WO2012071039A1 - Événement dp-061061-7 de brassica gat et compositions et procédés pour l'identifier et/ou le détecter - Google Patents

Événement dp-061061-7 de brassica gat et compositions et procédés pour l'identifier et/ou le détecter Download PDF

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WO2012071039A1
WO2012071039A1 PCT/US2010/058002 US2010058002W WO2012071039A1 WO 2012071039 A1 WO2012071039 A1 WO 2012071039A1 US 2010058002 W US2010058002 W US 2010058002W WO 2012071039 A1 WO2012071039 A1 WO 2012071039A1
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polynucleotide
seq
plant
herbicide
dna
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PCT/US2010/058002
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WO2012071039A8 (fr
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Lomas Tulsieram
Yongping Zhang
Jayantilal Devabhai Patel
David George Charne
Wenpin Chen
Ferdinand Gerard Thoonen
Chadwick Bruce Koscielny
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Pioner Hi-Bred International, Inc.
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Priority to CA2818918A priority Critical patent/CA2818918A1/fr
Priority to PCT/US2010/058002 priority patent/WO2012071039A1/fr
Publication of WO2012071039A1 publication Critical patent/WO2012071039A1/fr
Publication of WO2012071039A8 publication Critical patent/WO2012071039A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • This invention is in the field of molecular biology. More specifically, this invention pertains to expression of a sequence that confers tolerance to glyphosate.
  • sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 399082seqlist.txt, a creation date of November 24, 2010, and a size of 40 Kb.
  • ASCII American Standard Code for Information Interchange
  • sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
  • the expression of foreign genes in plants is known to be influenced by their location in the plant genome, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) close to the integration site (Weising, et ah, (1988) Ann. Rev. Genet 22:421-477).
  • chromatin structure e.g., heterochromatin
  • transcriptional regulatory elements e.g., enhancers
  • transgene insertion can affect the endogenous gene expression. For these reasons, it is common to produce hundreds to thousands of different events and screen those events for a single event that has desired transgene expression levels and patterns for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
  • weeds unwanted plants
  • An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unharmed.
  • One such treatment system would involve the use of crop plants which are tolerant to an herbicide so that when the herbicide was sprayed on a field of herbicide-tolerant crop plants, the crop plants would continue to thrive while non-herbicide -tolerant weeds were killed or severely damaged.
  • compositions and methods related to transgenic glyphosate-tolerant Brassica plants are provided.
  • the present invention provides Brassica plants containing a transgene which imparts tolerance to glyphosate.
  • the event may be, for example, DP-061061-7.
  • the Brassica plant harboring the transgene at the recited chromosomal location comprises unique genomic/transgene junctions having at least the polynucleotide sequence of SEQ ID NO: 2 or at least the polynucleotide sequence of SEQ ID NO: 12 and/or 13. Further provided are the seeds deposited as Patent Deposit Number
  • PTA- and plants, plant cells, plant parts, seed and plant products derived therefrom are examples of plants, plant cells, plant parts, seed and plant products derived therefrom.
  • Characterization of the genomic insertion site of DP-061061-7 or any other event comprising integration of the glyphosate -tolerance transgene provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof.
  • Various methods and compositions for the identification, detection, and use of the glyphosate-N-acetyltransferase ("GAT" or "glyat") transformation events in Brassica are provided.
  • Figure 1 shows synthesis of plasmid PHP28181. Plasmid PHP28181 was used to produce the GAT Brassica lines.
  • Figure 2 provides a schematic map of plasmid PHP28181.
  • Figure 3 provides a schematic map of insertion DNA, fragment PHP28181A.
  • Figure 5 provides a schematic representation of fragment A from PHP28181 (PHP28181A), specifically a schematic map of Hind IIVNot I fragment (PHP28181A) containing the gat4621 gene cassette that was used for plant transformation to generate DP-061061-7 canola.
  • the fragment size is 2112 bp.
  • the construct-specific primer locations of 09-0-3290/09-0-3288 are indicated on the map.
  • Figure 6 Southern analysis of Construct Specific PCR of Leaf DNA from DP- 061061-7 Brassica and Non-Genetically Modified Control Brassica. PCR amplification with primer set 09-0-3290/09-0-3288 targeting the unique ubiquitin promoter and gatA62 ⁇ junction present in DP-061061-7 canola. Expected PCR amplicon size is 675 bp.
