position for inducing pollination by human-mediated pollinating insects (e.g., placing beehives in an orchard or field or putting the plants in a cage with pollinating insects), or by liberating or removing the flower part of the forage beet by human to allow or cause foreign pollen from a plant containing or comprising event Bv_CSM63713 to be placed on or contact the style or stigma of the forage beet plant, selective placement of plants (e.g., intentionally planting plants in close proximity for pollination), and / or applying chemicals to promote flowering or to promote receptivity (of the stigma to pollen). Except for inducing cross-pollination, synchronizing the flowering of sugar beet and forage beet including the sugar beet event Bv_CSM63713 to occur very close in time to achieve random cross-pollination, and then selecting the offspring including the sugar beet event Bv_CSM63713 can be done.

[0100] The sugar beet event Bv_CSM63713 confers resistance to benzoic acid-type auxin herbicides, such as dicamba, inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), such as glyphosate, and inhibitors of glutamine synthetase, such as glufosinate, in sugar beet cells, plants, and seeds containing the event. The sugar beet event Bv_CSM63713 contains three expression cassettes. Table 15 of Example 7 provides a list of the elements contained in SEQ ID NO: 10, shown in Figure 1.

[0101] As used herein, the terms "derived from" or "deriving from" in connection with a particular DNA molecule, amplicon or sequence in relation to sugar beet plants, plant parts, seeds, progeny, cells and / or sugar beet plant products, such as commercial products, means that the DNA molecule, amplicon or sequence is taken, purified, isolated or manufactured directly or indirectly from such sugar beet plants, plant parts, seeds, progeny, cells and / or sugar beet plant products, such as commercial products. Alternatively, in connection with sugar beet plants, plant parts, seeds, progeny, or cells, the terms "derived from" or "deriving from" in relation to sugar beet plant products, such as commercial products, means that the sugar beet plant products are taken, purified, isolated or manufactured directly or indirectly from such sugar beet plants, plant parts, seeds, progeny, or cells.

[0102] "Capable of being detected" refers to the ability to detect a particular DNA molecule, segment or sequence in a sample by amplification and determination of its presence, size or sequence, such as DNA sequence analysis, and / or binding of a probe to the target DNA molecule, segment or sequence.

[0103] "Sample" is intended to refer to any composition that directly or indirectly contains or is derived from a biological sample, source, or material. The sample can generally contain sugar beet DNA and / or substantially or completely pure, purified, or isolated sugar beet DNA. "Biological sample" includes, but is not limited to, biological materials directly or indirectly obtained from or derived from the genome of sugar beet cells (s), tissues (s), seeds (s), plants (s), plant parts (s) and / or sugar beet plant products (s), such as commercial products (s), DNA. Such sugar beet cells (s), tissues (s), seeds (s), plants (s), plant parts (s) and / or sugar beet plant products (s), such as commercial products (s), can contain the sugar beet event Bv_CSM63713, or DNA molecules (s) and / or DNA segments (s) containing the sugar beet event Bv_CSM63713. In some embodiments, the sample or biological sample is one in which the cell or cell membrane has been disrupted (e.g., fragmented or released), the contents of the sugar beet cell (s) containing genomic DNA or protein have been released, and / or the contents of the sugar beet cell (s), sugar beet tissue (s), sugar beet seed (s), sugar beet plant (s), sugar beet plant part (s) and / or sugar beet plant product (s) containing genomic DNA or protein have been made more accessible or usable for an assay or test. "Directly" means, to those skilled in the art, directly obtaining DNA by disrupting sugar beet cells (or obtaining a sugar beet sample containing disrupted sugar beet cells) from the sugar beet genome and exposing or using the genomic DNA or protein derived from the sugar beet cells for detection."Indirectly" means that a person skilled in the art obtains the target or a specific reference DNA (e.g., the novel and unique junction segment(s) described herein used for the diagnosis of the presence of event Bv_CSM63713) contained in a specific sample by a method other than directly obtaining it through the destruction of sugar beet cells or obtaining a sugar beet sample containing the destroyed sugar beet cells. Such indirect means include amplification of a DNA segment containing a DNA sequence designed to specifically bind to a target sequence or targeted by a specific probe(s) and / or primer set(s) in the vicinity of the target sequence, or measurement and characterization thereof (e.g., measured by transfer or separation from other DNA segments and / or identification on an effective matrix such as an agarose or acrylamide gel, or by direct sequence analysis of the amplicon(s) or cloning the amplicon(s) into a vector(s) and characterizing the inserted amplicon(s) present in such vector(s) by direct sequencing), including but not limited to amplification of a DNA segment containing all or part of the target sequence.

[0104] As used herein, the term "recombinant" refers to non-natural DNA, proteins, or organisms created by human intervention that are not normally found in nature. As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that do not naturally occur together and are the result of human intervention, for example, a DNA molecule composed of a combination of at least two DNA molecules that are heterologous to each other, such as a DNA molecule containing a transgene and plant genomic DNA adjacent to the transgene. An example of a recombinant DNA molecule is a DNA molecule containing at least one sequence selected from SEQ ID NOs: 1-10. As used herein, a "recombinant plant" is a plant that does not normally exist in nature and is the result of human intervention and contains a transgenic DNA molecule. As a result of such genomic changes, the recombinant plant is novel and distinct from the related wild-type plant. An example of a recombinant plant is a sugar beet plant containing the sugar beet event Bv_CSM63713.

[0105] As used herein, the term "transgene" refers to a DNA molecule artificially integrated into the genome of an organism as a result of human intervention, for example, by a plant transformation method. The transgene can be heterologous to the organism. As used herein, the term "transgenic insert" refers to foreign DNA inserted into the sugar beet genome to produce the sugar beet event Bv_CSM63713 by plant transformation technology. The sequence of the transgenic insert of the sugar beet event Bv_CSM63713 is provided as SEQ ID NO: 9. The term "transgenic" refers to containing a transgene. For example, a "transgenic plant" refers to a plant containing a transgene.

[0106] As used herein, the term "heterologous" refers to a first molecule that is not normally associated in nature with a second molecule or organism. For example, a DNA molecule can be derived from a first species and inserted into the genome of a second species. Thus, the DNA molecule is heterologous to the genome and organism.

[0107] As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, where neither the first DNA molecule nor the second DNA molecule is normally found in the configuration in which it is fused to the other. Thus, the chimeric DNA molecule is a novel DNA molecule not normally found as such. An example of a chimeric DNA molecule is a DNA molecule comprising at least one sequence selected from SEQ ID NOs: 1-10.

[0108] As used herein, the term "isolated" refers to a molecule being separated from other molecules with which it is normally associated in its native or natural state. The term "isolated" can thus refer to a DNA molecule that has been separated from other DNA molecule(s) with which it is normally associated in its native or natural state. Such a DNA molecule can exist in a recombinant state, e.g., as a recombinant DNA molecule. Thus, a DNA molecule that has been removed from its natural state and fused to another DNA molecule with which it is not normally associated is an isolated DNA molecule. Such an isolated DNA molecule can be brought about by using biotechnology techniques, e.g., by making recombinant DNA or by integrating a foreign DNA molecule into the chromosome of a cell, plant, or seed.

[0109] Provided are DNA molecules, fragments, and their corresponding DNA sequences. As used herein, the terms "DNA" and "DNA molecule" refer to deoxyribonucleic acid (DNA) molecules. The DNA molecules can be of genomic origin or synthetic origin, and by convention, are from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule, i.e., the sequence of consecutive nucleotides within the DNA molecule. As used herein with respect to the nucleotides of a polynucleotide or DNA sequence or molecule, the terms "consecutive" and "contiguous" are interchangeable and synonymous, and refer to nucleotides in 5' to 3' order within a polynucleotide or DNA sequence, strand, or molecule that contain no gaps or interruptions between them. The nomenclature used is that required by 37 CFR 1.822 of the United States Code of Federal Regulations and defined in the tables of Annex 2, Tables 1 and 3 of WIPO Standard ST.25 (1998). By convention, DNA sequences and fragments thereof are disclosed by reference to only one strand of the two complementary DNA sequence strands. Implicitly and intentionally, the complementary sequence (the sequence of the complementary strand) of the sequences provided herein, also referred to in the art as the reverse complementary sequence, is within the scope of the present invention and is explicitly intended to be within the scope of the claimed subject matter. Accordingly, references herein to SEQ ID NOs: 1-10 and fragments thereof include and refer to the sequences of the complementary strand and fragments thereof. FIG. 1 is a diagram of transgenic DNA inserts in the genome of sugar beet plants containing event Bv_CSM63713, and the relative positions of SEQ ID NOs: 1-10 are arranged from 5' to 3'.

[0110] Also provided are nucleic acid molecules comprising polynucleotides having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NOs: 1 to 12.

[0111] For example, provided are nucleic acid molecules comprising polynucleotides having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9.

[0112] As used herein, the term "fragment" refers to a smaller piece or sequence of a larger or entire DNA molecule or sequence. For example, a fragment of any one of SEQ ID NOs: 1-12 can be at least about 10 consecutive nucleotides, at least about 11 consecutive nucleotides, at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, at least about 100 consecutive nucleotides, at least about 200 consecutive nucleotides, at least about 300 consecutive nucleotides, at least about 400 consecutive nucleotides, or at least about 500 consecutive nucleotides of the larger, whole or complete DNA molecule or sequence.

[0113] For example, a "fragment" of the transgenic insert array (SEQ ID NO: 9) of the sugar beet event Bv_CSM63713 can contain at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 consecutive nucleotides of SEQ ID NO: 9. Further, the present disclosure encompasses nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 9 or any fragment thereof.

[0114] Similarly, fragments of the 5' flank (SEQ ID NO: 11) or 3' flank (SEQ ID NO: 12) of the sugar beet event Bv_CSM63713 can contain at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 12. Further, the present disclosure encompasses nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or SEQ ID NO: 12, or any fragment thereof.

[0115] The DNA sequence of the transgenic insert of the sugar beet event Bv_CSM63713 is provided as SEQ ID NO: 9. The DNA sequence of the transgenic insert and the sugar beet genomic DNA flanking both sides of the transgenic insert is provided as SEQ ID NO: 10. The DNA sequences of a part of the flanking DNA and the 5' end of the transgenic insert are provided as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. The DNA sequences of a part of the flanking DNA and the 3' end of the transgenic insert are provided as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

[0116] The sugar beet event Bv_CSM63713 is characterized as a transgenic insertion into a single locus within the sugar beet genome, resulting in two new junctions (or joints or connection points). The region of DNA sequence spanning the connection by a phosphodiester bond to the adjacent sugar beet genomic DNA at one end of the transgenic insert is herein referred to as a "junction". A junction is the point of connection or covalent bond of one continuous molecule of one end of the transgenic insert and the adjacent genomic DNA, formed by the insertion of a heterologous nucleic acid molecule into the sugar beet genomic DNA. One junction is found at the 5' end of the transgenic insert and the other is found at the 3' end of the transgenic insert, herein referred to as the 5' and 3' junctions, respectively. "Junction sequence" refers to the DNA sequence of any length of contiguous nucleotides spanning the 5' or 3' junction of an event within the plant genome. In the case of a "junction sequence" specific to the junction between a transgenic event and the adjacent genomic sequence, the junction sequence generally includes a sufficient number of contiguous nucleotides at one end of the insert and a sufficient number of contiguous nucleotides of the adjacent genomic sequence.According to some embodiments, a "junction sequence" may include (i) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, at least twenty-five (25) consecutive nucleotides, at least thirty (30) consecutive nucleotides, at least thirty-five (35) consecutive nucleotides, at least forty (40) consecutive nucleotides, at least forty-five (45) consecutive nucleotides, or at least fifty (50) consecutive nucleotides at one end of the insertion portion, and (ii) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, at least twenty-five (25) consecutive nucleotides, at least thirty (30) consecutive nucleotides, at least thirty-five (35) consecutive nucleotides, at least forty (40) consecutive nucleotides, at least forty-five (45) consecutive nucleotides, or at least fifty (50) consecutive nucleotides of the adjacent genomic DNA sequence. It is understood that any length of consecutive nucleotides across the junction of a transgenic event in a plant genome can be a junction sequence. The junction sequences of the sugar beet event Bv_CSM63713 are apparent to those skilled in the art, and the various junction sequences of the sugar beet event Bv_CSM63713 can be identified by those skilled in the art using SEQ ID NO: 10. In SEQ ID NO: 10, the 5' junction is at nucleotides 1000-1001, and the 3' junction is at nucleotides 12,722-12,723. Examples of the junction sequences of the sugar beet event Bv_CSM63713 are provided in SEQ ID NOs: 1-8. Figure 1 shows the physical arrangement of SEQ ID NOs: 1-10 arranged from 5' to 3'. The junction sequences of the sugar beet event Bv_CSM63713 can exist as part of the genome of a plant, seed, plant part, progeny, or plant cell containing the sugar beet event Bv_CSM63713.Identifying any one or more of SEQ ID NOs: 1-10 in a sample derived from a plant, plant part, seed, progeny or cell indicates that the DNA is from a sugar beet containing the sugar beet event Bv_CSM63713 and is used for diagnosing the presence of the sugar beet event Bv_CSM63713.

[0117] The junction sequences described herein are used for diagnosing the presence of all or part of the sugar beet event Bv_CSM63713. Thus, direct or indirect identification or detection of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10 in a sample or DNA molecule derived from a sugar beet plant, plant part, seed, progeny, cell, or commercial product is used for diagnosing whether the sugar beet plant, plant part, seed, progeny, cell, or commercial product has or contains all or part of the sugar beet event Bv_CSM63713. Direct or indirect identification or detection of the 5' junction sequence and / or 3' junction sequence (each provided or described herein) in a sample or DNA molecule derived from a sugar beet plant, plant part, seed, progeny, cell, or commercial product is used for diagnosing whether the sugar beet plant, plant part, seed, progeny, cell, or commercial product has or contains the sugar beet event Bv_CSM63713. The present disclosure thus provides a DNA molecule comprising at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Any segment of DNA derived from the transgenic sugar beet event Bv_CSM63713 that is sufficient to include at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 is within the scope of the present disclosure. Further, any DNA or polynucleotide molecule or sequence comprising a sequence complementary to any of the sequences described herein is also within the scope of the present disclosure.

[0118] The plant, seed, plant cell, plant part, progeny, and commodity product can be used for the detection of a DNA or protein molecule indicating the presence of the sugar beet event Bv_CSM63713. Provided is an exemplary DNA molecule that can be used as either a primer or a probe for detecting the presence of the sugar beet event Bv_CSM63713 in a sample. Such a primer or probe is specific to the target nucleic acid sequence and is thus useful for the identification of the sugar beet event Bv_CSM63713 by the methods described herein. The primer or probe can hybridize to the target polynucleotide sequence and enables the specific detection or amplification of a polynucleotide molecule that contains or is covalently linked and associated with the target polynucleotide sequence. The target polynucleotide sequence can include all or part of the sugar beet event Bv_CSM63713, the junction sequence, and / or the adjacent genomic DNA. The probes and primers according to the present disclosure have (i) complete or 100% sequence complementarity to the target polynucleotide sequence (i.e., 100% complementarity), or (ii) incomplete sequence complementarity to the target polynucleotide, such as at least 60% complementarity, at least 65% complementarity, at least 70% complementarity, at least 75% complementarity, at least 80% complementarity, at least 85% complementarity, at least 90% complementarity, at least 95% complementarity, or at least 99% complementarity to the target polynucleotide sequence, as long as the probe or primer has sufficient complementarity to the target polynucleotide sequence to hybridize to the target polynucleotide sequence under stringent hybridization conditions appropriate and necessary for the use of the probe or primer in the relevant amplification or detection assay, reaction, or method. As is understood in the art, if the length of the primer or probe is long and it depends on stringency and use, the percentage of complementarity of the primer or probe may be lower. Provided is an exemplary polynucleotide molecule that can be used as either a primer or a probe for detecting the presence of the sugar beet event Bv_CSM63713 in a sample.The detection of the presence of the sugar beet event Bv_CSM63713 can be carried out using methods known in the art, such as thermal or isothermal amplification of nucleic acids or nucleic acid hybridization techniques (e.g., Southern analysis).

[0119] A "primer" is a DNA molecule designed for use in annealing or hybridization methods involving an amplification reaction. The amplification reaction is an in vitro reaction that amplifies template DNA to produce an amplicon. As used herein, an "amplification product" or "amplified DNA" or "amplicon" is a DNA molecule synthesized using amplification techniques further described herein for a target nucleic acid or DNA molecule that is part of a template nucleic acid molecule. To amplify or amplification refers to making multiple copies of a target DNA molecule or segment from a template DNA. For example, to determine whether a sugar beet plant, plant part, seed, progeny or plant cell resulting from self-fertilization or outcrossing of a parent containing the sugar beet event Bv_CSM63713 contains the sugar beet event Bv_CSM63713, DNA is extracted from the sugar beet plant tissue sample and subjected to an amplification reaction or method using a pair of primers specific for a target sequence specifically related to or part of the sugar beet event Bv_CSM63713, for example, a first primer derived from a genomic DNA sequence in a region adjacent to the inserted heterologous DNA of the sugar beet event Bv_CSM63713 that is extended by polymerase in the 5' to 3' direction of the inserted DNA, and a second primer derived from the heterologous inserted DNA molecule that is extended by polymerase in the 5' to 3' direction of the adjacent genomic DNA from which the first primer is derived. The length of the amplicon can vary depending on the length of the polynucleotide or DNA sequence intervening between the target sequences of the two primers in the template DNA molecule. Alternatively, the primer pair can be derived from the genomic sequences flanking the inserted heterologous DNA, and an amplicon containing the entire inserted polynucleotide sequence is produced (e.g., a forward primer targeted to the genomic portion at the 5' end of SEQ ID NO: 10 (i.e., upstream of SEQ ID NO: 9) and a reverse primer targeted to the genomic portion at the 3' end of SEQ ID NO: 10 (i.e., downstream of SEQ ID NO: 9), which amplifies a DNA molecule containing the inserted DNA sequence (SEQ ID NO: 9) identified herein in the genome of the sugar beet event Bv_CSM63713).The use of the term "amplicon" specifically excludes primer dimers that may form during the DNA amplification reaction.

[0120] The amplicons described herein can include a DNA sequence that is a fragment of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, is at least 10 nucleotides in length, and includes one or more of the fragments that include nucleotides 1,000 - 1,001 or 12,722 - 12,723 of SEQ ID NO: 10. According to this embodiment, the sequence of the amplicon includes at least one junction sequence or two junction sequences for the sugar beet event Bv_CSM63713, for example, a 5' junction sequence and / or a 3' junction sequence. The amplification and detection of such amplicons are used for the implication or diagnosis of the sugar beet event Bv_CSM63713.

