Transgenic beet strain BVCSM63713 as well as detection method and application thereof

Abstract

A transgenic sugar beet line BvCSM63713 is provided. Also provided are transgenic plant cells, plant parts, plants, seeds, agricultural products, and commercial products comprising the strain BvCSM63713. Also provided are recombinant DNA molecules specific to the strain BvCSM63713, as well as methods of using and detecting the BvCSM63713. Beet plants containing the line BvCSM63713 exhibit tolerance to: a benzoic acid auxin, such as dicamba; an EPSPS inhibitor, such as glyphosate; and a glutamine synthetase inhibitor, such as glufosinate-ammonium.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 340,278, filed on May 10, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation of Sequence Listing

[0004] The file contains a sequence listing named "MONS531WO_ST26.xml", which is 113 kilobytes in size (measured in MS-WINDOWS), created on April 5, 2023, submitted electronically with this application, and incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates generally to the fields of agriculture, plant biotechnology and molecular biology. More specifically, the present disclosure relates to compositions and methods for providing herbicide tolerance in transgenic sugar beet plants. More specifically, recombinant DNA molecules of sugar beet strain Bv_CSM63713 are provided. Also provided are transgenic sugar beet plants, plant parts, seeds, cells and agricultural products comprising sugar beet strain Bv_CSM63713, as well as methods of using transgenic sugar beet plants, plant parts, seeds, cells and agricultural products comprising sugar beet strain Bv_CSM63713; methods of detecting sugar beet strain Bv_CSM63713; and methods of controlling weeds. Transgenic sugar beet plants, plant parts, seeds and cells comprising sugar beet line Bv_CSM63713 exhibit tolerance to: benzoate auxins, such as dicamba; 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, such as glyphosate; and glutamine synthetase inhibitors, such as glufosinate. Background Art

[0006] Sugar beet (Beta vulgaris) is an important cash crop in many countries, and the important role of herbicides in controlling weeds in crop production has been well documented. Weeds compete with crops for space, nutrients, water and light, and may contaminate harvests, so weed control is essential for achieving successful crop yields. Biotechnology methods have proven to be extremely useful in producing transgenic sugar beets that are tolerant to specific herbicides by expressing heterologous genes (transgenics). Transgenic herbicide tolerance enables the use of herbicides in crop growth environments without causing damage to the crop or minimal damage to the crop (e.g., damage less than about 10%). Transgenic traits of sugar beet have been used to confer tolerance to glyphosate and are widely used for weed control in commercial sugar beet production. Transgenic glyphosate tolerance is produced by inserting a variant (i.e., enzyme 5-enolpyruvate-3-phosphoshikimate synthase (EPSPS)) that produces a glyphosate target in the sugar beet genome. This variant is tolerant to glyphosate and is CP4-EPSPS from Agrobacterium strain CP4.

[0007] The herbicide tolerance trait can be used alone, or combined with other traits (such as tolerance to another herbicide). For providing weed control selection, the combination of herbicide tolerance traits is ideal, which will increase the flexibility of the grower and can use multiple herbicide action modes to control challenging weeds. Combining multiple desired traits in the genome can be achieved in the following ways: by hybridizing between two parents with desired traits separately, and identifying the progeny plant with the desired trait combination, or by using one or more genes providing additional desired traits, through random integration or through one or more targeted integration of genes providing additional desired traits to re-transform the transgenic plant containing one or more desired traits. Alternatively, the combination of multiple desired traits can be achieved by inserting multiple genes into a position or locus in the genome in the form of a single DNA molecule. Combining multiple herbicide tolerance traits at a single locus in beet will provide useful weed control means, making it simpler and less costly to maintain in the subsequent breeding process of hybrid plants with a series of different superior germplasms.

[0008] The expression of a transgene in a transgenic plant, part, seed or cell and therefore its effectiveness may be affected by many factors, such as the regulatory elements used in the transgenic expression cassette, the combination and / or interaction of these regulatory elements, the chromosomal location of the transgenic insertion site, the chromatin structure of the genome at or near the transgenic insertion site, and the presence or proximity of any endogenous cis- and / or trans-regulatory elements or genes near the transgenic insertion site. In addition, the expression of traits in transgenic plants becomes more complex when the transgenic insert comprises multiple expression cassettes, each with a different transgene conferring a different trait. These differences or factors may result in variations in the level of transgene expression or the spatial or temporal pattern of transgene expression between different transgenic insertion lines in the same expression cassette. In addition, different transgenic lines may also differ in the molecular mass of these lines. For example, a transgenic line may contain two or more copies of a transgenic insert at one or more chromosomal locations, or a transgenic insert may be truncated or contain vector backbone sequences relative to the intended insert, or a transgene may be inserted into an endogenous gene or repeat region. These features may result in undesirable consequences, 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 different lines.

[0009] Even in the case of targeted sequence insertions, variations in transgene expression levels between independent but genetically identical target sequence insertion (TSI) lines have been observed in subpopulations of transgenic lines (Verkest et al., 2019). This expression variation and silencing occurs independently of the transgene sequence and can be attributed to DNA methylation, which in turn is associated with different DNA methylation mechanisms. The fact that significant variations in transgene expression are observed in subpopulations of pure TSI lines suggests that even when integrated lines are targeted, selection is still required to identify TSI lines with stable expression of the gene of interest over several generations, similar to what is practiced for random integrated lines.

[0010] Commercially useful multi-gene transgenic lines require that each transgene in the transgenic insert is expressed in a manner required to successfully obtain the trait, and involve rigorous and careful testing, evaluation, and selection. Once a tolerance trait is selected, each expression cassette is designed and tested in vitro and / or in plant body to select the best expression cassette for each trait. These tests include testing different regulatory elements (such as promoters, introns, leader sequences, and 3'UTR) and combinations of different regulatory elements for the expected spatial and temporal expression of transgenics, and checking whether the transgenic product (protein) is targeted to a subcellular compartment, such as chloroplasts. Then, the expression cassette selected for each trait is combined in a construct, and the construct is tested to ensure that all expression cassettes work well together and each transgenic is correctly expressed. Then, the selected expression cassette combination is used for transformation to produce transgenic plants. Since the T-DNA construct comprising one or more transgenic cassettes is used for Agrobacterium-mediated transformation, there is great variability and randomness in the position where one or more transgenics can be inserted in the plant genome, each transgenic line is unique, and transgenic DNA is randomly and uniquely inserted into different plant genome positions. Thus, the selected combination of expression cassettes is used to generate hundreds of unique multigenic transgenic lines, each of which is the result of a random insertion of foreign DNA into a different location in the plant genome.

[0011] For these reasons, the performance of different transformed lines from the same transformation construct may vary, and the transformed lines that confer the most favorable traits or characteristics without other potential abnormalities or problems need to be identified to select superior lines for commercial use. Therefore, a large number of independent transformed lines must be generated and analyzed to select lines with superior commercial characteristics, which would be a major project involving analysis and selection among many different transformed lines.

[0012] In order to produce multi-gene strains for commercial use, strict and meticulous molecular characterization, greenhouse testing and multi-year field trials of different germplasms at multiple locations and under multiple conditions are required, thereby allowing the acquisition of a large amount of agronomic, phenotypic and molecular data. Then, the obtained data are analyzed to select strains suitable for commercial purposes. After being identified as having desired transgenic expression, molecular characteristics, efficacy and field performance, commercial multi-gene strains can be infiltrated into other beet gene backgrounds as single loci with multiple herbicide tolerance traits using plant breeding methods. The resulting beet variety contains new traits and other desired qualities, such as natural traits, disease tolerance traits, high-yield germplasms and / or one or more other transgenic herbicide tolerance traits. Summary of the invention

[0013] Recombinant DNA molecules are provided herein. Examples of such recombinant DNA molecules include sequences selected from the group consisting of: 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 the complete complementary sequence of any of the foregoing. In some embodiments, the recombinant DNA molecule is derived from a plant, seed, plant part, plant cell, progeny plant, or commercial product comprising the sugar beet strain Bv_CSM63713, a representative sample of seeds comprising the strain has been deposited with ATCC Accession No. PTA-127098. In some embodiments, the recombinant DNA molecule is contained in a plant, seed, plant part, plant cell, or progeny plant comprising the sugar beet strain Bv_CSM63713, a representative sample of seeds comprising the strain has been deposited with 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 for diagnosing the presence of the sugar beet strain Bv_CSM63713.

[0014] A DNA molecule that acts as a DNA probe is provided. An example of such a DNA molecule is a DNA molecule that comprises a polynucleotide segment that is long enough to act as a DNA probe, which specifically hybridizes with the sugar beet strain Bv_CSM63713 DNA in a sample under stringent hybridization conditions. Detection of hybridization of the DNA molecule under stringent hybridization conditions will diagnose the presence of sugar beet strain Bv_CSM63713 in the sample.

[0015] Also provided is a DNA molecule comprising a polynucleotide segment of sufficient length to act as a DNA probe, which specifically detects at least one of the following in a sample: a 5' junction sequence between the flanking sugar beet genomic DNA and the transgenic insert of the sugar beet variety Bv_CSM63713; a 3' junction sequence between the transgenic insert of the sugar beet variety Bv_CSM63713 and the flanking sugar beet genomic DNA; SEQ ID NO:9; and a fragment of SEQ ID NO:9, which comprises a sufficient length of contiguous nucleotides of SEQ ID NO:9 to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713.

[0016] The DNA probe may comprise SEQ ID NO: 36. Alternatively, the DNA molecule serving 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 complementary sequence of any of the foregoing. The sample may be derived from a beet plant, seed, plant part, plant cell, progeny plant, or commercial product.

[0017] A DNA molecule pair is provided. The DNA molecule pair comprises a first DNA molecule and a second DNA molecule. The first DNA molecule and the second DNA molecule comprise a fragment of SEQ ID NO: 10 or a complementary sequence thereof, and act as DNA primers when used in an amplification reaction with DNA comprising the sugar beet strain Bv_CSM63713 to produce an amplicon of the sugar beet strain Bv_CSM63713 for use in a diagnostic sample. For example, the first DNA molecule and the second DNA molecule may comprise 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 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 a fragment 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 comprises nucleotides 1,000–1,001 or 12,722–12,723 of SEQ ID NO: 10.

[0018] Provided is a method for detecting the presence of sugar beet strain 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 comprises: a) contacting the sample with any DNA molecule described herein that acts as a probe; b) subjecting the sample and the DNA molecule that acts as a probe to stringent hybridization conditions; and c) detecting hybridization of the DNA molecule that acts as a probe with a DNA molecule in the sample. Hybridization of the DNA molecule that acts as a probe with a DNA molecule in the sample will diagnose the presence of sugar beet strain Bv_CSM63713 in the sample.

[0019] Another method for detecting the presence of sugar beet strain Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants or commercial products is provided. The method comprises: contacting the sample with any DNA molecule pair 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 comprises at least one of: a 5' junction sequence between the sugar beet genomic DNA and the transgenic insert of sugar beet strain Bv_CSM63713; a 3' junction sequence between the sugar beet genomic DNA and the transgenic insert of sugar beet strain Bv_CSM63713; SEQ ID NO: 9; and a fragment of SEQ ID NO: 9, the fragment comprising a sufficient length of contiguous nucleotides of SEQ ID NO: 9 to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713. The presence of the DNA amplicon indicates the presence of sugar beet strain Bv_CSM63713 in the sample. The DNA amplicon can be at least 10 nucleotides long, at least 11 nucleotides long, at least 12 nucleotides long, at least 13 nucleotides long, at least 14 nucleotides long, at least 15 nucleotides long, at least 16 nucleotides long, at least 17 nucleotides long, at least 18 nucleotides long, at least 19 nucleotides long, at least 20 nucleotides long, at least 25 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long, at least 40 nucleotides long, at least 45 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long, or at least 100 nucleotides long. 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 a fragment of any one 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, wherein the fragment is at least 10 nucleotides in length and comprises nucleotides 1,000-1,001 or 12,722-12,723 of SEQID NO:10.

[0020] Another method for detecting the presence of sugar beet line Bv_CSM63713 in a DNA sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants or commercial products is provided. The method comprises: a) contacting the sample with any DNA molecule described herein that acts as a probe; and b) performing a sequencing reaction to generate a target sequence. The 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 complete complementary sequence of any one thereof; and a fragment 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, SEQ ID NO:8 and SEQ ID NO:10, wherein the fragment is at least 10 nucleotides in length and comprises nucleotides 1,000-1,001 or 12,722-12,723 of SEQ ID NO:10.

[0021] Another method for detecting the presence of sugar beet strain Bv_CSM63713 in a sample derived from sugar beet seeds, plants, plant parts, plant cells, progeny plants or commercial products is provided. The method comprises: a) contacting the sample with at least one antibody, the at least one antibody being specific for at least one protein encoded by sugar beet strain 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 sugar beet strain Bv_CSM63713 in the sample.

[0022] A DNA detection kit for detecting the presence of sugar beet strain Bv_CSM63713 in a sample is provided. An example of such a DNA detection kit is a kit comprising any DNA primer pair described herein. Another example of a DNA detection kit is a kit comprising any DNA molecule acting as a probe described herein.

[0023] Also provided is a protein detection kit for detecting the presence of sugar beet strain Bv_CSM63713 in a sample. An example of such a kit is a kit comprising at least one antibody that is specific for at least one protein encoded by sugar beet strain Bv_CSM63713. Detection of binding of the at least one antibody to at least one protein encoded by sugar beet strain Bv_CSM63713 in a sample is diagnostic for the presence of sugar beet strain Bv_CSM63713 in the sample.

[0024] A sugar beet seed, plant, plant part or plant cell is provided. The beet seed, plant, plant part or plant cell contains a recombinant DNA molecule, which contains a 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; 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 the complete complementary sequence of any of the foregoing. The beet seed, plant, plant part or plant cell may 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 beet seed, plant, plant part or plant cell may be tolerant to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate and any combination thereof. The beet plant, plant seed, plant part or plant cell may comprise the beet strain Bv_CSM63713, and a representative sample of the seeds comprising the strain has been deposited with ATCC accession number PTA-127098. The beet plant, plant seed, plant part or plant cell may also be defined as a progeny plant of any generation of a beet plant comprising the beet line Bv_CSM63713, or a beet plant part, plant seed or plant cell obtained therefrom.

[0025] Also provided are beet plants, plant parts, plant seeds and plant cells. The beet plants, plant parts, plant seeds and plant cells comprise the beet strain Bv_CSM63713, a representative sample of seeds comprising the beet strain Bv_CSM63713 has been deposited under ATCC Accession No. PTA-127098.

[0026] Any of the Beet plant parts described herein may comprise roots, beets, microspores, pollen, anthers, ovules, ovaries, flowers, embryos, stems, leaves, protoplasts, or callus.

[0027] Provided is a method for controlling or preventing weeds in a region. An example of this method includes planting beets comprising strain Bv_CSM63713 in the region, and applying an effective amount of at least one herbicide to control the weeds in the region, while not causing damage to the beets or less than about 10% damage to the beets, the herbicide being selected from the group consisting of: dicamba, glyphosate, glufosinate and any combination thereof. Applying at least one herbicide of an effective amount may include applying at least two or more herbicides in the growing season, the herbicide being selected from the group consisting of: dicamba, glyphosate, glufosinate and any combination thereof. In the growing season, the effective amount of dicamba can be about 0.5 pounds of acid equivalents / acre (lb ae / acre) to about 2 pounds of acid equivalents / acre. In the growing season, the effective amount of glufosinate can be about 0.4 pounds of active ingredients / acre (lb ai / acre) to about 2.16 pounds of active ingredients / acre. An effective amount of glyphosate may be from about 0.75 pounds acid equivalent per acre to about 2.25 pounds acid equivalent per acre during the growing season.

[0028] Provided is a method for controlling the volunteer beet comprising beet strain Bv_CSM63713 in a region. An example of this method includes applying at least one herbicide except dicamba, glyphosate or glufosinate or ammonium phosphine in a herbicidally effective amount, wherein the application of the herbicide will prevent the growth of the beet comprising beet strain Bv_CSM63713. The herbicide except dicamba, glyphosate or glufosinate can 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 sulfonylureas (SUs), imidazolinones, triazolopyrimidines, pyrimidineoxybenzoates and sulfonamidocarbonyltriazolinones.

[0029] A method for obtaining a seed or beet plant having tolerance to glyphosate, dicamba, glufosinate or any combination thereof is provided. 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 beet strain Bv_CSM63713; and b) identifying at least one first progeny seed or plant grown therefrom comprising beet strain Bv_CSM63713. Identifying a progeny seed or plant grown therefrom comprising beet strain Bv_CSM63713 may 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 having tolerance to the at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate and any combination thereof. Alternatively or additionally, identifying progeny seeds comprising Sugar Beet Line Bv_CSM63713 or plants grown therefrom may comprise detecting the presence of Sugar Beet Line Bv_CSM63713 in a sample derived from the progeny seeds or plants grown therefrom. Alternatively or additionally, identifying progeny seeds comprising Sugar Beet Line Bv_CSM63713 or plants grown therefrom may comprise detecting the presence of at least one protein encoded by Sugar Beet Line Bv_CSM63713 in a sample derived from the progeny seeds or plants grown therefrom.

[0030] Provided is a method for determining the zygosity of a beet plant, plant part, plant seed or plant cell comprising beet strain Bv_CSM63713. An example of such a method comprises: a) contacting a sample comprising DNA derived from a beet plant, plant part, plant seed or plant cell with a primer set capable of producing a first amplicon for diagnosing the presence of beet strain Bv_CSM63713 and a second amplicon for diagnosing wild-type beet genomic DNA that does not contain beet bean strain Bv_CSM63713; b) performing a nucleic acid amplification reaction; and c) detecting the first amplicon and the second amplicon. The presence of two amplicons indicates that the sample is heterozygous for beet strain Bv_CSM63713, while the presence of only the first amplicon indicates that the sample is homozygous for beet strain Bv_CSM63713. Illustrative examples of primer sets that can be used are primer sets comprising the following sequences: 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.

[0031] Another method for determining the zygosity of a beet plant, plant part, plant seed or plant cell comprising beet strain Bv_CSM63713 is provided. The method comprises: a) contacting a sample comprising DNA derived from a beet plant, plant part, plant seed or plant cell with a probe set, the probe set comprising at least one first probe that specifically hybridizes with beet strain Bv_CSM63713, and at least one second probe that specifically hybridizes with beet genomic DNA and does not hybridize with beet strain Bv_CSM63713, wherein the beet genomic DNA is disrupted by the insertion of heterologous DNA of beet strain Bv_CSM63713; and b) hybridizing the probe set with the sample under stringent hybridization conditions. Detection of only the first probe hybridizing under hybridization conditions will diagnose that the beet plant, plant part, seed or plant cell is homozygous for beet strain Bv_CSM63713. Detection of hybridization of both the first probe and the second probe under hybridization conditions is diagnostic for the Sugar Beet plant, plant part, seed or plant cell being heterozygous for Sugar Beet line Bv_CSM63713.

[0032] A DNA construct is provided. An example of such a DNA construct 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 connection: i) a chlorophyll AB binding protein (Cab1) promoter and leader sequence from Arabidopsis thaliana; ii) a phosphinothricin N-acetyltransferase (PAT) coding sequence; and iii) a small heat shock protein (Hsp20) 3'UTR from Medicago truncatula. The second expression cassette comprises in operable connection: i) a ubiquitin (Ubq1) promoter, leader sequence, and intron from Cucumis melo; ii) a ribulose bisphosphate carboxylase small subunit (RbcS) chloroplast transit peptide coding sequence from Pisum sativum; iii) a dicamba monooxygenase coding sequence (DMO); and iv) a putative protein 3'UTR from Medicago truncatula. The third expression cassette comprises in operable connection: i) an inclusion matrix protein enhancer from Dahlia Mosaic Virus; ii) a S-adenosyl-L-methionine synthetase (SAMS2) promoter, leader sequence and intron from melon; 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 hypothetical protein 3'UTR from Medicago truncatula. For example, the DNA construct may comprise SEQ ID NO: 9.

[0033] A Sugar Beet plant, plant seed, plant part or plant cell comprising any of the DNA constructs described herein is provided.

[0034] A method for increasing the tolerance of a sugar beet plant to at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate and any combination thereof is provided. The method comprises: a) inserting any DNA construct described herein into the genome of a sugar beet cell; b) producing a sugar beet plant from the sugar beet cell; and c) selecting a sugar beet plant comprising the DNA construct. The selection may comprise treating the sugar beet cell or plant with an effective amount of at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate and any combination thereof.

[0035] Also provided is a Sugar Beet plant, plant seed, plant part or plant cell tolerant to herbicides with three different herbicide modes of action at a single genomic location.The Sugar Beet plant, plant seed, plant part or plant cell may comprise any of the DNA constructs described herein.

[0036] Provided are beet seeds, plants, plant parts or plant cells tolerant to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate and any combination thereof.Beet seeds, plants, plant parts or plant cells contain any DNA construct provided herein.

[0037] Any sugar beet seed, plant, plant part or cell can be obtained by any method provided herein for increasing the tolerance of a sugar beet plant to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate and any combination thereof.

[0038] A method for producing a progeny sugar beet plant comprising sugar beet line Bv_CSM63713 is provided. The method comprises: a) sexually crossing a first sugar beet plant comprising sugar beet line Bv_CSM63713 with itself or a second sugar beet plant; b) collecting one or more seeds produced by the crossing; c) growing the one or more seeds to produce one or more progeny plants; and d) selecting at least one first progeny plant or seed comprising sugar beet line Bv_CSM63713. Selfed and hybrid sugar beet plants and seeds comprising sugar beet line Bv_CSM63713 produced by the method are also provided.

[0039] Also provided are non-living sugar beet plant material and non-renewable sugar beet plant material.The material may comprise any recombinant DNA molecule or any DNA construct described herein.

[0040] Also provided is non-living sugar beet plant material or non-renewable sugar beet plant material comprising the sugar beet line Bv_CSM63713, a representative sample of seeds comprising the sugar beet line Bv_CSM63713 having been deposited under ATCC Accession No. PTA-127098.

[0041] Commodity products are also provided. An example of such commodity products is a commodity product comprising any recombinant DNA molecule described herein or any DNA construct. The commodity product can be produced by a transgenic beet plant, plant part, plant seed or plant cell comprising beet strain Bv_CSM63713. The commodity product can include, for example, complete or processed seeds, inactive seeds, processed plant parts, processed plant tissues, dehydrated plant tissues, dehydrated plant parts, frozen plant tissues, frozen plant parts, plant parts, fiber, pulp, pulp particles, pulp fragments, tailings, fruit juice, syrup, molasses, extracts, raffinate, betaine, separation molasses solubles (separatormolasses soluble, SMS) or any other food, vigorous seeds, vigorous plant parts (such as roots and leaves) or vigorous plant cells for human consumption.

[0042] A method for producing a commercial product is provided. The method comprises: a) obtaining a transgenic sugar beet plant, plant part or plant seed comprising the sugar beet line Bv_CSM63713; and b) producing a commercial product from the transgenic sugar beet plant, plant part or plant seed.

[0043] A method for controlling, preventing or reducing the development of herbicide tolerant weeds is provided. The method comprises growing a sugar beet plant comprising a transgene in a crop growing environment that provides tolerance to herbicides having three different herbicide modes of action at a single genomic location. The three different herbicide modes of action may be selected from the group consisting of: inhibition of glutamine synthetase, benzoic acid auxin, and inhibition of EPSPS.

[0044] Also provided is a method for controlling, preventing or reducing the development of herbicide-tolerant weeds. The method comprises: a) cultivating a sugar beet plant in a crop growing environment, the sugar beet plant comprising any of the DNA constructs described herein for providing tolerance to herbicides having three different herbicide modes of action at a single genomic location; and b) applying at least one herbicide selected from the group consisting of dicamba, glufosinate, and any combination thereof to the crop growing environment, wherein the sugar beet plant is tolerant to the at least one herbicide.