  • Figure 7 Southern analysis of Brassica FatA gene PCR of leaf DNA from DP- 061061-7 Canola and Non-Genetically Modified Control Brassica. PCR amplification of endogenous canola FatA gene with primer set 09-0-2812/09-02813 as positive control for PCR amplification. Expected PCR amplicon size is 506 bp.
  • compositions and methods related to transgenic glyphosate-tolerant Brassica plants are provided.
  • the present invention provides Brassica plants having event DP-061061-7 or another event comprising PHP28181A or an operable fragment or variant thereof.
  • a Brassica plant having event DP-061061-7 for example, has been modified by the insertion of the glyphosate acetyltransferase (glyat4621) gene derived from Bacillus licheniformis .
  • the glyat4621 gene was functionally improved by a gene shuffling process to optimize the kinetics of glyphosate acetyltransferase (GLYAT) activity for acetylating the herbicide glyphosate.
  • a Brassica plant having the event DP-061061-7 is tolerant to glyphosate.
  • the polynucleotides conferring the glyphosate tolerance are inserted at a specific position in the Brassica genome and thereby produce, for example, the DP-061061-7 event.
  • a Brassica plant harboring the DP-061061-7 event at a specific chromosomal location comprises genomic/transgene junctions having a unique polynucleotide sequence exemplified by SEQ ID NO: 2 or at least the polynucleotide sequence of SEQ ID NO: 12 and/or 13, or at least the polynucleotide sequence of SEQ ID NO: 14 and/or 15, or at least the polynucleotide sequence of SEQ ID NO: 16 and/or 17.
  • a brassica plant having in its genome in the following order: a polynucleotide comprising SEQ ID NO: 12, a polynucleotide encoding a glyphosate-N-acetyltransferase and a polynucleotide comprising SEQ ID NO: 13 is provided.
  • Various methods and compositions for the identification, detection, and use of the Brassica DP-061061-7 event are provided herein.
  • vent DP-061061-7 specific refers to a polynucleotide sequence which is suitable for discriminatively identifying event DP-061061-7 in plants, plant material, or in products such as, but not limited to, oil produced from the seeds, or food or feed products (fresh or processed) comprising, or derived from, plant material.
  • Compositions further include seed deposited as Patent Deposit Numbers PTA- and plants, plant cells, and seed derived therefrom.
  • Applicant(s) have made a deposit of at least 2500 seeds of Brassica event DP-061061-7 with the American Type Culture Collection (ATCC), Manassas, VA 20110-2209 USA on November 24, 2010 and the deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Deposits are made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the deposit of seed comprising Brassica event DP-061061-7 will be maintained in the ATCC depository, which is a public depository, for a period of 30 years or 5 years after the most recent request or for the enforceable life of the patent, whichever is longer and will be replaced if it becomes nonviable during that period. Additionally, Applicant(s) will have satisfied all the requirements of 37 C.F.R. ⁇ 1.801 - 1.809, including providing an indication of the viability of the sample upon deposit. Applicant(s) have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce.
  • the term "Brassica” means any Brassica plant and includes all plant varieties that can be bred with Brassica.
  • the term plant includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, stalks, roots, root tips, anthers, and the like. Mature seed produced may be used for food, feed, fuel or other commercial or industrial purposes or for purposes of growing or reproducing the species. Progeny, variants and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise a DP-061061-7 event.
  • a transgenic "event” is produced by transformation of plant cells with a heterologous DNA construct(s) including a nucleic acid expression cassette that comprises a transgene of interest, the regeneration of a population of plants from cells which each comprise the inserted transgene and selection of a particular plant characterized by insertion into a particular genome location.
  • An event is characterized phenotypically by the expression of the transgene(s).
  • an event is part of the genetic makeup of a plant.
  • the term “event” also refers to progeny, produced by a sexual outcross between the transformant and another variety, that include the heterologous DNA.
  • vent also refers to DNA from the original transformant comprising the inserted DNA and flanking sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
  • flanking regions of the DP-061061-7 event comprise polynucleotide sequences that are set forth in SEQ ID NOS: 2, 8 and/or 9, and variants and fragments thereof.
  • junction DNA refers to DNA that comprises a junction point.