[0121] Primers are typically designed to hybridize to complementary target DNA strands and form hybrids between the primer and the target DNA strand. The presence of the primer is the recognition point for a polymerase to initiate the extension of the primer (i.e., the polymerization of additional nucleotides into an extended nucleotide molecule) using the target DNA strand as a template. A primer pair refers to the use of two primers that bind to opposite strands of a double-stranded nucleotide segment for the purpose of amplifying the polynucleotide segment between the positions targeted for binding by each of the primer pair, usually in a thermal amplification reaction or other conventional nucleic acid amplification method.

[0122] To detect the presence or absence of the sugar beet event Bv_CSM63713, the target position and / or intervening region or sequence of the template DNA molecule may include at least one junction sequence and / or at least a part of the insert of the sugar beet event Bv_CSM63713. To detect the absence of the sugar beet event Bv_CSM63713, the target position and / or intervening region or sequence of the template DNA molecule may include sugar beet genomic DNA that does not include the junction sequence of the insert of the sugar beet event Bv_CSM63713 nor any part of the insert. Thus, the presence or absence of an amplicon having a primer pair may be used, in some cases, to diagnose the presence or absence of the sugar beet event Bv_CSM63713 in a DNA molecule or sample, and in other cases, vice versa. This may also be possible with two or more primer pairs. For example, when the sugar beet event Bv_CSM63713 is present, a first primer pair may produce a first amplicon, and when the sugar beet event Bv_CSM63713 is absent or not present, a second primer pair may produce a second amplicon. As another method, the size of the amplicon produced in the amplification reaction may be used to diagnose the presence or absence of the sugar beet event Bv_CSM63713 in a DNA molecule or sample. For example, the primer pair may produce a first amplicon of a first size when the sugar beet event Bv_CSM63713 is present, or a second amplicon of a second size when the sugar beet event Bv_CSM63713 is absent or not present, or the first primer pair may produce a first amplicon of a first size when the sugar beet event Bv_CSM63713 is present, and the second primer pair may produce a second amplicon of a second size when the sugar beet event Bv_CSM63713 is absent or not present. According to some of these embodiments, at least two primer pairs may be used, in which case at least one primer pair is used as an internal control and is not related to the sugar beet event Bv_CSM63713.

[0123] According to this embodiment, a primer pair for detecting the presence or absence of all or part of the sugar beet event Bv_CSM63713 in a DNA molecule or sample includes a first primer and a second primer, the first primer being complementary to the 5'-adjacent genomic DNA sequence, and the second primer being complementary to a sequence within the transgenic insert, or the first primer being complementary to the 5'-adjacent genomic DNA sequence, and the second primer being complementary to the 3'-adjacent genomic DNA sequence, or the first primer being complementary to a sequence within the transgenic insert, and the second primer being complementary to the 3'-adjacent genomic DNA sequence. In this paragraph, each reference to a primer complementary to the 5'-adjacent genomic DNA sequence, 3'-adjacent genomic DNA sequence, or sequence within the transgenic insert of the sugar beet event Bv_CSM63713 is also intended to potentially include a primer complementary to the reverse complement or opposite strand of the 5'-adjacent genomic DNA sequence, 3'-adjacent genomic DNA sequence, or sequence within the transgenic insert of the sugar beet event Bv_CSM63713, respectively.

[0124] Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 19 / 20 and SEQ ID NO: 23, SEQ ID NO: 14 / 15 and SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 26 and SEQ ID NO: 25 / 31, and SEQ ID NO: 28 / 33 and SEQ ID NO: 29. For example, exemplary event-specific primers for PCR to identify event Bv_CSM63713 are provided as SEQ ID NO: 14 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 23, and SEQ ID NO: 34 and SEQ ID NO: 35. Exemplary primers that can be used for analysis of the 5' (left) junction region are provided as SEQ ID NO: 14 and SEQ ID NO: 18, and exemplary primers that can be used for analysis of the 3' (right) junction region are provided as SEQ ID NO: 19 and SEQ ID NO: 23, and SEQ ID NO: 34 and SEQ ID NO: 35. Exemplary primers that can be used for the zygosity test of event Bv_CSM63713 are provided as SEQ ID NO: 26, SEQ ID NO: 25 and SEQ ID NO: 24, and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0125] The primer pairs provided as SEQ ID NO: 19 / 20 and SEQ ID NO: 23, SEQ ID NO: 14 / 15 and SEQ ID NO: 18, and SEQ ID NO: 34 and SEQ ID NO: 35 are useful as a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is a fragment of the DNA sequence of the transgenic insert of SEQ ID NO: 10, and the second DNA molecule is a fragment of the adjacent DNA sequence of SEQ ID NO: 10, each of which functions as a DNA primer when used together in an amplification reaction with DNA containing the sugar beet event Bv_CSM63713 and is of sufficient length to produce an amplicon used for the diagnosis of the sugar beet event Bv_CSM63713 in a sample. The amplicon used for the diagnosis of event Bv_CSM63713 contains sequences not naturally found within the sugar beet genome. The primer pair may also be defined as comprising a first and a second DNA molecule, wherein the first DNA molecule is a fragment of the sugar beet genomic portion of SEQ ID NO: 10, and the second DNA molecule is a fragment of the transgene portion of SEQ ID NO: 10 (or fragment 9 of the sequence), each of which functions as a DNA primer when used together in an amplification reaction with DNA containing the sugar beet event Bv_CSM63713 and is of sufficient length to produce an amplicon used for the diagnosis of the sugar beet event Bv_CSM63713 in a sample. The primer may further include an oligo-tail sequence, for example, those used in the Kompetitive Allele-Specific PCR (KASP™) method. Each of the allele-specific primers carries a unique tail sequence corresponding to a universal FRET (fluorescence resonance energy transfer) cassette, one of which is labeled with the FAM™ dye and the other is labeled with the HEX™ dye. During thermal cycling, as the relevant allele-specific primer binds to and extends from the template, the tail sequence attaches to the newly synthesized strand. Next, the complement of the allele-specific tail sequence is generated during subsequent rounds of PCR, allowing the FRET cassette to bind to the DNA. The FRET cassette is no longer quenched and emits fluorescence. Examples of primers containing an oligo-tail sequence are SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 27.Examples of primers corresponding to SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 27 but not containing the oligo tail sequence are SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

[0126] A "probe" is a nucleic acid molecule that is complementary to a strand of a target nucleic acid and is useful in a hybridization detection method. The probe includes not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence, and the detection of such binding can be useful for detecting the presence or absence of the target DNA sequence. The probe can be linked to a conventional detectable label or reporter molecule, such as a radioisotope, a ligand, a chemiluminescent agent, or an enzyme.

[0127] An exemplary DNA sequence useful as a probe for detecting the sugar beet event Bv_CSM63713 is provided as SEQ ID NO: 36. In some embodiments, the DNA molecule functioning as a probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, a complement of any of the foregoing, or a fragment of any of the foregoing. In other embodiments, the DNA molecule comprises a polynucleotide segment of sufficient length to function as a DNA probe specific for at least one of the following: a) the 5' junction sequence between the adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713; b) the 3' junction sequence between the transgenic insert of the sugar beet event Bv_CSM63713 and the adjacent sugar beet genomic DNA; c) SEQ ID NO: 9; or d) a fragment of SEQ ID NO: 9 comprising a continuous nucleotide of SEQ ID NO: 9 of sufficient length to identify the sequence as a fragment of the transgenic insert of the sugar beet event Bv_CSM63713 in a DNA sample.

[0128] Methods of designing and using primers and probes are well known in the art. DNA molecules containing the fragments of SEQ ID NOs: 1-10 are useful as primers and probes for detecting the sugar beet event Bv_CSM63713 and can be readily designed by those skilled in the art using the sequences provided herein. DNA probes and DNA primers generally are at least ten (10) nucleotides in length, often at least fifteen (15) nucleotides in length, at least twenty (20) nucleotides in length, or at least thirty (30) nucleotides in length. Such probes and primers are selected to have a length sufficient to specifically hybridize to a target sequence under stringent hybridization conditions.

[0129] Probes and primers can have perfect sequence identity with the target sequence, but different primers and probes that retain the ability to preferentially hybridize to the target sequence can be designed by conventional methods. For a nucleic acid molecule to function as a primer or probe, it is only necessary that the sequences be sufficiently complementary and / or of sufficient length to allow for the formation of a stable double-stranded structure under specific hybridization or reaction conditions. The presence of transgenic DNA derived from sugar beet event Bv_CSM63713 in a sample can be identified using any conventional nucleic acid hybridization or amplification method. A polynucleotide molecule referred to as a "polynucleotide segment of sufficient length" or "continuous nucleotides of sufficient length" can thus specifically hybridize to a target DNA sequence under certain hybridization or reaction conditions. As used herein, the term "sufficient length" refers to a length sufficient to be useful in an optimal detection method. Probes and primers are generally at least about 8 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 14 nucleotides, at least about 16 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 24 nucleotides, at least about 26 nucleotides, at least about 28 nucleotides, or at least about 30 nucleotides or longer in length. Such probes and primers specifically hybridize to the target DNA sequence under stringent hybridization conditions. Conventional stringency conditions are described in MR Green and J Sambrook, Molecular cloning: a laboratory manual, 4 th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0130] As used herein, two nucleic acid molecules are capable of specifically hybridizing to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is a "complement" of another nucleic acid molecule if they exhibit complete complementarity. As used herein, two molecules exhibit "complete complementarity" if, when aligned, all of the nucleotides of the first molecule are complementary to all of the nucleotides of the second molecule. Two molecules are "minimally complementary" if they can hybridize to each other with sufficient stability to remain annealed to each other, at least under conventional "low stringency" conditions. Similarly, the molecules are "complementary" if they can hybridize to each other with sufficient stability to remain annealed to each other under conventional "high stringency" conditions. Deviations from complete complementarity are thus tolerated as long as such deviations do not completely preclude the ability of the molecules to form a double-stranded structure.

[0131] As used herein, "stringent hybridization conditions" refers to conditions under which a polynucleotide hybridizes to its target sequence, typically in a complex mixture of nucleic acids, essentially without hybridizing to other sequences. "Stringent conditions" or "stringent hybridization conditions" when referring to polynucleotide probes refers to conditions under which the probe hybridizes to its target sequence to a detectably high degree (e.g., at least 2-fold over background) relative to other sequences. Stringent conditions are sequence-dependent and vary depending on the environment. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 - 10 °C lower than the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, at the Tm, 50% of the probe is occupied at equilibrium). Stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 - 1.0 M sodium ion concentration (or other salts), pH 7.0 - 8.3, and the temperature is at least about 30 °C for short probes (e.g., 10 - 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing). Alternatively, the stringency conditions can be adjusted to allow some mismatches in the sequence, resulting in the detection of a lower degree of identity (non-homologous probing).

[0132] As used herein, a substantially complementary or identical sequence is a polynucleotide that specifically hybridizes to a nucleic acid molecule being compared under high stringency conditions, or to its complement, respectively. Appropriate stringency conditions that promote DNA hybridization, for example, 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash in 2× SSC at 50°C, are known to those of ordinary skill in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

[0133] The polynucleotide molecules of the present disclosure, such as primers or probes, have a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or at least one nucleic acid molecule selected from the group consisting of the exact complement or fragment of any of the foregoing, and specifically hybridize under stringent conditions or, if the sequence of the polynucleotide molecule is not identical to the at least one nucleic acid molecule, under moderately stringent hybridization conditions. An exemplary DNA sequence useful as a probe for detecting the sugar beet event Bv_CSM63713 is provided as SEQ ID NO: 36. Hybridization of the probe to the target DNA molecule can be detected by any method known to those of ordinary skill in the art, including but not limited to fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

[0134] Regarding the amplification of a target polynucleotide using a specific amplification primer pair (e.g., by PCR), "stringent conditions" or "stringent hybridization conditions" mean that in the thermal amplification reaction of DNA, the primer pair hybridizes to the target polynucleotide to which a primer having the corresponding wild-type sequence (or its complement) binds, and is capable of producing an identifiable amplification product (amplicon) having a region specific to the Beta vulgaris Bv_CSM63713 event.

[0135] The term "specific for" a target sequence indicates that a probe or primer hybridizes only to the target sequence in a sample containing the target sequence under stringent hybridization conditions.

[0136] Suitable stringency conditions for promoting DNA hybridization, such as washing with 6.0x sodium chloride / sodium citrate (SSC) at about 45°C followed by 2.0x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from low stringency of about 2.0x SSC at 50°C to high stringency of about 0.2x SSC at 50°C. Further, the temperature of the washing step can be increased from low stringency conditions at room temperature, i.e., about 22°C, to high stringency conditions at about 65°C. Both temperature and salt can be varied, or one of temperature or salt concentration can be kept constant while the other variable element is changed.

[0137] Diagnostic amplicons produced by the methods described herein can be detected by a plurality of techniques known in the art, such as sequencing, restriction enzyme mapping, Southern analysis, or any other suitable polynucleotide or DNA hybridization, blotting, polymerization and / or amplification-based approaches or techniques. One method is genetic bit analysis (Nikiforov, et al., 1994), where DNA oligonucleotides that overlap both the adjacent genomic DNA sequence and the inserted DNA sequence, i.e., junction sequences, are designed. The oligonucleotides are immobilized in the wells of a microtiter plate. Following PCR of the region of interest (e.g., using one primer in the inserted sequence and one in the adjacent genomic sequence), the single-stranded PCR product can hybridize to the immobilized oligonucleotide and function as a template for a single-base extension reaction using DNA polymerase and a labeled dideoxynucleotide triphosphate (ddNTP) specific for the expected next base. The readout can be by fluorescence or ELISA-based. The signal indicates the presence of the transgene / genomic junction sequence by virtue of the achievement of amplification, hybridization, and single-base extension.

[0138] Another method is pyrosequencing technology, as described by Winge (2000). In this method, oligonucleotides that overlap the adjacent genomic DNA and the insert DNA junction are designed. The oligonucleotides are hybridized to single-stranded PCR products from the region of interest (one primer in the inserted sequence and one in the adjacent genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate, and luciferin. The dNTPs are added individually and their incorporation results in a light signal that is measured. The light signal indicates the presence of the transgene / genomic sequence by virtue of the achievement of amplification, hybridization, and single-base or multi-base extension.

[0139] The fluorescence polarization described by Chen, et al., (1999) is a method that can be used to detect the amplicons of the present invention. Using this method, oligonucleotides that overlap with genomic flanks and the inserted DNA junction are designed. The oligonucleotides are hybridized to single-stranded PCR products from the region of interest (one primer in the inserted DNA and one in the adjacent genomic DNA sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTPs. Incorporation of the ddNTP occurs by single-base extension. Incorporation can be measured as a change in polarization using a fluorometer. The change in polarization indicates the presence of the transgene / genomic sequence due to the achievement of amplification, hybridization, and single-base extension.

[0140] In real-time polymerase chain reaction (PCR), it is possible to observe the progress of PCR as it occurs (i.e., in real-time). Data are collected throughout the PCR process rather than at the end of PCR. In real-time PCR, the reaction is characterized by the point in the cycle at which target amplification is first detected, rather than the amount of target accumulated after a fixed number of cycles. In a real-time PCR assay, a positive reaction is detected by the accumulation of a fluorescent signal. The greater the starting copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed. The cycle threshold (Ct value) is defined as the number of cycles required for the fluorescent signal to exceed a threshold (i.e., above the background level). The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct value, the greater the amount of target nucleic acid in the sample).

[0141] Taqman® (PE Applied Biosystems, Foster City, CA) is a method for detecting and quantifying the presence of DNA sequences using real-time PCR and is well understood in the manufacturer's instructions. Briefly, FRET oligonucleotide probes that overlap the genomic flanks and the inserted DNA junction are designed. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one in the adjacent genomic sequence) are subjected to cycles in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety of the FRET probe. The fluorescent signal indicates the presence of the transgene / genomic sequence by achieving amplification and hybridization.

[0142] Molecular beacons are described for use in sequence detection and are described in Tyangi, et al. (1996). Briefly, FRET oligonucleotide probes that overlap the adjacent genomic and inserted DNA junction are designed. The unique structure of the FRET probe results in a secondary structure that maintains the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one in the adjacent genomic sequence) are subjected to cycles in the presence of a thermostable polymerase and dNTPs. Following achievement of PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe's secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal is generated, which indicates the presence of the adjacent / transgene insertion sequence by achieving amplification and hybridization.

[0143] Other detection methods known in the art may also be used. For example, microfluidic technology (see, e.g., U.S. Patent Publication No. 2006 / 068398, U.S. Patent No. 6,544,734) provides methods and devices that can be used to separate and amplify DNA samples or molecules. Optical dyes can be used to detect and measure specific DNA molecules (see, e.g., WO / 05017181). Next, nanotube devices containing electronic sensors for detecting DNA molecules or nanobeads that bind to specific DNA molecules (see, e.g., WO / 06024023) can be detected. Nanopore sequencing technologies, such as those described in Wang et al. (2021), Tyler et al. (2018), or Pearson et al. (2019), can also be used for the detection of events.

[0144] Provided is a protein that can be used to produce an antibody for detecting the presence of the sugar beet event Bv_CSM63713 in a sample. Such an antibody is specific for one or more of the proteins encoded by the sugar beet event Bv_CSM63713. Methods for preparing polyclonal or monoclonal antibodies are well known to those skilled in the art and can be used to generate antibodies specific for one or more of the proteins encoded by the sugar beet event Bv_CSM63713. For example, U.S. Patent No. 7,838,729 and Wang et al. (2016) describe antibodies against DMO, and Harrison et al. (1996) and Chinnaduura et al. (2018) describe antibodies against CP4 EPSPS. U.S. Patent No. 9,371,394 describes antibodies against the PAT enzyme. The DNA sequence encoding such a protein is provided in SEQ ID NO: 10, and the start and stop positions of the coding sequence are shown in Table 15 in Example 7. The DNA sequence encoding each protein, and the protein encoded by the sequence, are useful for producing an antibody for detecting the presence of the sugar beet event Bv_CSM63713 by the methods described herein. Detection of the presence of the sugar beet event Bv_CSM63713 can be performed using any protein detection technique known in the art, such as Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), antibody binding to a detectable label or reporter molecule (e.g., radioisotope, ligand, chemiluminescent agent, or enzyme), or enzymatic action on a reporter molecule. One method provides contacting a sample with an antibody that binds to the PAT, DMO, or CP4-EPSPS protein encoded by the sugar beet event Bv_CSM63713 and then detecting the presence or absence of antibody binding. Binding of such an antibody is used for diagnosing the presence of one or more proteins encoded by the sugar beet event Bv_CSM63713.