[0045] A method for reducing loci for beet breeding is provided by inserting a transgene at a single genomic location to provide tolerance to three different classes of herbicides. The transgene can be inserted as a single molecule-linked transgenic insert. The single molecule-linked transgenic insert can provide commercial levels of tolerance to at least one herbicide for each herbicide mode of action.

[0046] Also provided are beet plants, plant cells, plant parts and plant seeds. Also provided are beet plants, plant cells, plant parts and plant seeds comprising a recombinant DNA construct integrated into chromosome 4. The recombinant DNA construct confers tolerance to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate and any combination thereof. The recombinant DNA construct is integrated into the chromosome at a position flanking SEQ ID NO: 11 and SEQ ID NO: 12. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1The sequence of the sugar beet strain Bv_CSM63713 is shown. The horizontal lines correspond to the positions 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: 11 and SEQ ID NO: 12 relative to SEQ ID NO: 10. The horizontal arrows labeled SEQ ID NO: 14 / 15 and SEQ ID NO: 18, SEQ ID NO: 19 / 20 and SEQ ID NO: 23, and SEQ ID NO: 34 and SEQ ID NO: 35 represent the approximate positions of three exemplary primer pairs that can be used to detect the sugar beet strain Bv_CSM63713. The horizontal bar without an arrow labeled SEQ ID NO: 36 represents the approximate position of an exemplary probe. The dotted horizontal arrows labeled SEQ ID NO:24 / 30, SEQ ID NO:25 / 31, and SEQ ID NO:26 indicate the approximate location of an exemplary set of primers used to determine the zygosity of a sugar beet plant comprising line Bv_CSM63713. The hollow horizontal arrows labeled SEQ ID NO:27 / 32, SEQ ID NO:28 / 33, and SEQ ID NO:29 indicate the approximate location of another exemplary set of primers used to determine the zygosity of a sugar beet plant comprising line Bv_CSM63713. Where applicable, when a " / " separates the two numbers following "SEQ ID NO:", the first number represents the SEQ ID NO of the primer with the labeled tail, and the second number after the " / " represents the SEQ ID NO of the same primer without the labeled tail. "RB" refers to the right border of the Agrobacterium T-DNA; "LB" refers to the left border of the Agrobacterium T-DNA. "P" represents a promoter element; "L" represents a leader sequence (5'UTR) element; "I" represents an intron element; "CTP" represents a chloroplast transit peptide element; "T" represents a 3'UTR. "PAT" represents a phosphinothricin N-acetyltransferase encoding element; "DMO" represents a dicamba monooxygenase encoding element; and "CP4" represents a 5-enolpyruvylshikimate-3-phosphate synthase encoding element.

[0049] Figure 2Provided are photos showing the effects of herbicide treatment on beet. R0 plants grown in greenhouses at the 4-6 leaf stage were treated with glyphosate (4x), glufosinate (4x), dicamba (4x), dicamba+glyphosate (4x each) and glyphosate+glufosinate+dicamba (2.4x each). Photos were taken before and two weeks after the herbicide was applied. Figures A1 and A2: Wild-type beet before and after the herbicide was applied; Figures B1 and B2: Line Bv_CSM63713 before and after the herbicide was applied; Figures C1 and C2: Another line before and after the herbicide was applied. Herbicide treatments (from left to right) are: 1. glyphosate+glufosinate+dicamba triple mixture; 2. glyphosate+dicamba double mixture; 3. dicamba; 4. glufosinate; 5. glyphosate; and 6. untreated controls.

[0050] Sequence Description

[0051] SEQ ID NO:1 is a 30 nucleotide sequence representing the 5' junction region of the sugar beet genomic DNA and the integrated transgenic insert. SEQ ID NO:1 corresponds to nucleotide positions 986-1015 of SEQ ID NO:10.

[0052] SEQ ID NO:2 is a 30 nucleotide sequence representing the 3' junction region of the integrated transgenic insert and the sugar beet genomic DNA. SEQ ID NO:2 corresponds to nucleotide positions 12708-12737 of SEQ ID NO:10.

[0053] SEQ ID NO:3 is a 60 nucleotide sequence representing the 5' junction region of the sugar beet genomic DNA and the integrated transgenic insert. SEQ ID NO:3 corresponds to nucleotide positions 971-1030 of SEQ ID NO:10.

[0054] SEQ ID NO:4 is a 60 nucleotide sequence representing the 3' junction region of the integrated transgenic insert and the sugar beet genomic DNA. SEQ ID NO:4 corresponds to nucleotide positions 12693-12752 of SEQ ID NO:10.

[0055] SEQ ID NO:5 is a 100 nucleotide sequence representing the 5' junction region of the sugar beet genomic DNA and the integrated transgenic insert. SEQ ID NO:5 corresponds to nucleotide positions 951-1050 of SEQ ID NO:10.

[0056] SEQ ID NO:6 is a 100 nucleotide sequence representing the 3' junction region of the integrated transgenic insert and the sugar beet genomic DNA. SEQ ID NO:6 corresponds to nucleotide positions 12673-12772 of SEQ ID NO:10.

[0057] SEQ ID NO:7 is a 1050 nucleotide sequence representing the 5' genomic flanking region of the sugar beet genomic DNA and 50 nucleotides of the integrated transgenic insert. SEQ ID NO:7 corresponds to nucleotide positions 1-1050 of SEQ ID NO:10.

[0058] SEQ ID NO:8 is a 1050 nucleotide sequence representing 50 nucleotides of the integrated transgenic insert and the 3' genomic flanking region of the sugar beet genomic DNA. SEQ ID NO:8 corresponds to nucleotide positions 12673-13722 of SEQ ID NO:10.

[0059] SEQ ID NO:9 is a sequence of 11722 nucleotides corresponding to the transgenic insert of Sugar beet line Bv_CSM63713. SEQ ID NO:9 corresponds to nucleotide positions 1001-12722 of SEQ ID NO:10.

[0060] SEQ ID NO: 10 is a sequence of 13722 nucleotides corresponding to the contiguous nucleotide sequence of the 5' sugar beet genomic DNA sequence (SEQ ID NO: 11), the transgenic insert in line Bv_CSM63713 (SEQ ID NO: 9) and the 3' sugar beet genomic DNA sequence (SEQ ID NO: 12).

[0061] SEQ ID NO:11 is a 1000 nucleotide sequence representing the 5' flanking sugar beet genomic DNA up to the transgenic insert (SEQ ID NO:9). SEQ ID NO:11 corresponds to nucleotide positions 1-1000 of SEQ ID NO:10.

[0062] SEQ ID NO: 12 is a 1000 nucleotide sequence representing the 3' flanking sugar beet genomic DNA following the transgenic insert (SEQ ID NO: 9). SEQ ID NO: 12 corresponds to nucleotide positions 12723-13722 of SEQ ID NO: 10.

[0063] SEQ ID NO: 13 is a 2007 nucleotide sequence representing the wild-type sugar beet genomic DNA at the location where the transgenic sequence was inserted in line Bv_CSM63713. The transgenic insert (SEQ ID NO: 9) was integrated into the sugar beet genome, resulting in a seven-nucleotide deletion (ACCTCGC) in the genomic sequence of sugar beet line Bv_CSM63713, such as Figure 1 shown.

[0064] SEQ ID NO: 14 is a 47 nucleotide sequence corresponding to a forward primer called txht024d01-X, used to identify the sugar beet line Bv_CSM63713 DNA in a sample. The primer contains a 5' end 21 nucleotide oligonucleotide sequence for competitive allele-specific PCR (KASP). Nucleotides 20-47 of SEQ ID NO: 14 correspond to positions 972-999 of SEQ ID NO: 10 (nucleotides 20-21 (CT) and nucleotides 22-47 of the oligonucleotide tail are also aligned with SEQ ID NO: 10).

[0065] SEQ ID NO: 15 is a sequence having 26 nucleotides, corresponding to the forward primer txht024d01-X described in SEQ ID NO: 14, but without the 21 nucleotide oligonucleotide sequence.

[0066] SEQ ID NO: 16 is a 41 nucleotide sequence corresponding to a reverse primer called sxcp4xxxs1-X, which is used in competitive allele-specific PCR (KASP) to detect the presence of the CP4 EPSPS gene in regenerated sugar beet lines for line selection. Nucleotides 1-21 of SEQ ID NO: 16 are the oligonucleotide tail sequence.

[0067] SEQ ID NO: 17 is a 27 nucleotide sequence corresponding to the forward primer designated sxcp4xxx s1-C, which was used in KASP to detect the presence of the CP4 EPS PS gene in regenerating sugar beet lines for line selection.

[0068] SEQ ID NO 18 is a 26-nucleotide sequence corresponding to a reverse primer designated txht024d01-R, used to identify Sugar beet line Bv_CSM63713 DNA in a sample when combined with SEQ ID NO: 14 or 15; it corresponds to positions 1004-1029 of SEQ ID NO: 10 in reverse orientation.

[0069] SEQ ID NO: 19 is a 48 nucleotide sequence corresponding to a forward primer called txht024d02-X, which was used to identify the sugar beet line Bv_CSM63713 DNA in a sample. The primer contains an oligonucleotide sequence of 21 nucleotides at the 5' end for competitive allele-specific PCR (KASP). Nucleotides 22-48 of SEQ ID NO: 19 correspond to positions 12697-12723 of SEQ ID NO: 10.

[0070] SEQ ID NO:20 is a sequence having 27 nucleotides, corresponding to the forward primer txht024d02-X described in SEQ ID NO:19, but without the 21 nucleotide oligonucleotide sequence.

[0071] SEQ ID NO:21 is a 43 nucleotide sequence corresponding to a reverse primer called sxpatxxx s2-X, which was used in competitive allele-specific PCR (KASP) to detect the presence of the PAT gene in regenerated sugar beet lines for line selection. Nucleotides 1-21 of SEQ ID NO:21 are the oligonucleotide tail sequence.

[0072] SEQ ID NO:22 is a 25 nucleotide sequence corresponding to the forward primer designated sxpatxxx s2-C, which was used in KASP to detect the presence of the PAT gene in regenerated sugar beet lines for line selection.

[0073] SEQ ID NO:23 is a 29 nucleotide sequence corresponding to a reverse primer designated txht024d02-R, which is used to identify Sugar beet line Bv_CSM63713 DNA in a sample when combined with SEQ ID NO:19 or 20; it corresponds to positions 12727-12755 of SEQ ID NO:10 in reverse orientation.

[0074] SEQ ID NO:24 is a 44-nucleotide sequence corresponding to a reverse primer called txht024s01-X, which was used in a zygosity assay for the sugar beet line Bv_CSM63713 DNA in a sample and hybridizes to a region of the sugar beet genome. The primer contains a 5' 21-nucleotide oligonucleotide sequence for competitive allele-specific PCR (KASP). Nucleotides 22-37 of SEQ ID NO:24 correspond to positions 12723–12738 of SEQ ID NO:10 in a reverse orientation. Nucleotides 38-44 of SEQ ID NO:24 correspond to a wild-type sugar beet genomic DNA sequence (ACCTCGC) of seven nucleotides that is missing in the sugar beet line Bv_CSM63713, as shown in FIG. Figure 1 As shown in SEQ ID NO:13 depicted in .

[0075] SEQ ID NO:25 is a 45 nucleotide sequence corresponding to a reverse primer called txht024s01-Y, which was used in a zygosity assay for the sugar beet line Bv_CSM63713 DNA in a sample. The primer contains an oligonucleotide sequence of 21 nucleotides at the 5' end for competitive allele-specific PCR (KASP). Nucleotides 21-45 of SEQ ID NO:25 correspond to positions 1001-1025 of SEQ ID NO:10 in a reverse orientation (nucleotides 21 (T) and nucleotides 22-45 of the oligonucleotide tail are also aligned with SEQ ID NO:10).

[0076] SEQ ID NO:26 is a 30 nucleotide sequence corresponding to the forward primer called txht024s01-R, which was used in the zygosity assay for the sugar beet line Bv_CSM63713 DNA in the sample. It corresponds to positions 957-986 in SEQ ID NO:10.

[0077] SEQ ID NO:27 is a 42 nucleotide sequence corresponding to a forward primer called txht024s02-X, which was used in a zygosity assay for the sugar beet line Bv_CSM63713 DNA in a sample and hybridizes to a region of the sugar beet genome. The primer contains a 5' end 21 nucleotide oligonucleotide tail sequence for competitive allele-specific PCR (KASP). Nucleotides 21-35 of SEQ ID NO:27 correspond to positions 986-1000 of SEQ ID NO:10. (Nucleotide 21, i.e., "T", and nucleotides 22-35 of the oligonucleotide tail also align with SEQ ID NO:10). Nucleotides 36-42 of SEQ ID NO:27 correspond to a wild-type sugar beet genomic DNA sequence (ACCTCGC) of seven nucleotides, which is missing in the sugar beet line Bv_CSM63713, as described in SEQ ID NO:13.

[0078] SEQ ID NO:28 is a 48 nucleotide sequence corresponding to a forward primer called txht024s02-Y, which was used in a zygosity assay for the sugar beet line Bv_CSM63713 DNA in a sample. Nucleotides 22-48 of SEQ ID NO:28 correspond to positions 12696-12722 of SEQ ID NO:10, and nucleotides 1-21 of SEQ ID NO:28 are the oligonucleotide tail sequence.

[0079] SEQ ID NO:29 is a 30 nucleotide sequence corresponding to the reverse primer called txht024s02-R, used in the zygosity assay for the sugar beet line Bv_CSM63713 DNA in the sample. It corresponds to positions 12743-12772 of SEQ ID NO:10 in reverse orientation.

[0080] SEQ ID NO:30 is a 23 nucleotide sequence corresponding to SEQ ID NO:24 but without the 21 nucleotide oligonucleotide sequence. It was used for zygosity determination of the sugar beet line Bv_CSM63713 DNA in the sample and hybridized to a region of the sugar beet genome.

[0081] SEQ ID NO:31 is a 24 nucleotide sequence corresponding to SEQ ID NO:25 but without the 21 nucleotide oligonucleotide sequence. It was used in a zygosity assay for the Sugar Beet line Bv_CSM63713 DNA in the sample.

[0082] SEQ ID NO:32 is a 21 nucleotide sequence corresponding to SEQ ID NO:27 but without the 21 nucleotide oligonucleotide sequence. It was used for zygosity determination of the Sugar Beet line Bv_CSM63713 DNA in the sample and hybridized to a region of the Sugar Beet genome.

[0083] SEQ ID NO:33 is a 27 nucleotide sequence corresponding to SEQ ID NO:28, but without the 21 nucleotide oligonucleotide sequence. It was used in a zygosity assay for the Sugar Beet line Bv_CSM63713 DNA in the sample.

[0084] SEQ ID NO:34 is a 17 nucleotide sequence corresponding to the forward primer designated 2109_fwd1, which was used to identify the sugar beet line Bv_CSM63713 DNA in the sample. It corresponds to positions 12686-12702 in SEQ ID NO:10.

[0085] SEQ ID NO:35 is a 24 nucleotide sequence corresponding to the reverse primer designated 2109_rev1, which was used to identify the Sugar Beet line Bv_CSM63713 DNA in the sample; it corresponds to positions 12739-12762 of SEQ ID NO:10 in reverse orientation.

[0086] SEQ ID NO:36 is a 16-nucleotide sequence that corresponds to a probe designated 2109_pr obel and was used to identify the Sugar Beet line Bv_63713 DNA in a sample; it corresponds to positions 12710-12725 of SEQ ID NO:10.

[0087] SEQ ID NO:37 is a 40 nucleotide sequence corresponding to a forward primer called txdmoxx xxx-X, which was used in competitive allele-specific PCR (KASP) to detect the presence of the DMO gene in regenerated sugar beet lines for line selection. Nucleotides 1-21 of SEQ ID NO:40 are the oligonucleotide tail sequence.

[0088] SEQ ID NO:38 is a 29 nucleotide sequence corresponding to the reverse primer designated txdmoxx xxx-C, which was used in KASP to detect the presence of the DMO gene in regenerated sugar beet lines for line selection.

[0089] SEQ ID NO:39 and 40 are sequences of 39 and 25 nucleotides corresponding to primers called txmonlbxxx-X and txmonlbxxx-C, respectively, used in competitive allele-specific PCR (KASP) to detect the presence of Agrobacterium T-DNA left border (LB) overreads in regenerating sugar beet lines for line selection. Nucleotides 1-21 of each primer are oligonucleotide tail sequences.

[0090] SEQ ID NOs:41 and 42 are sequences of 40 and 28 nucleotides corresponding to primers designated txmonrbxxx-X and txmonrbxxx-C, respectively, used in competitive allele-specific PCR (KASP) to detect the presence of Agrobacterium T-DNA left border (LB) excess reads in regenerating sugar beet lines for line selection. Nucleotides 1–21 of each primer are oligonucleotide tail sequences.

[0091] SEQ ID NOs:43 and 44 are sequences of 42 and 23 nucleotides corresponding to primers designated txvir2dd04-X and txvir2dd04-C, respectively, used in competitive allele-specific PCR (KASP) to detect the presence of Agrobacterium in regenerating sugar beet lines for line selection. Nucleotides 1-21 of each primer are oligonucleotide tail sequences.

[0092] SEQ ID NO:45 is a 24 nucleotide sequence corresponding to the forward primer designated GluA3-F, used to amplify glutamine synthetase, used as an internal control for sugar beet PCR.

[0093] SEQ ID NO:46 is a 23 nucleotide sequence corresponding to the reverse primer designated GluA3-R, used to amplify glutamine synthetase, used as an internal control for sugar beet PCR.

[0094] SEQ ID NO:47 is a 27 nucleotide sequence corresponding to a probe called GluD1, used to amplify glutamine synthetase, used as an internal control for sugar beet PCR.

[0095] SEQ ID NOs:48-77 are the nucleotide sequences of the genetic elements in the transgenic insert of Sugar Beet line Bv_CSM63713 and are further described in Table 15 below. DETAILED DESCRIPTION

[0096] The following definitions, descriptions and methods are provided to better define the present invention and guide those skilled in the art in the practice of the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the relevant art.

[0097] Herbicide tolerance is an important agronomic trait for effective weed control to maintain good crop growing conditions and crop yields, and is achieved through transgenic engineering of herbicide tolerance in crops using modern plant biotechnology. Sugar beet line Bv_CSM61723 provides tolerance to three different herbicide chemistries acting through different modes of action, thereby achieving weed control and herbicide-resistant weed management.

[0098] Plant transformation techniques are used to randomly insert foreign DNA (also known as transgenic DNA) into the chromosomes of the cell genome to produce genetically engineered cells, also known as "transgenic" or "recombinant" cells. Using this technique, many individual cells are transformed, each of which produces a unique transgenic strain due to the random insertion of foreign DNA into the genome. Transgenic plants are then regenerated from each individual transgenic cell. This allows each cell of the transgenic plant to contain a uniquely inserted transgenic strain as a stable part of its genome. This transgenic plant can then be used to produce progeny plants, each of which contains a unique transgenic strain.

[0099] Provided is a sugar beet strain Bv_CSM63713.Strain Bv_CSM63713 is produced by Agrobacterium-mediated transformation of sugar beet bud meristems, which is achieved by (i) transforming tens of thousands of sugar beet cells with four different DNA constructs, each of which includes three expression cassettes (each selected after testing alone and then in combination with the other two expression cassettes); (ii) regenerating a population of transgenic plants, each of which contains a unique transgenic strain; and (iii) rigorous and meticulous multi-year strain selection, involving tens of thousands of plant tests and analysis of hundreds of strains in a variety of genetic backgrounds for molecular characteristics, herbicide tolerance efficacy and agronomic characteristics. Sugar beet strain Bv_CSM63713 is thus produced and selected as a unique superior strain that can be used for large-scale agronomic commercial purposes.

[0100] As used herein, "expression cassette" or "box" or "transgenic cassette" is a recombinant DNA molecule or sequence comprising a combination of different elements, for expressing RNA and / or protein encoded by the coding sequence of the transgenic in a transformed plant cell or transformed plant comprising a transgenic. As provided herein, "expression cassette" or "box" or "transgenic cassette" includes one or more regulatory elements operably connected to a coding or transcribable DNA sequence. Regulatory elements may include promoters, leader sequences, 5' untranslated regions (5'UTR), introns and / or 3' untranslated regions (3'UTR) regions. "Expression cassette" or "box" or "transgenic cassette" is recombinant and heterologous relative to the transformed plant cell genome. For the purposes of this disclosure, such "expression cassette" or "box" or "transgenic cassette" is a recombinant DNA molecule or sequence that encodes a protein that confers tolerance to at least one class of herbicides described herein. Table 15 provides a list of genetic elements contained in three transgenic cassettes in the transgenic insert (SEQ ID NO:9) of sugar beet strain Bv_CSM63713.

[0101] The operation of inserting transgenic DNA into the genome of the sugar beet plant is accomplished by plant transformation methods known in the art, and produces new transgenic genomic DNA sequences, referred to as "transgenic lines" or "lines". The DNA sequence of the line includes the inserted foreign DNA (referred to as "transgenic inserts") and genomic DNA adjacent to or "flanking" the transgenic inserts on both sides of the insertion position. As used herein, the term "flanking" with respect to transgenic lines refers to one or more plant genomic sequences adjacent to the transgenic DNA insert in the genome of the transformed plant, plant part, plant tissue or plant cell comprising the transgenic line at the 5' end and / or 3' end of the transgenic line insert. Similarly, "flanking DNA" refers to a genomic DNA sequence adjacent to the transgenic DNA insert in the genome of the transformed line at the 5' end and / or 3' end of the insert. Therefore, "5' flanking sequence" means a sugar beet genomic DNA sequence adjacent to the transgenic DNA insert and located upstream (or at the 5' end) of the transgenic DNA insert. For example, a "5' flanking sequence" may include a sugar beet genomic DNA sequence that is adjacent to a transgenic insertion and upstream thereof (at the 5' end), or any sugar beet genomic DNA sequence that is upstream of a transgenic insertion (at the 5' end), which sequence is not adjacent to the transgenic insertion, but is within about 5000 nucleotides, within about 3000 nucleotides, or within about 1000 nucleotides upstream of the transgenic insertion. Similarly, a "3' flanking sequence" means a sugar beet genomic DNA sequence that is adjacent to a transgenic insertion and is located downstream thereof (or at the 3' end). For example, a "3' flanking sequence" may include a sugar beet genomic DNA sequence that is adjacent to a transgenic insertion and is located downstream thereof (at the 3' end), or any sugar beet genomic DNA sequence that is downstream of a transgenic insertion (at the 3' end), which sequence is not adjacent to a transgenic insertion, but is within about 5000 nucleotides, within about 3000 nucleotides, or within about 1000 nucleotides downstream of the transgenic insertion. The DNA sequence of a line is unique and specific to said line and can be easily identified when compared to other DNA sequences, such as sequences of other lines or untransformed sugar beet genomic DNA. Sugar beet line Bv_CSM63713 has a novel and unique DNA sequence provided as SEQ ID NO: 10, which comprises a continuous 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, sugar beet line Bv_CSM63713 is a DNA molecule that is an integral part of the chromosomes of transgenic sugar beet cells and plants comprising said line and is therefore static and can be passed on to daughter cells and plants.