  • Non-limiting examples of junction DNA from the DP-061061-7 event set are forth in SEQ ID NO: 2, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or variants and fragments thereof.
  • a DP-061061-7 plant can be bred by first sexually crossing a first parental Brassica plant grown from the transgenic DP-061061-7 Brassica plant (or progeny thereof derived from transformation with the expression cassettes of the embodiments of the present invention that confer herbicide tolerance) and a second parental Brassica plant that lacks the herbicide tolerance phenotype, thereby producing a plurality of first progeny plants and then selecting a first progeny plant that displays the desired herbicide tolerance and selfing the first progeny plant, thereby producing a plurality of second progeny plants and then selecting from the second progeny plants which display the desired herbicide tolerance.
  • steps can further include the back-crossing of the first herbicide tolerant progeny plant or the second herbicide tolerant progeny plant to the second parental Brassica plant or a third parental Brassica plant, thereby producing a Brassica plant that displays the desired herbicide tolerance. It is further recognized that assaying progeny for phenotype is not required.
  • Various methods and compositions, as disclosed elsewhere herein, can be used to detect and/or identify the DP-061061-7 or other event.
  • Two different transgenic plants can also be sexually crossed to produce offspring that contain two independently-segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back- crossing to a parental plant and out-crossing with a non-trans genie plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcos, ed., American Society of Agronomy, Madison Wis. (1987).
  • germplasm refers to an individual, a group of individuals or a clone representing a genotype, variety, species or culture or the genetic material thereof.
  • a “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally isogenic or near isogenic.
  • Inbred lines tend to be highly homogeneous, homozygous and reproducible. Many analytical methods are available to determine the homozygosity and phenotypic stability of inbred lines.
  • hybrid plants refers to plants which result from a cross between genetically different individuals.
  • crossing means the fusion of gametes, e.g., via pollination to produce progeny (i.e., cells, seeds, or plants) in the case of plants.
  • progeny i.e., cells, seeds, or plants
  • the term encompasses both sexual crosses (the pollination of one plant by another) and, in the case of plants, selfing (self-pollination, i.e., when the pollen and ovule are from the same plant).
  • introduction refers to the transmission of a desired allele of a genetic locus from one genetic background to another.
  • the desired alleles can be introgressed through a sexual cross between two parents, wherein at least one of the parents has the desired allele in its genome.
  • the polynucleotides conferring the brassica DP-061061-7 event of the invention are engineered into a molecular stack.
  • the molecular stack further comprises at least one additional polynucleotide that confers tolerance to a second herbicide.
  • the sequence confers tolerance to glufosinate, and in a specific embodiment, the sequence comprises pat gene.
  • the additional polynucleotide provides tolerance to ALS-inhibitor herbicides.
  • an event of the invention comprises one or more traits of interest, and in more specific embodiments, the plant is stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired combination of traits.
  • a trait refers to the phenotype derived from a particular sequence or groups of sequences.
  • herbicide -tolerance polynucleotides may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as Bacillus thuringiensis toxic proteins (described in US Patent Numbers 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al, (1986) Gene 48: 109; Lee, et al, (2003) Appl. Environ. Microbiol. 69:4648-4657 (Vip3A); Galitzky, et al., (2001) Acta Crystallogr. D. Biol. Crystallogr.
  • an event of the invention may be stacked with other herbicide-tolerance traits to create a transgenic plant of the invention with further improved properties.
  • Other herbicide-tolerance polynucleotides that could be used in such embodiments include those conferring tolerance to glyphosate by other modes of action, such as, for example, a gene that encodes a glyphosate oxido-reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175.
  • EPSPS 5- enolpymvylshikimate-3 -phosphate synthase
  • an event of the invention may be stacked with, for example, hydroxyphenylpyruvatedioxygenases which are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate.
  • HPP para-hydroxyphenylpyruvate
  • Molecules which inhibit this enzyme and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate are useful as herbicides. Traits conferring tolerance to such herbicides in plants are described in US Patent Numbers 6,245,968 Bl; 6,268,549 and 6,069,115 and International Publication Number WO 99/23886.
  • herbicide-tolerance traits that could be stacked with an event of the invention include aryloxyalkanoate dioxygenase polynucleotides (which reportedly confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in International Publication WO 05/107437) and dicamba-tolerance polynucleotides as described, for example, in Herman, et al., (2005) J. Biol. Chem. 280:24759-24767.