[0145] Provided are protein and nucleic acid detection kits for detecting the presence of the sugar beet event Bv_CSM63713. Variations of such kits can also be developed using the compositions and methods disclosed herein, as well as methods well known in the art for the detection of proteins and nucleic acids for identifying the sugar beet event Bv_CSM63713. The protein and nucleic acid detection kits can be applied to methods for breeding plants containing the sugar beet event Bv_CSM63713. Such kits include primers and / or probes or antibodies specific for the sugar beet event Bv_CSM63713. Such DNA primers and / or probes can include one or more fragments of SEQ ID NOs: 1-10, or antibodies specific for the protein encoded by the sugar beet event Bv_CSM63713. The kit can also include instructions for the use of the primers, probes, or antibodies for detecting the presence of the sugar beet event Bv_CSM63713. The kit can also optionally include reagents for performing the detection or diagnostic reactions described herein.

[0146] An example of a detection kit includes at least one DNA molecule of a contiguous nucleotide of SEQ ID NO: 10 that is of sufficient length to function as a DNA probe useful for detecting the presence or absence of the sugar beet event Bv_CSM63713 in a sample. DNA derived from a transgenic sugar beet plant containing the event Bv_CSM63713 includes a DNA molecule having at least one sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, the complement of any of the foregoing, or a fragment of any of the foregoing. An exemplary DNA molecule sufficient for use as a probe includes one having the sequence provided as SEQ ID NO: 36. Other probes can be readily designed by those skilled in the art. The probe can include a junction sequence spanning the 5' or 3' junction between the sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713.

[0147] Another example of a detection kit includes at least one primer pair that specifically hybridizes to a target DNA and amplifies a diagnostic amplicon under appropriate reaction conditions useful for detecting the presence or absence of the sugar beet event Bv_CSM63713 in a sample. Kits containing DNA primers that are homologous or complementary to any portion of the sugar beet event region shown in SEQ ID NO: 10 and any portion of the inserted transgenic DNA shown in SEQ ID NO: 9 are within the scope of the present disclosure. The kit can provide an agarose gel-based detection method or various methods for detecting the amplicon(s) known in the art. Such methods can also include sequencing of the amplicon or a fragment thereof. Exemplary DNA molecules that are sufficient for use as primer pairs include those containing the sequences provided as SEQ ID NO: 19 / 20 and SEQ ID NO: 23, SEQ ID NO: 14 / 15 and SEQ ID NO: 18, and SEQ ID NO: 34 and SEQ ID NO: 35. The primer pairs of SEQ ID NO: 19 / 20 and SEQ ID NO: 23, SEQ ID NO: 14 / 15 and SEQ ID NO: 18, and SEQ ID NO: 34 and SEQ ID NO: 35 produce an amplicon used for diagnosing the presence of event Bv_CSM63713 in a sample. Other primer pairs can be readily designed by those skilled in the art.

[0148] Another example of a detection kit includes at least one antibody specific for at least one protein encoded by the sugar beet event Bv_CSM63713. For example, such a kit can utilize a lateral flow strip that includes a reagent that is activated when the tip of the strip contacts an aqueous solution. Exemplary proteins that are sufficient for use in antibody production are those encoded by the sequence provided as SEQ ID NO: 10 or any fragment thereof. Detection of the binding of at least one antibody to at least one protein encoded by the sugar beet event Bv_CSM63713 in a sample is used for diagnosing the presence of the sugar beet event Bv_CSM63713 in the sample.

[0149] The detection kit provided herein is useful, inter alia, for identifying the sugar beet event Bv_CSM63713, selecting plant species or hybrids containing the sugar beet event Bv_CSM63713, detecting the presence of DNA derived from transgenic sugar beet plants containing the sugar beet event Bv_CSM63713 in a sample, and observing a sample for the presence and / or absence of sugar beet plants containing the sugar beet event Bv_CSM63713 or a plant part derived from a sugar beet plant containing the sugar beet event Bv_CSM63713.

[0150] Provided are sugar beet plants, progeny, seeds, cells, and plant parts containing the sugar beet event Bv_CSM63713, and commodity products manufactured using the same. These sugar beet plants, plant parts, plant cells, seeds, progeny plants, and commodity products contain or comprise, or are derived from transgenic sugar beet plants, plant parts, plant cells, seeds, progeny plants or commodity products containing or comprising the event Bv_CSM63713. These sugar beet plants, progeny, seeds, cells, plant parts, and commodity products contain a detectable amount of polynucleotides, such as at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, comprising at least one junction sequence and / or heterologous transgenic insert sequence of the sugar beet event Bv_CSM63713, polynucleotides, nucleic acids or DNA molecules comprising at least 21 consecutive nucleotides of SEQ ID NO: 1, at least 25 consecutive nucleotides of SEQ ID NO: 2, at least 33 consecutive nucleotides of SEQ ID NO: 3, at least 33 consecutive nucleotides of SEQ ID NO: 4, at least 53 consecutive nucleotides of SEQ ID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO: 6, nucleic acid molecules comprising polynucleotides having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9, and contain or comprise any of the above complete complements.In some embodiments, the sugar beet plant, plant part, plant cell, or seed is further defined as a progeny plant of any generation of a sugar beet plant comprising the sugar beet event Bv_CSM63713, or a sugar beet plant part, plant seed, or plant cell derived therefrom.

[0151] The sugar beet plant, plant part, plant cell, seed, progeny plant, and commodity product express or contain at least one herbicide tolerance gene selected from the group consisting of dicamba monooxygenase (DMO), phosphinothricin N-acetyltransferase (PAT), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and any combination thereof, and are tolerant to at least one herbicide selected from the group consisting of dicamba, glufosinate, glyphosate, and any combination thereof.

[0152] Also provided are sugar beet plants, plant seeds, plant parts, or plant cells that are resistant to herbicides having three different herbicide modes of action, wherein the genes conferring such herbicide resistance are present at a single genomic locus. To produce such sugar beet plants, plant seeds, plant parts, or plant cells, three transgenic cassettes containing herbicide resistance genes can be inserted into a single genomic locus within the sugar beet genome as a contiguous polynucleotide or a single molecularly linked transgenic insert. Alternatively, three transgenes cassettes containing herbicide resistance genes can be inserted into a single genomic locus by inserting separate cassettes containing herbicide resistance genes at the same position. "Single genomic locus" means that the gene is present at a single position on the chromosome, along with any regulatory sequences (e.g., promoters, introns, leader sequences, 5'-UTR, and / or 3'UTR, etc.), and / or sequences encoding a targeting peptide (e.g., a chloroplast transit peptide), and will be inherited as a single locus. Some intervening sequences may be present between each transgene cassette, but the length of such intervening sequences is limited such that the transgene cassettes are in close proximity to each other on the chromosome. For example, the intervening sequence between the transgene cassettes can be 500 nucleotides in length or less, 400 nucleotides in length or less, 300 nucleotides in length or less, 250 nucleotides in length or less, 200 nucleotides in length or less, 150 nucleotides in length or less, 100 nucleotides in length or less, or 50 nucleotides in length or less. For example, the sugar beet plants, plant seeds, plant parts, or plant cells can contain any of the DNA constructs described herein and can exhibit resistance to at least one herbicide selected from the group consisting of benzoic acid-type auxins, such as dicamba, glutamine synthetase inhibitors, such as glufosinate, EPSPS inhibitors, such as glyphosate, and any combination thereof.

[0153] As used herein, the terms "percent sequence identity" or "sequence identity %" refer to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference ("query") sequence (or its complementary strand) compared to a test ("subject") sequence (or its complementary strand), when the two sequences are optimally aligned (using appropriate nucleotides or amino acid insertions, deletions, or gaps that result in less than 20 percent total of the reference sequence over the comparison window). Optimal alignment of sequences for aligning a comparison window is well known to those of skill in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and by computer implementations of these algorithms, e.g., GAP, BESTFIT, FASTA, and TFASTA available as part of the sequence analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, Wis. 53715), and MUSCLE (version 3.6) (Edgar, "MUSCLE: multiple sequence alignment with high accuracy and high throughput" Nucleic Acids Research 32(5):1792-7 (2004)), using, e.g., default parameters. The "identity fraction" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the segment of the aligned reference sequence, i.e., in the entire reference sequence or a smaller defined portion of the reference sequence. The percent sequence identity is represented as the identity fraction multiplied by 100. Comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.Sugar beet plants, progeny, seeds, cells, plant parts, and commodity products containing a detectable amount of a polynucleotide having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9 are within the scope of the present disclosure.

[0154] The present disclosure provides plants, progeny, seeds, plant cells, and plant parts, such as roots, beets, pollen, anthers, ovaries, embryos, ovules, flowers, stems, leaves, microspores, protoplasts, and calli, derived from transgenic sugar beet plants containing event Bv_63713. Representative samples of seeds containing event Bv_63713 have been deposited in accordance with the Budapest Treaty for the purpose of enabling the present disclosure. The ATCC Repository has assigned accession number PTA-127098 to seeds containing event Bv_CSM63713.

[0155] The present disclosure provides a microorganism. The microorganism contains a polynucleotide molecule having the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 9. Examples of such microorganisms are Agrobacterium cells. Another example of such microorganisms is E. coli cells.

[0156] Provided are plant cells comprising the polynucleotide molecules described herein. For example, a nucleotide sequence selected from the group consisting of nucleic acid molecules comprising a polynucleotide having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or the full length of SEQ ID NO: 9.

[0157] The plant cells and microorganisms of the present disclosure are useful in many industrial applications including, but not limited to: (i) use as research tools for scientific or industrial research, (ii) use in culture to produce endogenous or recombinant carbohydrate, lipid, nucleic acid, enzyme or protein products, or small molecules that can subsequently be used for scientific research or as industrial products, and (iii) in the case of the plant cells of the present disclosure, use with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures that can subsequently be used in agricultural research or production. The production and use of such transgenic plant cells utilizes modern microbiological techniques and human intervention to produce unique artificial plant cells. In this process, recombinant DNA is inserted into the genome of the plant cell, creating a unique transgenic plant cell distinct from the naturally occurring plant cell. This transgenic plant cell can then be cultured, using modern microbiological techniques, to closely resemble bacterial and yeast cells and can exist in an undifferentiated single cell state. The genetic constitution and phenotype of this novel plant cell are the technical effects created by integrating heterologous DNA into the genome of the cell.

[0158] Provided is a method of using plant cells, such as transgenic plant cells. These include (i) a method of generating transgenic cells by integrating recombinant DNA into the genome of the cells and then using these cells to derive further cells having the same heterologous DNA, (ii) a method of culturing cells containing recombinant DNA using modern microbiological techniques, (iii) a method of producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, enzyme or protein products from the cultured cells, and (iv) a method of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.

[0159] Plants, progeny, seeds, cells, and plant parts may also contain one or more additional desirable trait(s). Such desirable traits can be transgenic traits, natural traits, or traits produced by other methods such as genome editing, base editing, prime editing or other conventional mutagenesis methods. Desirable traits may be combined with the sugar beet event Bv_CSM63713, for example, by crossing a sugar beet plant containing the sugar beet event Bv_CSM63713 with another sugar beet plant containing additional trait(s) or a transgenic event. Such traits or transgenic events include, but are not limited to, improved insect pest resistance, improved water use efficiency, improved yield, improved drought tolerance, improved disease resistance, improved seed quality, improved nutritional value, hybrid seed production, and / or improved herbicide tolerance, and the trait is measured relative to a sugar beet plant lacking such transgenic trait.

[0160] The plants described herein can be used to produce progeny that contain the sugar beet event Bv_CSM63713. As used herein, "progeny" includes any plant, seed, and cell and / or renewable plant part that contains the sugar beet event Bv_CSM63713 inherited from an ancestral plant, and at least one polynucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, at least 21 consecutive nucleotides of SEQ ID NO:1, at least 25 consecutive nucleotides of SEQ ID NO:2, at least 33 consecutive nucleotides of SEQ ID NO:3, at least 33 consecutive nucleotides of SEQ ID NO:4, at least 53 consecutive nucleotides of SEQ ID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6, or a polynucleotide having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO:10 or the full length of SEQ ID NO:9.

[0161] Plants, seeds, progeny, plant parts, and cells can be either homozygous or heterozygous for the sugar beet event Bv_CSM63713. Progeny plants can be grown from seeds produced by sugar beet plants containing the sugar beet event Bv_CSM63713, or from seeds produced by sugar beet plants pollinated with pollen containing the sugar beet event Bv_CSM63713.

[0162] The progeny plants may be self-pollinated (also known as "selfing") to produce plants of a pure breeding line, i.e., plants that are homozygous for the DNA of the sugar beet event Bv_CSM63713. Alternatively, the progeny plants may be crossbred, i.e., mated with another plant, to produce variant or hybrid seeds or plants. The other plant may be transgenic or non-transgenic. The variant or hybrid seeds or plants of the present disclosure may thus be obtained by crossing a first parent lacking the specific and unique DNA of event Bv_CSM63713 with a second parent containing event Bv_CSM63713, resulting in a hybrid containing the specific and unique DNA of event Bv_CSM63713.

[0163] The variant or hybrid seeds or plants of the present disclosure may be produced via three-way crossing (Poehlman J.M., 1987). In such cases, three parental lines, i.e., a first parental line, a second parental line, and a pollinator, are used for crossing. The F1 line produced by crossing a first parent containing event Bv_CSM63713 with a second parent also containing event Bv_CSM63713 is crossed with a third parent lacking the specific and unique DNA of event Bv_CSM63713. The parent containing event Bv_CSM63713 for the hybrid seeds or plants may be either the maternal or paternal parent. The maternal parent is preferably the parent containing event Bv_CSM63713 as it is cytoplasmic male sterile and does not produce transgenic pollen during the process of sugar beet variant or hybrid seed production.

[0164] Each parent may be a hybrid or an inbred / variety plant as long as the mating or breeding results in seeds having a polynucleotide having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to at least 21 consecutive nucleotides of SEQ ID NO: 1, at least 25 consecutive nucleotides of SEQ ID NO: 2, at least 33 consecutive nucleotides of SEQ ID NO: 3, at least 33 consecutive nucleotides of SEQ ID NO: 4, at least 53 consecutive nucleotides of SEQ ID NO: 5, at least 51 consecutive nucleotides of SEQ ID NO: 6, or the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9, and / or at least one allele containing the specific and unique DNA of the disclosed plant or seed, i.e., Event Bv_CSM63713.

[0165] Crossing one plant with another, i.e., outcrossing, may be achieved or facilitated by human intervention, for example, collecting the pollen of one plant by hand and contacting this pollen with the style or stigma of a second plant, removing, destroying, or covering the stamens or anthers of a plant (e.g., by manual intervention or by application of a chemical emasculant) by hand and / or by human action to prevent natural self-pollination and to effect pollination, outcrossing must be performed, placement in a position for "pollination induction" of pollen-carrying insects by humans (e.g., placing beehives in an orchard or field or enclosing a plant with pollen-carrying insects in a cage), releasing or removing parts of the flower by humans so that foreign pollen is placed on or contacts the style or stigma, selective placement of plants (e.g., intentionally planting plants in close proximity for pollination), and / or by applying a chemical to promote flowering or to promote receptivity (of the stigma to pollen) may be achieved or facilitated.

[0166] Provides a plant part. As used herein, "plant part" refers to any part of a plant composed of or derived directly from material from a plant including the sugar beet event Bv_CSM63713. Plant parts include, but are not limited to, roots, beets, pollen, anthers, ovaries, ovules, embryos, flowers, stems, leaves, microspores, protoplasts, and whole or parts of callus. A plant part can be growing or non-growing, renewable, and / or non-renewable.

[0167] Provided is a commercial product produced from a plant containing the sugar beet event Bv_CSM63713. The commercial product contains a detectable amount of DNA comprising a polynucleotide having a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 10, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9. As used herein, "commercial product" refers to any composition or product composed of materials derived from plants, seeds, cells, or plant parts containing the sugar beet event Bv_CSM63713. The commercial product may be viable plant material or non-viable plant material, which is not alive and is derived from a plant, seed, cell, or plant part containing the sugar beet event Bv_CSM63713. Non-viable commercial products include, but are not limited to, non-viable seeds, whole seeds or processed seeds, processed plant tissues or plant parts, dried plant tissues or parts, frozen plant tissues or parts, plant parts processed for animal feed, fibers, pulp pellets, pulp fragments, tailings, squeezed juice, syrup, molasses, extracts, raffinates, betaine, separator molasses soluble fraction (SMS), or any other food for human consumption. Viable commercial products include, but are not limited to, viable seeds, viable plant parts (e.g., roots and leaves), and viable plant cells. Thus, a plant containing the event Bv_CSM63713 can be used to produce any commercial product normally obtained from sugar beet plants.Any such commercial product derived from a plant containing the event Bv_CSM63713 may contain at least a detectable amount of the specific and unique DNA corresponding to the event Bv_CSM63713, in particular, at least 21 consecutive nucleotides of SEQ ID NO: 1, at least 25 consecutive nucleotides of SEQ ID NO: 2, at least 33 consecutive nucleotides of SEQ ID NO: 3, at least 33 consecutive nucleotides of SEQ ID NO: 4, at least 53 consecutive nucleotides of SEQ ID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO: 6, or a polynucleotide having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9. Any standard detection method for polynucleotide molecules, including the detection methods disclosed herein, can be used.

[0168] Plants resistant to herbicides include polynucleotides having the nucleotide sequences of SEQ ID NOs: 1 to 10, at least 21 consecutive nucleotides of SEQ ID NO: 1, at least 25 consecutive nucleotides of SEQ ID NO: 2, at least 33 consecutive nucleotides of SEQ ID NO: 3, at least 33 consecutive nucleotides of SEQ ID NO: 4, at least 53 consecutive nucleotides of SEQ ID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO: 6, and polynucleotides having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9. It can be produced by cross-breeding a plant containing event Bv_CSM63713 with another plant and thereby producing seeds, which are then grown into progeny plants. These progeny plants can be analyzed using a diagnostic method for selecting progeny plants containing the DNA of event Bv_CSM63713 or progeny plants resistant to the herbicides dicamba, glyphosate, glufosinate, and any combination thereof. The other plants used may or may not be transgenic. The progeny plants and / or seeds produced can be variant or hybrid seeds.