[0102] Also provided is a progeny of the original transformed cells and plants comprising the beet strain Bv_CSM63713. This progeny can be produced by cell tissue culture, by selfing of a beet plant comprising the beet strain Bv_CSM63713, or by sexual outcrossing between a beet plant comprising the beet strain Bv_CSM63713 and another plant containing or not containing the strain, or by any other method known in the art (including any plant cell or tissue culture method), wherein the progeny comprises the beet strain Bv_CSM63713. Such other plants can be transgenic plants or non-transgenic plants comprising the same or different strains, such as from different varieties. The beet strain Bv_CSM63713 is passed from the original parent to the progeny by each passage. Thus, a "transgenic plant" or "plant" can be the original transformant plant that is regenerated from the transformed plant cell and contains the transgenic DNA and strain, or a progeny plant of the original transformant plant that can be separated from the transformant by one or more passages, retaining the transgenic DNA and strain in the same specific location and sequence context in the plant genome.

[0103] As used herein, the term "beet" refers to Beta vulgaris, including all subspecies of Beta vulgaris, and in addition, all plant species that can be bred with Beta vulgaris. It includes Beta vulgaris subsp. adanensis, Beta vulgaris subsp. maritima and Beta vulgaris subsp. vulgaris, Beta macrocarpa and Beta patula. Within red beet, it includes: 1) the Altissima group, such as sugar beet; 2) the Cicla group, such as spinach beet or chard; 3) the Flavescens group, such as swiss chard; 4) the Conditiva group, such as beetroot or garden beet; and 5) the Crassa or fodder beet group, such as mangelwurzel.

[0104] The present disclosure describes the introduction of line Bv_CSM63713 into sugar beet, and therefore the term "sugar beet line Bv_CSM63713" is used herein to refer to the line. However, it will be appreciated by those skilled in the art that line Bv_CSM63713 can be easily introduced into other sugar beets and plant species that can be bred with sugar beets by sexually crossing or outcrossing a sugar beet plant containing sugar beet line Bv_C SM63713 with another plant that does not contain sugar beet line Bv_CSM63713, or by any other method known in the art. For example, in order to introduce line Bv_CSM63713 into fodder beet, a plant containing or comprising line Bv_CSM63713 can be cross-pollinated with a fodder beet plant, which can be achieved or promoted by human intervention, for example, by manually collecting pollen from a plant containing line Bv_CSM63713 and placing the pollen on the style or stigma of a fodder beet plant that does not contain line Bv_CSM63713, wherein line Bv_CSM63713 is transmitted from a male parent containing Bv_CSM63713 to a fodder beet progeny; or by manually and / or manually removing, damaging or covering the stamens or anthers of a fodder beet plant (for example, by human intervention or application of a chemical maleicide), Thus preventing natural self-pollination and cross-pollinating with pollen from plants containing line Bv_CSM63713 for fertilization; or by artificially placing pollinators in a position for directional pollination (e.g. by placing beehives in orchards or fields or by placing plants in cages with pollinators); or by artificially opening or removing parts of the fodder beet flowers to allow foreign pollen from plants containing or including line Bv_CSM63713 to be placed on or in contact with the style or stigma of the fodder beet plant; by selectively placing plants (e.g. intentionally planting plants near a pollination environment); and / or by applying chemicals to accelerate flowering or promote receptivity (stigma receptivity to pollen). In addition to directional cross-pollination, beets containing beet line Bv_CSM63713 and fodder beets can also be allowed to flower at synchronized times and in close proximity to achieve random cross-pollination, followed by selection of offspring containing beet line Bv_CSM63713.

[0105] Sugar beet line Bv_CSM63713 provides sugar beet cells, plants and seeds containing the line with tolerance to: benzoic acid auxin herbicides, such as dicamba; 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, such as glyphosate; and glutamine synthetase inhibitors, such as glufosinate. Sugar beet line Bv_CSM63713 contains three expression cassettes. Table 15 in Example 7 provides a list of elements contained in SEQ ID NO: 10, such as Figure 1 as shown in .

[0106] As used herein, the term "source" or "derived from" with respect to a particular DNA molecule, amplicon or sequence of a sugar beet plant, plant part, seed, progeny, cell and / or sugar beet plant product (e.g., commercial product) means that the DNA molecule, amplicon or sequence is obtained, purified, isolated or made directly or indirectly from such sugar beet plant, plant part, seed, progeny, cell and / or sugar beet plant product (e.g., commercial product). Alternatively, the term "source" or "derived from" with respect to a sugar beet plant product (e.g., commercial product) of a sugar beet plant, plant part, seed, progeny or cell means that the sugar beet plant product is obtained, purified, isolated or made directly or indirectly from such sugar beet plant, plant part, seed, progeny or cell.

[0107] "Capable of detecting" refers to the ability to detect a specific DNA molecule, segment or sequence in a sample, such as by amplification, and determining its presence, size or sequence, such as by DNA sequence analysis, and / or the binding of the probe to the target DNA molecule, segment or sequence.

[0108] "Sample" is intended to mean any composition comprising or directly or indirectly derived from a biological sample, source or material. A sample may generally comprise sugar beet DNA and / or substantially or completely pure, purified or isolated sugar beet DNA. A "biological sample" contains biological material, including but not limited to DNA obtained or obtained directly or indirectly from the genome of one or more sugar beet cells, tissues, seeds, plants, plant parts and / or sugar beet plant products (e.g., one or more commercial products). The one or more sugar beet cells, tissues, seeds, plants, plant parts and / or sugar beet plant products (e.g., one or more commercial products) may comprise sugar beet strain Bv_CSM63713, or one or more DNA molecules and / or DNA segments comprising sugar beet strain Bv_CSM63713. In some embodiments, the sample or biological sample may comprise one or more beet cells, beet tissues, beet seeds, beet plants, beet plant parts and / or beet plant products, the cells or cell membranes of which have been ruptured (e.g., broken or opened) to release the contents of the one or more beet cells, including genomic DNA or proteins, and / or to make the contents of the one or more beet cells, including genomic DNA or proteins, accessible or available for determination or testing. "Directly" means that the technician obtains DNA directly from the beet genome by rupturing the beet cells (or by obtaining a beet sample containing ruptured beet cells) and exposing the genomic DNA or proteins in the beet cells, or using the genomic DNA or proteins for detection purposes. "Indirectly" means that the technician obtains the target DNA or specific reference DNA in a specific sample (e.g., the novel and unique junction segment described herein as being diagnostic for the presence of strain Bv_CSM63713) by a certain means, and the means is not directly obtained by rupturing the beet cells or obtaining a beet sample containing ruptured beet cells. Such indirect means include, but are not limited to, amplifying a DNA segment containing a DNA sequence that is the target of one or more specific probes and / or primer sets that are designed to specifically bind to the target sequence or the vicinity of the target sequence; or amplifying a DNA segment that includes all or part of the target sequence, which target sequence can be measured or characterized (e.g., by migration or separation from other DNA fragments and / or identification in an effective matrix such as agarose or acrylamide gel, or by direct sequence analysis of one or more amplicons, or by cloning one or more amplicons into one or more vectors and directly sequencing the one or more amplicons inserted into the one or more vectors).

[0109] As used herein, the term "recombination" refers to non-natural DNA, protein or organism, which does not usually exist in nature and is produced by human intervention. As used herein, "recombinant DNA molecule" is a DNA molecule comprising a combination of DNA molecules that do not appear simultaneously in nature and are produced by human intervention, for example, a DNA molecule composed of a combination of at least two mutually heterogeneous DNA molecules, such as a DNA molecule comprising a transgenic and a plant genomic DNA adjacent to the transgenic. An example of a recombinant DNA molecule is a DNA molecule comprising at least one sequence selected from SEQ ID NO.1-10. As used herein, "recombinant plant" is a plant that does not usually exist in nature, is the result of human intervention, and contains transgenic DNA molecules. Due to this genome change, recombinant plants are new things and are significantly different from related wild-type plants. An example of a recombinant plant is a beet plant containing beet strain Bv_CSM63713.

[0110] As used herein, the term "transgenic" refers to a DNA molecule that is artificially incorporated into the genome of an organism due to human intervention, such as by a plant transformation method. The transgene can be heterologous to the organism. As used herein, the term "transgenic insert" refers to foreign DNA that is inserted into the sugar beet genome by plant transformation technology to produce sugar beet strain Bv_CSM63713. The sequence of the transgenic insert of sugar beet strain Bv_CSM63713 is provided as SEQ ID NO:9. The term "transgenic" refers to a plant that contains a transgene, for example, a "transgenic plant" refers to a plant that contains a transgene.

[0111] As used herein, the term "heterologous" refers to a first molecule that is not generally associated with a second molecule or organism in nature. For example, a DNA molecule can be from a first species and inserted into the genome of a second species. Thus, the DNA molecule is heterologous to both the genome and the organism.

[0112] As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first DNA molecule nor the second DNA molecule fuses to each other in a normally seen configuration. Thus, a chimeric DNA molecule is a new DNA molecule not typically found in nature. An example of a chimeric DNA molecule is a DNA molecule comprising at least one sequence selected from SEQ ID NO: 1-10.

[0113] As used herein, the term "isolated" refers to separating a molecule from other molecules that are usually associated with it in the original or natural state. Therefore, the term "isolated" can refer to that a DNA molecule has been separated from other DNA molecules associated with it in its original or natural state. This DNA molecule can exist in a recombinant state, such as a recombinant DNA molecule. Therefore, a DNA molecule that is taken out from its natural state and fused to another DNA molecule that is not normally associated with it is an isolated DNA molecule. This isolated DNA molecule may be the result of using biotechnology, such as preparing recombinant DNA or integrating foreign DNA molecules into the chromosome of a cell, plant or seed.

[0114] DNA molecules, fragments and their corresponding DNA sequences are provided. As used herein, the terms "DNA" and "DNA molecules" refer to deoxyribonucleic acid (DNA) molecules. DNA molecules can be of genomic or synthetic origin, and are conventionally from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the polynucleotide sequence of a DNA molecule, that is, the sequence of adjacent nucleotides in a DNA molecule. As used herein with respect to the nucleotides of a polynucleotide or DNA sequence or molecule, the terms "adjacent" and "continuous" are interchangeable and synonymous, and refer to the 5' to 3' order of nucleotides in a polynucleotide or DNA sequence, chain or molecule, and there is no space or interruption between them. The nomenclature used is the nomenclature required by Section 37 of Article 1.822 of the U.S. Code of Federal Regulations, and is set forth in the table in Appendix 2, Tables 1 and 3 of WIPO Standard ST.25 (1998). Conventionally, DNA sequences and fragments thereof are disclosed with reference to only one of the two complementary DNA sequence chains. By implication and intent, the complementary sequences of the sequences provided herein (sequences of complementary strands), also referred to in the art as reverse complementary sequences, are within the scope of the present invention and are expressly intended to be within the scope of the claimed subject matter. Thus, as used herein, reference to SEQ ID NOs: 1-10 and fragments thereof include and refer to sequences of complementary strands and fragments thereof. Figure 1 The transgenic DNA inserted into the genome of a sugar beet plant comprising line Bv_CSM63713 is shown, along with the relative positions of SEQ ID NOs: 1-10 arranged from 5' to 3'.

[0115] Also provided is a nucleic acid 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 NOs: 1-12.

[0116] For example, a nucleic acid molecule is provided, which comprises 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 to the full length of SEQ ID NO: 9.

[0117] As used herein, the term "fragment" refers to a smaller piece or sequence of a larger or complete DNA molecule or sequence. For example, a fragment of any of SEQ ID NOs: 1-12 may include at least about 10 contiguous nucleotides, at least about 11 contiguous nucleotides, at least about 12 contiguous nucleotides, at least about 13 contiguous nucleotides, at least about 14 contiguous nucleotides, at least about 15 contiguous nucleotides, at least about 16 contiguous nucleotides, at least about 17 contiguous nucleotides, at least about 18 contiguous nucleotides, at least about 19 contiguous nucleotides, at least about 20 contiguous nucleotides, at least about 21 contiguous nucleotides, at least about 22 contiguous nucleotides, at least about 23 contiguous nucleotides, or at least about 24 contiguous nucleotides of a larger, complete or complete DNA molecule or sequence. , a sequence of at least about 24 contiguous nucleotides, at least about 25 contiguous nucleotides, at least about 30 contiguous nucleotides, at least about 35 contiguous nucleotides, at least about 40 contiguous nucleotides, at least about 45 contiguous nucleotides, at least about 50 contiguous nucleotides, at least about 60 contiguous nucleotides, at least about 70 contiguous nucleotides, at least about 80 contiguous nucleotides, at least about 90 contiguous nucleotides, at least about 100 contiguous nucleotides, at least about 200 contiguous nucleotides, at least about 300 contiguous nucleotides, at least about 400 contiguous nucleotides, or at least about 500 contiguous nucleotides.

[0118] For example, a "fragment" of the transgenic insert sequence of the sugar beet line Bv_CSM63713 (SEQ ID NO:9) can comprise 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 contiguous nucleotides of SEQ ID NO:9. In addition, 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.

[0119] Similarly, a fragment of the 5' flanking sequence (SEQ ID NO: 11) or the 3' flanking sequence (SEQ ID NO: 12) of the sugar beet line Bv_CSM63713 may comprise SEQ ID NO: 11 or SEQ ID 12, 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 contiguous nucleotides of NO:12. In addition, the present disclosure also 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 12, or a fragment of either one thereof.

[0120] The DNA sequence of the transgenic insert of Sugar Beet line Bv_CSM63713 is provided as SEQ ID NO: 9. The DNA sequence of the transgenic insert and the Sugar Beet genomic DNA flanking each side of the transgenic insert is provided as SEQ ID NO: 10. The DNA sequence of a portion of the flanking DNA and the 5' end of the transgenic insert is provided as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. The DNA sequence of a portion of the flanking DNA and the 3' end of the transgenic insert is provided as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

[0121] The beet strain Bv_CSM63713 is characterized in that transgenic is inserted into a single locus in the beet genome, thereby producing two new junctions (or connection points or junctions). The DNA sequence across the region of the phosphodiester bond connecting one end of the transgenic insert and the side-joined beet genomic DNA is referred to as "junction" in this article. The junction is a junction or covalent linkage that connects one end of the transgenic insert to the side-joined genomic DNA as a continuous molecule, and is formed by inserting a heterologous nucleic acid molecule into the beet genomic DNA. One junction is found in the 5' end of the transgenic insert, and the other is found in the 3' end of the transgenic insert, which is referred to as 5' junction sequence and 3' junction sequence, respectively, herein." junction sequence" refers to the DNA sequence of adjacent nucleotides of any length of the 5' or 3' junction sequence of the strain across the plant genome. For the unique "junction sequence" of the junction between the transgenic strain and the side-joined genomic sequence, the junction sequence will generally be included in a sufficient number of adjacent nucleotides at one end of the insertion and a sufficient number of adjacent nucleotides of the side-joined genomic sequence. According to some embodiments, the "junction sequence" may include: (i) at least five (5) contiguous nucleotides, at least ten (10) contiguous nucleotides, at least fifteen (15) contiguous nucleotides, at least twenty (20) contiguous nucleotides, at least twenty-five (25) contiguous nucleotides, at least thirty (30) contiguous nucleotides, at least thirty-five (35) contiguous nucleotides, at least forty (40) contiguous nucleotides, at least forty-five (45) contiguous nucleotides, or at least fifty (50) contiguous nucleotides at one end of the insert; and (ii) flanking genomic DNA. At least five (5) contiguous nucleotides, at least ten (10) contiguous nucleotides, at least fifteen (15) contiguous nucleotides, at least twenty (20) contiguous nucleotides, at least twenty-five (25) contiguous nucleotides, at least thirty (30) contiguous nucleotides, at least thirty-five (35) contiguous nucleotides, at least forty (40) contiguous nucleotides, at least forty-five (45) contiguous nucleotides, or at least fifty (50) contiguous nucleotides of the A sequence, but it should be understood that any length of contiguous nucleotides spanning the junction of the transgenic line in the plant genome can be a junction sequence. The junction sequence of the sugar beet line Bv_CSM63713 is obvious to those skilled in the art, and those skilled in the art can determine various junction sequences of the sugar beet line Bv_CSM63713 using SEQ ID NO: 10. In SEQ ID NO: 10, the 5' junction is located at nucleotides 1000-1001, and the 3' junction is located at nucleotides 12,722-12,723. Examples of junction sequences of Sugar beet line Bv_CSM63713 are provided as SEQ ID NOs: 1-8. Figure 1The physical arrangement of SEQ ID NOs: 1-10 arranged from 5' to 3' is shown. The junction sequence of the sugar beet strain Bv_CSM63713 may be present as part of the genome of a plant, seed, plant part, progeny or plant cell containing the sugar beet strain Bv_CSM63713. The identification of any one or more of SEQ ID NOs: 1-10 in a sample from a plant, plant part, seed, progeny or cell indicates that the DNA was obtained from sugar beet containing the sugar beet strain Bv_CSM63713 and is used to diagnose the presence of the sugar beet strain Bv_CSM63713.

[0122] The junction sequences described herein are diagnostic for the presence of all or a portion of the sugar beet strain 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 commodity product can be diagnostic that the sugar beet plant, plant part, seed, progeny, cell, or commodity product has or contains all or a portion of the sugar beet strain Bv_CSM63713. Direct or indirect identification or detection of a 5' junction sequence and / or a 3' junction sequence (each as provided or described herein) in a sample or DNA molecule derived from a sugar beet plant, plant part, seed, progeny, cell, or commodity product can be diagnostic that the sugar beet plant, plant part, seed, progeny, cell, or commodity product has or contains the sugar beet strain Bv_CSM63713. Thus, the present disclosure provides a DNA molecule comprising at least one of the nucleotide sequences provided in 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 DNA segment derived from the transgenic sugar beet line Bv_CSM63713 is within the scope of the present disclosure, and the DNA segment is sufficient to include at least one of the sequences provided in 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. In addition, any DNA or polynucleotide molecule or sequence comprising a sequence complementary to any sequence described herein is also within the scope of the present disclosure.

[0123] The plant, seed, plant cell, plant part and commercial product can be used for detecting the DNA or protein molecule of the existence of sugar beet strain Bv_CSM63713. An exemplary DNA molecule is provided, which can be used as a primer or probe to detect the existence of sugar beet strain Bv_CSM63713 in a sample. Such primers or probes are specific to the target nucleic acid sequence, and therefore can be used to identify sugar beet strain Bv_CSM63713 by the method described herein. Primers or probes can hybridize with target polynucleotide sequences, thereby allowing specific detection or amplification of polynucleotide molecules comprising target polynucleotide sequences or covalently connected and associated with target polynucleotide sequences. The target polynucleotide sequence can include all or part of, a junction sequence and / or a flanking genomic DNA of sugar beet strain Bv_CSM63713. Probes and primers according to the present disclosure may (i) have complete or 100% sequence complementarity (i.e., 100% complementarity) with a target polynucleotide sequence or (ii) have incomplete sequence complementarity with a target polynucleotide sequence, 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 with a target polynucleotide sequence, as long as the probe or primer has sufficient complementarity with the target polynucleotide sequence to hybridize with the target polynucleotide sequence under stringent hybridization conditions, which are suitable and necessary for use of the probe or primer in a related amplification or detection assay, reaction, or method. It is understood in the art that the complementarity percentage of a primer or probe may be lower if the length of the primer or probe is longer and depends on stringency and use. Exemplary polynucleotide molecules are provided that can be used as primers or probes to detect the presence of sugar beet strain Bv_CSM63713 in a sample. Detection of the presence of Sugar Beet line Bv_CSM63713 can be performed using methods known in the art, such as thermal or isothermal amplification of nucleic acids, or nucleic acid hybridization techniques (such as Southern analysis).

[0124] "Primers" are DNA molecules designed to participate in annealing or hybridization methods for amplification reactions. An amplification reaction is an in vitro reaction that amplifies a template DNA to produce an amplicon. As used herein, an "amplification product" or "amplified DNA" or "amplicon" refers to a DNA molecule synthesized using an amplification technique described elsewhere herein that is directed to a target nucleic acid or DNA molecule that is part of a template nucleic acid molecule. Amplification or amplifying refers to the preparation of multiple copies of a target DNA molecule or segment from a template DNA. For example, to determine whether a beet plant, plant part, seed, progeny or plant cell produced by selfing or outcrossing a parent comprising beet line Bv_CSM63713 contains beet line Bv_CSM63713, DNA can be extracted from a beet plant tissue sample and an amplification reaction or method can be performed using a primer pair, the primer being specific for a target sequence that is uniquely associated with or is part of the beet line Bv_CSM63713, for example, a first primer derived from a genomic DNA sequence in a region of a flanking inserted heterologous DNA of beet line Bv_CSM63713, the first primer being extended by a polymerase from 5' to 3' in the direction of the inserted DNA; and a second primer derived from the inserted heterologous DNA molecule, the second primer being extended by a polymerase from 5' to 3' in the direction of the flanking genomic DNA from which the first primer originated. The length range of the amplicon can depend on the length of the inserted polynucleotide or DNA sequence between the two primer target sequences in the template DNA molecule. Alternatively, the primer pairs can be derived from genomic sequences on both sides of the inserted heterologous DNA, thereby generating an amplicon that includes the entire inserted polynucleotide sequence (e.g., a forward primer targeting the genomic portion at the 5' end of SEQ ID NO: 10 (i.e., upstream of SEQ ID NO: 9) and a reverse primer targeting 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 comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the genome of the sugar beet strain Bv_CSM63713). The use of the term "amplicon" explicitly excludes primer dimers that can be formed in a DNA amplification reaction.

[0125] The amplicon described herein may comprise a DNA sequence comprising 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, SEQ ID NO: 9, SEQ ID NO: 10, or 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, or SEQ ID NO: 10, wherein the fragment is at least 10 nucleotides long and comprises nucleotides 1,000-1,001 or 12,722-12,723 of SEQ ID NO: 10. According to an embodiment of the present invention, the sequence of the amplicon comprises at least one junction sequence or two junction sequences, for example, the 5' junction sequence and / or the 3' junction sequence of the sugar beet line Bv_CSM63713. Amplification and detection of this amplicon would be indicative or diagnostic of the sugar beet line Bv_CSM63713.

[0126] Primers are usually designed to hybridize with complementary target DNA strands to form a hybrid between the primer and the target DNA strand. The presence of a primer is the recognition point for the start of primer extension by a polymerase (i.e., polymerizing additional nucleotides into an elongated nucleotide molecule) using the target DNA strand as a template. A primer pair refers to a polynucleotide segment between the positions targeted for binding by each member of the primer pair, usually in a thermal amplification reaction or other conventional DNA amplification methods, using two primers that bind to the opposite strands of a double-stranded nucleotide segment.

[0127] In order to detect the presence or absence of sugar beet strain Bv_CSM63713, the target position and / or insertion region or sequence of the template DNA molecule can include at least one junction sequence and / or at least a portion of the insert of sugar beet strain Bv_CSM63713. In order to detect the absence of sugar beet strain Bv_CSM63713, the target position and / or insertion region or sequence of the template DNA molecule can include the sugar beet genomic DNA of any part of the junction sequence or insert of the sugar beet strain Bv_CSM63713. Therefore, the presence or absence of the amplicon using the primer pair can be respectively diagnosed for the presence or absence of sugar beet strain Bv_CSM63713 in the DNA molecule or sample, or vice versa. It is also possible to use more than one primer pair. For example, if sugar beet strain Bv_CSM63713 exists, the first primer pair can produce the first amplicon, and if sugar beet strain Bv_CSM63713 lacks or does not exist, the second primer pair can produce the second amplicon. Alternatively, the size of the amplicon produced in the amplification reaction can also diagnose the presence or absence of sugar beet strain Bv_CSM63713 in the DNA molecule or sample, for example, if sugar beet strain Bv_CSM63713 is present, the primer pair can produce a first amplicon of a first size, or if sugar beet strain Bv_CSM63713 is lacking and not present, the primer pair can produce a second amplicon of a second size; or if sugar beet strain Bv_CSM63713 is present, the first primer pair can produce a first amplicon of a first size, and if sugar beet strain Bv_CSM63713 is lacking or not present, the second primer pair can produce a second amplicon of a second size. According to some embodiments in these embodiments, at least two primer pairs can be used, wherein at least one of the primer pairs is used as an internal control and does not associate with sugar beet strain Bv_CSM63713.