  • aryloxyalkanoate dioxygenase polynucleotides which reportedly confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in International Publication WO 05/107437
  • dicamba-tolerance polynucleotides as described, for example, in Herman, et al.,
  • herbicide-tolerance traits that could be combined with an event disclosed herein include those conferred by polynucleotides encoding an exogenous phosphinothricin acetyltransferase, as described in US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903. Plants containing an exogenous phosphinothricin acetyltransferase can exhibit improved tolerance to glufosinate herbicides, which inhibit the enzyme glutamine synthase.
  • herbicide-tolerance traits that could be combined with an event disclosed herein include those conferred by polynucleotides conferring altered protoporphyrinogen oxidase (protox) activity, as described in US Patent Numbers 6,288,306 Bl; 6,282,837 Bl and 5,767,373 and International Publication Number WO 01/12825. Plants containing such polynucleotides can exhibit improved tolerance to any of a variety of herbicides which target the protox enzyme (also referred to as "protox inhibitors").
  • an ALS inhibitor-tolerant trait is combined with the event disclosed herein.
  • an "ALS inhibitor-tolerant polypeptide” comprises any polypeptide which when expressed in a plant confers tolerance to at least one ALS inhibitor.
  • ALS inhibitors include, for example, sulfonylurea, imidazolinone, triazolopyrimidines, pryimidinyoxy(thio)benzoates, and/or
  • ALS mutations fall into different classes with regard to tolerance to sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidinyl(thio)benzoates, including mutations having the following characteristics: (1) broad tolerance to all four of these groups; (2) tolerance to imidazolinones and pyrimidinyl(thio)benzoates; (3) tolerance to sulfonylureas and triazolopyrimidines; and (4) tolerance to sulfonylureas and imidazolinones.
  • the ALS inhibitor-tolerant polypeptides can be employed.
  • the ALS inhibitor-tolerant polynucleotides contain at least one nucleotide mutation resulting in one amino acid change in the ALS polypeptide.
  • the change occurs in one of seven substantially conserved regions of acetolactate synthase. See, for example, Hattori et al. (1995) Molecular Genetics and Genomes 246:419-425; Lee et al. (1998) EMBO Journal 7: 1241-1248; Mazur et al. (1989) Ann. Rev. Plant Phys. 40:441-470; and U.S. Patent No. 5,605,011, each of which is incorporated by reference in their entirety.
  • the ALS inhibitor-tolerant polypeptide can be encoded by, for example, the SuRA or SuRB locus of ALS.
  • the ALS inhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALS mutant, the S4 mutant or the S4/HRA mutant or any combination thereof.
  • Different mutations in ALS are known to confer tolerance to different herbicides and groups (and/or subgroups) of herbicides; see, e.g., Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Patent No. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which is herein incorporated by reference in their entirety.
  • SEQ ID NO: 65 comprising a soybean HRA sequence
  • SEQ ID NO:66 comprising a maize HRA sequence
  • SEQ ID NO:67 comprising an Arabidopsis HRA sequence
  • SEQ ID NO: 86 comprising an HRA sequence used in cotton.
  • the HRA mutation in ALS finds particular use in one embodiment of the invention. The mutation results in the production of an acetolactate synthase polypeptide which is resistant to at least one ALS inhibitor chemistry in comparison to the wild-type protein.
  • a plant expressing an ALS inhibitor- tolerant polypeptide may be tolerant of a dose of sulfonylurea, imidazolinone, triazolopyrimidines, pryimidinyloxy(thio)benzoates, and/or
  • an ALS inhibitor-tolerant polypeptide comprises a number of mutations. Additionally, plants having an ALS inhibitor polypeptide can be generated through the selection of naturally occurring mutations that impart tolerance to glyphosate. In some embodiments, the ALS inhibitor-tolerant polypeptide confers tolerance to sulfonylurea and imidazolinone herbicides.
  • Sulfonylurea and imidazolinone herbicides inhibit growth of higher plants by blocking aceto lactate synthase (ALS), also known as, acetohydroxy acid synthase (AHAS).