[0169] Plants resistant to herbicides include polynucleotides having the nucleotide sequences of SEQ ID NOs: 1-10, at least 21 consecutive nucleotides of SEQ ID NO: 1, at least 25 consecutive nucleotides of SEQ ID NO: 2, at least 33 consecutive nucleotides of SEQ ID NO: 3, at least 33 consecutive nucleotides of SEQ ID NO: 4, at least 53 consecutive nucleotides of SEQ ID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO: 6, and polynucleotides having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9. The self-propagation of plants containing event Bv_CSM63713, and the production of seeds thereby, can be produced by growing these seeds into progeny plants. These progeny plants can then be analyzed using a diagnostic method for selecting progeny plants containing the DNA of event Bv_CSM63713 or progeny plants resistant to the herbicides dicamba, glyphosate, glufosinate, and any combination thereof. The sugar beet event Bv_CSM63713 contains three expression cassettes that together provide resistance to benzoic acid-type auxins, inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and inhibitors of glutamine synthetase.

[0170] The sugar beet event Bv_CSM63713 contains three expression cassettes that together provide resistance to benzoic acid-type auxins such as dicamba, inhibitors of glutamine synthetase such as glufosinate, and inhibitors of EPSPS such as glyphosate.

[0171] In the present specification, inhibitors of glutamine synthetase include, but are not limited to, phosphinothricin, glufosinate, glufosinate salts, glufosinate ammonium, glufosinate sodium, glufosinate-P, L-glufosinate ammonium, and L-glufosinate sodium.

[0172] In the present specification, benzoic acid-type herbicides include, but are not limited to, dicamba (3,6-dichloro-2-methoxybenzoic acid), dicamba salts, dicamba-butotyl, dicamba diglycolamine salt, dicamba dimethylammonium, dicamba diethanolammonium, dicamba isopropylammonium, dicamba potassium, dicamba sodium, and dicamba trolamine.

[0173] In the present specification, inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) include, but are not limited to, glyphosate, glyphosate salts, glyphosate isopropylammonium, glyphosate ammonium, glyphosate dimethylammonium, glyphosate trimessium (=sulfosate), glyphosate diammonium, glyphosate potassium, and glyphosate sodium.

[0174] As used herein, "herbicide-tolerant" or "herbicide tolerance" or "tolerance" means not being wholly or partially affected by the presence or application of one or more herbicides, e.g., the ability to resist the toxic effects of the herbicide when applied. A cell, seed, or plant is "herbicide-tolerant" or has "improved tolerance" if it can maintain at least some normal growth or phenotype in the presence of one or more herbicides. A trait is a herbicide tolerance trait if its presence can confer an improvement in herbicide tolerance in a cell, plant, or seed as compared to a wild-type or control cell, plant, or seed. A crop plant containing a herbicide tolerance trait can continue to grow in the presence of the herbicide and may be minimally affected by the presence of the herbicide. A protein confers "herbicide tolerance" if its expression can confer an improvement in herbicide tolerance in a cell, plant, or seed as compared to a wild-type or control cell, plant, or seed. Examples of herbicide tolerance proteins are phosphinothricin N-acetyltransferase, dicamba monooxygenase, and glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase from strain CP4 of Agrobacterium species. Herbicide tolerance can be complete or partial insensitivity to a particular herbicide and can be expressed as a percentage (%) of tolerance or insensitivity to a particular herbicide.

[0175] As used herein, "herbicide phytotoxicity" or "phytotoxicity" refers to phytotoxicity to plants caused by the application of herbicides. "Phytotoxicity rate" or "phytotoxicity percentage" refers to the visual evaluation of phytotoxicity caused by herbicides, and is expressed as a percentage based on the visual evaluation of the leaf area of plants showing damage caused by the application of herbicides, such as necrosis (brown or necrotic tissue), chlorosis (yellow tissue or yellow spots), and malformations (abnormalities in leaf or plant structure, upward growth or twisting of the stem, cupping of the leaf). This is measured on a scale of 0 to 100, where "0" represents no phytotoxicity and "100" represents complete phytotoxicity (death). In the case of sugar beet plants containing the sugar beet event Bv_CSM63713, the phytotoxicity of the plants decreases after the application of one or more of benzoic acid-type auxins (e.g., dicamba), inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (e.g., glyphosate), or inhibitors of glutamine synthetase (e.g., glufosinate), or any combination thereof. For example, sugar beet plants containing the sugar beet event Bv_CSM63713 have less than about 5% phytotoxicity, less than about 10% phytotoxicity, less than about 15% phytotoxicity, or less than about 20% phytotoxicity compared to sugar beet plants that are otherwise identical except for not containing the sugar beet event Bv_CSM63713 after the application of a benzoic acid-type auxin such as dicamba, an EPSPS inhibitor such as glyphosate, or a glutamine synthetase inhibitor such as glufosinate.

[0176] As used herein, "weed" is any undesirable plant. A plant may generally be considered undesirable for agricultural or horticultural purposes (e.g., Amaranthus species), or may be considered undesirable in certain situations (e.g., certain crops in a field of a different species, also known as volunteer plants). Weeds are generally known in the art and vary depending on topography, season, growing environment, and time. Lists of weed species are available from agricultural and scientific societies and initiatives (e.g., Weed Science Society of America, Weed Science Society of Canada, Weed Science Society of Brazil, International Weed Science Society, and International Survey of Herbicide-Resistant Weeds), government agencies (e.g., United States Department of Agriculture and Australian Department of Environment and Energy), and industry and agricultural producer groups (e.g., United States Sugarbeet Growers Association). Weeds that are mainly troublesome in sugarbeet production include waterhemp (Amaranthus tuberculatus (Moq.) J.D. Sauer), Kochia (Bassia scoparia (L.) A.J. Scott or Kochia scoparia (L.)), lambsquarters (Chenopodium album L.), horseweed / marestail (Erigeron canadensis L.), Palmer amaranth (Amaranthus palmeri), redroot pigweed (Amaranthus retroflexus L.), velvetleaf (Abutilon theophrasti Medik.), and yellow nutsedge (Cyperus esculentus L.) (Soltani et al., 2018).

[0177] Provided is a method for weed control in a sugar beet cultivation area. The method includes applying at least one herbicide selected from the group consisting of (i) an inhibitor of glutamine synthetase, such as glufosinate, (ii) a benzoic acid type auxin, such as dicamba, and (iii) the EPSPS inhibitor glyphosate, wherein seeds or plants containing the sugar beet event Bv_CSM63713 are planted in the area before, during, or after the application of the herbicide, and the application of the herbicide prevents or inhibits the growth of weeds without phytotoxicity to the sugar beet plants. The growth area of the plants may or may not contain weed seeds or plants at the time of herbicide application. The herbicide(s) used in the method described herein can be applied alone or in combination with one or more herbicide(s) during the growth period. The herbicide(s) used in the method described herein can be applied in combination with one or more herbicide(s) temporally (e.g., as a tank mix or sequential application), spatially (e.g., at different times during the growth period including before and after planting of sugar beet seeds), or both. For example, provided is a method for controlling weeds including planting seeds containing the sugar beet event Bv_CSM63713 in an area and applying, during the growth period, an herbicidally effective amount of one or more of dicamba, glyphosate, or glufosinate alone or in any combination with another herbicide for the purpose of controlling weeds without phytotoxicity or with less than about 10% phytotoxicity to the plants containing the sugar beet event Bv_CSM63713 in the area. Such application of the herbicide(s) can be before planting (application to weeds germinated or present before planting seeds containing the sugar beet event Bv_CSM63713, including for eradication purposes, i.e., before sowing of the plants), pre-emergence (any time after seeds containing the sugar beet event Bv_CSM63713 are planted and before the plants containing the sugar beet event Bv_CSM63713 germinate), or post-emergence (any time after the plants containing the sugar beet event Bv_CSM63713 germinate).Multiple applications of one or more herbicides, or combinations of herbicides applied together or individually, can be used throughout the growing season, for example, two applications (e.g., an application before planting and an application after germination, or an application before germination and an application after germination), or three or more applications (e.g., an application before planting and two applications after germination).

[0178] The application of the herbicide in the practice of the methods described herein may be at the recommended commercial application rate or any fraction or multiple thereof, for example, twice the recommended commercial application rate. The application rate of the herbicide can be expressed as pounds acid equivalent per acre (lb ae / acre), pounds active ingredient per acre (lb ai / acre) or pounds active ingredient per hectare (lb ai / ha), depending on the herbicide and formulation. The use of acre in the herbicide application rates provided herein is for indication only, and herbicide application rates equivalent to any of the application rates provided herein may be used in areas larger or smaller than an acre. The herbicide application comprises at least one herbicide selected from the group consisting of (i) inhibitors of glutamine synthetase, such as glufosinate, (ii) benzoic acid-type auxins, such as dicamba, and (iii) the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate. The plant growth area may or may not contain weed plants at the time of herbicide application. The herbicidally effective amount of the glutamine synthetase inhibitor used in the area for weed control can range from about 0.1 lb ae / acre to about 10 lb ae / acre throughout the growing season (for example, glufosinate can be applied at application rates from about 0.4 lb ai / acre to about 2.16 lb ai / acre). The herbicidally effective amount of the benzoic acid-type auxin herbicide used in the area for weed control can range from about 0.1 lb ae / acre to about 16 lb ae / acre throughout the growing season (for example, dicamba can be applied at application rates from about 0.5 lb ae / acre to about 2.0 lb ae / acre). The herbicidally effective amount of the EPSPS inhibitor used in the area for weed control ranges from about 0.5 lb ae / ac to about 12 lb ae / ac throughout the growing season (for example, glyphosate can be applied at application rates from about 0.75 lb ae / acre to about 2.25 lb ae / acre).

[0179] A method for controlling volunteer sugar beets containing the sugar beet event Bv_CSM63713 for crop cultivation in a certain area is provided. The method includes applying a herbicidally effective amount of at least one herbicide, such as paraquat, clethodim, clopyralid, desmedipham, triflusulfuron, 2,4-dichlorophenoxyacetic acid (2,4-D), and acetolactate synthase (ALS) inhibitors, such as sulfonylurea (SU), imidazolinone, triazolopyrimidine, pyrimidinyl oxybenzoate, and sulfonylaminocarbonyl triazolinone, and any combination thereof, and by applying the herbicide, the growth of sugar beets containing the sugar beet event Bv_CSM63713 is prevented.

[0180] Provided are methods for producing plants and seeds comprising the sugar beet event Bv_CSM63713. The plants can be bred using any method known in the art. For example, descriptions of commonly used breeding methods can be found in WR Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison WI (1987). The plants may be self-pollinated (also known as "selfing") or cross-pollinated (known as "crossing"). Plants comprising the sugar beet event Bv_CSM63713 may be self-pollinated to generate plants of a pure-breeding line that are homozygous for the sugar beet event Bv_CSM63713. Selfing results in progeny known as "inbred lines" and can be used to produce genetically uniform inbred lines. As another method, plants comprising the sugar beet event Bv_CSM63713 may be cross-pollinated (crossed with another plant, which may be transgenic or non-transgenic) to produce variant or hybrid seeds. As further described herein, the sugar beet event Bv_CSM63713 comprises three independent expression cassettes or transgenes encoding a dicamba monooxygenase (DMO), a phosphinothricin N-acetyltransferase (PAT), and an EPSPS, respectively. Transgenic sugar beet plants (s) comprising the event Bv_CSM63713 are resistant to benzoic acid-type auxins, such as dicamba, inhibitors of glutamine synthetase, such as glufosinate, inhibitors of EPSPS, such as glyphosate, or any combination thereof, as compared to non-transgenic control plants. The transgenic sugar beet plants used in these methods can be homozygous or heterozygous for the transgene.The progeny plants produced by these methods may be variant or hybrid plants, and may be grown from seeds produced by plants containing the sugar beet event Bv_CSM63713 and / or from seeds produced by plants pollinated with pollen derived from plants containing the sugar beet event Bv_CSM63713, and may be homozygous or heterozygous for the transgene and / or the event Bv_CSM63713.

[0181] Furthermore, a plant containing the sugar beet event Bv_CSM63713 may be produced via a three-way cross in which an F1 line is produced by crossing a first parental line containing the event Bv_CSM63713 with a second parental line also containing the event Bv_CSM63713, which is then crossed with a third parent lacking the specific and unique DNA of the event Bv_CSM63713. The parent containing the event Bv_CSM63713 for the hybrid seeds or plants may be either the maternal or the paternal parent. The maternal parent is cytoplasmically male sterile and does not produce transgenic pollen during the process of producing variant or hybrid seeds, and thus preferably, the parent containing the event Bv_CSM63713 is the maternal parent.

[0182] The production of doubled haploids may be used to produce sugar beet plants and seeds that are homozygous for the DNA of event Bv_CSM63713 in a breeding program. Doubled haploids are produced by doubling a set of chromosomes (1N) from a heterozygous plant to produce completely homozygous individuals. See, for example, Wan, et al., (1989) and U.S. Patent No. 7,135,615. This process can be advantageous because it omits the generations of selfing required to obtain homozygous plants from a heterozygous source. One method of producing haploid and doubled haploid sugar beet plants containing event Bv_CSM63713 is by ovule culture of unpollinated flowers containing event Bv_CSM63713 (Gurel et al., 2021, Weich and Levall, 2003). Other methods, such as natural polyembryony, induction by irradiated pollen, crosses with polyploid plants or wild species, and anther and microspore culture, can also be applied to produce haploid and doubled haploid sugar beet plants containing event Bv_CSM63713.

[0183] Seeds and progeny plants produced by the methods described herein contain the sugar beet event Bv_CSM63713. Application of one or more herbicides to which the sugar beet event Bv_CSM63713 confers tolerance may be used in the selection of progeny containing the sugar beet event Bv_CSM63713. Alternatively, progeny may be analyzed using diagnostic methods for selecting plants or seeds containing the sugar beet event Bv_CSM63713. Progeny may be variant or hybrid plants and may be grown from seeds produced by plants containing the sugar beet event Bv_CSM63713 or from seeds produced by plants pollinated with pollen from plants containing the sugar beet event Bv_CSM63713, and may be homozygous or heterozygous for the sugar beet event Bv_CSM63713.

[0184] Transgenic events in sugar beets are known to those skilled in the art. For example, a list of such traits is provided by the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) and can be found on the website at www.aphis.usda.gov. Thus, two or more transgenic events may be combined in progeny seeds or plants by crossing two parent plants, each containing one or more transgenic events (s), collecting the progeny seeds, and selecting progeny seeds or plants containing two or more transgenic events. These steps may then be repeated until the desired combination of transgenic events in the progeny is achieved. Backcrossing to the parent plant and outcrossing to non-transgenic plants are also contemplated, as is vegetative propagation.

[0185] As used herein, the term "comprising" means "including but not limited to".

[0186] Provided is a method for detecting the presence of DNA derived from sugar beet plants, plant parts, plant cells, or seeds containing the sugar beet event Bv_CSM63713 in a sample. One method includes (i) extracting a DNA sample from at least one sugar beet plant, plant part, plant cell, or seed, (ii) contacting the DNA sample with at least one primer capable of producing a DNA sequence specific for the DNA of event Bv_CSM63713 under conditions appropriate for DNA sequencing, (iii) performing a DNA sequencing reaction, and then (iv) confirming that the nucleotide sequence includes a nucleotide sequence specific for event Bv_CSM63713 of the transgenic insert contained therein, for example, selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0187] Another method involves (i) extracting a DNA sample from at least one sugar beet plant, plant part, plant cell, or seed, (ii) contacting the DNA sample with a primer pair capable of producing an amplicon from the DNA of event Bv_CSM63713 under conditions appropriate for DNA amplification, (iii) performing a DNA amplification reaction, and then (iv) detecting the amplicon molecule and / or confirming that the nucleotide sequence of the amplicon contains a nucleotide sequence specific to event Bv_CSM63713, for example, one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The amplicon should be specific to event Bv_CSM63713 and contain junctions at nucleotide positions 1000 - 1001 and / or 12,722 - 12,723 of SEQ ID NO: 10, and include, for example, an amplicon containing SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10. Detection of the nucleotide sequence specific to event Bv_CSM63713 in the amplicon is used for determining and / or diagnosing the presence of sugar beet event Bv_CSM63713-specific DNA in the sample. Exemplary primer pairs capable of producing an amplicon from the DNA of event Bv_CSM63713 under conditions appropriate for DNA amplification are provided as SEQ ID NO: 14 and SEQ ID NO: 18. Other primer pairs for producing an amplicon for use in diagnosing sugar beet event Bv_CSM63713 can be readily designed by those skilled in the art. Such primer pairs include at least one primer in the genomic region adjacent to the insert and a second primer within the insert, provided that any primer pair that produces an amplicon containing the junction sequence and / or all or part of the insert or transgene sequence can be designed and used.The detection of amplicons can be based on any suitable method, such as sequencing, measurement of the fragment size or migration of the amplicons in a matrix or gel, or hybridization-based methods.

[0188] Another method for detecting the presence of DNA derived from sugar beet plants, plant parts, plant cells, or seeds containing the sugar beet event Bv_CSM63713 in a sample comprises: (i) extracting a DNA sample from at least one sugar beet plant, plant part, plant cell, or seed; (ii) contacting the DNA sample with a DNA probe specific for the DNA of event Bv_CSM63713; (iii) hybridizing the probe to the DNA sample under stringent hybridization conditions; and then (iv) detecting the hybridization between the probe and the target DNA sample. An example of the sequence of a DNA probe specific for event Bv_CSM63713 is provided as SEQ ID NO: 36. Other probes can be readily designed by those skilled in the art. Detection of hybridization of the probe to the DNA sample is used to diagnose the presence of sugar beet event Bv_CSM63713-specific DNA in the sample. The absence of hybridization is used as an alternative diagnosis for the absence of sugar beet event Bv_CSM63713-specific DNA in the sample.