[0128] According to an embodiment of the present invention, a primer pair for detecting the presence or absence of all or part of the sugar beet strain Bv_CSM63713 in a DNA molecule or sample comprises a first primer and a second primer, wherein the first primer is complementary to the 5' flanking genomic DNA sequence and the second primer is complementary to the sequence within the transgenic insert; or wherein the first primer is complementary to the 5' flanking genomic DNA sequence and the second primer is complementary to the 3' flanking genomic DNA sequence; or wherein the first primer is complementary to the sequence within the transgenic insert and the second primer is complementary to the 3' flanking genomic DNA sequence. Each reference in this paragraph to a primer complementary to a 5' flanking genomic DNA sequence, a 3' flanking genomic DNA sequence, or a sequence within the transgenic insert of the sugar beet strain Bv_CSM63713 is also intended to potentially include a primer complementary to the reverse complementary sequence or the relative strand of the corresponding 5' flanking genomic DNA sequence, the 3' flanking genomic DNA sequence, or a sequence within the transgenic insert of the sugar beet strain Bv_CSM63713.

[0129] Exemplary DNA molecules that can be used 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 strain-specific primers for PCR to identify strain 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 to analyze the 5' (left) junction region are provided as SEQ ID NO: 14 and SEQ ID NO: 18; and exemplary primers that can be used to analyze 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 zygosity testing of strain 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.

[0130] 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 can be used as a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is a fragment of the transgenic insert DNA sequence of SEQ ID NO: 10, and the second DNA molecule is a fragment of the flanking DNA sequence of SEQ ID NO: 10, and when used in an amplification reaction with DNA containing the sugar beet line Bv_CSM63713, each DNA molecule has a sufficient length to be used as a DNA primer to produce an amplicon of the sugar beet line Bv_CSM63713 in a diagnostic sample. The amplicon of the diagnostic line Bv_CSM63713 contains sequences that do not naturally occur in the sugar beet genome. A primer pair can also be defined as comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is a fragment of the sugar beet genome portion of SEQ ID NO: 10 and the second DNA molecule is a fragment of the transgenic portion of SEQ ID NO: 10 (or SEQ ID NO: 9), and each DNA molecule has a sufficient length to be used as a DNA primer when used in an amplification reaction with DNA containing sugar beet line Bv_CSM63713 to produce an amplicon of sugar beet line Bv_CSM63713 in a diagnostic sample. The primer can also contain an oligonucleotide tail sequence, such as in a competitive allele-specific PCR (KASP TM ) sequences used in the method. Each allele-specific primer carries a unique tail sequence that corresponds to a universal fluorescence resonance energy transfer (FRET) box; one uses FAM TM dye labeled, and the other with HEX TMDye labeling. During the thermal cycle, the relevant allele-specific primer binds to the template and extends, thereby connecting the tail sequence to the newly synthesized chain. Then, in the subsequent rounds of PCR, the complementary sequence of the allele-specific tail sequence is generated, allowing the FRET box to bind to the DNA. The FRET box no longer quenches and emits fluorescence. Examples of primers containing oligonucleotide tail sequences 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 oligonucleotide tail sequences are SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.

[0131] A "probe" is a nucleic acid molecule that is complementary to a target nucleic acid strand and can be used in a hybridization detection method. Probes include not only deoxyribonucleic acids or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence, and detection of this binding can be used to detect the presence or absence of a target DNA sequence. The probe can be connected to a conventional detectable label or reporter molecule, such as a radioisotope, a ligand, a chemiluminescent agent, or an enzyme.

[0132] An exemplary DNA sequence that can be used as a probe to detect sugar beet line Bv_CSM63713 is provided as SEQ ID NO: 36. In some embodiments, the DNA molecule used 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 for use as a DNA probe, which is specific for at least one of the following: a) a 5' junction sequence between the transgenic insert of the sugar beet line Bv_CSM63713 and the transgenic insert of the sugar beet line Bv_CSM63713; b) a 3' junction sequence between the transgenic insert of the sugar beet line Bv_CSM63713 and the transgenic insert of the sugar beet line Bv_CSM63713; c) SEQ ID NO:9; or d) a fragment of SEQ ID NO:9, which comprises a sufficient length of contiguous nucleotides of SEQ IDNO:9 to identify the sequence as a fragment of the transgenic insert of the sugar beet line Bv_CSM63713 in the DNA sample.

[0133] Methods for designing and using primers and probes are well known in the art. DNA molecules comprising fragments of SEQ ID NO:1-10 can be used as primers and probes for detecting beet strain Bv_CSM63713, and can be easily designed by those skilled in the art using the sequences provided herein. The length of DNA probes and DNA primers is generally ten (10) nucleotides or longer, usually fifteen (15) nucleotides or longer, twenty (20) nucleotides or longer, or thirty (30) nucleotides or longer. Such probes and primers selected should have enough length to specifically hybridize with the target sequence under stringent hybridization conditions.

[0134] Probe and primer can have complete sequence identity with target sequence, but can utilize conventional method design and target sequence different and retain the primer and probe of the ability of preferential hybridization with target sequence.In order to make nucleic acid molecule serve as primer or probe, only need nucleic acid molecule fully complementary and / or have enough length so that can form stable double-stranded structure under specific hybridization condition or reaction condition in sequence.Any conventional nucleic acid hybridization or amplification method can be used for the existence of transgenic DNA from beet strain Bv_CSM63713 in identification sample.Therefore, the polynucleotide molecule called " polynucleotide segment with enough length " or " continuous nucleotide of enough length " can hybridize with target DNA sequence specificity under certain hybridization condition or reaction condition.As used herein, term " having enough length " refers to any length that is enough for selected detection method. The length of probes and primers is 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. Such probes and primers specifically hybridize with target DNA sequences under stringent hybridization conditions. Conventional stringency conditions are described by MR Green and J Sambrook in Molecular cloning: a laboratory manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0135] As used herein, if two molecules can form an antiparallel double-stranded nucleic acid structure, the two molecules can specifically hybridize to each other. If two nucleic acid molecules show complete complementarity, it is the "complementarity" of another nucleic acid molecule. As used herein, if each nucleotide of the first molecule is complementary to each nucleotide of the second molecule when compared, the two molecules show "complete complementarity". If two molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under at least conventional "low stringency" conditions, they are "minimally complementary". Similarly, if molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under conventional "high stringency" conditions, they are "complementary". Therefore, deviations from complete complementarity are allowed as long as these deviations do not completely exclude the ability of molecules to form double-stranded structures.

[0136] As used herein, "stringent hybridization conditions" refers to conditions under which a polynucleotide is hybridized to its target sequence (usually in the form of a complex nucleic acid mixture), but substantially not to other sequences. When referring to polynucleotide probes, "stringent conditions" or "stringent hybridization conditions" refers to conditions under which the degree of hybridization of the probe to its target sequence is detectably higher than the degree of hybridization to other sequences (e.g., at least 2 times the background). Stringent conditions are sequence-dependent and will be different in different environments. Longer sequences will hybridize specifically at higher temperatures. In general, the stringent conditions selected are those that are higher than the thermal melting temperature (T) of a specific sequence at a defined ionic strength pH. m ) is about 5-10℃ lower. m At equilibrium, 50% of the probes complementary to the target are hybridized to the target sequence (when the target sequence is present in excess, at T m Stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides), and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequence, thereby detecting lower degrees of identity (heterologous probing).

[0137] As used herein, substantially complementary or identical sequences are polynucleotides that specifically hybridize to a compared nucleic acid molecule or its complementary sequence under high stringency conditions. Suitable stringency conditions for promoting DNA hybridization are known to those skilled in the art, such as 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by washing with 2×SSC at 50° C., or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0138] The polynucleotide molecules of the present disclosure, such as primers or probes, will specifically hybridize to at least one nucleic acid molecule 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; having a sequence similar to SEQ ID NO: 11; NO: 10 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, at least 99.9% identical nucleotide sequence; or under stringent hybridization conditions, or if the sequence of the polynucleotide molecule is not identical to at least one of the nucleic acid molecules, under moderately stringent hybridization conditions, it is a completely complementary sequence or fragment of any of the foregoing. An exemplary DNA sequence that can be used as a probe to detect sugar beet strain Bv_CSM63713 is provided as SEQ ID NO: 36. The hybridization of the probe to the target DNA molecule can be detected by a variety of methods known to those skilled in the art, which methods may include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

[0139] For amplifying a target polynucleotide sequence (e.g., by PCR) using a specific amplification primer pair, "stringent conditions" or "stringent hybridization conditions" are conditions that allow the primer pair to hybridize to the bound target polynucleotide with a primer having the corresponding wild-type sequence (or its complementary sequence) and produce an identifiable amplification product (amplicon) having a sugar beet Bv_CSM63713 strain-specific region in a DNA thermal amplification reaction.

[0140] The term "specific for a target sequence" indicates that the probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample containing the target sequence.

[0141] Suitable stringent conditions to promote DNA hybridization are known to those skilled in the art, such as 6.0× sodium chloride / sodium citrate (SSC) at about 45° C., followed by 2.0×SSC wash at 50° C., or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected between a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from a low stringency condition of room temperature of about 22° C. to a high stringency condition of about 65° C. Both the temperature and the salt can be varied, or the temperature or the salt concentration can be kept constant while the other variable is changed.

[0142] The diagnostic amplicon produced using the methods described herein can be detected by a variety of techniques known in the art, such as sequencing, restriction mapping, Southern analysis or any other suitable polynucleotide or DNA hybridization, blotting, polymerization and / or amplification-based methods or techniques. One method is genetic bit analysis (Genetic Bit Analysis) (Nikiforov et al., 1994), in which DNA oligonucleotides are designed to overlap adjacent lateral genomic DNA sequences and inserted DNA sequences (i.e., junction sequences). Oligonucleotides are fixed in the wells of microtiter plates. After PCR is performed on the region of interest (e.g., one primer is used in the inserted sequence and one primer is used in the adjacent lateral genomic sequence), the single-stranded PCR product can be hybridized with the fixed oligonucleotide and used as a template for a single base extension reaction using DNA polymerase and a marker dideoxynucleotide triphosphate (ddNTP) specific to the expected next base. The read segment may be based on fluorescence or ELISA. The signal indicates that the transgenic / genomic junction sequence exists due to successful amplification, hybridization and single base extension.

[0143] Another method is the pyrophosphate sequencing technique described by Winge (2000). In this method, an oligonucleotide that is connected to adjacent genomic DNA and insert DNA is designed. Oligonucleotides are hybridized with single-stranded PCR products from the region of interest (a primer in the insert sequence and a primer in the lateral genomic sequence), and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. DNTP is added separately, and the light signal generated and measured is mixed. The light signal represents the presence of transgenic / genomic sequence due to successful amplification, hybridization and single base or multi-base extension.

[0144] The fluorescence polarization described by Chen et al. (1999) is a method that can be used to detect the amplicon of the present invention. Using this method, an oligonucleotide overlapping with the genomic side joint sequence and the inserted DNA junction sequence is designed. The oligonucleotide is hybridized with the single-stranded PCR product from the region of interest (one primer in the inserted DNA and one primer in the side joint genomic DNA sequence), and incubated in the presence of a DNA polymerase and fluorescently labeled ddNTPs. Single base extension leads to the incorporation of ddNTPs. Incorporation can be measured as a polarized change using a fluorometer. The polarized change represents the presence of transgenic / genomic sequence due to successful amplification, hybridization and single base extension.

[0145] Real-time polymerase chain reaction (PCR) is able to monitor the progress of PCR as it occurs (i.e., in real time). Data is collected throughout the PCR method, rather than at the end of PCR. In real-time PCR, the reaction is characterized by the time point at which target amplification is first detected during the cycle rather than the amount of target accumulated after a fixed number of cycles. In real-time PCR assays, positive reactions are detected by the accumulation of fluorescent signals. The higher the starting copy number of the nucleic acid target, the faster 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 cross the threshold (i.e., exceed the background level). The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the smaller the Ct value, the greater the amount of target nucleic acid in the sample).

[0146] (PE Applied Biosystems, Foster City, CA) is described as a method for detecting the presence and quantification of DNA sequences using real-time PCR, and is fully understood in the instructions provided by the manufacturer. In short, a FRET oligonucleotide probe overlapping with the genome side joint sequence and the insertion DNA junction sequence is designed. FRET probe and PCR primer (a primer in the insertion DNA sequence and a primer in the side joint genome sequence) are circulated in the presence of a thermostable polymerase and dNTP. The hybridization of the FRET probe causes the fluorescent part to crack and release from the quenching part on the FRET probe. Fluorescent signal indicates that there is a transgenic / genomic sequence due to successful amplification and hybridization.

[0147] Molecular beacons have been described for sequence detection, as described in Tyangi et al. (1996). In short, a FRET oligonucleotide probe is designed that overlaps the flanking genomic sequence and the inserted DNA junction sequence. The unique structure of the FRET probe allows it to contain a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one primer in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal is generated and indicates the presence of the flanking / transgenic insert sequence due to successful amplification and hybridization.

[0148] Other detection methods known in the art can be used. For example, microfluidics (see, for example, 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, for example, WO / 05017181). Then, a nanotube device (see, for example, WO / 06024023) can be detected, which includes an electronic sensor for detecting DNA molecules or nanobeads that bind specific DNA molecules. Nanopore sequencing technology, such as the technology described in Wang et al. (2021), Tyler et al. (2018) or Pearson et al. (2019), can also be used for strain detection.

[0149] Proteins are provided that can be used to produce antibodies that detect the presence of sugar beet strain Bv_CSM63713 in a sample. Such antibodies are specific to one or more proteins encoded by sugar beet strain Bv_CSM63713. Methods for preparing polyclonal or monoclonal antibodies are well known to those skilled in the art and can be used to prepare antibodies that are specific to one or more proteins encoded by sugar beet strain Bv_CSM63713. For example, U.S. Patent No. 7,838,729 and Wang et al. (2016) describe antibodies against DMO; Harrison et al. (1996) and Chinnadurai et al. (2018) describe antibodies against CP4 EPSPS. U.S. Patent No. 9,371,394 describes antibodies against PAT enzymes. The DNA sequence encoding such proteins is provided as SEQ ID NO: 10, and the start and end 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 can be used to produce antibodies to detect the presence of sugar beet strain Bv_CSM63713 by the method described herein. The presence of sugar beet strain Bv_CSM63713 can be detected by using any protein detection technology known in the art, such as Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), antibodies connected to detectable markers or reporter molecules (such as radioisotopes, ligands, chemiluminescent agents or enzymes) or the enzymatic action of reporter molecules. A method is provided, which contacts a sample with antibodies that bind to DMO, PAT, FT_T or CP4-EPSPS proteins encoded by sugar beet strain Bv_CSM63713, and then detects the presence or absence of antibody binding. Such antibody binding will diagnose the presence of one or more proteins encoded by sugar beet strain Bv_CSM63713.

[0150] Protein and nucleic acid detection kits for detecting the presence of sugar beet strain Bv_CSM63713 are provided. Variants of these kits can also be developed using the compositions and methods disclosed herein and protein and nucleic acid detection methods well known in the art for identifying sugar beet strain Bv_CSM63713. Protein and nucleic acid detection kits can be used for methods of breeding plants containing detection strain Bv_CSM63713. These kits contain primers and / or probes or antibodies that are specific to sugar beet strain Bv_CSM63713. Such DNA primers and / or probes can contain fragments of one or more of SEQ ID NO:1-10, or antibodies that are specific to proteins encoded by sugar beet strain Bv_CSM63713. The kit may also contain instructions for detecting the presence of sugar beet strain Bv_CSM63713 using primers, probes or antibodies. The kit may also optionally contain reagents for performing detection or diagnostic reactions described herein.

[0151] An example of a test kit comprises at least one DNA molecule having a sufficient length of contiguous nucleotides of SEQ ID NO: 10 to be used as a DNA probe that can be used to detect the presence or absence of sugar beet strain Bv_CSM63713 in a sample. The DNA derived from a transgenic sugar beet plant comprising strain Bv_CSM63713 will comprise 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, a complementary sequence of any of the foregoing, or a fragment of any of the foregoing. An exemplary DNA molecule sufficient for use as a probe is a DNA molecule comprising a sequence provided as SEQ ID NO: 36. Other probes can be easily designed by those skilled in the art. The probe can include a junction sequence that spans the 5' or 3' junction sequence between the sugar beet genomic DNA and the transgenic insert of sugar beet strain Bv_CSM63713.

[0152] Another example of a test kit comprises at least one primer pair that specifically hybridizes to the target DNA and amplifies a diagnostic amplicon under appropriate reaction conditions, which can be used to detect the presence or absence of the sugar beet strain Bv_CSM63713 in a sample. An object of the present disclosure is a test kit containing DNA primers that are homologous or complementary to any portion of the sugar beet genomic region shown in SEQ ID NO: 10 and to any portion of the transgenic DNA insert shown in SEQ ID NO: 9. The test kit can provide agarose gel-based detection methods or various methods of detecting amplicons known in the art. Such methods can also include sequencing the amplicon or a fragment thereof. Exemplary DNA molecules sufficient for use as primer pairs are DNA molecules comprising 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, respectively, wherein the primer pairs 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 will produce amplicons for the presence of strain Bv_CSM63713 in a sample. Other primer pairs can be readily designed by those skilled in the art.

[0153] Another example of a test kit comprises at least one antibody specific for at least one protein encoded by beet strain Bv_CSM63713. For example, such a kit may utilize a lateral flow strip comprising a reagent that is activated when the tip of the strip is in contact with an aqueous solution. An exemplary protein sufficient for antibody production is a protein encoded by the sequence provided in SEQ ID NO: 10 or any fragment thereof. Detection of binding of the at least one antibody to at least one protein encoded by beet strain Bv_CSM63713 in a sample will diagnose the presence of beet strain Bv_CSM63713 in the sample.

[0154] The detection kit provided in this article can be used in particular for identifying the sugar beet line Bv_CSM63713; selecting plant varieties or hybrids containing the sugar beet line Bv_CSM63713; detecting the presence of DNA derived from a transgenic sugar beet plant containing the sugar beet line Bv_CSM63713 in a sample; and monitoring the presence and / or absence of sugar beet plants containing the line Bv_CSM63713 or plant parts derived from sugar beet plants containing the sugar beet line Bv_CSM63713 in a sample.

[0155] Provided are sugar beet plants, progeny, seeds, cells and plant parts containing the proposed strain Bv_CSM63713, and commercial products produced using these. These sugar beet plants, plant parts, plant cells, seeds, progeny plants and commercial products contain or include sugar beet strain Bv_CSM63713, or are derived from transgenic sugar beet plants, plant parts, plant cells, seeds, progeny plants or commercial products containing or including strain Bv_CSM63713. These beet plants, progeny, seeds, cells, plant parts and commercial products contain or include a detectable amount of a polynucleotide comprising at least one junction sequence and / or a heterologous transgenic insert sequence of the beet variety Bv_CSM63713, such as a polynucleotide or nucleic acid or DNA molecule comprising at least one of the sequences provided below: 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 polynucleotide comprising at least 21 contiguous nucleotides of SEQ ID NO:1, at least 25 contiguous nucleotides of SEQ ID NO:2, at least 33 contiguous nucleotides of SEQ ID NO:3, at least 33 contiguous nucleotides of SEQ ID NO:4, at least 53 contiguous nucleotides of SEQ ID NO:5, or at least 51 contiguous nucleotides of SEQ ID NO:6; a polynucleotide having a full length or SEQ ID NO:10; NO:9 full length 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 nucleic acid molecules, and the complete complementary sequence of any of the foregoing. In some embodiments, the beet plant, plant part, plant cell or seed is further defined as a progeny plant of any generation of a beet plant comprising the beet line Bv_CSM63713, or a beet plant part, plant seed or plant cell obtained therefrom.

[0156] Sugar beet plants, plant parts, plant cells, seeds, progeny plants and commercial products 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.

[0157] Also provided are beet plants, plant seeds, plant parts and plant cells that are tolerant to herbicides with at least three different herbicide action modes, wherein the gene that confers herbicide tolerance is present at a single genomic location. In order to make such beet plants, plant seeds, plant parts or plant cells, three transgenic boxes containing herbicide tolerance genes can be inserted into a single genomic location in the beet genome in the form of transgenic inserts connected by continuous polynucleotides or single molecules. Alternatively, three transgenic boxes containing herbicide tolerance genes can be inserted at a single genomic location by inserting a separate box containing herbicide tolerance genes at the same position. "Single genomic location" means that the gene is present at a single location on the chromosome together with any regulatory sequence (e.g., promoter, intron, leader sequence, 5'-UTR and / or 3'UTR, etc.) and / or a sequence encoding a target peptide (e.g., chloroplast transit peptide), and will be inherited as a single locus. Although a certain intervening sequence may exist between each transgenic box, the length of the intervening sequence is limited, so that the transgenic boxes are close to each other on the chromosome. For example, the intervening sequence between the transgenic cassettes can be 500 nucleotides or less in length, 400 nucleotides or less in length, 300 nucleotides or less in length, 250 nucleotides or less in length, 200 nucleotides or less in length, 150 nucleotides or less in length, 100 nucleotides or less in length, or 50 nucleotides or less in length. For example, a beet plant, plant seed, plant part, or plant cell can comprise any of the DNA constructs described herein and can exhibit tolerance to at least one herbicide selected from the group consisting of: a benzoic acid auxin, such as dicamba; a glutamine synthetase inhibitor, such as glufosinate; an EPSPS inhibitor, such as glyphosate; and any combination thereof.

[0158] As used herein, the term "sequence identity percentage" or "sequence identity %" refers 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 ("target") sequence (or its complementary strand) when two sequences are optimally aligned (with less than 20% of the appropriate nucleotide or amino acid insertions, deletions or gaps in the comparison window). The optimal alignment of sequences for aligning the comparison window is well known to those skilled in the art and can be performed by tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and computer implementations of these algorithms, such as GAP, BESTFIT, FASTA and TFASTA, which can be used as part of the following sequence analysis software packages: Wisconsin (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 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)), for example, using default parameters. The "identity score" of the alignment segment of the test sequence and the reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the aligned portion of the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The percentage of sequence identity is expressed as the identity score multiplied by 100. One or more sequences can be compared to a full-length sequence or a portion thereof or to a longer sequence. Sugar beet plants, progeny, seeds, cells, plant parts and commercial 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.

[0159] The present disclosure provides plants, progeny, seeds, plant cells and plant parts derived from transgenic sugar beet plants comprising strain Bv_63713, such as roots, beets, pollen, anthers, ovaries, embryos, ovules, flowers, stems, leaves, microspores, protoplasts and callus. In order to enable the present disclosure, representative samples of seeds comprising sugar beet strain Bv_63713 have been deposited under the Budapest Treaty. The ATCC repository has assigned accession numbers. PTA-127098 is a seed containing strain Bv_CSM63713.

[0160] A microorganism is provided. The microorganism comprises a polynucleotide molecule having a nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence identical to the full length of SEQ ID NO: 9 of 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%. An example of this microorganism is an Agrobacterium cell. Another example of this microorganism is an Escherichia coli (E. coli) cell.

[0161] A plant cell is provided, the plant cell comprising a polynucleotide molecule described herein. For example, a plant cell is provided, the plant cell having 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 a nucleic acid molecule 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: 10 or the full length of SEQ ID NO: 9 present in its genome.