  • ALS aceto lactate synthase
  • AHAS acetohydroxy acid synthase
  • plants containing particular mutations in ALS e.g., the S4 and/or HRA mutations
  • AHAS acetohydroxy acid synthase
  • the ALS inhibitor-tolerant polypeptide comprises a sulfonamide-tolerant acetolactate synthase (otherwise known as a sulfonamide-tolerant acetohydroxy acid synthase) or an imidazolinone-tolerant acetolactate synthase (otherwise known as an imidazolinone-tolerant acetohydroxy acid synthase).
  • herbicide-tolerance traits that could be combined with an event disclosed herein include those conferring tolerance to at least one herbicide in a plant such as, for example, a brassica plant or horseweed.
  • Herbicide-tolerant weeds are known in the art, as are plants that vary in their tolerance to particular herbicides. See, e.g., Green and Williams, (2004) "Correlation of Corn (Zea mays) Inbred Response to Nicosulfuron and Mesotrione," poster presented at the WSSA Annual Meeting in Kansas City, Missouri, February 9-12, 2004; Green, (1998) Weed Technology 12:474-477; Green and Ulrich, (1993) Weed Science 41 :508-516.
  • the trait(s) responsible for these tolerances can be combined by breeding or via other methods with an event disclosed herein to provide a plant of the invention as well as methods of use thereof.
  • An event disclosed herein can also be combined with at least one other trait to produce plants of the present invention that further comprise a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil content (e.g., US Patent Number 6,232,529); balanced amino acid content (e.g., hordothionins (US Patent Numbers 5,990,389; 5,885,801; 5,885,802 and 5,703,409; US Patent Number 5,850,016); barley high lysine (Williamson, et ah, (1987) Eur. J. Biochem. 165:99-106 and WO 98/20122) and high methionine proteins (Pedersen, et ah, (1986) J.
  • traits desirable for animal feed such as high oil content (e.g., US Patent Number 6,232,529); balanced amino acid content (e.g., hordothionins (US Patent Numbers 5,990,389; 5,885,801;
  • Desired trait combinations also include LLNC (low linolenic acid content; see, e.g., Dyer, et al, (2002) Appl. Microbiol. Biotechnol. 59:224-230) and OLCH (high oleic acid content; see, e.g., Fernandez-Moya, et al, (2005) J. Agric. Food Chem. 53:5326-5330).
  • An event disclosed herein may also be combined with other desirable traits such as, for example, fumonisin detoxification genes (US Patent Number 5,792,931), avirulence and disease resistance genes (Jones, et al, (1994) Science 266:789; Martin, et al, (1993) Science 262: 1432; Mindrinos, et al, (1994) Cell 78: 1089) and traits desirable for processing or process products such as modified oils (e.g., fatty acid desaturase genes (US Patent Number 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)) and polymers or bioplastics (e.g., US Patent Number 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA
  • PHAs polyhydroxyalkanoates
  • agronomic traits such as male sterility (e.g., see, US Patent Number 5.583,210), stalk strength, flowering time or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364 and WO 99/25821), the disclosures of which are herein incorporated by reference.
  • an event disclosed herein can also be combined with the Rcgl sequence or biologically active variant or fragment thereof.
  • the Rcgl sequence is an anthracnose stalk rot resistance gene in corn. See, for example, US Patent Application Serial Number 11/397,153, 11/397,275 and 11/397,247, each of which is herein incorporated by reference.
  • stacked combinations can be created by any method including, but not limited to, breeding plants by any conventional methodology or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order.
  • the traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest.
  • polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, W099/25821, W099/25854, WO99/25840, W099/25855 and W099/25853, all of which are herein incorporated by reference.
  • polynucleotide is not intended to limit the present invention to polynucleotides comprising DNA.
  • polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • the polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • a DP-061061-7 Brassica plant comprises an expression cassette having an optimized glyphosate acetyltransferase polynucleotide.
  • the cassette can include 5' and 3' regulatory sequences operably linked to the glyat polynucleotides.
  • "Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for the expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a coding region and a transcriptional and translational termination region functional in plants.
  • a transcriptional and translational initiation region i.e., a promoter
  • a coding region i.e., a coding region
  • a transcriptional and translational termination region functional in plants.
  • Promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence can comprise proximal and more distal upstream elements, the latter elements are often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types or at different stages of development or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters".
  • the expression cassettes may also contain 5' leader sequences. Such leader sequences can act to enhance translation.