[0189] A method is provided for determining the zygosity of the sugar beet event Bv_CSM63713 and the transgene by genomic DNA derived from at least one sugar beet plant, plant part, plant cell or seed comprising said event in a sample. One method comprises (i) extracting a DNA sample from at least a sugar beet plant, plant part, plant cell or seed, (ii) contacting the DNA sample with a first primer pair capable of producing a first amplicon used for diagnosing the event Bv_CSM63713, (iii) contacting the DNA sample with a second primer pair capable of producing a second amplicon used for diagnosing wild-type genomic DNA not comprising the event Bv_CSM63713, (iv) performing a DNA amplification reaction, and then (v) detecting the amplicon, wherein the presence of only the first amplicon is diagnostic of homozygous event Bv_CSM63713 DNA in the sample, the presence of both the first amplicon and the second amplicon is diagnostic of a sugar beet plant heterozygous for the event Bv_CSM63713, and the presence of only the second amplicon is diagnostic of the absence of event Bv_CSM63713 DNA in the sample. Exemplary primer pair sets are shown as SEQ ID NO: 26 and SEQ ID NO: 25, which produce an amplicon for diagnosis of event Bv_CSM63713, and SEQ ID NO: 26 and SEQ ID NO: 24, which produce an amplicon for diagnosis of wild-type sugar beet genomic DNA that does not contain event Bv_CSM63713. The probe set can also be incorporated into such an amplification method for use in a real-time PCR format using the above-mentioned primer pair sets.

[0190] Another method for determining zygosity comprises: (i) extracting a DNA sample from at least one sugar beet plant, plant part, plant cell or seed; (ii) contacting the DNA sample with a probe set comprising at least a first probe that specifically hybridizes to the DNA of event Bv_CSM63713 and at least a second probe that specifically hybridizes to sugar beet genomic DNA disrupted by the insertion of the foreign DNA of event Bv_CSM63713 and does not hybridize to the DNA of event Bv_CSM63713; (iii) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is used for diagnosing a sugar beet plant, plant part, plant cell or seed that is homozygous for the DNA of event Bv_CSM63713 in the sample, detecting hybridization of both the first probe and the second probe under the hybridization conditions is used for diagnosing a sugar beet plant, plant part, plant cell or seed that is heterozygous for event Bv_CSM63713 in the DNA sample, and detecting hybridization of only the second probe under the hybridization conditions is used for diagnosing the absence of the DNA of event Bv_CSM63713 in the sample.

[0191] Yet another method for determining zygosity involves: (i) extracting a DNA sample from at least one sugar beet plant, plant part, plant cell, or seed; (ii) contacting the DNA sample with a first primer pair capable of producing a first amplicon used in the diagnosis of event Bv_CSM63713; (iii) contacting the DNA sample with a second primer pair capable of producing a second amplicon of an internal standard known to be in single copy and homozygous in the sugar beet plant; (iv) contacting the DNA sample with a probe set comprising at least a first probe that specifically hybridizes to the first amplicon and at least a second probe that specifically hybridizes to the second amplicon; (v) performing a DNA amplification reaction using real-time PCR to determine the cycle threshold (Ct value) of the first and second amplicons; (vi) calculating the difference (ΔCt) between the Ct values of the first amplicon and the second amplicon; and (vii) determining zygosity, where ΔCt of approximately zero (0) indicates homozygosity of the event or the inserted T-DNA, and ΔCt of approximately one (1) indicates heterozygosity of the event or the inserted T-DNA. Heterozygous and homozygous events are distinguished by a ΔCt value unit of approximately one (1). Considering multiple factors, such as amplification efficiency and normal variations observed in real-time PCR due to the ideal annealing temperature, the range of "approximately one (1)" is defined as ΔCt 0.75 - 1.25, and the range of "approximately zero (0)" is defined as ΔCt -0.25 - 0.25 (or 0.0 - 0.25 if the ΔCt is measured as an absolute value). The primer pairs and probes for the above method for determining zygosity are capable of amplifying and detecting amplicons from the transgene or event DNA and the internal DNA standard.

[0192] Provided is a DNA construct comprising a first expression cassette, a second expression cassette, and a third expression cassette. The first expression cassette comprises, in operable linkage, i) a promoter and leader sequence of a chlorophyll A-B (Cab1) binding protein gene derived from Arabidopsis thaliana, ii) a codon-optimized phosphinothricin N-acetyltransferase (PAT) coding sequence derived from Streptomyces viridochromogene for conferring resistance to a glutamine synthetase inhibitor, such as glufosinate, and iii) a 3’ untranslated region (UTR) of a small heat shock protein (Hsp20) gene derived from Medicago truncatula. The second expression cassette comprises, in operable linkage, i) an enhancer of an inclusion body matrix protein derived from Dahlia mosaic virus, ii) a promoter, leader, and intron sequence of a ubiquitin protein gene derived from Cucumis melo, iii) a chloroplast targeting sequence of a ribulose bisphosphate carboxylase small subunit (RbcS) gene derived from Pisum sativum, iv) a codon-optimized dicamba monooxygenase coding sequence (DMO) derived from Stenotrophomonas maltophilia for conferring resistance to a benzoic acid type auxin, such as dicamba, and v) a 3’UTR of a putative protein gene derived from Medicago truncatula. The third expression cassette comprises, in operable linkage, i) a promoter, leader, and intron sequence of an S-adenosyl-L-methionine synthetase (SAMS2) gene derived from Cucumis melo, ii) a chloroplast targeting sequence of a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene derived from Arabidopsis thaliana, iii) a codon-optimized coding sequence of EPSPS derived from the CP4 strain of Agrobacterium species for conferring resistance to a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor, such as glyphosate, and iv) a 3’UTR of a hypothetical protein derived from Medicago truncatula.The nucleotide sequences of the three expression cassettes were contained in SEQ ID NO: 9 and SEQ ID NO: 10 of the sugar beet event Bv_CSM63713. The expression of the DMO, PAT, and EPSPS in transgenic plants confers herbicide resistance through three different mechanisms of action. For example, plants, plant parts, plant cells, or seeds containing or comprising the sugar beet event Bv_CSM63713 are resistant to dicamba (benzoic acid type herbicide), glufosinate (glutamine synthetase inhibitor), and glyphosate (EPSPS inhibitor).

[0193] Provided is a method for improving herbicide resistance. The method comprises: i) inserting a DNA construct comprising a first expression cassette, a second expression cassette, and a third expression cassette described herein into the genome of a plant cell; ii) generating a plant from the plant cell; and iii) selecting a regenerated transgenic plant comprising the DNA construct. The transgenic plants produced by the method contain a unique combination of three transgene expression cassettes in terms of their orientation and position relative to each other, each having a unique combination of expression elements for optimal expression of the transgene. Further, the transgenic plants produced by the method described herein acquire herbicide resistance through three different herbicide mechanisms of action. The selection of the regenerated plants comprising the DNA construct can be performed using the DNA detection method or protein detection method described in this disclosure. Alternatively or additionally, the selection can consist of treating the transgenic plant or plant cell with an effective amount of at least one herbicide selected from the group consisting of benzoic acid type auxins such as dicamba, inhibitors of glutamine synthetase such as glufosinate, inhibitors of EPSPS such as glyphosate, and any combination thereof.

[0194] Provided is a method for controlling, preventing, or reducing the occurrence of herbicide-resistant weeds. The method includes: a) cultivating a sugar beet plant comprising a DNA molecule conferring resistance to a herbicide having three different herbicide modes of action at a single genomic locus, or a transgene or event Bv_CSM63713 of the present disclosure, in the growth environment of a crop; and b) applying at least one herbicide selected from the group consisting of dicamba, glufosinate, glyphosate, and any combination thereof to the growth environment of the crop, wherein the sugar beet plant is resistant to the at least one herbicide. In some embodiments, the three different herbicide modes of action are inhibition of glutamine synthetase, benzoic acid-type auxin, and inhibition of EPSPS. A sugar beet plant grown from a seed comprising the DNA molecule or transgene of the present disclosure, or event Bv_CSM63713, is resistant to dicamba, glufosinate, glyphosate, or any combination thereof.

[0195] The herbicide(s) used in the methods described herein can be applied alone, sequentially with, or in combination with one or more herbicide(s) during the growth period. The herbicide(s) used in the methods described herein can be combined with one or more herbicide(s) temporally (e.g., as a tank mix or sequential application), spatially (e.g., at different times during the growth period including before and after planting of sugar beet seeds), or both. For example, a method for controlling the expression of herbicide tolerance in weeds is provided, which includes planting seeds including the sugar beet event Bv_CSM63713 in an area, and applying, alone or in combination with another herbicide, one or more of a benzoic acid type auxin, such as dicamba, an inhibitor of glutamine synthetase, such as glufosinate, and an inhibitor of EPSPS, such as glyphosate, in a herbicidally effective amount throughout the growth period for the purpose of controlling the expression of herbicide tolerance in the weeds of the area. Such application of the herbicide(s) can be before planting (at any time before planting seeds including the sugar beet event Bv_CSM63713 for eradication purposes, i.e., application to weeds germinated or present before sowing of the plant), pre-emergence (at any time after seeds including the sugar beet event Bv_CSM63713 are planted and before plants including the sugar beet event Bv_CSM63713 germinate), or post-emergence (at any time after plants including the sugar beet event Bv_CSM63713 germinate). Multiple applications of one or more herbicides, or combinations of herbicides applied together or individually, can be used throughout the growth period, e.g., 2 times (e.g., an application before planting and an application after emergence, or an application before emergence and an application after emergence), or 3 or more times (e.g., an application before planting and 2 applications after emergence).

[0196] Also provided is a method of reducing the number of loci for sugar beet breeding by inserting multiple transgenes at a single genomic position, thereby providing three different modes of action for herbicide tolerance. The sugar beet event Bv_CSM63713 contains or comprises a transgenic insert containing three independent transgene cassettes. The first expression cassette encodes phosphinothricin N-acetyltransferase (PAT), the second expression cassette encodes dicamba monooxygenase (DMO), and the third expression cassette encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). These three transgene cassettes are inserted at a single genomic position as a contiguous polynucleotide or DNA molecule, or a single molecularly linked transgenic insert, conferring commercial-level tolerance to at least one herbicide, such as glufosinate, dicamba, glyphosate, and any combination thereof, for each mode of action of the herbicide in the field. The nucleotide sequences of the three expression cassettes are contained in SEQ ID NO: 9 and SEQ ID NO: 10. As used herein, the term "commercial level" with respect to a herbicide refers to the commercial application rate (1X) recommended for the application of the herbicide for a particular herbicide. For example, for post-emergence application, the 1X application rate of dicamba is 0.5 lb / acre, the 1X application rate of glufosinate is 0.54 lb / acre, and the 1X application rate of glyphosate is 1 lb / acre. As used herein, "commercial-level tolerance" refers to tolerance to one or more herbicides at or above the recommended commercial application rate as a result of transgene expression from one or more of the four expression cassettes in plants containing the event Bv_CSM63713.

[0197] The sugar beet event Bv_CSM63713, which has a combination of consistent excellent efficacy, including inherent characteristics such as a single insertion site, stable integration and expression of the PAT, DMO, and EPSPS transgenes, herbicide tolerance, and agricultural productivity among multiple environmental conditions and over multiple environmental conditions, can be bred or introgressed into elite lines or varieties as a single locus by conventional breeding methods and is maintained across generations according to Mendelian inheritance of a single locus. Thus, the methods of the present disclosure enable the rapid introgression of multiple transgenes in terms of trait integration for separating materials, compared to cases where selecting plants containing multiple genes requires multiple generations of monotonous and laborious crosses by inserting individual transgenes into two or more loci, saving time and resources in breeding programs and enabling the rapid development of lines. The newly introgressed or integrated DNA molecule or polynucleotide of event Bv_CSM63713, which contains SEQ ID NO: 9 and / or SEQ ID NO: 10, maintains the expression characteristics of the transgene, as well as the genomic flanking sequences and chromosomal location, which confers tolerance to at least one herbicide, such as glufosinate, dicamba, glyphosate, and any combination thereof, for each mode of action of the herbicide in the field.

[0198] Deposit Information The deposit of a representative sample of sugar beet seeds containing the event Bv_CSM63713 was made on August 10, 2021, in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110, USA. The ATCC patent deposit designation (accession number) for the seeds containing the sugar beet event Bv_CSM63713 is accession number PTA-127098. Access to the deposit is available during the pendency of the application to the Commissioner of Patents and Trademarks and to those determined by the Commissioner to have a right to access upon request. When the patent issues, all restrictions on availability to the public are irrevocably removed. The deposit will be maintained by the depository institution for thirty (30) years, or for five (5) years from the last request, or for the life of the patent, whichever is longer, and will be replaced as necessary during that period.

Example

[0199] Examples are given below to explain the present invention in more detail. To summarize, it is a five-year analysis of thousands of individual plants through the strict molecular, agronomic, and field tests required for the construction and testing of four different transformation constructs, the generation of 1,803 unique transformation events, and the creation, identification, and final selection of the sugar beet event Bv_CSM63713.

[0200] It should be understood by those skilled in the art that many changes can be made in the specific examples disclosed herein and still obtain similar results. As long as the same or similar results are achieved, certain chemically and physiologically related agents may replace the agents described herein. All such substitutions and modifications that are obvious to those skilled in the art are considered to be within the scope of the present invention.

[0201] Example 1: Design of Constructs, Generation of Events, Testing of R0 Plants This example describes the design of four different expression constructs for resistance to dicamba, glyphosate, and glufosinate herbicides via a vector stack, the generation of 1,803 unique sugar beet events, and the testing and analysis of the resulting transgenic sugar beet plants over multiple generations.

[0202] Four expression constructs were designed and constructed. Each of these four constructs contained three expression cassettes in the same orientation to drive the expression of the dicamba monooxygenase (DMO), CP4 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and phosphinothricin N-acetyltransferase (PAT) transgenes. However, each construct was designed to have a unique combination of 5' and 3' expression elements of the DMO, CP4 EPSPS, and PAT transgenes operably linked to enable testing of different promoters, chloroplast targeting sequences, and 3'UTRs in sugar beet plants for optimal productivity of the transgenes. The constructs are shown in Table 1. These four expression constructs were then each cloned into plant transformation vectors and introduced into the AGL1 strain of Agrobacterium.

[0203] Agrobacterium-mediated transformation of four sugar beet lines was performed using these four constructs. Lines 1 and 2 were transformed using a slightly modified transformation protocol (Lindsey and Gallois, 1990) using shoot meristems, and lines 3 and 4 were transformed using a callus-based transformation protocol modified after Kishchenko, Komarnitskii, Kuckuk (2005) using hypocotyl and cotyledon explants. Phosphinothricin (DL-PPT, Duchefa, Haarlem - The Netherlands) was used as the selection agent at a concentration of 6 mg / L.

[0204] A total of 1803 unique transformation events were generated across all four lines from four transformation constructs. Each transformation event was generated by random insertion of T-DNA containing three expression cassettes into the sugar beet genome at unique positions. Rooted plants from tissue culture were transferred to soil and acclimated for four weeks under a photoperiod of 20 °C during the day / 17 °C at night, 16 h light period / 8 h dark period. Subsequently, the plants were transferred to a greenhouse at 19 °C during the day and 17 °C at night and grown hydroponically under a photoperiod of 18 h light period / 6 h dark period.

[0205] Tables 2 and 3 provide an overview of the total number of events produced by each line, or each construct, respectively. Line 1 is closest to the elite germplasm and contains all the disease resistances required for field cultivation in the United States, and since it is the most established line in tissue culture, it was used in most experiments and thus produced the most transgenic events.

Table 1

Table 2

Table 3

[0206] DNA of all 1803 primary transformants was isolated using the modified Doyle and Doyle (1990) method and screened for the presence or absence of the CP4, PAT, and DMO genes, the left / right border overlaps of the Agrobacterium T-DNA, and the presence of the VirD2 gene using the Competitive Allele Specific PCR (KASP) genotyping assay (LGC, Teddinton, UK) according to the manufacturer's standard protocol. The VirD2 gene is a gene present in Agrobacterium tumefaciens AGL1, and thus, the detection of VirD2 indicates the presence of Agrobacterium tumefaciens AGL1. This assay is based on competitive allele-specific PCR and enables the scoring of two alleles for single nucleotide polymorphisms (SNPs) as well as insertions and deletions (indels) at specific loci. The KASP assay mix contained assay-specific unlabeled oligos, namely allele-specific primers ("AlleleX" or "X") and one common primer ("Primer Common" or "C"). The CP4, PAT, and DMO genes, as well as the primer sets used for Agrobacterium, are listed in Table 4.

Table 4

[0207] Presumptive transformants that were negative for CP4, PAT, and DMO were discarded. Furthermore, after the tissue culture stage, transformants that showed positive signals for the left border (LB) or / and right border (RB) overlaps or read-throughs, or the presence of Agrobacterium tumefaciens AGL1 were also discarded. Only events that were positive for CP4, PAT, and DMO, negative for the LB and / or RB overlaps or read-throughs, and negative for the presence of Agrobacterium tumefaciens AGL1 were maintained for further growth and testing.

[0208] The remaining events were further analyzed with respect to the copy number of the transgene, the integrity of the transgenic insert, the absence of the vector backbone sequence, and the transgenic insertion site in the genome, and normal fertile plants were screened. From this initial molecular analysis and phenotypic screening, 622 R0 events were examined for the effectiveness of the R0 trait (dicamba, glyphosate, and glufosinate tolerance) at the 4- to 6-leaf stage in the greenhouse.

[0209] Glyphosate was applied at a rate of 0.4 lb / acre of Roundup PowerFlex herbicide (4x rate). Glufosinate was applied at a rate of 2.16 lb / acre of Basta herbicide (4x rate). Dicamba was applied at a rate of 2 lb / acre of Mais Banvel WG herbicide (4x rate) (Table 5). Double mixes of glyphosate + dicamba were applied at 4x rates each, and triple mixes of glyphosate + dicamba + glufosinate were applied at 2.4x rates each. For these double and triple mixes, different herbicide products were mixed prior to application. Phytotoxicity of the herbicides to the plants was visually evaluated. Events that did not show good tolerance to all three herbicides were discarded.

[0210] Representative results are shown in Figure 2. Photos were taken before and 2 weeks after herbicide application. Panels A1 and A2 show wild-type sugarbeets before and after herbicide application, panels B1 and B2 show event Bv_CSM63713 before and after herbicide application, and panels C1 and C2 show another event before and after herbicide application. The herbicide treatments were (from left to right): 1. glyphosate + glufosinate + dicamba triple mix, 2. glyphosate + dicamba double mix, 3. dicamba, 4. glufosinate, 5. glyphosate, and 6. untreated control. As shown in Figure 2, wild-type sugarbeet plants did not tolerate any of the herbicide treatments, and the plants shown in panels C1 and C2 tolerated only the glufosinate treatment. In contrast, the Bv_CSM63713 event showed excellent tolerance to all herbicide treatments. Single plants of the Bv_CSM63713 event showed some damage after glyphosate treatment, but this was likely mostly due to damage during potting before herbicide treatment.