[0162] Plant cells and microorganisms disclosed herein can be used for many industrial applications, including but not limited to: (i) as research tools for scientific exploration or industrial research; (ii) used in culture to produce endogenous or recombinant carbohydrates, lipids, nucleic acids, enzymes or protein products or small molecules that can be used for subsequent scientific research or as industrial products; and (iii) for plant cells disclosed herein, used together with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures, which can then be used for agricultural research or production. The production and use of such transgenic plant cells will utilize modern microbiological technology and human intervention to produce artificial unique plant cells. In this process, recombinant DNA is inserted into the genome of the plant cell to produce transgenic plant cells that are separate and unique from naturally occurring plant cells. Then, such transgenic plant cells can be cultured using modern microbiological techniques like bacterial and yeast cells, and can exist in an undifferentiated single cell state. The genetic composition and phenotype of the new plant cell are technical effects produced by integrating heterologous DNA into the cell genome.

[0163] Methods for using plant cells (e.g., transgenic plant cells) are provided. These methods include (i) methods for producing transgenic cells by integrating recombinant DNA into the cell genome and then using such cells to produce additional cells with the same heterologous DNA; (ii) methods for culturing cells containing recombinant DNA using modern microbiological techniques; (iii) methods for producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, enzyme or protein products from cultured cells; and (iv) methods for producing transgenic plants or transgenic plant tissue cultures using modern plant tissue culture techniques with transgenic plant cells.

[0164] Plant, progeny, seed, cell and plant part can also contain one or more additional desired traits.These desired 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.By for example, the beet plant comprising beet strain Bv_CSM63713 is hybridized with another beet plant containing additional traits or transgenic strain, the desired traits can be combined with beet strain Bv_CSM63713.Such traits or transgenic strain include but are not limited to the insect resistance of increase, the water use efficiency of increase, the yield performance of increase, the drought resistance of increase, the disease resistance of increase, the seed quality of increase, the nutritional quality of improvement, the hybrid seed yield and / or the herbicide tolerance of increase, wherein the trait is measured relative to the beet plant lacking this transgenic trait.

[0165] The plants described herein can be used to produce progeny comprising the sugar beet line Bv_CSM63713. As used herein, "progeny" includes any plant, seed and cell and / or regenerable plant part comprising the Beta vulgaris line Bv_CSM63713 inherited from an ancestral plant, which is indicated by a plant comprising a polynucleotide molecule having 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; a polynucleotide comprising at least 21 contiguous nucleotides of SEQ ID NO: 1, at least 25 contiguous nucleotides of SEQ ID NO: 2, at least 33 contiguous nucleotides of SEQ ID NO: 3, at least 33 contiguous nucleotides of SEQ ID NO: 4, at least 53 contiguous nucleotides of SEQ ID NO: 5, or at least 51 contiguous nucleotides of SEQ ID NO: 6; or a polynucleotide having the full length of SEQ ID NO: 10 or SEQ ID NO: 11. NO:9 is a polynucleotide of 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 NO:9.

[0166] Plants, seeds, progeny, plant parts and cells may be homozygous or heterozygous for the sugar beet line Bv_CSM63713. Progeny plants may be grown from seeds produced by a sugar beet plant comprising the sugar beet line Bv_CSM63713 or from seeds produced by fertilizing a sugar beet plant with pollen containing the sugar beet line Bv_CSM63713.

[0167] The progeny plants can be self-pollinated (also known as "selfing") to produce a true plant breeding line, i.e., a plant homozygous for the Sugar Beet Line Bv_CSM63713 DNA. Alternatively, the progeny plants can be outcrossed, i.e., bred with another plant to produce a variant or hybrid seed or plant. The other plant can be transgenic or non-transgenic. Thus, a hybrid comprising DNA unique and distinctive to Line Bv_CSM63713 can be produced by crossing a first parent lacking DNA unique and distinctive to Line Bv_CSM63713 with a second parent comprising Line Bv_CSM63713, thereby obtaining a variant or hybrid seed or plant of the present disclosure.

[0168] Varieties or hybrid seeds or plants disclosed herein can also be produced by triadic hybridization (Poehlman JM, 1987). In this case, three parental lines are used for hybridization: a first parental line, a second parental line, and a pollinator. A first parent comprising line Bv_CSM63713 is hybridized with a second parent also comprising line Bv_CSM63713 to produce an F1 line, which is hybridized with a third parent lacking DNA that is specific and unique to line Bv_CSM63713. The parent comprising line Bv_CSM63713 for hybrid seeds or plants can be a female parent or a male parent. Preferably, the parent comprising line Bv_CSM63713 is a female parent, because this parent is cytoplasmic male sterile and will not produce transgenic pollen during the production of beet varieties or hybrid seeds.

[0169] Each parent can be a hybrid or an inbred line / variety, so long as the hybridization or breeding produces a plant or seed of the present disclosure, i.e., a seed having at least one allele comprising DNA that is unique and distinctive to line Bv_CSM63713 and / or at least 21 contiguous nucleotides of SEQ ID NO: 1, at least 25 contiguous nucleotides of SEQ ID NO: 2, at least 33 contiguous nucleotides of SEQ ID NO: 3, at least 33 contiguous nucleotides of SEQ ID NO: 4, at least 53 contiguous nucleotides of SEQ ID NO: 5, at least 51 contiguous nucleotides of SEQ ID NO: 6, or a contiguous nucleotide sequence having the full length of SEQ ID NO: 10 or SEQ ID NO: 11. NO:9 is a polynucleotide of 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 NO:9.

[0170] The sexual crossing of one plant with another, i.e., cross-pollination, can be achieved or promoted by human intervention, for example: by collecting pollen from one plant by hand and bringing this pollen into contact with the style or stigma of a second plant; by removing, damaging or covering the stamens or anthers of a plant by hand and / or human action (e.g., by human intervention or by the application of chemical gametocides), thereby preventing natural self-pollination and necessitating cross-pollination for fertilization to occur; by artificially placing pollinating insects in a position for "directed pollination" (e.g., by placing beehives in an orchard or field or by housing plants with pollinating insects); by artificially opening or removing parts of a flower to allow foreign pollen to be placed on or come into contact with the style or stigma; by selectively placing plants (e.g., intentionally planting plants near pollination); and / or by applying chemicals to accelerate flowering or promote receptivity (pollen receptivity of the stigma).

[0171] A plant part is provided. As used herein, "plant part" refers to any part of a plant comprising material directly from or derived from a plant comprising beet strain Bv_CSM63713. Plant parts include, but are not limited to, all or part of roots, beets, pollen, anthers, ovaries, ovules, embryos, flowers, stems, leaves, microspores, protoplasts, and callus. Plant parts can be vigorous or inactive, regenerable and / or non-regenerable.

[0172] A commodity product produced by a plant comprising the sugar beet strain Bv_CSM63713 is provided. The commodity product contains a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1-10, or 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. As used herein, a "commodity product" refers to any composition or product comprising material from a plant, seed, cell or plant part comprising the sugar beet strain Bv_CSM63713. Commodity products can be vigorous or inanimate plant materials, that is, not vigorous and derived from materials of plants, seeds, cells or plant parts comprising sugar beet strain Bv_CSM63713. Inactive commodity products include but are not limited to inactive seeds, complete or processed seeds, processed plant tissues or plant parts, dehydrated plant tissues or parts, frozen plant tissues or parts, plant parts processed for animal feed, fibers, pulp particles, pulp fragments, tailings, fruit juices, syrups, molasses, extracts, raffinates, betaine, separation molasses solubles (separator molasses soluble, SMS) or any other food for human consumption. Vigorous commodity products include but are not limited to vigorous seeds, vigorous plant parts (such as roots and leaves) and vigorous plant cells. Therefore, plants comprising strain Bv_CSM63713 can be used to manufacture any commodity products commonly obtained from sugar beet plants.Any such commercial product derived from a plant comprising line Bv_CSM63713 may contain at least a detectable amount of the characteristic and unique DNA corresponding to line Bv_CSM63713, and specifically may contain a detectable amount of a polynucleotide having a nucleotide sequence of at least 21 contiguous nucleotides of SEQ ID NO: 1, at least 25 contiguous nucleotides of SEQ ID NO: 2, at least 33 contiguous nucleotides of SEQ ID NO: 3, at least 33 contiguous nucleotides of SEQ ID NO: 4, at least 53 contiguous nucleotides of SEQ ID NO: 5, or at least 51 contiguous nucleotides of SEQ ID NO: 6, or having a full length of SEQ ID NO: 10 or SEQ ID NO: 11. The polynucleotides of the total length of 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 sequence of NO:9. Any standard polynucleotide molecule detection method can be used, including detection method disclosed herein.

[0173] Plants tolerant to herbicides can be produced by sexually crossing a plant comprising line Bv_CSM63713 with another plant and producing seeds therefrom, and then growing the seeds into progeny plants, wherein the line comprises a polynucleotide having a nucleotide sequence of SEQ ID NOs: 1-10, at least 21 contiguous nucleotides of SEQ ID NO: 1, at least 25 contiguous nucleotides of SEQ ID NO: 2, at least 33 contiguous nucleotides of SEQ ID NO: 3, at least 33 contiguous nucleotides of SEQ ID NO: 4, at least 53 contiguous nucleotides of SEQ ID NO: 5, or at least 51 contiguous nucleotides of SEQ ID NO: 6, and a polynucleotide having a full length of SEQ ID NO: 10 or SEQ ID NO: 11. The polynucleotides of the total length of NO:9 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 sequence. These progeny plants can be analyzed using diagnostic methods to select the progeny plants that comprise strain Bv_CSM63713DNA, or the progeny plants that have tolerance to herbicides Mediben, glyphosate, ammonium phosphine and any combination thereof. Other plants used may or may not be genetically modified. Progeny plants and / or the seed produced can be variants or hybrid seeds.

[0174] Plants tolerant to herbicides can be produced by selfing a plant comprising DNA of line Bv_CSM63713 and producing seeds therefrom, and then growing the seeds into progeny plants, the line comprising a polynucleotide having a nucleotide sequence of SEQ ID NOs: 1-10, at least 21 contiguous nucleotides of SEQ ID NO: 1, at least 25 contiguous nucleotides of SEQ ID NO: 2, at least 33 contiguous nucleotides of SEQ ID NO: 3, at least 33 contiguous nucleotides of SEQ ID NO: 4, at least 53 contiguous nucleotides of SEQ ID NO: 5, or at least 51 contiguous nucleotides of SEQ ID NO: 6, and a polynucleotide having a full length of SEQ ID NO: 10 or SEQ ID NO: 11. The polynucleotides of the total length of NO:9 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 sequence. Then, diagnostic methods can be used to analyze these progeny plants to select the progeny plants comprising strain Bv_CSM63713 DNA, or the progeny plants with tolerance to herbicides Mediben, glyphosate, glufosinate and any combination thereof. Beet strain Bv_CSM63713 contains three expression cassettes, which together provide tolerance to benzoic acid growth hormone, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and glutamine synthetase inhibitor.

[0175] Sugar beet line Bv_CSM63713 contains three expression cassettes that together provide tolerance to: benzoate auxins, such as dicamba; glutamine synthetase inhibitors, such as glufosinate; and EPSPS inhibitors, such as glyphosate.

[0176] As used herein, glutamine synthetase inhibitors include, but are not limited to, phosphinothricin, glufosinate, glufosinate salts, glufosinate-ammonium, glufosinate-sodium, glufosinate-P, L-glufosinate-ammonium, and L-glufosinate-sodium.

[0177] As used herein, benzoic acid herbicides include, but are not limited to, dicamba (3,6-dichloro-2-methoxybenzoic acid), dicamba salts, dicamba-butoxyethyl ester, dicamba-diglycolamine salts, dicamba-dimethylammonium, dicamba-diethanolammonium, dicamba-isopropylammonium, dicamba-potassium, dicamba-sodium, and dicamba-triethanolamine.

[0178] As used herein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors include, but are not limited to, glyphosate, glyphosate salts, glyphosate-isopropylammonium, glyphosate-ammonium, glyphosate-dimethylammonium, glyphosate-trimethylsulfonium salt (= sulfate), glyphosate-diammonium, glyphosate-potassium, and glyphosate-sodium.

[0179] As used herein, "herbicide tolerance or herbicide tolerance" or "tolerance" means the ability to be completely or partially unaffected by the presence or application of one or more herbicides, such as the ability to resist toxic effects when a herbicide is applied. If a cell, seed, or plant is able to maintain at least a certain degree of normal growth or phenotype in the presence of one or more herbicides, it is "herbicide tolerant" or has "improved tolerance". If the presence of a trait confers improved herbicide tolerance to a cell, seed, or plant compared to a wild-type or control cell, seed, or plant, the trait is a herbicide tolerance trait. Crops comprising a herbicide tolerance trait can continue to grow in the presence of a herbicide and are minimally affected by the presence of the herbicide. If the expression of a protein confers improved herbicide tolerance to a cell, plant, or seed compared to a wild-type or control cell, plant, or seed, the protein confers "herbicide tolerance". Examples of herbicide tolerance proteins include phosphinothricin N-acetyltransferase, dicamba monooxygenase, and glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium strain CP4. 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.

[0180] As used herein, "herbicide damage" or "damage" refers to damage to plants caused by the application of herbicides. "Injury rate" or "percentage of damage" refers to the damage caused by herbicides as visually assessed, and is expressed as the percentage of plant leaf area that exhibits damage caused by the application of herbicides, such as necrosis (brown or dead tissue), chlorosis (yellow tissue or yellow spots), and deformity (deformation of leaves or plant structures, upward growth or twisting of stems, curling of leaves) based on visual assessment. The injury rate or percentage of damage is measured on a scale of 0 to 100, where "0" means that the crop is not damaged, and "100" means that the crop is completely damaged (dead). For beet plants containing beet strain Bv_CSM63713, the damage to the plant will be reduced after applying one or more of the following: benzoic acid auxin (e.g., dicamba); 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (e.g., glyphosate); or glutamine synthetase inhibitors (e.g., glufosinate); or any combination thereof. For example, after application of a benzoic acid auxin (e.g., dicamba), an EPSPS inhibitor (e.g., glyphosate), or a glutamine synthetase inhibitor (e.g., glufosinate), a sugar beet plant containing sugar beet line Bv_CSM63713 will have less than about 5% damage, less than about 10% damage, less than about 15% damage, or less than about 20% damage, compared to a sugar beet plant that does not contain sugar beet line Bv_CSM63713 and is otherwise identical.

[0181] As used herein, "weeds" are any undesirable plants. A plant may be generally considered to be undesirable (e.g., Amaranthus species) for agricultural or horticultural purposes, or may be considered to be undesirable (e.g., a crop plant of a species in a field of different species, also referred to as a volunteer plant) in a particular case. Weeds are generally known in the art and vary with geography, season, growth environment, and time. Lists of weed species can be obtained from agricultural and scientific societies (such as the American Weed Science Society, the Canadian Weed Science Society, the Brazilian Weed Science Society, the International Weed Science Society, and the International Herbicide Resistant Weed Survey), government agencies (such as the U.S. Department of Agriculture and the Australian Department of Environment and Energy), and industry and farmer associations (such as the American Sugar Beet Growers Association). The major noxious weeds in sugar beet production include waterhemp (Amaranthus tuberculatus (Moq.) JD Sauer), kochia (Bassiascoparia (L.) AJ Scott or Kochia scoparia (L.)), common lambsquarters (Chenopodium album L.), horseweed / marestail (Erigeroncanadensis L.), palmer amaranth (Amaranthus palmeri), redroot pigweed (Amaranthus retroflexus L.), velvetleaf (Abutilon theophrasti Medik.) and yellow nutsedge (Cyperus culentus L.))(Soltani et al., 2018).

[0182] A method for controlling weeds in a beet cultivation area is provided. The method comprises applying at least one herbicide selected from the group consisting of: (i) a glutamine synthetase inhibitor, such as glufosinate; (ii) a benzoic acid auxin, such as dicamba; and (iii) a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate, wherein the seeds or plants comprising the beet strain Bv_CSM63713 are planted in the area before, during or after the application of the herbicide, and the application of the herbicide will prevent or inhibit weed growth and will not damage the beet plant. When the herbicide is applied, the plant growth area may or may not contain weed seeds or plants. The one or more herbicides used in the method described herein may be applied alone during the growing season, or applied in combination with one or more herbicides. One or more herbicides used in the methods described herein can be applied with one or more herbicides in time (e.g., in a tank mix or in a sequential application), in space (e.g., at different times during the growing season, including before and after beet seed planting), or in combination. For example, a method for controlling weeds is provided, the method comprising: planting seeds containing beet strain Bv_CSM63713 in an area, and applying a herbicidally effective amount of dicamba, glyphosate, or glufosinate alone, or any combination of dicamba, glyphosate, or glufosinate with another herbicide during the growing season to achieve the purpose of controlling weeds in the area without damaging plants containing beet strain Bv_CSM63713 or causing only less than about 10% damage to the plants. The application of the one or more herbicides may be pre-planting (at any time before planting seeds comprising sugar beet strain Bv_CSM63713, including for burndown purposes, i.e., application to weeds that have emerged or are present before seed planting), pre-emergence (at any time after planting seeds comprising sugar beet strain Bv_CSM63713 and before plants comprising sugar beet strain Bv_CSM63713 emerge), or post-emergence (at any time after plants comprising sugar beet strain Bv_CSM63713 emerge). Multiple applications of one or more herbicides or a combination of herbicides applied together or separately may be applied throughout the growing season, for example two applications (such as a pre-planting application and a post-emergence application, or a pre-emergence application and a post-emergence application) or three or more applications (such as a pre-planting application and two post-emergence applications).

[0183] In the implementation of the methods described herein, the application of the herbicide can be the recommended commercial amount or any fraction or multiple thereof, such as twice the recommended commercial amount. The herbicide rate can be expressed as pounds of acid equivalent per acre (lb ae / acre), pounds of active ingredient per acre (lb ai / acre), or pounds of active ingredient per hectare (lb bai / ha) according to the herbicide and the distribution. The herbicide application rates in acres provided herein are only indicative; for areas greater than or less than one acre, herbicide application rates of dosages equivalent to any ratio provided herein can be used. The herbicide application includes at least one herbicide selected from the group consisting of: (i) glutamine synthetase inhibitors, such as glufosinate; (ii) benzoic acid growth hormones, such as dicamba; and (iii) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate. When the herbicide is applied, the plant growth area may or may not contain weed plants. In the growing season, the herbicidal effective amount of the glutamine synthetase inhibitor used to control weeds in the region is within the range of about 0.1 lb ae / acre to about 10 lb ae / acre (for example, the application rate of glufosinate can be about 0.4 lb ae / acre to about 2.16 lb ae / acre). In the growing season, the herbicidal effective amount of the benzoic acid auxin herbicide used to control weeds in the region is within the range of about 0.1 lb ae / acre to about 16 lb ae / acre (for example, the application rate of dicamba can be about 0.5 lb ae / acre to about 2.0 lb ae / acre). In the growing season, the herbicidal effective amount of the EPSPS inhibitor used to control weeds in the region is within the range of about 0.5 lb ae / ac to about 12 lb ae / ac (for example, the application rate of glyphosate can be about 0.75 lb ae / acre to about 2.25 lb ae / acre).

[0184] A method for controlling volunteer sugar beets comprising sugar beet strain Bv_CSM63713 in a crop cultivation area is provided. The method comprises applying a herbicidally effective amount of at least one herbicide, such as paraquat, clethodim, clopyralid, betadine, flumethalin, 2,4-dichlorophenoxyacetic acid (2,4-D) and acetolactate synthase (ALS) inhibitors (e.g., sulfonylureas (SU)), imidazolinones, triazolopyrimidines, pyrimidinoxybenzoates and sulfonamidocarbonyltriazolinones, and any combination thereof, wherein the application of the herbicide will prevent the growth of sugar beets comprising sugar beet strain Bv_CSM63713.

[0185] Provided is a method for producing plants and seeds comprising sugar beet strain Bv_CSM63713. Plants can be bred using any method known in the art. For example, descriptions of commonly used breeding methods can be found in, for example, WR Fehr, Breeding Methods for Cultivar Development, Wilcox J., American Society of Agronomy, Madison WI (1987). Plants can be self-pollinated (also referred to as "selfing") or cross-pollinated (also referred to as "hybridization"). Plants comprising sugar beet strain Bv_CSM63713 can be self-pollinated to produce true breeding lines of plants that are isozygous for sugar beet strain Bv_CSM63713. Self-pollination produces offspring referred to as "inbreeding", and can be used to produce genetically uniform inbred strains. Alternatively, plants containing sugar beet strain Bv_CSM63713 can be cross-pollinated (breeding with another plant of transgenic or non-transgenic) to produce variants or hybrid seeds. As further described herein, beet strain Bv_CSM63713 comprises three independent expression cassettes or transgenics, encoding dicamba monooxygenase (DMO), phosphinothricin N-acetyltransferase (PAT) and EPSPS, respectively. Relative to non-transgenic control plants, transgenic beet plants comprising strain Bv_CSM63713 have tolerance to the following: benzoic acid growth hormone, such as dicamba; Glutamine synthetase inhibitors, such as glufosinate; EPSPS inhibitors, such as glyphosate; or any combination thereof. The transgenic beet plants used in these methods can be homozygous or heterozygous for transgenics. The progeny plants produced by these methods can be variants or hybrid plants; seeds produced by plants containing beet strain Bv_CSM63713 and / or by plants from pollen fertilization containing beet strain Bv_CSM63713 Seed growth; and can be homozygous or heterozygous for transgenics and / or strain Bv_CSM63713.

[0186] In addition, plants comprising sugar beet line Bv_CSM63713 can be produced by a three-way cross, i.e., a first parental line comprising line Bv_CSM63713 is crossed with a second parental line also comprising line Bv_CSM63713 to produce an F1 line, which is then crossed with a third parent lacking DNA specific and unique to line Bv_CSM63713. The parent comprising line Bv_CSM63713 for hybrid seed or plant can be either the female parent or the male parent. Preferably, the parent comprising line Bv_CSM63713 is the female parent, as this parent is cytoplasmic male sterile and will not produce transgenic pollen during the production of variant or hybrid seed.

[0187] The production of double haploids can also be used in breeding programs to produce beet plants and seeds homozygous for strain Bv_CSM63713 DNA. Double haploids are produced by doubling a set of chromosomes (1N) of heterozygous plants, thereby producing completely homozygous individuals. For example, see Wan et al. (1989) and U.S. Patent No. 7,135,615. This will be advantageous because this process omits the selfing algebra required to obtain homozygous plants from heterozygous sources. One way to produce haploid and double haploid beet plants comprising strain Bv_CSM63713 is by culturing ovules of unfertilized flowers comprising strain Bv_CSM63713 (Gurel et al., 2021; Weich and Levall, 2003). Other methods, such as natural polyembryony, induction with irradiated pollen, hybridization with polyploid plants or wild species, and anther and microspore culture can also be used to produce haploid and doubled haploid sugar beet plants comprising line Bv_CSM63713.

[0188] Seeds and progeny plants produced using the methods described herein comprise beet strain Bv_CSM63713. One or more herbicides to which tolerance is imparted by applying beet strain Bv_CSM63713 can be used to select progeny comprising beet strain Bv_CSM63713. Alternatively, progeny can be analyzed using diagnostic methods to select plants or seeds comprising beet strain Bv_CSM63713. Progeny can be variants or hybrid plants; seeds produced by plants comprising beet strain Bv_CSM63713 or by plants fertilized by pollen of plants comprising beet strain Bv_CSM63713 can be grown; and can be homozygous or heterozygous for beet strain Bv_CSM63713.

[0189] Sugar beet transgenic lines are known to those skilled in the art; for example, the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS) provides a list of such traits, and the table can be found on its website www.aphis.usda.gov. Thus, two or more transgenic lines can be combined in a progeny seed or plant by crossing two parent plants each containing one or more transgenic lines, collecting progeny seeds and selecting progeny seeds or plants containing two or more transgenic lines; then, these steps can be repeated until the desired combination of transgenic lines is obtained in the progeny. As with vegetative propagation, backcrossing with parental plants and outcrossing with non-transgenic plants are also covered.