  • the regulatory regions i.e., promoters, transcriptional regulatory regions, RNA processing or stability regions, introns, polyadenylation signals, transcriptional termination regions and translational termination regions
  • the coding region may be native/analogous or heterologous to the host cell or to each other.
  • the "translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect numerous parameters including, processing of the primary transcript to mRNA, mRNA stability and/or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster, (1995) Mol. Biotechnol. 3:225-236).
  • the "3' non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3 ' end of the mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht, et ah, (1989) Plant Cell 1 :671-680.
  • heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus or the promoter is not the native promoter for the operably linked polynucleotide.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Isolated polynucleotides are provided that can be used in various methods for the detection and/or identification of the brassica DP061061-7 event.
  • An "isolated” or “purified” polynucleotide or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the polynucleotide as found in its naturally occurring environment.
  • an isolated or purified polynucleotide is substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
  • the polynucleotides of the invention comprise the junction DNA sequence set forth in SEQ ID NO: 2 or variants and/or fragments thereof, or the junction DNA sequence set forth in SEQ ID NO: 12 and/or 13. In other embodiments, the polynucleotides of the invention comprise the junction DNA sequences set forth in SEQ ID NO: 14, 15, 16, 17, 18 and/or 19 or variants and fragments thereof. In specific embodiments, methods of detection described herein amplify a polynucleotide comprising a junction of the specific DP-061061-7 event. Fragments and variants of junction DNA sequences are suitable for discriminatively identifying either event DP- 061061-7. As discussed elsewhere herein, such sequences find use as primers and/or probes.
  • the polynucleotides of the invention comprise polynucleotides that can detect a DP-061061-7 event or a region specific to DP-061061-7.
  • sequences include any polynucleotide set forth in SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19, or variants and fragments thereof. Fragments and variants of polynucleotides that detect a DP-061061-7 event or a region specific to DP- 061061-7 are suitable for discriminatively identifying event DP-061061-7. As discussed elsewhere herein, such sequences find use as primers and/or probes.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "probe” is an isolated polynucleotide to which is attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, enzyme, etc.
  • a probe is complementary to a strand of a target polynucleotide.
  • the probe is complementary to a strand of isolated DNA from Brassica event DP-061061-7, whether from a Brassica plant or from a sample that includes DNA from the event.
  • Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that can specifically detect the presence of the target DNA sequence.
  • primer pairs of the invention refer to their use for amplification of a target polynucleotide, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
  • PCR polymerase chain reaction
  • polymerase chain reaction is a technique used for the amplification of specific DNA segments (see, US Patent Numbers 4,683,195 and 4,800,159, herein incorporated by reference). Any combination of primers can be used such that the pair allows for the detection of a DP- 061061-7 event or a region specific to DP-061061-7.
  • Probes and primers are of sufficient nucleotide length to bind to the target DNA sequence and specifically detect and/or identify a polynucleotide having a DP-061061-7 event. It is recognized that the hybridization conditions or reaction conditions can be determined by the operator to achieve this result. This length may be of any length that is of sufficient length to be useful in a detection method of choice.
  • Probes and primers can hybridize specifically to a target sequence under high stringency hybridization conditions.
  • Probes and primers according to embodiments of the present invention may have complete DNA sequence identity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to specifically detect and/or identify a target DNA sequence may be designed by conventional methods.
  • probes and primers can share about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity or complementarity to the target polynucleotide, or can differ from the target sequence by 1, 2, 3, 4, 5, 6 or more nucleotides.
  • Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.
  • a probe can comprises a polynucleotide encoding the glyat4621 sequence or any variant or fragment thereof.
  • Specific primers can be used to amplify an integration fragment to produce an amplicon that can be used as a "specific probe” or can itself be detected for identifying event DP-061061-7 in biological samples.
  • a probe of the invention can be used during the PCR reaction to allow for the detection of the amplification event (i.e., a TaqmanTM probe or an MGB probe, so called real-time PCR).
  • the probe is hybridized with the polynucleotides of a biological sample under conditions which allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of event DP061061-7 in the biological sample.
  • Such identification of a bound probe has been described in the art.
  • the specific probe is a sequence which, under optimized conditions, hybridizes specifically to a region within the 5' or 3' flanking region of the event and also comprises a part of the foreign DNA contiguous therewith.