[0211] Combining molecular analysis data with the R0 herbicide tolerance test, 44 unique events were selected from the first 1803 unique transformation events produced using four transformation constructs and four transformation genotypes and advanced to the first-year field trial. [Table 5]

[0212] Example 2: First-year field trial This example describes the first-year field trial of plants containing each of the 44 selected unique events. For each unique event, thousands of plants were examined for trait efficacy (herbicide tolerance) and agricultural productivity. These data were analyzed to compare the productivity of each event under field conditions across all plants and all locations. The first-year field productivity data were then combined with the molecular analysis data to select excellent events for advancement to the second-year field trial.

[0213] In the first-year field trial, 44 unique events were selected from the original 1803 events. These 44 events represented the best events from four constructs, namely, 6 events from HT1, 7 events from HT2, 11 events from HT3, and 20 events from HT4. Hybrid seeds were produced from the selected events via open pollination with sterile females from the partitions (the pollinators were either homozygous or heterozygous for the transgene). The field trial for trait efficacy was conducted at two locations in the United States with two replicates per location using a split-plot design (the main plots were the chemical treatments and the subplots were the individual events). The individual plots were 3.5 feet x 10 feet in size and had a 5-foot alley between plots. Each plot contained two rows and the seeding interval was 2 inches.

[0214] In the first-year trait efficacy trial, F1 hybrid plants were evaluated for resistance to dicamba, glyphosate, and glufosinate. Herbicides were applied to all plants at three different stages of sugar beet plant development, namely, Treatment 1: two-leaf stage, Treatment 2: 6 - 8 leaf stage, and Treatment 3: 8 - 12 leaf stage. The efficacy trial included a total of nine treatments, namely, 1) untreated control with weeds removed by hand, 2) 2x glyphosate, 3) 4x glyphosate, 4) 2x glufosinate, 5) 4x glufosinate, 6) 2x dicamba, 7) 4x dicamba, 8) tank mix of 2x dicamba and glyphosate, and 9) tank mix of 4x dicamba and glyphosate (the 1x and 4x application rates for each herbicide are provided in Table 5). Ten to fourteen days after each herbicide application, the percentage of phytotoxicity of the plants due to the herbicide was visually evaluated based on the estimated growth reduction (poor growth), chlorosis, and necrosis.

[0215] Data on the effectiveness of traits from the first-year field trials were summarized. For each unique event, a meta-analysis of the aggregated data across both locations and all individual plants was conducted for comparison of the herbicide injury ratings of the hybrids. Table 6 provides the average herbicide injury rating for each event across both locations for the herbicide treatment program. Table 7 provides an explanation of the herbicide injury rating scale. These results showed that plants from all events had, on average, low herbicide injury.

Table 6-1

Table 6-2

Table 7

[0216] Example 3: Molecular Characterization This example describes an extensive molecular characterization that was conducted concurrently with the field trials of the selected events. The molecular characterization of each event was used to evaluate whether the event should be selected to proceed further.

[0217] DNA and RNA analyses of the events were performed using various techniques known in the art. Southern blot analysis and qPCR were performed on genomic DNA to confirm the presence of a single copy of the entire transgene without the vector backbone sequence. DNA amplification and sequencing were used to confirm the composition and integrity of the sequences of the insertions in the transgenic insertions for each event. DNA adjacent to each end (5' and 3' ends) of the transgenic insertions was sequenced to identify each junction. Northern analysis was performed to detect and measure the mRNA transcripts of the CP4 gene, DMO gene, and PAT gene in the transgenic plants for each event.

[0218] Protein analysis of plants containing each event was performed using techniques known in the art. N-terminal protein sequencing of CP4, DMO, and PAT proteins purified from transgenic plants containing each event was performed to confirm the sequences of the recombinant proteins. Western blot analysis was performed on protein extracts from plants containing each event to confirm the production of CP4, DMO, and PAT proteins.

[0219] The insertion sites of each event in the genome were analyzed. The flanking sequences were used for bioinformatics analysis of the chromosomal positions of the inserts, and the insertion sites for each event were mapped to the soybean genome. DNA amplification at the wild-type alleles in the genome was performed using primers specific to the flanking regions of each event. The unique site of transgenic integration for the event was mapped to the soybean reference genome using the wild-type insertion site sequence.

[0220] Combined with the trait efficacy productivity data from the first-year field trials, a total of 17 unique events out of 44 examined in the first-year field trials were selected and advanced based on comprehensive and detailed molecular characterization evaluations for each event.

[0221] Example 4: Second-year field trials This example describes the second-year field trials in plants containing each of the 17 unique events advanced from the first-year field trials for constructs HT1, HT2, HT3, and HT4. For each unique event, multiple individual plants were field-tested at two locations with respect to trait efficacy (dicamba, glyphosate, and glufosinate tolerance), agricultural productivity, and yield. These data were analyzed to compare the productivity of each event under field conditions across all plants and both locations. The data from this second-year field trial were then used to select superior events for advancement to the third-year field trial.

[0222] The F1 hybrid seeds for the second-year field trial (hemizygous for the event) were produced by open pollination from the selected events of the sterile female parent from the partition. The advanced 5 lines (homozygous for the event) of R2 seeds were also produced by open pollination in the partition for examination in the field trial. The field trial for trait efficacy was conducted at two locations in the United States with 2 replicates at each location using a split-plot design (main plots were chemical treatments and subplots were individual events). Individual plots were 3.5 feet x 10 feet in size and had a 5-foot alley between plots. Each plot contained 2 rows and the seeding interval was 2 inches.

[0223] In the second-year trait efficacy test, 17 unique events were selected for the test, representing 1 event for construct HT1, 3 events for construct HT2, 4 events for construct HT3, and 9 events for construct HT4. Plants from these events were evaluated for tolerance to dicamba, glyphosate, and glufosinate. Herbicides were applied to all plants at three different stages of sugar beet plant development, namely, Treatment 1: two-leaf stage, Treatment 2: 6 - 8 leaf stage, and Treatment 3: 8 - 12 leaf stage. The efficacy test included a total of 10 treatments, namely, 1) untreated control, 2) 2x dicamba, 3) 2x glufosinate, 4) 2x glyphosate, 5) tank mix of 2x glyphosate and dicamba, 6) 2x glyphosate and dicamba tank mix followed by 2x glufosinate, 7) 4x dicamba, 8) 1x glufosinate, 9) 4x glufosinate, and 10) 4x glyphosate (the 1x and 4x application rates for each herbicide are provided in Table 5). 10 - 14 days after each herbicide application, the percentage of phytotoxicity of the plants by the herbicide was visually evaluated based on the estimated growth reduction (poor growth), chlorosis, and necrosis.

[0224] Data on the effectiveness of traits from the second-year field trials were summarized. Using the phytotoxicity scale provided in Table 7, a meta-analysis of the aggregated data across both locations and all individual plants was performed for the comparison of phytotoxicity grades of hybrids and homozygous lines (where applicable) for each unique event. Table 8 provides the average phytotoxicity grade for each event across both locations for the herbicide treatment program. Meta-analysis of the field trials on trait effectiveness showed that plants from all four constructs had, on average, low herbicide phytotoxicity and very good productivity.

Table 8

[0225] In the second-year field trials, field trials on agricultural productivity were also conducted to evaluate whether the introduced genes had any effect on the yields of these events compared to conventional counterparts. Additionally, the effect of post-emergence glufosinate application on the yields of these events was evaluated. Six unique events were included in this field trial on agricultural productivity. These included 1 event from construct HT2, 2 events from construct HT3, and 3 events from construct HT4. These trials were conducted at three locations in the United States using a split-plot design with five replicates at each location. Individual plots were 5.5 feet x 27 feet in size. Each plot contained 3 rows with a seeding interval of 2 inches. Plots were thinned to 6-inch stands.

[0226] In the agricultural productivity trials of the six unique events, sugar beet root yields were measured from the beets harvested per individual plot and expressed as tons per hectare of weight. Polarization / sugar content (POL) was calculated for each individual plot by near-infrared spectroscopy (NIRS) analysis as a percentage of fresh matter. Sugar yields were calculated in tons per hectare based on these two components.

[0227] For all three test sites, in the combined test analysis of Series A (conventional counterparts and null segregants tested without glyphosate treatment) and Series B (glyphosate treatment to select segregating hybrids), no significant differences in sugar yield were observed between any of the six unique events and their conventional counterparts. Event Bv_CSM63713 was the most productive event among them. The sugar content was significantly higher in the unique events compared to the conventional counterparts depending on the site for all three sites tested. In the combined test analysis, there was a tendency for higher sugar yield in the unique events. However, the difference was not significant (Table 9). "Check" in Table 9 refers to a well-known variety (reference material) commonly used to relativize the data.

[0228] In the agronomic field trials, the effects of glufosinate treatment (1x) and glyphosate treatment (1x) on yield after germination were also evaluated for the six unique events. No phenotypic differences were observed between the two different herbicide treatments. However, a decrease in sugar yield was observed in all six events with glufosinate treatment. The decrease in sugar yield was a result of the decrease in root yield (Table 10). However, the sugar content was not affected by the treatment.

[0229] In Tables 9 and 10, LSD represents the least significant difference, which enables the determination of the amount by which two levels (e.g., varieties, treatments) that are considered to be significantly different from each other at the 5% error level need to differ. LSD measures the variation / difference in this test that can be attributed to a specific treatment(s), e.g., the presence / absence of a transgenic insert in the sugar beet genome (Table 9) or glyphosate treatment versus glufosinate treatment (Table 10). This value of LSD means that the difference in a parameter (e.g., sugar yield or sugar content in Table 9) needs to be greater than this value of LSD shown between two different treatments for it to be considered significant. For example, in Table 9, the sugar yield for the hybrid of "Tester 1xBv_CSM63713" is 13.1, and the sugar yield for the hybrid of "Tester 1xLine E" is 12.7. Since the difference between 13.1 and 12.7 is <1.3 (LSD value), it is considered not significant (Table 9).

Table 9

Table 10

[0230] Example 5: Field Test in the Third Year This example describes the third-year field test in plants containing each of six unique events advanced from the second-year field test for constructs HT3 and HT4. For each unique event, many individual plants were field-tested at one location with respect to the effectiveness of traits (dicamba, glyphosate, and glufosinate tolerance), agricultural productivity, and yield. These data were analyzed to compare the productivity of each event under field conditions across all plants. Next, the data from the third-year field test were combined with molecular analysis data to select the last event.

[0231] The hybrid F1 seeds for the third-year field trial (hemizygous for the event) were produced by open pollination from the selected event of the sterile female parent from the partition. Additionally, seeds that were homozygous for event Bv_CSM63713 were also produced by open pollination in an isolated field. These seeds were used in the field trial for the efficacy of the trait. These tests were conducted at one location in the United States in two replicates using a split-plot design (the main plots were the chemical treatments and the subplots were the individual events). The individual plots were 60 inches x 8 feet in size and had a 22-inch alley between plots. Each plot contained four rows and the seeding interval was 2 inches.

[0232] In the third-year efficacy test of the trait, six unique events were selected for the test, representing three events for construct HT3 and three events for construct HT4. Plants from these events were evaluated for tolerance to dicamba, glyphosate, and glufosinate. Herbicides were applied to all plants at three different stages of sugar beet plant development, namely, Treatment 1: two-leaf stage, Treatment 2: six to eight-leaf stage, and Treatment 3: eight to twelve-leaf stage.

[0233] The efficacy test included a total of 10 treatments, namely, 1) 2x glyphosate, 2) 4x glyphosate, 3) 1x glufosinate, 4) 2x glufosinate, 5) 4x glufosinate, 6) untreated control, 7) 2x dicamba, 8) 4x dicamba, 9) tank mix of 2x dicamba and glyphosate, and 10) tank mix of 2x dicamba and glyphosate followed by 2x glufosinate (the 1x and 4x application rates for each herbicide are provided in Table 5). Ten to fourteen days after each herbicide application, the percentage of phytotoxicity of the plants due to the herbicide was visually evaluated based on the estimated growth reduction (poor growth), chlorosis, and necrosis.

[0234] Data on the effectiveness of traits from the third-year field trial were summarized. For each unique event, a meta-analysis of the aggregated data across replicates and all individual plants was performed to compare the herbicide injury ratings of the hybrids and homozygous lines (where applicable). Table 11 provides the average herbicide injury rating for each event for the herbicide treatment program using the injury scale provided in Table 7. Meta-analysis of the field trials for trait effectiveness showed that plants from both constructs had, on average, lower herbicide injury ratings and very good productivity.

Table 11

[0235] In the third-year field trial, field trials of agricultural productivity were also conducted to evaluate whether the introduced genes had any effect on the yields of these events compared to the conventional counterparts. Four unique events were included in this field trial of agricultural productivity. These included two events from construct HT3 and two events from construct HT4. These trials were conducted at four locations in the United States using a randomized block design with three replicates per location. The individual plots were 5.5 feet x 27 feet in size and had an 8-foot alley between plots. Each plot contained three rows with a seeding interval of 2 inches. The plots were thinned to a 6-inch stand.

[0236] In the agricultural productivity trials of four distinct events, the yield of sugar beet roots was measured from the harvested beets per single plot and expressed as tons of weight / hectare. The polarization / sugar content (POL) was calculated for each single plot by NIRS analysis as a percentage of the fresh matter. The sugar yield was calculated in tons / hectare based on these two components. In the year prior to the agronomic trials, an increase in sugar content was observed in the events examined compared to the conventional counterparts. One difference in these trials was that the herbicide-tolerant events were treated with glyphosate, whereas the conventional counterparts were not treated with glyphosate. In the third-year field trials, two sets of the four distinct events that were included in the previous year's yield trials were examined in the yield trials at four locations. The conventional counterparts and a null segregant from one of the events were included as controls in these trials. Glyphosate was not applied to the plants. Instead, classical sugar beet herbicides and hand weeding were applied to control weeds. As shown in Table 12, a significant increase in sugar content was observed in all four events, consistent with the previous year's observations. The null segregant also showed an increase in sugar content. No significant differences were detected between the transgenic events and the conventional counterparts with respect to sugar yield and root yield, except for event HT4-22 (Table 12). The LSD and "check" in Table 12 are defined in the same manner as those described above for Table 9.

Table 12

[0237] Among the unique events in the two-year agronomic trials, Event Bv_CSM63713 showed the highest productivity. This event was further tested to evaluate the effect of different herbicide treatments on its yield performance. These herbicide treatments included: 1) 1x dicamba pre-emergence treatment (PRE), 2) 2x dicamba application PRE, 3) 1x glufosinate, 4) 2x glufosinate, 5) 1x glyphosate, 6) 2x glyphosate, 7) 1x dicamba / glyphosate PRE, 8) 2x dicamba / glyphosate PRE, 9) 1x dicamba / glyphosate + 1x glufosinate PRE, 10) 2x dicamba / glyphosate + 2x glufosinate PRE (the 1x application rate for each herbicide is provided in Table 5). The pre-emergence treatment was carried out only at 2 out of 4 locations due to weather conditions. However, since the materials investigated responded similarly at all 4 locations, a combined analysis for all 4 test locations was possible. This combined analysis showed that there were no significant differences in sugar yield, sugar content, and root yield for this event between hand weeding and different herbicide treatments, except that a decrease in sugar yield and root yield was observed with the pre-emergence treatment of 2x dicamba / glyphosate + 2x glufosinate (Table 13). The LSD in Table 13 is defined in the same way as described above for Tables 9, 10, and 12.

Table 13

[0238] Example 6: Final Selection of the Sugar Beet Event Bv_CSM63713 As described in the previous examples, active event selection was carried out over several years, and events were selected and characterized from 1803 unique transformation events generated from four different transformation constructs. Event selection included molecular characterization, as well as greenhouse and field trials regarding efficacy, agronomic productivity, and yield. For molecular characterization, only events that met the criteria of the absence of Agrobacterium and left / right border overlaps, the presence of a single copy and complete transgene located at a predetermined genomic position, and no insertion of the transgene within or near endogenous genes were selected and advanced to further testing. Similarly, events showing the efficacy of traits that do not adversely affect plant growth and development, and agronomic productivity and yield over multiple years, at multiple locations, and under various growth conditions were selected. Table 14 summarizes the results of such an extensive and intensive selection process that led to the identification and selection of sugar beet event Bv_CSM63713 as the best event for commercial development, showing the reasons for the rejection of most events.

Table 14

[0239] Example 7: Molecular Characterization of Sugar Beet Event Bv_CSM63713 This example describes the extensive molecular characterization of sugar beet event Bv_CSM63713. This characterization included confirmation of a single-copy insertion at a "neutral" insertion site within the sugar beet genome, identification of the insertion site on the chromosome, identification of the flanking sequences of the transgene, confirmation that the sequence of the insert matches the transformation construct, and confirmation of no transgene read-through at either the left or right border. The transgenic insert of sugar beet event Bv_CSM63713 contains the elements and sequences described in Table 15.

[0240] DNA sequence analysis of the sugar beet event Bv_CSM63713 was performed. Southern blot analysis was carried out to confirm that plants containing the sugar beet event Bv_CSM63713 contain a complete single copy of the entire transgenic insert and do not contain the backbone sequence of the transformation vector. The insertion site of the transgene was identified by targeted locus amplification (TLA) (Paula JP de Vree et al., 2014) based on the publicly accessible sugar beet genome (Beta vulgaris resource, http: / / bvseq.boku.ac.at / index.shtml). DNA flanking both the 5’ and 3’ ends of the transgene insert was PCR amplified and sequenced. The sequence of the flanking DNA of the sugar beet event Bv_CSM63713 was then mapped to the physical assembly of the elite wild-type genome obtained by whole-genome sequencing. The sequence information of the insertion site was used for the bioinformatics analysis of the chromosomal location of the event. The integrity of the insertion site was determined by PCR with wild-type alleles using primers specific to the flanking region of the sugar beet event Bv_CSM63713. Using the wild-type insertion site sequence, the unique site of transgenic integration of the sugar beet event Bv_CSM63713 was mapped to the sugar beet reference genome. To ensure that no changes or mutations were introduced into any region of the transgene insert during the transformation process, the entire transgenic insert of the sugar beet event Bv_CSM63713 was isolated from the plant and sequenced. By aligning the recovered sequences with the sequences from the transformation vector, it was confirmed that they were identical. The sequence information of the 5’ junction, 3’ junction, and the transgenic insert is provided herein as SEQ ID NOs: 1-12. The wild-type sequence at the insertion site is provided as SEQ ID NO: 13. As shown in the depiction of SEQ ID NO: 13 in Figure 1, a 7-nucleotide deletion was observed at the insertion site.