[0190] As used herein, the term "including" means "including but not limited to".

[0191] Methods are provided for detecting the presence of DNA derived from a sugar beet plant, plant part, plant cell or seed comprising sugar beet line Bv_CSM63713 in a sample. One method 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 at least one primer capable of producing a DNA sequence specific for line Bv_CSM63713 DNA under conditions suitable for DNA sequencing; (iii) performing a DNA sequencing reaction; and then (iv) confirming that the nucleotide sequence comprises a nucleotide sequence specific for line Bv_CSM63713 of a transgenic implant contained therein, such as 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.

[0192] Another method comprises (i) extracting a DNA sample from at least one 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 the line Bv_CSM63713 under conditions suitable for DNA amplification; (iii) performing a DNA amplification reaction; and then (iv) detecting the amplicon molecules and / or confirming that the nucleotide sequence of the amplicon contains a nucleotide sequence specific to the line Bv_CSM63713, for example, selected from one of 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 an amplicon specific for strain Bv_CSM63713 and contain a junction sequence at nucleotide positions 1000-1001 and / or nucleotide positions 12,722-12,723 of SEQ ID NO: 10, 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 a nucleotide sequence specific for strain Bv_CSM63713 in the amplicon will determine and / or diagnose the presence of sugar beet strain Bv_CSM63713 specific DNA in the sample. An exemplary primer pair capable of generating an amplicon from strain Bv_CSM63713 DNA under conditions suitable for DNA amplification is provided as SEQ ID NO: 14 and SEQ ID NO: 18. Other primer pairs can be easily designed by those skilled in the art to generate an amplicon for diagnosis of the beet strain Bv_CSM63713, wherein such primer pairs include at least one primer within the genomic region flanking the insert and a second primer within the insert, as long as any primer pair that generates an amplicon comprising a junction sequence and / or all or part of the insert or transgenic sequence can be designed and used. Detection of the amplicon can be based on any suitable method, such as sequencing, determining the fragment size or migration of the amplicon in a matrix or gel, or based on hybridization methods.

[0193] Another method for detecting the presence of DNA derived from a sugar beet plant, plant part, plant cell or seed comprising sugar beet strain 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 strain Bv_CSM63713 DNA; (iii) hybridizing the probe with the DNA sample under stringent hybridization conditions; and then (iv) detecting hybridization between the probe and the target DNA sample. An example of a sequence of a DNA probe specific for strain Bv_CSM63713 is provided as SEQ ID NO:36. Other probes can be easily designed by one skilled in the art. Hybridization of the detection probe with the DNA sample will diagnose the presence of sugar beet strain Bv_CSM63713 specific DNA in the sample. Alternatively, the absence of hybridization will diagnose the absence of sugar beet strain Bv_CSM63713 specific DNA in the sample.

[0194] Methods are provided for determining the zygosity of the lines and transgenes with genomic DNA in a sample derived from at least one Sugar Beet plant, plant part, plant cell or seed comprising Sugar Beet line Bv_CSM63713. A method comprises: (i) extracting a DNA sample from at least one beet plant, plant part, plant cell or seed; (ii) contacting the DNA sample with a first primer pair, the first primer pair being capable of producing a first amplicon that is diagnostic for line Bv_CSM63713; (iii) contacting the DNA sample with a second primer pair, the second primer pair being capable of producing a second amplicon that is diagnostic for wild-type genomic DNA that does not contain line Bv_CSM63713; (iv) performing a DNA amplification reaction; and then (v) detecting the amplicons, wherein the presence of only the first amplicon will diagnose homozygous line Bv_CSM63713 DNA in the sample, the presence of both the first amplicon and the second amplicon will diagnose a beet plant that is heterozygous for line Bv_CSM63713, and the presence of only the second amplicon will diagnose the absence of line Bv_CSM63713 DNA in the sample. An exemplary set of primer pairs is as follows: SEQ ID NO: 26 and SEQ ID NO: 25, which produces an amplicon diagnostic for line Bv_CSM63713; and SEQ ID NO: 26 and SEQ ID NO: 24, which produces an amplicon diagnostic for wild-type sugar beet genomic DNA that does not contain line Bv_CSM63713. A set of probes can also be incorporated into this amplification method for use in a real-time PCR format using the primer pair sets described above.

[0195] 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 one first probe that specifically hybridizes to DNA of line Bv_CSM63713 and at least one second probe that specifically hybridizes to sugar beet genomic DNA and does not hybridize to DNA of line Bv_CSM63713, wherein the sugar beet genomic DNA is interrupted by the insertion of heterologous DNA of line Bv_CSM63713; (iii) hybridizing the probe set to the sample under stringent hybridization conditions, wherein detection of only the first probe hybridizing under the hybridization conditions is diagnostic for the presence of a zygosity in the sample for line Bv_CSM63713. The DNA of a sugar beet plant, plant part, plant cell or seed is homozygous; wherein detection of hybridization of both the first probe and the second probe under hybridization conditions will diagnose the sugar beet plant, plant part, plant cell or seed as heterozygous for the variety Bv_CSM63713 in the DNA sample; and detection of only the second probe hybridizing under hybridization conditions will diagnose the absence of variety Bv_CSM63713 DNA in the sample.

[0196] Yet 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 first primer pair capable of producing a first amplicon that is diagnostic for line Bv_CSM63713; (iii) contacting the DNA sample with a second primer pair capable of producing a second amplicon that is an internal standard known to be single copy and homozygous in sugar beet plants; (iv) contacting the DNA sample with a probe set containing a probe that is identical to the first amplicon; (v) performing a DNA amplification reaction using real-time PCR and determining a cycle threshold (Ct value) of the first amplicon and the second amplicon; (vi) calculating a difference (ΔCt) between the Ct values ​​of the first amplicon and the second amplicon; and (vii) determining zygosity, wherein a ΔCt of about zero (0) indicates homozygosity of the strain or the inserted T-DNA, and a ΔCt of about one (1) indicates heterozygosity of the strain or the inserted T-DNA. Heterozygous strains are distinguished from homozygous strains by a ΔCt value of about one (1). Taking into account the normal variability observed in real-time PCR due to factors such as amplification efficiency and ideal annealing temperature, the range of "about one (1)" is defined as a ΔCt of 0.75 to 1.25, and the range of "about zero (0)" is defined as a ΔCt of -0.25 to 0.25 (or 0.0 to 0.25 if the ΔCt is measured in absolute terms). The primer pairs and probes used in the above methods of determining zygosity can amplify and detect amplicons from transgenic or line DNA and internal DNA standards.

[0197] A DNA construct is provided, the DNA construct comprising a first expression cassette, a second expression cassette and a third expression cassette, wherein the first expression cassette comprises in operable connection i) a promoter and a leader sequence of a chlorophyll AB (Cab1) binding protein gene from Arabidopsis thaliana; ii) a codon-optimized phosphinothricin N-acetyltransferase (PAT) coding sequence from Streptomyces viridis for conferring tolerance to glutamine synthetase inhibitors such as glufosinate; and iii) a 3'UTR (untranslated region) of a small heat shock protein (Hsp20) gene from Medicago truncatula; the second expression cassette comprises in operable connection i) an enhancer of an inclusion matrix protein from dahlia mosaic virus; ii) a promoter, a leader sequence and an intron sequence of a ubiquitin protein gene from melon; iii) a ribulose bisphosphate carboxylase small subunit (RbcS) gene from pea iv) a codon-optimized coding sequence for dicamba monooxygenase (DMO) from Stenotrophomonas maltophilia for conferring tolerance to benzoic acid auxins such as dicamba; and v) a 3'UTR of a putative protein gene from Medicago truncatula; the third expression cassette comprises in operable connection i) a promoter, leader and intron sequences of the S-adenosyl-L-methionine synthetase (SAMS2) gene from melon; ii) a chloroplast targeting sequence of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Arabidopsis thaliana; iii) a codon-optimized coding sequence for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium strain CP4 for conferring tolerance to EPSPS inhibitors such as glyphosate; and iv) a 3'UTR of a putative protein from Medicago truncatula. The nucleotide sequences of the three expression cassettes are contained in SEQ ID NO:9 and SED ID NO:10 of the sugar beet strain Bv_CSM63713. The expression of DMO, PAT and EPSPS in the transgenic plant confers tolerance to herbicides with at least three different modes of action. For example, plants, plant parts, plant cells or seeds containing or comprising the sugar beet strain Bv_CSM63713 are tolerant to dicamba (a benzoic acid herbicide), glufosinate (a glutamine synthetase inhibitor) and glyphosate (an EPSPS inhibitor).

[0198] A method for improving tolerance to herbicides is provided. The method consists of: i) inserting a DNA construct into the genome of a plant cell, the DNA construct comprising a first expression cassette, a second expression cassette, and a third expression cassette described herein; ii) producing a plant from a plant cell; and iii) selecting a regenerated transgenic plant comprising the DNA construct. The transgenic plant produced by the method comprises a combination of three transgenic expression cassettes that are unique in orientation and position relative to each other, each of which has a unique combination of expression elements to achieve optimal expression of the transgene. In addition, the transgenic plants produced by the method described herein also obtain tolerance to herbicides with three different herbicide action modes. The DNA or protein detection methods described in the present disclosure can be used to select regenerated plants comprising the DNA construct. Alternatively or in addition, the selection can consist of: treating the transgenic plant or plant cell with an effective amount of at least one herbicide, the herbicide being selected from the group consisting of: benzoic acid auxin, such as dicamba; glutamine synthetase inhibitors, such as glufosinate; EPSPS inhibitors, such as glyphosate; and any combination thereof.

[0199] A method for controlling, preventing or reducing the development of herbicide-tolerant weeds is provided. The method comprises: a) cultivating a beet plant in a crop growth environment, the beet plant comprising a DNA construct or transgenic disclosed herein, or strain Bv_CSM63713, thereby providing tolerance to herbicides with three different herbicide action modes at a single genomic position; and b) applying at least one herbicide to the crop growth environment, the herbicide being selected from the group consisting of: dicamba, glufosinate, glyphosate, and any combination thereof, wherein the beet plant is tolerant to the at least one herbicide. In some embodiments, the three different herbicide action modes are glutamine synthetase inhibition, benzoic acid auxin, and EPSPS inhibition. The beet plant grown from seeds comprising a DNA molecule or transgenic disclosed herein, or strain Bv_CSM63713, is tolerant to dicamba, glufosinate, glyphosate, or any combination thereof.

[0200] The herbicide used in the method described herein can be applied alone during the growing season, or applied sequentially or in combination with one or more herbicides. The one or more herbicides used in the method described herein can be applied with one or more herbicides in time (e.g., in the form of a tank mix or in a sequential application mode), space (e.g., at different times during the growing season, including before and after beet seed planting), or in combination with both. For example, a method for controlling the development of herbicide resistance in weeds is provided, the method comprising: planting seeds containing beet strain Bv_CSM63713 in a certain area, and applying a herbicidally effective amount of one or more of the following alone or in any combination with another herbicide during the growing season to achieve the purpose of controlling the development of herbicide resistance in the weeds in the area: benzoic acid auxin, such as dicamba; glutamine synthetase inhibitors, such as glufosinate; and EPSPS inhibitors, such as glyphosate. The application of the one or more herbicides may be pre-planting (at any time before planting seeds comprising sugar beet strain Bv_CSM63713, including for burndown purposes, i.e., application to weeds that have emerged or are present before seed planting), pre-emergence (at any time after planting seeds comprising sugar beet strain Bv_CSM63713 and before plants comprising sugar beet strain Bv_CSM63713 emerge), or post-emergence (at any time after plants comprising sugar beet strain Bv_CSM63713 emerge). Multiple applications of one or more herbicides or a combination of herbicides applied together or separately may be applied throughout the growing season, for example two applications (such as a pre-planting application and a post-emergence application, or a pre-emergence application and a post-emergence application) or three or more applications (such as a pre-planting application and two post-emergence applications).

[0201] Also provided is a method for reducing locus to carry out beet breeding by inserting multiple transgenics in a single genome position to provide three different modes of action for herbicide tolerance.Sugar beet strain Bv_CSM63713 contains or comprises a transgenic insert, which comprises three independent transgenic boxes: a first expression box encoding phosphinothricin N-acetyltransferase (PAT), a second expression box encoding dicamba monooxygenase (DMO) and a third expression box encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). These three transgenic boxes are inserted into a single genome position in the form of a transgenic insert connected with continuous polynucleotides or DNA molecules or single molecules, and provide commercial tolerance of at least one herbicide for each herbicide mode of action in the field, the herbicide being for example glufosinate, dicamba, glyphosate and any combination thereof. The nucleotide sequence of the three expression boxes is included in SEQ ID NO:9 and SED ID NO:10. As used herein, the term "commercial level" with respect to herbicides refers to the recommended commercial herbicide application rate (1X) 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; 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 the recommended commercial application rate or higher doses due to transgenic expression of one or more of the four expression cassettes in plants comprising strain Bv_CSM63713.

[0202] The beet strain Bv_CSM63713 with unique characteristics can be bred or introgressed into superior strains or variants in the form of a single locus by conventional breeding methods, and maintained in subsequent generations following the Mendelian inheritance of a single locus, wherein the unique characteristics are, for example, a single insertion site; stable integration and expression of PAT, DMO and EPSPS transgenes; and consistent and superior efficacy combinations in a variety of environmental conditions in different geographic regions, including herbicide tolerance and agronomic performance. Therefore, compared with the situation where a single transgene is inserted into two or more loci, the method disclosed in the present invention allows multiple transgenes to be rapidly integrated on separate materials, saving time and resources in breeding programs and enabling rapid development of strains, while the insertion of a single transgene requires multiple generations of tedious and laborious multiple crosses to select plants containing multiple genes. The newly introgressed or integrated DNA molecule or polynucleotide of Line Bv_CSM63713 comprising SEQ ID NO: 9 and / or SEQ ID NO: 10 will maintain the expression characteristics of the transgene, as well as the genomic flanking sequences and chromosomal location, wherein it will confer tolerance to at least one herbicide in each herbicide mode of action in the field, such as glufosinate, dicamba, glyphosate, and any combination thereof.

[0203] Collection Information

[0204] Pursuant to the Budapest Treaty with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia, 20110, USA, a representative sample of sugar beet seeds comprising strain Bv_CSM63713 was deposited on August 10, 2021. The ATCC patent deposit name (accession number) for seeds comprising sugar beet strain Bv_CSM63713 is accession number PTA-127098. During the pendency of this application, the Commissioner of Patents and Trademarks and persons who are qualified personnel of the Commissioner may obtain the deposit upon request. Upon issuance of the patent, all restrictions on public availability will be irrevocably lifted. The deposit will be kept at the depository for a period of thirty (30) years, or five (5) years after the last request, or for the effective period of the preservation patent, whichever is longer.

[0205] Example

[0206] The following examples are included to more fully describe the present invention. The rigorous molecular, agronomic and field testing required to generate, identify and ultimately select the sugar beet line Bv_CSM63713 summarizes the construction and testing of four different transformation constructs, the generation of 1803 unique transformation lines, and the analysis of hundreds of thousands of individual plants over seven years.

[0207] It will be appreciated by those skilled in the art that many modifications may be made in the disclosed specific embodiments and still similar results may be obtained. Certain chemically and physiologically related reagents may replace the reagents described herein and obtain identical or similar results. All such substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the invention.

[0208] Example 1: Construct design, line generation, R0 plant testing

[0209] This example describes the design of four different expression constructs tolerant to dicamba, glyphosate, and glufosinate herbicides by vector stacking; the generation of 1803 unique sugar beet lines; and the testing and analysis of multiple generations of the resulting transgenic sugar beet plants.

[0210] Design and construct four kinds of expression constructs.These four kinds of constructs each contain three expression cassettes in the same orientation, for driving the expression of Mediben monooxygenase (DMO), CP45-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and phosphinothricin N-acetyltransferase (PAT) transgenic.However, each construct is designed with unique 5 ' and 3 ' expression element combination, for operably connected DMO, CP4 EPSPS and PAT transgenic, so that different promoters, chloroplast targeting sequences and 3 ' UTR are tested in beet plants, thus realizing the best performance of transgenic.Construct is as shown in Table 1.Then, these four kinds of expression constructs are cloned into plant transformation vectors respectively and introduced in Agrobacterium strain AGL1.

[0211] Four sugar beet strains were transformed with Agrobacterium-mediated transformation using these four constructs. Strain 1 and 2 were transformed using shoot meristems following a slightly modified transformation protocol (Lindsey and Gallois, 1990), while strains 3 and 4 were transformed using hypocotyl and cotyledon explants using a callus-based transformation protocol modified by Kishchenko, Komarnitskii, Kuckuk (2005). Phosphinothricin (DL-PPT; Duchefa, Haarlem-The Netherlands) was used as a selection agent at a concentration of 6 mg / L.

[0212] All four strains obtained by the four transformation constructs produced a total of 1803 unique transformation lines. Each transformation line was generated by randomly inserting the T-DNA containing three expression cassettes into a unique position of the sugar beet genome. Rooted plants from tissue culture were transplanted into soil and cultivated for four weeks at 20°C during the day / 17°C at night, with a photoperiod of 16 hours of light / 8 hours of darkness. Then, the plants were transferred to a greenhouse and planted under a photoperiod of 19°C during the day and 17°C at night, with a photoperiod of 18 hours of light / 6 hours of darkness.

[0213] A summary of the total number of lines generated for each line or construct, respectively, is provided in Tables 2 and 3. Because line 1 was closest to elite germplasm and contained all the disease resistance required for field cultivation in the United States, and because it was the most established line in tissue culture, it was used in the majority of experiments and thus generated the greatest number of transgenic lines.

[0214] Table 1. Description of transformation constructs

[0215]

[0216] Table 2. Summary of transgenic lines generated by genotype

[0217]

[0218] Table 3. Summary of transgenic lines generated by constructs

[0219]

[0220] DNA from all 1803 primary transformants was isolated using a modified Doyle and Doyle (1990) method and screened for the presence or absence of CP4, PAT and DMO genes, Agrobacterium T-DNA left / right border overreading and the presence of the VirD2 gene using a 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 detection of VirD2 indicates the presence of Agrobacterium tumefaciens AGL1. The assay is based on competitive allele-specific PCR and is capable of biallelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci. The KASP assay mix contains assay-specific unlabeled oligonucleotides: an allele-specific primer ("AlleleX" or "X") and a universal primer ("Primer Common" or "C"). The primer sets used for CP4, PAT and DMO genes and Agrobacterium are listed in Table 4.

[0221] Table 4. Primer sets for CP4, PAT, DMO and Agrobacterium KASP

[0222]

[0223]

[0224] Putative transformants that were negative for CP4, PAT, and DMO were discarded. In addition, transformants that showed positive signals for overreading or read-through of the left border (LB) or / and the right border (RB), or the presence of A. tumefaciens AGL1 after the tissue culture stage were also discarded. Only lines that were positive for CP4, PAT, and DMO, negative for overreading or read-through of LB and / or RB, and negative for the presence of A. tumefaciens AGL1 were retained for further growth and testing.

[0225] The remaining lines were further analyzed to determine transgene copy number, integrity of the transgene insert, absence of vector backbone sequences, transgene insertion site in the genome, and to screen for normal fertile plants. From this initial molecular analysis and phenotypic screening, 622 R0 lines were tested for R0 trait efficacy (tolerance to dicamba, glyphosate, and glufosinate) in the greenhouse at the 4-6 leaf stage.

[0226] Glyphosate is applied at a rate of 4lb / acre's Roundup PowerFlex herbicide (4x application rate). Glufosinate is applied at a rate of 2.16lb / acre's Basta herbicide (4x application rate). Dicamba is applied at a rate of 2lb / acre's MaisBanvel WG herbicide (4x application rate) (Table 5). Glyphosate+dicamba double mixtures are each applied at a 4x rate, while glyphosate+dicamba+glufosinate triple mixtures are applied at a 2.4x rate. For the double mixture and triple mixture, different herbicide products are co-mixed before application. Plant damage caused by herbicides is assessed visually. Lines that do not show good resistance to all three herbicides are discarded.

[0227] Representative results are shown in Figure 2 As shown. The photos were taken two weeks before and after the herbicide application. Figures A1 and A2 show wild-type beets before and after the herbicide application; Figures B1 and B2 show the line Bv_CSM63713 before and after the herbicide application; Figures C1 and C2 show another line before and after the herbicide application. The herbicide treatments (from left to right) are: 1. A triple mixture of glyphosate + glufosinate + dicamba; 2. A double mixture of glyphosate + dicamba; 3. dicamba; 4. glufosinate; 5. glyphosate; and 6. Untreated control. As shown Figure 2 As shown in Figures C1 and C2, wild-type beet plants failed to withstand any herbicide treatment, and only the plants depicted in Figures C1 and C2 survived after the glufosinate treatment. In contrast, the Bv_CSM63713 line showed excellent tolerance to all herbicide treatments. A plant of the Bv_CSM63713 line showed partial damage after glyphosate treatment, but this was most likely due to damage caused during potting before the herbicide treatment.

[0228] Combining the molecular analysis data and R0 herbicide tolerance testing, 44 unique lines were selected from the initial 1803 unique transformed lines generated using the four transformation constructs and four transformed genotypes to enter the first year of field trials.

[0229] Table 5. Herbicide concentrations

[0230]

[0231] Example 2: First year field trial

[0232] This example describes the first year of field trials containing plants from each of the 44 selected unique lines. For each unique line, thousands of plants were tested for trait efficacy (herbicide tolerance) and agronomic performance. These data were analyzed to compare the performance of each line across all plants and all locations under field conditions. The first year field performance data and molecular analysis data were then used to select superior lines for the second year of field trials.

[0233] In the first year of field trials, 44 unique lines were selected from the original 1803 lines. These 44 lines represent the best lines in four constructs: 6 lines from HT1, 7 lines from HT2, 11 lines from HT3, and 20 lines from HT4. Hybrid seeds were produced from the selected lines by open pollination of the corresponding lines with sterile female parents from the partition (the pollinators are homozygous or heterozygous for the transgene). The trait efficacy field trial adopted a split-plot design (the main plot was chemically treated, and the sub-plot was a single line) and was tested at two locations in the United States, with two replicates at each location. The size of a single plot was 3.5 feet × 10 feet, with a 5-foot wide path between each plot. Each plot had 2 rows and the planting distance was 2 inches.

[0234] In the first year of the trait efficacy trial, the tolerance of F1 hybrid plants to dicamba, glyphosate and glufosinate was evaluated. Herbicides were applied to all plants at three different stages of beet plant development: treatment 1: 2 true leaves stage; treatment 2: 6-8 leaves stage; treatment 3: 8-12 leaves stage. The efficacy trial included a total of nine treatments: 1) untreated control, where weeds were manually removed; 2) 2x glyphosate; 3) 4x glyphosate; 4) 2x glufosinate; 5) 4x glufosinate; 6) 2x dicamba; 7) 4x dicamba; 8) 2x dicamba and glyphosate tank mix; and 9) 4x dicamba and glyphosate tank mix (1x and 4x ratios for each herbicide are provided in Table 5). The percentage of plant damage caused by the herbicide was visually assessed 10-14 days after each herbicide application based on estimated growth reduction (stunting), chlorosis and necrosis.

[0235] Trait efficacy data from the first year of field trials were compiled. For each unique line, the pooled data from both locations and all individual plants were meta-analyzed to compare hybrid injury ratings. Table 6 provides the mean injury ratings for each line across the herbicide treatment regimens at both locations. Table 7 provides a description of the injury rating scale. The results showed that, on average, plants from all lines experienced low herbicide injury.