[0241] Furthermore, using a high-quality pre-trained Augustus gene model for sugar beet, detailed gene prediction was performed for the integration site and an additional 50 kb of flanking regions on both sides. These results showed that there were no genes at the insertion site (therefore, the insertion site is called a "neutral insertion site") and no functional gene features were present in the 5' and 3' flanking regions.

Table 15-1

Table 15-2

Table 15-3

[0242] RNA analysis of plants containing the sugar beet event Bv_CSM63713 was performed. Northern blot analysis was carried out using total RNA isolated from leaf tissues of plants containing the sugar beet event Bv_CSM63713. From these results, it was confirmed that the mRNA transcripts of CP4, DMO, and PAT were expressed in the exact sizes and amounts predicted for the event Bv_CSM63713.

[0243] Protein analysis of plants containing the event Bv_CSM63713 was performed. N-terminal protein sequencing of the expressed CP4-EPSPS, DMO, and PAT proteins was carried out using immunopurified protein extracts from leaf tissues to confirm the authentic N-terminal amino acid sequences. Western blot analysis was performed using protein extracts from leaf tissues of plants containing the sugar beet event Bv_CSM63713, and it was confirmed that proteins of a single predicted size were produced for CP4-EPSPS, DMO, and PAT, respectively. ELISA was also used to measure the protein levels in the leaves of plants for CP4-EPSPS, DMO, and PAT proteins.

[0244] Example 8: Detection of the sugar beet event Bv_CSM63713 This example describes the detection of the sugar beet event Bv_CSM63713. Detection of the event in a sample can be achieved using DNA, RNA, or protein detection techniques. Specific, robust, and easily extensible qualitative detection methods enable the tracking of specific events in research and breeding programs and help maintain product integrity. Exemplary detection methods and materials are provided below. Detection can determine the presence or absence of the sugar beet event Bv_CSM63713 in a sample. The DNA sequence information of the sugar beet event Bv_CSM63713 is provided herein as SEQ ID NOs: 1-12. The transgenic insert of the sugar beet event Bv_CSM63713 contains the elements described in Table 15.

[0245] 1) Detection of the sugar beet event Bv_CSM63713 by KASP To identify the sugar beet event Bv_CSM63713 in a sample, a Kompetitive Allele Specific PCR (KASP, LGC, Teddinton, UK) genotyping assay was developed. This assay is based on competitive allele-specific PCR and enables the scoring of two alleles for single nucleotide polymorphisms (SNPs) as well as insertions and deletions (indels) at specific loci.

[0246] The KASP assay mix contained three assay-specific unlabeled oligos, namely, two allele-specific forward primers and one common reverse primer. Two sets of primer combinations were developed for the event-specific KASP assay. These primers and the corresponding SEQ ID NOs are described in Table 16. Txht024d01 of primer set 1 was targeted to the 5' region of SEQ ID NO: 10, and txht024d02 of primer set 2 was targeted to the 3' region of SEQ ID NO: 10. The event-specific assay can be performed using these two sets of primers independently or using only one set of the primer combination.

[0247] The KASP assay mix and the Universal KASP master mix were added to the DNA sample, followed by performing a thermal cycle reaction and subsequently an endpoint fluorescence read. Each of the allele-specific primers carried a unique tail sequence (underlined in Table 16) corresponding to the universal FRET (fluorescence resonance energy transfer) cassette, one labeled with the FAM dye and the other labeled with the HEX dye. The KASP master mix contained, in an optimized buffer solution, the universal FRET cassette, the ROX passive reference dye, Taq polymerase, free nucleotides, and MgCl 2 2 . In KASP PCR, during the thermal cycle, as the relevant allele-specific primer binds to and extends on the template, the tail sequence attaches to the newly synthesized strand. Next, the complement of the allele-specific tail sequence is generated during subsequent rounds of PCR, enabling the FRET cassette to bind to the DNA. The FRET cassette then no longer quenches and emits fluorescence. When the sugar beet event Bv_CSM63713 was present in the sample, a fluorescence signal was generated. However, when the event was not present in the sample (e.g., wild-type or non-transgenic plants), no fluorescence signal was generated.

[0248] Therefore, when the primer sets txht024d01, or txht024d02, or both are used independently with these reaction methods, DNA amplicons for the diagnosis of the sugar beet event Bv_CSM63713 are produced. The controls for this analysis shall include a positive control containing the sugar beet event Bv_CSM63713, a negative control from non-transgenic sugar beet, and a negative control without template DNA. Alternatively, each set of primers without the underlined tail can be used independently or in combination for PCR amplification and detection of event-specific amplicons using techniques known in the art.

Table 16

[0249] 2) Detection of Sugar Beet Event Bv_CSM63713 Using Antibodies Another example of the detection of Sugar Beet Event Bv_CSM63713 is a detection kit that includes at least one antibody specific for at least one protein encoded by Sugar Beet Event Bv_CSM63713. For example, such a kit may utilize a lateral flow strip that includes a reagent that is activated when the tip of the strip contacts an aqueous solution. Exemplary proteins sufficient for use in antibody production are those encoded by the sequence provided as SEQ ID NO: 10, or any fragment thereof.

[0250] Develop a protein detection method to determine whether a sample is derived from a plant, seed, cell, or plant part (e.g., root) that contains Sugar Beet Event Bv_CSM63713. Use at least one antibody specific for at least one protein encoded by Sugar Beet Event Bv_CSM63713 to detect the protein encoded by Sugar Beet Event Bv_CSM63713 in the sample. A detection kit that includes one or more antibodies specific for one or more proteins encoded by Sugar Beet Event Bv_CSM63713 may utilize a lateral flow strip that includes a reagent that is activated when the tip of the strip contacts an aqueous solution. The sugar beet tissue sample may be ground and the protein for analysis may be extracted using water or an aqueous buffer (e.g., phosphate buffered saline containing a surfactant and bovine serum albumin). After centrifugation, the aqueous supernatant is analyzed using a sandwich ELISA format on a lateral flow strip that includes an absorbent pad. Detection is initiated by dipping the tip of the strip into an aqueous solution containing the sample to be examined.

[0251] This aqueous solution moves along the strip by capillary action and dissolves the gold-labeled antibody on the strip. The gold-labeled antibody is specific for at least one protein encoded by the sugar beet event Bv_CSM63713, binds to the epitope on the protein in the sample, and forms an antibody-antigen complex. This gold-labeled antibody-antigen complex then moves along the strip to the nitrocellulose membrane. This membrane contains a test line of immobilized antibody that binds to a second, different epitope on the protein encoded by the sugar beet event Bv_CSM63713, and if the protein encoded by the sugar beet event Bv_CSM63713 is present in the sample, a visible line across the test strip appears.

[0252] 3) Detection of the sugar beet event Bv_CSM63713 by real-time PCR A qualitative real-time event-specific PCR method was developed to identify the sugar beet event Bv_63713 in samples. Qualitative detection of sugar beet genomic DNA was performed using forward primer 2109_fwd1 (SEQ ID NO: 34), reverse primer 2109_rev1 (SEQ ID NO: 35), and probe 2109_probe1 (SEQ ID NO: 36). The 5′ end of the probe was labeled with 6-carboxy-fluorescein (6-FAM™), and the 3′ end was labeled with Minor Grove Binder Non-Fluorescent-Quencher (MGBNFQ). The 5′ exonuclease activity of Taq DNA polymerase cleaves the probe from the 5′ end between the fluorophore and the quencher. When hybridized to the target DNA strand, fluorescence is emitted because the quencher and the fluorophore are sufficiently separated to generate a fluorescent signal. Primers 2109_fwd1 and 2109_rev1 generated DNA amplicons used for the diagnosis of the sugar beet event Bv_63713 when used with these reaction methods and probe 2109_probe1. Appropriate controls including a positive control containing the sugar beet event Bv_63713, a negative control derived from non-transgenic sugar beet, and a negative control without template DNA were used in this analysis. Furthermore, this analysis included internal control primers and probes specific for a single-copy gene within the sugar beet genome.

[0253] An example of the components of the reaction mix for the sugar beet Bv_63713 event-specific PCR method is shown in Table 17. The PCR amplification reaction was performed using a Bio-Rad CFX96 Touch™ real-time PCR detection system according to the PCR cycle conditions described in Table 18. [Table 17] [Table 18]

[0254] An example of an endogenous sugar beet gene used as an internal control is glutamine synthetase (GS). Using GS-specific primers and a GS-specific probe with the 5' end labeled with the reporter dye 6-FAM and the 3' end labeled with the quencher BHQ1, a 121 bp fragment of the glutamine synthetase gene was amplified. The sequences of the primers and probe are shown in Table 19. The components of the reaction mix are described in Table 20. The PCR amplification reaction was carried out using a Bio-Rad CFX96 Touch™ real-time PCR detection system according to the PCR cycle conditions described in Table 18. [Table 19] [Table 20]

[0255] Example 9: Zygosity assay for sugar beet event Bv_CSM63713 In this example, a method useful for determining the zygosity of event Bv_CSM63713 is described. In this zygosity assay, plants containing the sugar beet event Bv_CSM63713 are determined to be heterozygous or homozygous for that event or the wild-type allele. Exemplary detection methods and materials are provided below.

[0256] 1) KASP zygosity assay A KASP genotyping assay for zygosity was developed based on the competitive allele-specific (KASP) PCR described in Example 8. Discrimination of two alleles is enabled through the competitive binding of two allele-specific forward primers. If a plant containing the sugar beet event Bv_CSM63713 is homozygous, only one of two possible fluorescent signals is generated. If a plant containing the sugar beet event Bv_CSM63713 is heterozygous, a mixed fluorescent signal is generated.

[0257] The KASP assay mix and Universal KASP master mix were added to the DNA samples, followed by performing a thermal cycle reaction and then endpoint fluorescence reading. Each of the allele-specific primers carried a unique tail sequence (underlined in Table 21) corresponding to the universal FRET (fluorescence resonance energy transfer) cassette, one labeled with the FAM dye and the other with the HEX dye. The KASP master mix contained the universal FRET cassette, ROX passive reference dye, Taq polymerase, free nucleotides, and MgCl 2 in the optimized buffer solution. During the thermal cycle, as the relevant allele-specific primers bind to and extend on the template, the tail sequence attaches to the newly synthesized strand. Next, the complement of the allele-specific tail sequence is generated during subsequent rounds of PCR, enabling the FRET cassette to bind to the DNA. The FRET cassette then no longer quenched and emitted fluorescence.

[0258] Two sets of primer combinations were developed for the zygosity assay of the event. These primers and their corresponding SEQ ID NOs are provided in Table 21. In Table 21, "AlleleX" refers to the wild-type allele, and "AlleleY" refers to the allele containing the event. Thus, the AlleleX primer detects the wild-type sequence, and the AlleleY primer detects the event. The zygosity assay can be performed using these two sets of primers independently or using only one set of the primer combination. As a result of these assays, it was found that homozygous wild-type plants produced only one signal, while transgenic plants homozygous for this event produced only different signals. However, plants heterozygous for this event produced signals for both alleles.

Table 21

[0259] 2) Further zygosity assayDevelop another zygosity assay to determine whether a plant containing the sugar beet event Bv_CSM63713 is heterozygous or homozygous for that event or the wild-type allele. An amplification reaction assay using the sequence information provided herein can be designed. For example, such a PCR assay includes the design of at least three primers, primer 1, primer 2, and primer 3, where primer 1 is specific for sugar beet genomic DNA of the 3' flanking DNA of the sugar beet event Bv_CSM63713, primer 2 is specific for the sugar beet event Bv_CSM63713 transgenic insert, and primer 3 is specific for the wild-type allele. When used as primer pairs in an amplification reaction, primer 1 and primer 2 produce a PCR amplicon specific for the sugar beet event Bv_CSM63713. When used as primer pairs in an amplification reaction, primer 1 and primer 3 produce a PCR amplicon specific for the wild-type allele. In a PCR reaction performed on sugar beet genomic DNA, the PCR amplicon generated from primer 1 + primer 2 and that generated from primer 1 + primer 3 differ in amplicon sequence and size. When these three primers are included in a PCR reaction with DNA extracted from a plant homozygous for the sugar beet event Bv_CSM63713, only the amplicon of primer 1 + primer 2 (specific for the sugar beet event Bv_CSM63713) is generated. When these three primers are included in a PCR reaction with DNA extracted from a plant heterozygous for the sugar beet event Bv_CSM63713, both the amplicon of primer 1 + primer 2 (specific for the sugar beet event Bv_CSM63713 insert) and the amplicon of primer 1 + primer 3 (specific for the absence of the wild-type allele or the sugar beet event Bv_CSM63713 insert) are generated. When these three primers are mixed together with DNA extracted from a plant null for the sugar beet event Bv_CSM63713 (wild-type plant) in a PCR reaction, only the amplicon of primer 1 + primer 3 (specific for the wild-type allele) is generated.The amplicons produced using these PCR reactions can be identified or distinguished using any method known in the art.

[0260] Another method for detecting the presence and zygosity of the sugar beet event Bv_CSM63713 in a plant sample is Southern blot analysis. Those skilled in the art will understand how to design a first Southern hybridization probe(s) specific for the sugar beet event Bv_CSM63713 and a second Southern hybridization probe specific for sugar beet plants that are null (wild type) for the sugar beet event Bv_CSM63713. In Southern blot analysis, a signal detected only from the first Southern hybridization probe indicates a plant that is homozygous for the sugar beet event Bv_CSM63713, a signal detected from both the first and second hybridization probes indicates a plant that is heterozygous for the sugar beet event Bv_CSM63713, and a signal detected only from the second Southern hybridization probe indicates that the DNA was extracted from a plant that is null (wild type) for the sugar beet event Bv_CSM63713. References X.Chen,L.Levine and P.-Y.Kwok(1999).Fluorescence polarization in homogeneous nucleic acid analysis.Genome Res.9:492-498. P.Chinnadurai,D.Stojsin,K.Liu,G.E.Frierdich,K.C.Glenn,T.Geng,A.Schapaugh,K.Huang,A.E.Deffenbaugh,Z.L.Liu,L.A.Burzio(2018).“Variability of CP4 EPSPS expression in genetically engineered soybean(Glycine max L.Merrill).“Transgenic Research,27:511-524. J.J. Doyle and J.L. Doyle (1990). “Isolation of Plant DNA from Fresh Tissue”. Focus, 12(1): 13 - 15. R.C. Edgar (2004). “MUSCLE: multiple sequence alignment with high accuracy and high throughput”. Nucleic Acids Research 32(5): 1792 - 7. S. Gurel, A. Pazuki, F. Aflaki, E. Gurel (2021). “Production of doubled haploid sugar beet (Beta vulgaris L.) plants trough gynogenesis. Methods Mol. Biol. 2289: 313 - 323. L.A. Harrison, M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. Hammond, D.L. Nida, B.L. Burnette, T.E. Nickson, T.A. Mitsky, M.L. Taylor, R.L. Fuchs, S.R. Padgette (1996). “The Expressed Protein in Glyphosate - Tolerant Soybean, 5 - Enolypyruvylshikimate - 3 - Phosphate Synthase from Agrobacterium sp. Strain CP4, Is Rapidly Digested In Vitro and Is not Toxic to Acutely Gavaged Mice”. Journal of Nutrition, 126(3): 728 - 740. E.M. Kishchenko, I.K. Komarnitskii, N.V. Kuchuk (2005). “Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin”. Cell Biology International 29: 15 - 19. K. Lindsey and P. Gallois (1990). “Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens”. J. of Experimental Botany 41(5):529 - 536. T. T. Nikiforov, R. B. Rendie, P. Goelet, Y.-H. Rogers, M. L. Kotewicz, S. Anderson, G. L. Trainor and M. R. Knapp (1994). Genetic bit analysis: a solid phase method for typing single nucleotide polymorphisms. Nucleic Acids Res. 22:4167 - 4175. M. D. Pearson, L. Nguyen, Y. Zhao, W. L. McKenna, T. J. Morin and W. B. Dunbar (2019). Past and accurate quantification of insertion - site specific transgene levels from raw seed samples using solid - state nanopore technology. PloS One 14(12):e0226719. J. M. Poehlman (1987) Breeding Sugar Beets. In: Breeding Field Crops. Springer, Dordrecht. pp 592 - 624. N. Soltani, J. A. Dille, D. E. Robinson, C. L. Sprague, D. W. Morishita, N. C. Lawrence, A. R. Kniss, P. Jha, J. Felix, R. E. Nurse and P. H. Sikkema (2018). “Potential yield loss in sugar beet due to weed interference in the United States and Canada”. Weed Technology 32:749 - 753. A.D. Taylor, L. Mataseje, C.J. Urfano, L. Schmidt, K.S. Antonation, M.R. Mulvey and C.R. Corbett (2018). Evaluation of Oxford Nanopore’s MinION Sequencing Device for Microbial Whole Genome Sequencing Applications. Scientific Reports 8:10931. S. Tyagi and F.R. Kramer (1996). Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology 14:303 - 308. P.J.P. de Vree, E. de Wit, M. Yilmaz et al. (2014). “Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping”. Nature Biotechnology 32(10):1019 - 1025. Y. Wan, J.F. Petolino and J.M. Widholm. (1989). “Efficient Production of Doubled Haploid Plants Through Colchicine Treatment of Anther - Derived Maize Callus”, Theor. Appl. Genet., 77:889 - 892. C. Wang, K.C. Glenn, C. Kessenich, E. Bell, L.A. Burzio, M.S. Koch, B. Li and A. Silvanovich (2016). “Safety assessment of dicamba mono - oxygenases that confer dicamba tolerance to various crops”. Regulatory Toxicology and Pharmacology, 81:171 - 182. Y. Wang, Y. Zhao, A. Bollas, Y. Wang and K. F. Au (2021). Nanopore sequencing technology, bioinformatics and applications. Nature Biotechnology 39: 1348 - 1365. M. Winge (2000). Pyrosequencing - a new approach to DNA analysis. Innovation in Pharma. Tech. 00: 18 - 24. E. W. Weich, M. W. Levall (2003). “Doubled haploid production of sugar beet (Beta vulgaris L.)”. In: Maluszynski M., Kasha K. J., Forster B. P., Szarejko I. (eds) Doubled Haploid Production in Crop Plants. Springer, Dordrecht.

Claims

1. Array number 10, array number 1, array number 2, array number 3, array number 4, array number 5, array number 6, array number 7, array number 8, array number 9, A polynucleotide having a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of array number 10 or the full length of array number 9, and A recombinant DNA molecule comprising a sequence selected from the group consisting of perfect complements of any of the above.

2. a. Derived from a sugar beet plant, seed, plant part, plant cell, progeny plant, or commercial product containing the sugar beet event Bv_CSM63713, and a representative sample of the seeds containing said event has been deposited as ATCC accession number PTA-127098, or b. Contained in a sugar beet plant, seed, plant part, plant cell, or progeny plant containing the sugar beet event Bv_CSM63713, or contained in a commercial product manufactured therefrom, and a representative sample of the seeds containing said event has been deposited as ATCC accession number PTA-127098, or c. Formed by insertion of a heterologous nucleic acid molecule into the genomic DNA of a sugar beet plant or sugar beet cell, or d. Containing an amplicon used for diagnosis of the presence of the sugar beet event Bv_CSM63713, The recombinant DNA molecule according to claim 1.

3. A DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe, a. Specifically hybridizes with the DNA of the sugar beet event Bv_CSM63713 in a sample under stringent hybridization conditions, and detection of the hybridization of said DNA molecule under said stringent hybridization conditions is used for diagnosis of the presence of the sugar beet event Bv_CSM63713 in said sample, or b. In a sample, at least one of the following: i) the 5' junction sequence between the adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713, ii) the 3' junction sequence between the transgenic insert of the sugar beet event Bv_CSM63713 and the adjacent sugar beet genomic DNA, iii) SEQ ID NO: 9, and iv) a fragment of SEQ ID NO: 9 containing a continuous nucleotide of SEQ ID NO: 9 of sufficient length to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713, wherein the DNA molecule is specific for the detection of **Claim 4** a. the DNA probe contains SEQ ID NO: 36, b. the DNA molecule contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and the complement of any of the above, and / or c. the sample is derived from a sugar beet plant, seed, plant part, plant cell, progeny plant, or commercial product, the DNA molecule according to claim 3. **Claim 5** A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein the first and the second DNA molecules contain a fragment of SEQ ID NO: 10 or its complement, and function as DNA primers when used together in an amplification reaction with DNA containing the sugar beet event Bv_CSM63713, and produce an amplicon used for the diagnosis of the sugar beet event Bv_CSM63713 in a sample. **Claim 6** a. the first and the second DNA molecules contain SEQ ID NO: 14 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 23, SEQ ID NO: 20 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 31 and SEQ ID NO: 26, SEQ ID NO: 33 and SEQ ID NO: 29, SEQ ID NO: 28 and SEQ ID NO: 29, or SEQ ID NO: 34 and SEQ ID NO: 35, and / or b. the amplicon is i. SEQ ID NO: 1, ii. SEQ ID NO: 2, iii. SEQ ID NO: 3, iv. SEQ ID NO: 4, v. SEQ ID NO: 5, vi. SEQ ID NO: 6, vii. SEQ ID NO: 7, viii. SEQ ID NO: 8, ix. SEQ ID NO: 9, x. SEQ ID NO: 10, and A pair of DNA molecules according to claim 5, comprising a nucleotide sequence selected from the group consisting of fragments of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the fragment is at least 10 nucleotides in length and contains nucleotides 1000-1001 or 12,722-12,723 of SEQ ID NO:

10.

7. A method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commercial products, comprising: a. contacting the sample with a DNA molecule that functions as the DNA probe according to claim 3; b. subjecting the sample and the DNA molecule that functions as the probe to stringent hybridization conditions; and c. detecting hybridization of the DNA molecule in the sample to the DNA molecule that functions as the probe, wherein the hybridization of the DNA molecule in the sample to the DNA molecule that functions as the probe is used for diagnosing the presence of the sugar beet event Bv_CSM63713 in the sample.

8. A method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commercial products, comprising: a) contacting the sample with a pair of DNA molecules according to claim 5; b) performing an amplification reaction sufficient to produce a DNA amplicon; and c) detecting the presence of the DNA amplicon, wherein the DNA amplicon comprises: a 5' junction sequence between adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713; a 3' junction sequence between adjacent sugar beet genomic DNA and the transgenic insert of the sugar beet event Bv_CSM63713; SEQ ID NO: 9; and at least one of the fragments of SEQ ID NO: 9 comprising a continuous nucleotide sequence of SEQ ID NO: 9 of sufficient length to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713, wherein the presence of the DNA amplicon indicates the presence of the sugar beet event Bv_CSM63713 in the sample.

9. The DNA amplicon comprises a. at least 10 nucleotides in length, at least 11 nucleotides in length, at least 12 nucleotides in length, at least 13 nucleotides in length, at least 14 nucleotides in length, at least 15 nucleotides in length, at least 16 nucleotides in length, at least 17 nucleotides in length, at least 18 nucleotides in length, at least 19 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 30 nucleotides in length, at least 35 nucleotides in length, at least 40 nucleotides in length, at least 45 nucleotides in length, at least 50 nucleotides in length, at least 60 nucleotides in length, at least 70 nucleotides in length, at least 80 nucleotides in length, at least 90 nucleotides in length, or at least 100 nucleotides in length, and / or b. a nucleotide sequence comprising a fragment of any of SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, and SEQ ID NO:10, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:1, which is at least 10 nucleotides in length and comprises a fragment selected from the group consisting of said fragments containing nucleotides 1000 - 1001 or 12,722 - 12,723 of SEQ ID NO:10, the method according to claim 8. **Claim 10** A method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample of DNA derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants, or commercial products, comprising: a) contacting said sample with the DNA molecule according to claim 3, and b) performing a sequencing reaction to produce a target sequence, wherein said target sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, the perfect complement of any of them, and a fragment of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10, which is at least 10 nucleotides in length and comprises a fragment selected from the group consisting of said fragments containing nucleotides 1000 - 1001 or 12722 - 12723 of SEQ ID NO:10, said method. **Claim 11** A method for detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, cells, progeny plants, or commercial products, comprising: a) contacting the sample with at least one antibody specific for at least one protein encoded by the sugar beet event Bv_CSM63713; and b) detecting the binding of the antibody to the protein in the sample, wherein the binding of the antibody indicates the presence of the sugar beet event Bv_CSM63713 in the sample. **Claim 12** A DNA detection kit for detecting the presence of the sugar beet event Bv_CSM63713 in a sample, the kit comprising the pair of DNA primers according to claim 5. **Claim 13** A DNA detection kit for detecting the presence of the sugar beet event Bv_CSM63713 in a sample, the kit comprising a DNA molecule that functions as the probe according to claim 3. **Claim 14** A protein detection kit for detecting the presence of the sugar beet event Bv_CSM63713 in a sample, the kit comprising at least one antibody specific for at least one protein encoded by the sugar beet event Bv_CSM63713, wherein detecting the binding of the at least one antibody to the at least one protein encoded by the sugar beet event Bv_CSM63713 in the sample is used for diagnosing the presence of the sugar beet event Bv_CSM63713 in the sample. **Claim 15** SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 A sugar beet plant, plant seed, plant part, or plant cell comprising a polynucleotide having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the full length of SEQ ID NO: 10 or the full length of SEQ ID NO: 9, and a recombinant DNA molecule comprising a sequence selected from the group consisting of any of the above perfect complements. **Claim 16** a. expressing at least one herbicide tolerance gene selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), dicamba monooxygenase (DMO), phosphinothricin N-acetyltransferase (PAT), and any combination thereof; b. being resistant to at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof; c. comprising the sugar beet event Bv_CSM63713, a representative sample of seeds comprising said event having been deposited under ATCC accession number PTA-127098; d. further defined as a progeny plant of any generation of a sugar beet plant comprising the sugar beet event Bv_CSM63713, or a sugar beet plant part, seed, or plant cell derived therefrom, and / or e. the plant part comprising roots, beets, pollen, anthers, ovaries, ovules, flowers, embryos, stems, leaves, microspores, protoplasts, or callus, the sugar beet plant, plant seed, plant part, or plant cell according to claim 15. **Claim 17** A sugar beet plant, plant part, plant seed, or plant cell comprising the sugar beet event Bv_CSM63713, wherein a representative sample of seeds comprising said sugar beet event Bv_CSM63713 has been deposited under ATCC accession number PTA-127098, said sugar beet plant, plant part, plant seed, or plant cell. **Claim 18** A method for controlling or preventing weeds in an area, comprising planting sugar beets containing event Bv_CSM63713 in said area, and applying an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof, wherein there is no phytotoxicity to said sugar beets, or if there is any phytotoxicity, the phytotoxicity to said sugar beets is less than about 10% and said weeds in said area are controlled.

19. a. applying an effective amount of said at least one herbicide comprises applying at least two or more herbicides selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof throughout the growth period, or b. the effective amount of said dicamba is about 0.5 lb ae / acre to about 2 lb ae / acre of dicamba throughout the growth period, the effective amount of said glufosinate is about 0.4 lb ai / acre to about 2.16 lb ai / acre throughout the growth period, and / or the effective amount of said glyphosate is about 0.75 lb ae / acre to about 2.25 lb ae / acre throughout the growth period, the method according to claim 18.

20. A method for controlling volunteer sugar beets containing sugar beet event Bv_CSM63713 in an area, comprising applying a herbicidally effective amount of at least one herbicide other than dicamba, glyphosate, or glufosinate, wherein the application of said herbicide prevents the growth of sugar beets containing sugar beet event Bv_CSM63713.

21. The method according to claim 20, wherein the herbicide other than glyphosate, dicamba, or glufosinate is selected from the group consisting of paraquat, clethodim, clopyralid, desmedipham, triflusulfuron, 2,4-dichlorophenoxyacetic acid (2,4-D), and acetolactate synthase (ALS) inhibitors such as sulfonylurea (SU), imidazolinone, triazolopyrimidine, pyrimidinyl oxybenzoate, and sulfonylaminocarbonyl triazolinone, and any combination thereof.

22. A method for obtaining seeds or plants of sugar beet plants resistant to dicamba, glyphosate, glufosinate, or any combination thereof, comprising a) obtaining a population of progeny seeds or plants grown therefrom, at least one of which comprises the sugar beet event Bv_CSM63713; and b) identifying at least an initial progeny seed or a plant grown therefrom that comprises the sugar beet event Bv_CSM63713, said method. **Claim 23** Identifying a progeny seed or a plant grown therefrom that comprises the sugar beet event Bv_CSM63713 comprises a. i) growing said progeny seeds to produce progeny plants; ii) treating said progeny plants with an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof; and iii) selecting progeny plants that are resistant to at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof; b. detecting the presence of the sugar beet event Bv_CSM63713 in a sample derived from said progeny seed or a plant grown therefrom; or c. detecting the presence of at least one protein encoded by the sugar beet event Bv_CSM63713 in a sample derived from said progeny seed or a plant grown therefrom The method according to claim 22, comprising. **Claim 24** A method for determining the zygosity of a sugar beet plant, plant part, plant seed, or plant cell comprising the sugar beet event Bv_CSM63713, comprising a) contacting a sample containing DNA derived from said sugar beet plant, plant part, plant seed, or plant cell with a primer set capable of producing a first amplicon used for the diagnosis of the presence of the sugar beet event Bv_CSM63713 and a second amplicon used for the diagnosis of wild-type sugar beet genomic DNA that does not comprise the sugar beet event Bv_CSM63713; b) performing a nucleic acid amplification reaction; and c) detecting said first amplicon and said second amplicon, The presence of both amplicons indicates that said plant, plant part, seed or cell is heterozygous for the sugar beet event Bv_CSM63713, and the presence of only said first amplicon indicates that said plant, plant part, seed or cell is homozygous for the sugar beet event Bv_CSM63713, said method. **Claim 25** Said primer set is SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and The method according to claim 24, selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO:

29. **Claim 26** A method for determining the zygosity of a sugar beet plant, plant part, plant seed, or plant cell comprising a sugar beet event Bv_CSM63713, a) contacting a sample containing DNA derived from said sugar beet plant, plant part, plant seed, or plant cell with a probe set comprising at least a first probe that specifically hybridizes to sugar beet event Bv_CSM63713 and at least a second probe that specifically hybridizes to sugar beet genomic DNA disrupted by the insertion of heterologous DNA of sugar beet event Bv_CSM63713 but does not hybridize to sugar beet event Bv_CSM63713; and b) hybridizing said probe set with said sample under stringent hybridization conditions, Detection of hybridization of only said first probe under said hybridization conditions is used for the diagnosis of a sugar beet plant, plant part, seed or plant cell that is homozygous for sugar beet event Bv_CSM63713, and detection of hybridization of both said first probe and said second probe under said hybridization conditions is used for the diagnosis of a sugar beet plant, plant part, seed, or plant cell that is heterozygous for sugar beet event Bv_CSM63713. **Claim 27** A DNA construct comprising a first expression cassette, a second expression cassette, and a third expression cassette, a) said first expression cassette comprises, in operable linkage, i) a chlorophyll A-B binding protein (Cab1) promoter and leader from Arabidopsis thaliana, ii) a phosphinothricin N-acetyltransferase (PAT) coding sequence, and iii) a 3'UTR of a small heat shock protein (Hsp20) from Medicago truncatula, b) The second expression cassette comprises, in operable linkage, i) a ubiquitin (Ubc1) promoter, leader, and intron from Cucumis melo, ii) a chloroplast transit peptide coding sequence of ribulose bisphosphate carboxylase small subunit (RbcS) from Pisum sativum, iii) a dicamba monooxygenase coding sequence (DMO), and iv) a 3'UTR of a putative protein from Medicago truncatula, c) The third expression cassette comprises, in operable linkage, i) an inclusion body matrix protein enhancer from Dahlia mosaic virus, ii) a promoter, leader, and intron of S-adenosyl-L-methionine synthetase (SAMS2) from Cucumis melo, iii) a 5-enolpyruvylshikimate-3-phosphate synthase chloroplast transit peptide (EPSPS) coding sequence from Arabidopsis thaliana, iv) a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) coding sequence, and v) a 3'UTR of a hypothetical protein from Medicago truncatula, said DNA construct. Claim 28 The DNA construct according to claim 27, wherein the DNA construct comprises SEQ ID NO:

9. Claim 29 A method for improving tolerance to at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof in sugar beet plants, comprising: a) inserting the DNA construct according to claim 27 into the genome of sugar beet cells; b) generating sugar beet plants from said sugar beet cells; and c) selecting sugar beet plants comprising said DNA construct. Claim 30 The method according to claim 29, wherein said selecting comprises treating said sugar beet cells or plants with an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof. Claim 31 A sugar beet plant, plant seed, plant part, or plant cell that is resistant to herbicides having three different modes of action at a single genomic locus. Claim 32 A sugar beet plant, plant seed, plant part, or plant cell comprising the DNA construct according to claim 27. Claim 33 A sugar beet plant, plant seed, plant part, or plant cell obtained by the method according to claim 29.

34. A method for producing a progeny sugar beet plant comprising the sugar beet event Bv_CSM63713, comprising: a. selfing or cross-pollinating a first sugar beet plant comprising the sugar beet event Bv_CSM63713 with itself or a second sugar beet plant; b. collecting one or more seeds produced from said cross; c. growing the one or more seeds to produce one or more progeny plants; and d. selecting at least an initial progeny plant or seed comprising the sugar beet event Bv_CSM63713.

35. An inbred or hybrid sugar beet plant or seed comprising the sugar beet event Bv_CSM63713 produced by the method according to claim 34.

36. A non-living or non-regenerable sugar beet plant material comprising the recombinant DNA molecule according to claim 1.

37. a. the DNA construct according to claim 27, or b. a non-living or non-regenerable sugar beet plant material comprising the sugar beet event Bv_CSM63713, wherein a representative sample of seeds comprising said sugar beet event Bv_CSM63713 has been deposited under ATCC accession number PTA-127098.

38. A commercial product comprising the recombinant DNA molecule according to claim 1.

39. A commercial product comprising the DNA construct according to claim 27.

40. a. produced from a transgenic sugar beet plant, plant part, plant seed, or plant cell comprising the sugar beet event Bv_CSM63713, and / or b. the commercial product according to claim 38, comprising all seeds or processed seeds, non-growing seeds, processed plant parts, processed plant tissues, dried plant tissues, dried plant parts, frozen plant tissues, frozen plant parts, plant parts processed for animal feed, fibers, pulp, pulp pellets, pulp fragments, tailings, squeezed juice, syrup, molasses, extracts, raffinates, betaine, separator molasses soluble fraction (SMS), or any other human food, viable seeds, viable plant parts (e.g., roots and leaves), or viable plant cells.

41. A method for producing a commercial product, comprising: a) obtaining a transgenic sugar beet plant, plant part, or plant seed comprising the sugar beet event Bv_CSM63713, and b) producing a commercial product from said transgenic sugar beet plant, plant part, or plant seed, said method. **Claim 42** A method for controlling, preventing, or reducing the occurrence of herbicide-resistant weeds, comprising cultivating a sugar beet plant comprising a transgene conferring resistance to a herbicide, having the mode of action of three different herbicides at a single genomic locus, in the growth environment of a crop. **Claim 43** The method according to claim 42, wherein the modes of action of said three different herbicides are selected from the group consisting of inhibition of glutamine synthetase, inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and benzoic acid-type auxin. **Claim 44** A method for controlling, preventing, or reducing the occurrence of herbicide-resistant weeds, a. cultivating a sugar beet plant comprising the DNA construct according to claim 27 for conferring resistance to a herbicide, having the mode of action of three different herbicides at a single genomic locus, in the growth environment of a crop, and b. applying at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof to the growth environment of said crop, said sugar beet plant being resistant to said at least one herbicide. **Claim 45** A method for reducing the number of genetic loci for sugar beet breeding by inserting a transgene at a single genomic locus for resistance to three different classes of herbicides. **Claim 46** a. the transgene is inserted as a single molecularly linked transgenic insert, or b. the transgene is inserted as a single molecularly linked transgenic insert, said transgenic insert conferring commercial-level resistance to at least one herbicide for each mode of action of a herbicide, the method according to claim 45. **Claim 47** A sugar beet plant, plant cell, plant part, or plant seed comprising a recombinant DNA construct integrated into chromosome 4, wherein the recombinant DNA construct confers resistance to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof, and the recombinant DNA construct is integrated at the position of the chromosome where SEQ ID NO: 11 and SEQ ID NO: 12 are located horizontally, said sugar beet plant, plant cell, plant part, or plant seed.

Images