[0236]

[0237]

[0238]

[0239] Table 7. Description of the impairment scale

[0240] scale Trait damage (plot quality) 1 No symptoms / no stunting / no leaf yellowing 2 Only a few plants have a pale yellow color 3 <5% of plants yellowing / chlorotic / stunted 4 <15% of plants yellowing / chlorotic / stunted 5 <25% of plants yellowing / chlorotic / necrotic / stunted 6 <38% of plants yellowing / chlorotic / stunted 7 <50% of plants yellowing / chlorotic / necrotic / stunted 8 <75% of plants yellowing / chlorotic / stunted 9 Entire plot affected / dead

[0241] Example 3: Molecular Characterization

[0242] This example describes the extensive molecular characterization of selected lines that was performed in parallel with the field trials. The molecular characterization of each line was used to assess whether the line should be selected for the next step.

[0243] DNA and RNA analysis was performed on the strains 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 transgenic without any vector backbone sequence. DNA amplification and sequencing were used to confirm the composition and integrity of the insert sequence in the transgenic insert of each strain. The DNA at each end (5' end and 3' end) of the flanking transgenic insert was sequenced and the corresponding junction was determined. Northern analysis was performed to detect and measure the mRNA transcripts of the CP4 gene, DMO gene and PAT gene in the transgenic plants of each strain.

[0244] Protein analysis was performed on plants containing each strain using techniques known in the art. N-terminal protein sequencing was performed on CP4, DMO and PAT proteins purified from transgenic plants containing each strain to confirm the recombinant protein sequence. Western blot analysis was performed on protein extracts from plants containing each strain to confirm the production of CP4, DMO and PAT proteins.

[0245] Each strain was analyzed for its insertion site in the genome. Bioinformatics analysis was performed on the chromosomal position of the insert using flanking sequences, and the insertion site of each strain was mapped to the sugar beet genome. DNA amplification was performed on the wild-type allele in the genome using primers specific to the flanking regions of each strain. The unique site of transgenic integration of the strain was mapped to the sugar beet reference genome using the wild-type insertion site sequence.

[0246] A total of 17 unique lines were selected from the 44 lines tested in the first year of field trials for further investigation based on comprehensive and in-depth molecular characterization of each line and trait performance data obtained from the first year of field trials.

[0247] Embodiment 4: second year field test

[0248] The present embodiment has described the second year field test that the plant of each of 17 unique strains that contain the first year field test development from construct HT1, HT2, HT3 and HT4 is carried out.For each unique strain, the trait effect (medamba, glyphosate and glufosinate tolerance), agronomic performance and the output of many individual plants at two locations in the test field.Analyze these data to compare the performance of each strain of all plants and two locations under field conditions.Then, use the data of the second year field test to select excellent strains to enter the third year field test.

[0249] The hybrid F1 seeds (hemizygous for the strain) for the second year field trial are produced by open pollination of the sterile female parent in the partition and the selected strain. In addition, R2 seeds from five advanced strains (homozygous for the strain) are produced via open pollination in the partition to test in the field trial. The trait efficacy field trial adopts a split plot design (the main plot is chemically treated, and the sub-plot is a single strain), and is tested at two locations in the United States, and each location is tested twice. The size of a single plot is 3.5 feet × 10 feet, with a 5-foot wide trail between each plot. Each plot has 2 rows, and the planting distance is 2 inches.

[0250] In the second year of the trait efficacy trial, 17 unique lines were selected for testing, including one line of construct HT1, three lines of construct HT2, four lines of construct HT3, and nine lines of construct HT4. Plants from these lines were evaluated for tolerance to dicamba, glyphosate, and glufosinate. Herbicides were applied to all plants at three different stages of beet plant development: Treatment 1: 2 true leaf stage; Treatment 2: 6-8 leaf stage; Treatment 3: 8-12 leaf stage. The efficacy trial included a total of ten treatments: 1) untreated control; 2) 2x dicamba; 3) 2x glufosinate; 4) 2x glyphosate; 5) 2x glyphosate and dicamba tank mix; 6) 2x glyphosate and dicamba tank mix followed by 2x glufosinate; 7) 4x dicamba; 8) 1x glufosinate; 9) 4x glufosinate; and 10) 4x glyphosate (Table 5 provides the 1x and 4x rates for each herbicide). The percentage of plant damage caused by the herbicide was visually assessed 10-14 days after each herbicide application based on estimated growth reduction (stunting), chlorosis, and necrosis.

[0251] Trait efficacy data from the second year field trials were compiled. For each unique line, the pooled data for both locations and all individual plants were meta-analyzed, comparing injury ratings for hybrids and homozygous lines (if applicable) using the injury ratings provided in Table 7. Table 8 provides the average injury ratings for each line for the herbicide treatment regimens at both locations. The meta-analysis of the trait efficacy field trials showed that, on average, plants of all four constructs performed very well with low herbicide injury ratings.

[0252]

[0253] In the second year field trial, an agronomic performance field trial was also conducted to assess whether transgenics had any effect on yield compared to conventional counterparts. In addition, the effect of post-emergence application of glufosinate on the yield of each strain was evaluated. The agronomic performance field trial included six unique strains. These strains included one strain from construct HT2, two strains from construct HT3, and three strains from construct HT4. The trial was conducted in a split plot design at three locations in the United States, with five replicates at each location. The size of a single plot was 5.5 feet × 27 feet. Each plot had 3 rows, and the planting distance was 2 inches. These plots were sparsely divided into 6-inch squares.

[0254] In agronomic performance trials conducted on six unique lines, the beetroot yield was measured as beets harvested from each plot and expressed in tonnes / ha by weight. The polarization / sugar content (POL) of each individual plot was calculated by near infrared spectroscopy (NIRS) analysis and expressed as a percentage of fresh matter. From these two components the sugar yield was calculated in tonnes / ha.

[0255] In the comprehensive test analysis of series A (conventional counterparts and invalid segregants tested without glyphosate treatment) and series B (experience glyphosate treatment to select segregating hybrids) carried out in all three test sites, no significant differences in sugar yield were observed between all six unique strains and their conventional counterparts. Strain Bv_CSM63713 is one of the best performing strains. In all three test sites, the sugar content in the unique strain is significantly higher than the sugar content in the conventional counterpart based on the site. In the comprehensive test analysis, the unique strain presents a trend of higher sugar content. However, the difference is not significant (Table 9). "Inspection" in Table 9 refers to the well-known varieties (reference materials) that are usually used for relativized data.

[0256] In the agronomy field trial, the effect of post-emergence glufosinate treatment (1x) and glyphosate treatment (1x) on yield was also evaluated according to six unique strains. No phenotypic differences between the two different herbicide treatments were observed. However, in all six strains using glufosinate treatment, sugar yield reduction was observed. The reduction in sugar yield was caused by the reduction in root yield (Table 10). However, sugar content was not affected by the treatment.

[0257] In Table 9 and Table 10, LSD stands for the least significant difference, which allows to determine how much two levels (e.g., varieties, treatments) must differ to be considered significantly different from each other at a 5% error level. The LSD measures the variability / differences in the test, which may be due to specific treatments, such as the presence / absence of transgenic insertions in the sugar beet genome (as shown in Table 9) or the treatment of glyphosate versus glufosinate (as shown in Table 10). The value of LSD means that the difference in the parameter (e.g., sugar yield or sugar content in Table 9) must be greater than the LSD value indicated between the two different treatments to be considered significant. For example, in Table 9, the sugar yield of the hybrid variety of "test 1x Bv_CSM63713" is 13.1, while the sugar yield of the hybrid variety of "test 1x strain E" is 12.7. Since the difference between 13.1 and 12.7 is less than 1.3 (LSD value), the difference is not considered significant (Table 9).

[0258]

[0259] Table 10: Summary of yield trial data from combined analysis of three sites treated with glyphosate (Gly) or glufosinate (Glu)

[0260]

[0261] Embodiment 5: the third year field test

[0262] The present embodiment describes the third year field test that the plant of each of six unique strains containing the second year field test development from construct HT3 and HT4 is carried out.For each unique strain, the trait effect (medamba, glyphosate and glufosinate tolerance), agronomic performance and yield of many individual plants at one location in the test field.Analyze these data to compare the performance of each strain of all plants under field conditions.Then, utilize the data and molecular analysis data of the third year field test to select a final strain.

[0263] The hybrid F1 seeds (hemizygous for the strain) for the third year field test are produced by open pollination of the sterile female parent in the partition and the selected strain. In addition, the homozygous seeds of strain Bv_CSM63713 are also produced by open pollination in the isolated field. These seeds are used to carry out the trait efficacy field test. The test adopts the split plot design (the main plot is chemical treatment, and the subplot is a single strain), and is tested at a location in the U.S., wherein two replicate tests are carried out. The size of the single plot is 60 inches × 8 feet, wherein there is a 22-inch wide trail between each plot. Each plot has 4 rows, and the planting distance is 2 inches.

[0264] In the third year of the trait efficacy trial, six unique lines were selected for testing, three lines of construct HT3 and three lines of construct HT4. Plants from these lines were evaluated for tolerance to dicamba, glyphosate, and glufosinate. Herbicides were applied to all plants at three different stages of sugar beet plant development: Treatment 1: 2 true leaf stage; Treatment 2: 6-8 leaf stage; Treatment 3: 8-12 leaf stage.

[0265] The efficacy trial included a total of 10 treatments: 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) 2x dicamba and glyphosate tank mix; and 10) 2x dicamba and glyphosate tank mix followed by 2x glufosinate (1x and 4x rates for each herbicide are provided in Table 5). The percentage of plant injury caused by the herbicide was visually assessed 10-14 days after each herbicide application based on estimated growth reduction (stunting), chlorosis, and necrosis.

[0266] Trait efficacy data from the third year field trials were compiled. For each unique line, the combined data of replicates and all individual plants were meta-analyzed to compare injury ratings for hybrid lines and homozygous lines (when applicable). Table 11 provides the average injury rating for each line in the herbicide treatment regimen using the injury ratings provided in Table 7. The meta-analysis of the trait efficacy field trials showed that, on average, plants of both constructs performed very well with low herbicide injury ratings.

[0267]

[0268] In the third year of field trials, an agronomic performance field trial was also conducted to assess whether the transgenics had any effect on yield compared to conventional counterparts. The agronomic performance field trial included four unique lines. These lines included two lines from construct HT3 and two lines from construct HT4. The trial was conducted in a randomized complete block design at four locations in the United States, with three replicates at each location. The size of a single plot was 5.5 feet by 27 feet, with an 8-foot wide trail between each plot. Each plot had 3 rows, and the planting distance was 2 inches. The plots were sparsely divided into 6-inch squares.

[0269] In the agronomic performance test of four unique strains, the yield of beetroot was measured with beets harvested from each plot and expressed in tons / hectare weight. The polarization / sugar content (POL) of each single plot was calculated by NIRS analysis, expressed as a percentage of fresh matter. Sugar yield was calculated based on these two components, in tons / hectare. In the agronomic test of the previous year, it was observed that the sugar content of the test strain increased compared to the conventional counterpart. One difference in the test was that the herbicide-tolerant strain was treated with glyphosate, while the conventional counterpart did not. In the field test of the third year, a group of four unique strains were tested in the yield test at four locations, two of which were included in the yield test of the previous few years. The conventional counterpart and the invalid segregant from one of the strains were included in the test as controls. Glyphosate was not applied to the plants. In fact, weeds were controlled using classic beet herbicides and manual weeding. As shown in Table 12, it was observed that the sugar content in all four strains increased significantly, consistent with the observations of last year. The null segregants also showed an increase in sugar content. Except for line HT4-22, no significant differences in sugar yield and root yield were detected between the transgenic lines and the conventional counterparts (Table 12). The definitions of LSD and "check" in Table 12 are the same as those in Table 9 above.

[0270]

[0271] Among the unique strains in the two-year agronomic trial, strain Bv_CSM63713 showed the best performance. This strain was further tested to evaluate the effects of different herbicide treatments on its yield performance. Herbicide treatments included: 1) 1x dicamba pre-emergence treatment (PRE); 2) 2x dicamba, using 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 (1x ratio of each herbicide is provided in Table 5). Due to weather conditions, only two of the four locations were treated pre-emergence. However, the test material was similar in all four locations, allowing a comprehensive analysis of all four test locations. Comprehensive analysis showed that, except for the sugar yield and root yield reduction observed in the 2x Mediben / glyphosate + 2x glufosinate pre-emergence treatment, there was no significant difference in sugar yield, sugar content and root yield between manual weeding and different herbicide treatments (Table 13). The LSD in Table 13 is defined in the same manner as described above in Table 9, Table 10 and Table 12.

[0272]

[0273]

[0274] Example 6: Final selection of sugar beet line Bv_CSM63713

[0275] As described in the previous embodiment, through several years of strict and meticulous strain selection, select multiple strains and characterize from 1803 unique transformation strains produced by four different transformation constructs.Strain selection covers molecular characterization and greenhouse and field trials for effect, agronomy and yield performance.For molecular characterization, only the strain that meets the following standard will be selected for further testing: there is no Agrobacterium and left border / right border excessive reading, there is single copy and complete transgenic is in the definite genome position and transgenic is not inserted or close to any endogenous gene.Similarly, selected strain demonstrates trait efficacy, and does not have negative impact on plant growth and development, agronomy and yield performance under many years, multiple locations and various growth conditions.Table 14 summarizes the result of this extensive and intensive selection process, demonstrates the reason that most strains are discarded, identifies and selects beet strain Bv_CSM63713 as the best strain of commercial development thus.

[0276] Table 14. Summary of strain selection and reasons for discarding

[0277]

[0278]

[0279] Example 7: Molecular Characterization of Sugar Beet Line Bv_CSM63713

[0280] This example describes the extensive molecular characterization of the sugar beet line Bv_CSM63713. Characterization included confirmation of single copy insertion at a "neutral" insertion site within the sugar beet genome, determination of the insertion site on the chromosome, identification of transgenic flanking sequences, confirmation that the insert sequence matches the transformation construct, and confirmation that there is no read-through of the transgene on the left or right borders. The transgenic insert of the sugar beet line Bv_CSM63713 contains the elements and sequences described in Table 15.

[0281] Sugar beet strain Bv_CSM63713 was subjected to DNA sequence analysis. Southern blot analysis was performed to confirm that the plant containing sugar beet strain Bv_CSM63713 contained a single complete copy of the entire transgenic insert, without any transformation vector backbone sequence. According to the publicly accessible sugar beet genome (Beta vulgaris resources, http: / / bvseq.boku.ac.at / index.sh tml), the insertion site of the transgenic was determined by target locus amplification (TLA) (Paula JP de Vree et al., 2014). The flanking DNA at the 5' end and 3' end of the transgenic insert was PCR amplified and sequenced. Then, the flanking DNA sequence of sugar beet strain Bv_CSM63713 was mapped to the high-quality wild-type genome physical assembly obtained by whole genome sequencing. The chromosome position of the strain was subjected to bioinformatics analysis using the insertion site sequence information. The integrity of the insertion site was determined by PCR across the wild-type allele using primers specific for the flanking regions of the sugar beet line Bv_CSM63713. The unique transgenic integration site of sugar beet line Bv_CSM63713 was mapped to the sugar beet reference genome using the wild-type insertion site. To ensure that no alterations or mutations were introduced into any region of the transgenic insert during the transformation process, the entire transgenic insert of sugar beet line Bv_CSM63713 was isolated from the plant and sequenced. The recovered sequences were aligned with the sequences from the transformation vector to confirm that they were identical. The sequence information of the 5' junction, 3' junction, and 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 Figure 1 As shown in SEQ ID NO: 13, a seven nucleotide deletion was observed at the insertion site.

[0282] In addition, deep gene prediction was performed for the integration site and an additional 50 kb flanking region at both sites using a high-quality pre-trained sugar beet Augustus gene model. The results showed that no genes were present at the insertion site (hence, this insertion site was referred to as a "neutral insertion site"), and no markers for functional genes were present in the 5' and 3' flanking regions.

[0283] Table 15 Elements and description of sugar beet line Bv_CSM63713

[0284]

[0285]

[0286]

[0287] RNA analysis was performed on plants containing sugar beet line Bv_CSM63713. Northern blot analysis was performed on total RNA isolated from leaf tissue of plants containing sugar beet line Bv_CSM63713. The results confirmed that CP4, DMO and PAT mRNA transcripts were expressed in line Bv_CSM63713 with the expected correct size and amount.

[0288] Plants comprising strain Bv_CSM63713 are subjected to protein analysis. Protein extracts from leaf tissues using immunopurification are used to carry out N-terminal protein sequencing of expressed CP4-EPSPS, DMO and PAT proteins to confirm true N-terminal amino acid sequences. Protein extracts from leaf tissues comprising beet strain Bv_CSM63713 are subjected to Western blotting analysis to confirm that proteins of a single expected size of CP4-EPSPS, DMO and PAT have been produced respectively. ELISA is used to determine the protein level of CP4-EPSPS, DMO and PAT proteins in plant leaves.

[0289] Example 8: Detection of sugar beet line Bv_CSM63713

[0290] This embodiment describes the detection of sugar beet strain Bv_CSM63713. DNA, RNA or protein detection techniques can be used to detect the strain in a sample. A robust and easily expandable specific qualitative detection method can track specific strains 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 sugar beet strain Bv_CSM63713 in a sample. The DNA sequence information of sugar beet strain Bv_CSM63713 is provided herein as SEQ ID NO:1-12. The transgenic insert of sugar beet strain Bv_CSM63713 contains the elements described in Table 15.

[0291] 1) Using KASP to detect sugar beet line Bv_CSM63713

[0292] A competitive allele-specific PCR (KASP, LGC, Teddinton, UK) genotyping assay was developed to identify the sugar beet line Bv_CSM63713 in samples. The assay is based on competitive allele-specific PCR and is capable of biallelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci.

[0293] The KASP assay mix contains three assay-specific unlabeled oligonucleotides: two allele-specific forward primers and one universal reverse primer. Two sets of primer combinations were developed for strain-specific KASP assays. The primers and corresponding SEQ ID NOs are listed in Table 16. Primer set 1 txht024d01 targets the 5' region of SEQ ID NO: 10, while primer set 2 txht024d02 targets the 3' region of SEQ ID NO: 10. The strain-specific assay can be performed using both sets of primers independently or using only one set of primer combinations.

[0294] The KASP assay mix and the universal KASP master mix were added to the DNA sample and then subjected to a thermal cycling reaction followed by an endpoint fluorescence readout. Each of the allele-specific primers carries a unique tail sequence (underlined in Table 16) that corresponds to a universal fluorescence resonance energy transfer (FRET) box; one labeled with a FAM dye and the other labeled with a HEX dye. The KASP master mix contains a universal FRET box, a ROX passive reference dye, Taq polymerase, free nucleotides, and MgCl2 in an optimized buffer solution. In KASP PCR, during thermal cycling, the relevant allele-specific primer binds to the template and is extended, thereby attaching the tail sequence to the newly synthesized strand. The complementary sequence of the allele-specific tail sequence is then generated during subsequent rounds of PCR, enabling the FRET box to bind to the DNA. The FRET box is then no longer quenched and fluoresces. A fluorescent signal is generated when the sugar beet strain Bv_CSM63713 is present in the sample. However, when the strain is not present in the sample (eg, a wild-type or non-transgenic plant), no fluorescent signal is generated.

[0295] Thus, the primer set txht024d01 or txht024d02 or both, when used independently in the reaction method, will produce a DNA amplicon that can be used to diagnose the sugar beet strain Bv_CSM63713. Controls for this analysis should include a positive control containing sugar beet strain Bv_CSM63713, a negative control from non-transgenic sugar beets, and a negative control that does not contain template DNA. Alternatively, each set of primers without an underline tail can be used independently or in combination to perform PCR amplification and detect strain-specific amplicons using techniques known in the art.

[0296] Table 16. Strain-specific KASP primers

[0297]

[0298] 2) Use antibody to detect beet strain Bv_CSM63713

[0299] Another example of detecting sugar beet strain Bv_CSM63713 is a detection kit comprising at least one antibody specific for at least one protein encoded by sugar beet strain Bv_CSM63713. For example, such a kit can utilize a lateral flow strip comprising a reagent that is activated when the tip of the lateral flow strip is in contact with an aqueous solution. An exemplary protein sufficient for antibody generation is a protein encoded by the sequence provided in SEQ ID NO: 10 or any fragment thereof.

[0300] A protein detection method is developed to determine whether a sample is from a plant, seed, cell or plant part (e.g., root) containing beet strain Bv_CSM63713. At least one antibody specific to at least one protein encoded by beet strain Bv_CSM63713 is used to detect the protein encoded by beet strain Bv_CSM63713 in the sample. A detection kit comprising one or more antibodies specific to one or more proteins encoded by beet strain Bv_CSM63713 can utilize a lateral flow strip containing a reagent activated when the tip of the lateral flow strip contacts an aqueous solution. Samples of beet tissue can be ground and proteins are extracted using water or an aqueous buffer (e.g., phosphate buffered saline containing detergent and bovine serum albumin) for analysis. After centrifugation, an aqueous supernatant is analyzed in a sandwich format using an ELISA method on a lateral flow strip containing an absorbent pad. Activate the detection by immersing the tip of the strip in an aqueous solution containing the sample to be tested.

[0301] The aqueous solution is brought to the strip by capillary action and dissolves the gold-labeled antibody on the strip. The gold-labeled antibody is specific to at least one protein encoded by the beet strain Bv_CSM63713 and will bind to the epitope on the protein in the sample to form an antibody-antigen complex. Then, the gold-labeled antibody-antigen complex is brought to the nitrocellulose membrane by the strip. The membrane contains a test line fixed with an antibody, which binds to a second independent epitope on the protein encoded by the beet strain Bv_CSM63713, thereby a visible line will appear on the test strip when the protein encoded by the beet strain Bv_CSM63713 is present in the sample.

[0302] 3) Detection of sugar beet line Bv_CSM63713 using real-time PCR

[0303] A qualitative real-time strain-specific PCR method was developed to identify the sugar beet strain Bv_63713 in samples. The forward primer 2109_fwd1 (SEQ ID NO: 34), the reverse primer 2109_rev1 (SEQ ID NO: 35) and the probe 2109_probe1 (SEQ ID NO: 36) were used for qualitative detection of sugar beet genomic DNA. The probe was labeled with 6-carboxyfluorescein (6-FAM) at the 5' end. TM ), and is labeled with Minor Grove Binder non-fluorescent quencher (MGBNFQ) at the 3' end. The 5' exonuclease activity of Taq DNA polymerase cuts the probe from the 5' end between the fluorophore and the quencher. When hybridized with the target DNA chain, the quencher and the fluorophore are fully separated to produce a fluorescent signal, thereby releasing fluorescence. Primers 2109_fwd1 and 2109_rev1 produce a DNA amplicon that can be used to diagnose the sugar beet strain Bv_63713 when used in conjunction with the reaction method and probe 2109_probe1. Appropriate controls were used in the analysis, including a positive control containing the sugar beet strain Bv_63713, a negative control from non-transgenic sugar beets, and a negative control without template DNA. In addition, the analysis also contains internal control primers and probes that are specific to single-copy genes in the sugar beet genome.

[0304] An example of the composition of the reaction mixture for the sugar beet Bv_63713 line-specific PCR method is shown in Table 17. TM The real-time PCR detection system performs PCR amplification reaction according to the PCR cycle conditions listed in Table 18.

[0305] Table 17. Reaction mixture for Bv_63713 strain-specific PCR method

[0306] Components Stock concentration Volume (μl) Final concentration Sigma Jumpstart Taq Ready-to-Use Mix 2 X 10 1X ROX 50X 0.04 0.1X <![CDATA[MgCl2]]> 100mM 1.1 5.5mM 2109_fwd1 (SEQ ID NO: 34) 10μM 0.6 300nM 2109_rev1 (SEQ ID NO: 35) 10μM 0.6 300nM 2109_probe1 (SEQ ID NO: 36) 10μM 0.3 150nM Template DNA 2ng / μl 5 0.5ng / μl Nuclease-free water 2.36 total 20

[0307] Table 18. PCR Cycling Conditions

[0308]

[0309] An example of an endogenous gene of beet used as an internal control is glutamine synthetase (GS). A 121 bp fragment of the glutamine synthetase gene was amplified using GS-specific primers and a GS-specific probe (labeled with 6-FAM as a reporter dye at the 5' end and BHQ1 as a quencher at the 3' end). The primer and probe sequences are shown in Table 19. The components of the reaction mixture are listed in Table 20. The 121 bp fragment of the glutamine synthetase gene was amplified using a Bio-Rad CFX96 Touch TM The real-time PCR detection system performs PCR amplification reaction according to the PCR cycle conditions listed in Table 18.

[0310] Table 19. Primer and probe sequences for glutamine synthetase-specific PCR assay

[0311]

[0312] Table 20. Reaction mixture for glutamine synthetase specific PCR assay

[0313]

[0314] Example 9: Determination of zygosity of sugar beet line Bv_CSM63713

[0315] This example describes a method that can be used to determine the zygosity of line Bv_CSM63713. The zygosity assay will determine whether a plant comprising the sugar beet line Bv_CSM63713 is heterozygous or homozygous for the line or wild type alleles.

[0316] 1) KASP zygosity assay

[0317] As described in Example 8, a KASP genotyping assay for zygosity was developed based on competitive allele-specific (KASP) PCR. Bi-allelic discrimination is achieved by competitive binding of two allele-specific forward primers. If the plant comprising the sugar beet line Bv_CSM63713 is homozygous, only one of the two possible fluorescent signals will be generated. If the plant comprising the sugar beet line Bv_CSM63713 is heterozygous, a mixed fluorescent signal will be generated.

[0318] The KASP assay mix and the universal KASP master mix are added to the DNA sample and then subjected to a thermal cycling reaction followed by an endpoint fluorescence readout. The allele-specific primers each carry a unique tail sequence (underlined in Table 21) that corresponds to a universal fluorescence resonance energy transfer (FRET) box; one is labeled with a FAM dye and the other is labeled with a HEX dye. The KASP master mix contains a universal FRET box, a ROX passive reference dye, Taq polymerase, free nucleotides, and MgCl2 in an optimized buffer solution. During thermal cycling, the associated allele-specific primer binds to the template and is extended, thereby attaching the tail sequence to the newly synthesized strand. The complementary sequence of the allele-specific tail sequence is then generated during subsequent rounds of PCR, enabling the FRET box to bind to the DNA. The FRET box is then no longer quenched and emits fluorescence.

[0319] Two sets of primer combinations for strain zygosity determination were developed. Primers and 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 strain. Therefore, the AlleleX primer detects the wild-type sequence, while the AlleleY primer detects the strain. The zygosity determination can be performed using two sets of primers independently or only one set of primer combinations. The results of the determination show that the homozygous wild-type plants only produce one signal, while the homozygous transgenic plants for the strain only produce one different signal. However, the heterozygous plants for the strain produce signals for both alleles.

[0320] Table 21. Primers used for zygosity assay

[0321]

[0322] 2) Additional zygosity assay

[0323] Develop a zygosity assay to determine whether the plant comprising the beet strain Bv_CSM63713 is heterozygous or homozygous for the strain or wild-type allele. The sequence information design amplification reaction assay provided herein can be used. For example, this PCR assay will include the design of at least three primers: primer-1, primer-2 and primer-3, wherein primer-1 has specificity to the beet genomic DNA on the 3' side joint DNA of the beet strain Bv_CSM63713; primer-2 has specificity to the beet strain Bv_CSM63713 transgenic insert; and primer-3 has specificity to the wild-type allele. When used as primer pairs in the amplification reaction, primer-1 and primer-2 will produce a specific PCR amplicon to the beet strain Bv_CSM63713. When used as primer pairs in the amplification reaction, primer-1 and primer-3 will produce a specific PCR amplicon to the wild-type allele. In a PCR reaction performed on sugar beet genomic DNA, the corresponding PCR amplicon produced by primer-1 + primer-2 and the corresponding PCR amplicon produced by primer-1 + primer-3 will differ in the sequence and size of the amplicon. When these three primers are included in a PCR reaction performed using DNA extracted from a homozygous plant of sugar beet strain Bv_CSM63713, only the primer-1 + primer-2 amplicon (specific for sugar beet strain Bv_CSM63713) will be produced. When these three primers are included in a PCR reaction performed using DNA extracted from a heterozygous plant of sugar beet strain Bv_CSM63713, both the primer-1 + primer-2 amplicon (specific for the sugar beet strain Bv_CSM63713 insert) and the primer-1 + primer-3 amplicon (specific for the wild-type allele or the absence of the sugar beet strain Bv_CSM63713 insert) will be produced. When these three primers are mixed together in a PCR reaction using DNA extracted from a (wild-type) plant that does not contain beet strain Bv_CSM63713, only primer-1 + primer-3 amplicons (specific for the wild-type allele) will be produced. Any method known in the art can be used to identify or distinguish the amplicons produced using the PCR reaction.

[0324] Another method for detecting the presence and zygosity of sugar beet strain Bv_CSM63713 in plant samples is Southern blot analysis. Those skilled in the art will understand how to design a first Southern hybridization probe that is specific to sugar beet strain Bv_CSM63713 and a second Southern hybridization probe that is specific to sugar beet plants that do not contain sugar beet strain Bv_CSM63713 (wild type). By Southern blot analysis, the signal detected only from the first Southern hybridization probe will indicate that the plant is homozygous for sugar beet strain Bv_CSM63713; the signal detected from both the first hybridization probe and the second hybridization probe will indicate that the plant is heterozygous for sugar beet strain Bv_CSM63713; and the signal detected only from the second Southern hybridization probe will indicate that the DNA was extracted from a plant that does not contain sugar beet strain Bv_CSM63713 (wild type).

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Claims

1. A recombinant DNA molecule comprising a sequence selected from the group consisting of 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; SEQ ID NO: 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 SEQ ID NO: 10 or the full length of SEQ ID NO: 9; and the complete complementary sequence of any of the foregoing.

2. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule: a. A beet plant, seed, plant part, plant cell, progeny plant or commercial product derived from a beet strain Bv_CSM63713, a representative sample of seeds of said strain having been deposited under ATCC Accession No. PTA-127098; b. contained in a beet plant, seed, plant part, plant cell or progeny plant containing the beet strain Bv_CSM63713, or in a commercial product produced therefrom, a representative sample of seeds containing the strain having been deposited under ATCC Accession No. PTA-127098; c. formed by inserting a heterologous nucleic acid molecule into the genomic DNA of a beet plant or beet cell; or d. Contains an amplicon diagnostic for the presence of sugar beet line Bv_CSM63713.

3. A DNA molecule comprising a polynucleotide segment of sufficient length to serve as a DNA probe, wherein the polynucleotide segment: a. specifically hybridizing with the sugar beet strain Bv_CSM63713 DNA in the sample under stringent hybridization conditions, wherein the detection of hybridization of the DNA molecule under the stringent hybridization conditions will diagnose the presence of sugar beet strain Bv_CSM63713 in the sample; or b. Specific detection of at least one of the following in a sample: i) 5' junction sequence between the flanking sugar beet genomic DNA and the transgenic insert of sugar beet line Bv_CSM63713; ii) 3' junction sequence between the transgenic insert and the flanking sugar beet genomic DNA of sugar beet line Bv_CSM63713; iii) SEQ ID NO:9; and iv) a fragment of SEQ ID NO:9 comprising a sufficient length of contiguous nucleotides of SEQ ID NO:9 to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713.

4. The DNA molecule of claim 3, wherein: a. the DNA probe comprises SEQ ID NO:36; b. the DNA molecule 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 and the complementary sequence of any of the foregoing; and / or c. The sample originates from a sugar beet plant, seed, plant part, plant cell, progeny plant or commercial product.

5. A DNA molecule pair, comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule comprise a fragment of SEQ ID NO: 10 or its complementary sequence, and when used in an amplification reaction together with a DNA comprising the sugar beet strain Bv_CSM63713, act as DNA primers to produce an amplicon of the sugar beet strain Bv_CSM63713 in a diagnostic sample.

6. The DNA molecule pair according to claim 5, wherein: a. The first DNA molecule and the second DNA molecule comprise 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 comprises a nucleotide sequence selected from the group consisting of: 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 xi. A fragment 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, SEQ ID NO:8, wherein the fragment is at least 10 nucleotides long and comprises nucleotides 1000-1001 or 12,722-12,723 of SEQ ID NO:

10.

7. A method for detecting the presence of sugar beet line Bv_CSM63713 in a sample derived from a sugar beet seed, plant, plant part, plant cell, progeny plant or commercial product, the method comprising: a. contacting the sample with a DNA molecule acting as a DNA probe according to claim 3; b. subjecting the sample and the DNA molecule acting as a probe to stringent hybridization conditions; and c. detecting the hybridization of the DNA molecule acting as a probe with the DNA molecule in the sample, Wherein hybridization of said DNA molecule acting as a probe with said DNA molecule in said sample will be diagnostic for the presence of sugar beet line Bv_CSM63713 in said sample.

8. A method for detecting the presence of sugar beet line Bv_CSM63713 in a sample derived from a sugar beet seed, plant, plant part, plant cell, progeny plant or commercial product, the method comprising: a) contacting the sample with the DNA molecule pair according to claim 5; b) performing an amplification reaction sufficient to produce DNA amplicons; and c) detecting the presence of the DNA amplicon; Wherein the DNA amplicon comprises at least one of the following: The 5' junction sequence flanking the sugar beet genomic DNA and the transgenic insert of sugar beet line Bv_CSM63713, The 3' junction sequence flanking the sugar beet genomic DNA and the transgenic insert of sugar beet line Bv_CSM63713, SEQ ID NO:9, and A fragment of SEQ ID NO:9 comprising a sufficient length of contiguous nucleotides of SEQ ID NO:9 to identify the sequence as a fragment of the transgenic insert of Bv_CSM63713; and The presence of the DNA amplicon indicates the presence of sugar beet line Bv_CSM63713 in the sample.

9. The method of claim 8, wherein the DNA amplicon: a. is at least 10 nucleotides long, at least 11 nucleotides long, at least 12 nucleotides long, at least 13 nucleotides long, at least 14 nucleotides long, at least 15 nucleotides long, at least 16 nucleotides long, at least 17 nucleotides long, at least 18 nucleotides long, at least 19 nucleotides long, at least 20 nucleotides long, at least 25 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long, at least 40 nucleotides long, at least 45 nucleotides long, at least 50 nucleotides long, at least 60 nucleotides long, at least 70 nucleotides long, at least 80 nucleotides long, at least 90 nucleotides long or at least 100 nucleotides long; and / or b. comprising 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; SEQID NO:1; and a fragment of any one of SEQ ID NO:10, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:5, SEQID NO:4, SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:1, wherein the fragment is at least 10 nucleotides long and comprises nucleotides 1000–1001 or 12,722–12,723 of SEQ ID NO:

10.

10. A method for detecting the presence of sugar beet line Bv_CSM63713 in a DNA sample derived from a sugar beet seed, plant, plant part, plant cell, progeny plant or commercial product, the method comprising: a) contacting the sample with the DNA molecule according to claim 3; and b) performing a sequencing reaction to generate a target sequence, wherein the 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 complete complementary sequence of any one thereof; and a fragment 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, SEQ ID NO:8 and SEQ ID NO:10, wherein the fragment is at least 10 nucleotides in length and comprises nucleotides 1000–1001 or 12722–12723 of SEQ ID NO:

10.

11. A method for detecting the presence of sugar beet line Bv_CSM63713 in a sample derived from a sugar beet seed, plant, plant part, cell, progeny plant or commercial product, the method comprising: a) contacting the sample with at least one antibody specific for at least one protein encoded by the sugar beet line Bv_CSM63713; and b) detecting the binding of the antibody to the protein in the sample, Binding of the antibody indicates the presence of sugar beet line Bv_CSM63713 in the sample.

12. A DNA detection kit for detecting the presence of sugar beet line Bv_CSM63713 in a sample, wherein the kit comprises the DNA primer pair of claim 5.

13. A DNA detection kit for detecting the presence of sugar beet line Bv_CSM63713 in a sample, wherein the kit comprises the DNA molecule serving as a probe as claimed in claim 3.

14. A protein detection kit for detecting the presence of sugar beet strain Bv_CSM63713 in a sample, wherein the kit comprises at least one antibody, and the at least one antibody is specific to at least one protein encoded by the sugar beet strain Bv_CSM63713; wherein detection of the binding of the at least one antibody to at least one protein encoded by the sugar beet strain Bv_CSM63713 in the sample will diagnose the presence of the sugar beet strain Bv_CSM63713 in the sample.

15. A beet plant, plant seed, plant part or plant cell comprising a recombinant DNA molecule, wherein the recombinant DNA molecule comprises a 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 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 the complete complementary sequence of any of the foregoing.

16. The Sugar Beet plant, plant seed, plant part or plant cell of claim 15, wherein: a. The plant, plant seed, plant part or plant cell expresses 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. The plant, plant seed, plant part or plant cell is tolerant to at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate and any combination thereof; c. the plant, plant seed, plant part or plant cell comprises the beet strain Bv_CSM63713, a representative sample of seeds comprising the strain has been deposited under ATCC Accession No. PTA-127098; d. the plant, plant seed, plant part or plant cell is also defined as a progeny plant of any generation of a sugar beet plant comprising the sugar beet line Bv_CSM63713, or a sugar beet plant part, seed or plant cell obtained therefrom; and / or e. The plant part comprises a root, beet, pollen, anther, ovule, ovary, flower, embryo, stem, leaf, microspore, protoplast or callus.

17. A Sugar Beet plant, plant part, plant seed or plant cell comprising Sugar Beet Strain Bv_CSM63713, a representative sample of seeds comprising said Sugar Beet Strain Bv_CSM63713 having been deposited under ATCC Accession No. PTA-127098.

18. A method for controlling or preventing weeds in an area, the method comprising planting sugar beets comprising line Bv_CSM63713 in the area and applying an effective amount of at least one herbicide to control weeds in the area without causing damage to the sugar beets or causing less than about 10% damage to the sugar beets, the herbicide being selected from the group consisting of dicamba, glyphosate, glufosinate and any combination thereof.

19. The method of claim 18, wherein: a. applying said effective amount of at least one herbicide comprises applying at least two or more herbicides during the growing season, said herbicide being selected from the group consisting of: dicamba, glyphosate, glufosinate and any combination thereof; or b. wherein the effective amount of dicamba during the growing season is from about 0.5 lb ae / acre to about 2 lb ae / acre of dicamba; wherein the effective amount of glufosinate during the growing season is from about 0.4 lb ai / acre to about 2.16 lb ai / acre; and / or wherein the effective amount of glyphosate during the growing season is from about 0.75 lb ae / acre to about 2.25 lb ae / acre.

20. A method for controlling volunteer sugar beets comprising sugar beet line Bv_CSM63713 in an area, the method comprising 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 comprising sugar beet line Bv_CSM63713.

21. The method of claim 20, wherein the herbicide other than glyphosate, dicamba or glufosinate is selected from the group consisting of paraquat, clethodim, clopyralid, betaine, flumethoate, 2,4-dichlorophenoxyacetic acid (2,4-D), and acetolactate synthase (ALS) inhibitors such as sulfonylureas (SUs), imidazolinones, triazolopyrimidines, pyrimidinoxybenzoates and sulfonamidocarbonyltriazolinones, and any combination thereof.

22. A method of obtaining seeds or plants of a sugar beet plant that is tolerant to dicamba, glyphosate, glufosinate, or any combination thereof, the method comprising: a) obtaining a population of progeny seeds or plants grown therefrom, at least one of which comprises the sugar beet line Bv_CSM63713; as well as b) identifying at least one first progeny seed comprising or a plant grown therefrom of the Sugar Beet line Bv_CSM63713.

23. The method of claim 22, wherein identifying the progeny seed comprising Sugar Beet Line Bv_CSM63713 or a plant grown therefrom comprises: a. The following steps: i) growing the progeny seeds to produce progeny plants; ii) treating the progeny plants with an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate and any combination thereof; as well as iii) selecting progeny plants that are tolerant to the at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof; b. detecting the presence of beet line Bv_CSM63713 in a sample derived from said generation of seeds or plants grown therefrom; or c. Detecting the presence of at least one protein encoded by Sugar Beet Line Bv_CSM63713 in a sample derived from said progeny seeds or plants grown therefrom.

24. A method for determining the zygosity of a Sugar Beet plant, plant part, plant seed or plant cell comprising Sugar Beet line Bv_CSM63713, the method comprising: a) contacting a sample comprising DNA originating from said Sugar Beet plant, plant part, plant seed or plant cell with a primer set capable of producing a first amplicon diagnostic for the presence of Sugar Beet line Bv_CSM63713 and a second amplicon diagnostic for wild-type Sugar Beet genomic DNA that is free of Sugar Beet line Bv_CSM63713; b) performing a nucleic acid amplification reaction; and c) detecting the first amplicon and the second amplicon, wherein the presence of both amplicons indicates that the plant, plant part, seed or cell is heterozygous for the sugar beet line Bv_CSM63713, and the presence of only the first amplicon indicates that the plant, plant part, seed or cell is homozygous for the sugar beet line Bv_CSM63713.

25. The method of claim 24, wherein the primer set is selected from the group consisting of: 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.

26. A method for determining the zygosity of a Sugar Beet plant, plant part, plant seed or plant cell comprising Sugar Beet line Bv_CSM63713, the method comprising: a) contacting a sample comprising DNA derived from said sugar beet plant, plant part, plant seed or plant cell with a probe set, said probe set comprising at least one first probe that specifically hybridizes to sugar beet line Bv_CSM63713 and at least one second probe that specifically hybridizes to sugar beet genomic DNA but not to sugar beet line Bv_CSM63713, said sugar beet genomic DNA being disrupted by the insertion of heterologous DNA of sugar beet line Bv_CSM63713; and b) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detection of hybridization of only the first probe under the hybridization conditions will diagnose the sugar beet plant, plant part, seed or plant cell as being homozygous for the sugar beet line Bv_CSM63713, and wherein detection of hybridization of both the first probe and the second probe under the hybridization conditions will diagnose the sugar beet plant, plant part, seed or plant cell as being heterozygous for the sugar beet line Bv_CSM63713.

27. A DNA construct comprising a first expression cassette, a second expression cassette and a third expression cassette, wherein: a) the first expression cassette comprises in operable connection: i) a chlorophyll AB binding protein (Cab1) promoter and leader sequence from Arabidopsis thaliana; ii) a phosphinothricin N-acetyltransferase (PAT) coding sequence; and iii) a small heat shock protein (Hsp20) 3'UTR from Medicago truncatula; b) said second expression cassette comprises in operable connection: i) a ubiquitin (Ubq1) promoter, leader sequence and intron from melon; ii) a ribulose bisphosphate carboxylase small subunit (RbcS) chloroplast transit peptide coding sequence from pea; iii) a dicamba monooxygenase coding sequence (DMO); and iv) a putative protein 3'UTR from Medicago truncatula; and c) the third expression cassette comprises in operable connection: i) an inclusion matrix protein enhancer from dahlia mosaic virus; ii) an S-adenosyl-L-methionine synthase (SAMS2) promoter, leader sequence and introns from melon; 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 hypothetical protein 3'UTR from Medicago truncatula.

28. A DNA construct as claimed in claim 27, wherein the DNA construct comprises SEQ ID NO:

9.

29. A method of increasing the tolerance of a sugar beet plant to at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof, comprising: a) inserting the DNA construct according to claim 27 into the genome of a beet cell; b) producing a sugar beet plant from the sugar beet cell; as well as c) selecting a sugar beet plant comprising said DNA construct.

30. The method of claim 29, wherein said selecting comprises treating said sugar beet cell or plant with an effective amount of at least one herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and any combination thereof.

31. A Sugar Beet plant, plant seed, plant part or plant cell having tolerance to three herbicides with different modes of action at a single genomic location.

32. A Sugar Beet plant, plant seed, plant part or plant cell comprising the DNA construct of claim 27.

33. A Sugar Beet plant, plant seed, plant part or plant cell obtained by the method of claim 29.

34. A method for producing a progeny sugar beet plant comprising sugar beet line Bv_CSM63713, the method comprising: a. sexually crossing a first beet plant comprising beet line Bv_CSM63713 with itself or a second beet plant; b. collecting one or more seeds produced by said hybridization; c. growing one or more seeds to produce one or more progeny plants; and d. Selecting at least one first progeny plant or seed comprising the sugar beet line Bv_CSM63713.

35. An inbred or hybrid sugar beet plant or seed comprising the sugar beet line Bv_CSM63713 produced by the method of claim 34.

36. A non-living or non-renewable sugar beet plant material comprising the recombinant DNA molecule of claim 1.

37. A non-living or non-renewable sugar beet plant material comprising: a. The DNA construct of claim 27; or b. Sugar beet line Bv_CSM63713, a representative sample of seeds comprising said Sugar beet line Sugar beet line Bv_CSM63713 has been deposited under ATCC Accession No. PTA-127098.

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

39. A commercial product comprising the DNA construct of claim 27.

40. The commodity product of claim 38, wherein: a. the commercial product is produced by a transgenic beet plant, plant part, plant seed or plant cell comprising the beet line Bv_CSM63713; and / or b. The commercial product includes whole or processed seeds, non-viable seeds, processed plant parts, processed plant tissues, dehydrated plant tissues, dehydrated plant parts, frozen plant tissues, frozen plant parts, plant parts processed for animal feed, fibers, pulp, pulp particles, pulp chips, tailings, juices, syrups, molasses, extracts, raffinates, betaine, separated molasses solubles (SMS) or any other food for human consumption, viable seeds, viable plant parts (such as roots and leaves) or viable plant cells.

41. A method of producing a commodity product, the method comprising: a) obtaining transgenic sugar beet plants, plant parts or plant seeds comprising the sugar beet line Bv_CSM63713; as well as b) producing a commercial product from said transgenic sugar beet plants, plant parts or plant seeds.

42. A method of controlling, preventing or reducing the development of herbicide-tolerant weeds, the method comprising growing in a crop growing environment a sugar beet plant comprising a transgene providing tolerance to herbicides having three different herbicide modes of action at a single genomic location.

43. The method of claim 42, wherein the three different herbicide modes of action are selected from the group consisting of inhibition of glutamine synthetase, inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and benzoate auxin.

44. A method for controlling, preventing or reducing the development of herbicide-tolerant weeds, the method comprising: a. Cultivating a sugar beet plant comprising the DNA construct of claim 27 in a crop growing environment to provide tolerance to herbicides having three different herbicide modes of action at a single genomic location; as well as b. applying at least one herbicide to the crop growing environment, wherein the herbicide is selected from the group consisting of glufosinate, dicamba, glyphosate, and any combination thereof, wherein the sugar beet plant is tolerant to the at least one herbicide.

45. A method of reducing the number of genetic loci for sugar beet breeding by inserting a transgene at a single genomic location to provide tolerance to three different classes of herbicides.

46. ​​A method according to claim 45, wherein: a. The transgene is inserted in the form of a single molecule-linked transgene insert; or b. The transgene is inserted as a single linked transgenic insert and the transgenic insert provides commercial levels of tolerance to at least one herbicide per herbicide mode of action.

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 tolerance to at least one herbicide selected from the group consisting of glufosinate, dicamba, glyphosate and any combination thereof, and wherein the recombinant DNA construct is integrated in the chromosome at a position flanking SEQ ID NO: 11 or SEQ ID NO: 12.

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