  • the specific probe may comprise a sequence of at least 80%, between 80 and 85%, between 85 and 90%, between 90 and 95% and between 95 and 100% identical (or complementary) to a specific region of the DP061061-7 event.
  • amplified DNA refers to the product of polynucleotide amplification of a target polynucleotide that is part of a nucleic acid template.
  • DNA extracted from the Brassica plant tissue sample may be subjected to a polynucleotide amplification method using a DNA primer pair that includes a first primer derived from flanking sequence adjacent to the insertion site of inserted heterologous DNA and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the DP061061-7 event DNA.
  • the amplicon comprises a DP061061- 7 junction polynucleotide (i.e., a portion of SEQ ID NO: 2 which spans the junction site, such as, for example, SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19 or variants and fragments thereof).
  • a DP061061-7 event the use of any method or assay which discriminates between the presence or the absence of a DP061061-7 event in a biological sample is intended.
  • the second primer may be derived from the flanking sequence.
  • primer pairs can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert polynucleotide of the expression construct as well as the sequence flanking the transgenic insert. See, Figure 3.
  • the amplicon is of a length and has a sequence that is also diagnostic for the event (i.e., has a junction DNA from a DP061061-7 event).
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol.
  • a member of a primer pair derived from the flanking sequence may be located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to the limits of the amplification reaction or about twenty thousand nucleotide base pairs.
  • the use of the term "amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.
  • transgenic includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of a heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • the term "transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere, et al, (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein, et al, (1987) Nature (London) 327:70- 73; US Patent Number 4,945,050, incorporated herein by reference). Additional transformation methods are disclosed below.
  • isolated polynucleotides of the invention can be incorporated into recombinant constructs, typically DNA constructs, which are capable of introduction into and replication in a host cell.
  • a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide- encoding sequence in a given host cell.
  • vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels, et ah, (1985; Supp.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a promoterating inducible or constitutive, environmentally- or developmentally-regulated or cell- or tissue-specific expression
  • RNA processing signal e.g., a transcription termination site and/or a polyadenylation signal.
  • identifying event DP061061-7 find use in identifying and/or detecting a DP061061-7 event in any biological material. Such methods include, for example, methods to confirm seed purity and methods for screening seeds in a seed lot for a DP061061-7 event.
  • a method for identifying event DP061061-7 in a biological sample comprises contacting the sample with a first and a second primer; and, amplifying a polynucleotide comprising a DP061061-7 specific region.
  • a biological sample can comprise any sample in which one desires to determine if DNA having event DP061061-7 is present.
  • a biological sample can comprise any plant material or material comprising or derived from a plant material such as, but not limited to, food or feed products.
  • plant material refers to material which is obtained or derived from a plant or plant part.
  • the biological sample comprises a brassica tissue.
  • Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences.
  • the polynucleotide probes and primers of the present invention specifically detect a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample.
  • the polynucleotide can be used either as a primer to amplify a DP061061-7 specific region or the polynucleotide can be used as a probe that hybridizes under stringent conditions to a polynucleotide from a DP061061-7 event.
  • the level or degree of hybridization which allows for the specific detection of a DP061061-7 event or a specific region of a DP061061-7 event is sufficient to distinguish the polynucleotide with the DP061061-7 specific region from a polynucleotide lacking this region and thereby allow for discriminately identifying a DP061061-7 event.
  • “stringent conditions” are conditions that permit the primer pair to hybridize to the target polynucleotide to which one primer having the corresponding wild-type sequence (or its complement) and another primer having the corresponding DP061061-7 inserted DNA sequence would bind and preferably to produce an identifiable amplification product (the amplicon) having a DP061061-7 specific region in a DNA thermal amplification reaction.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify a DP061061-7 specific region.
  • the amplified polynucleotide can be of any length that allows for the detection of the DP061061-7 event or a DP061061-7 specific region.
  • the amplicon can be about 10, 50, 100, 200, 300, 500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in length or longer.
  • the specific region of the DP061061-7 event is detected.
  • the first primer comprises a fragment of a polynucleotide of SEQ ID NO: 2, 8 and/or 9, wherein the first or the second primer shares sufficient sequence identity or complementarity to the polynucleotide to amplify the DP061061-7 specific region.
  • the primer pair can comprise a fragment of SEQ ID NO: 2.
  • the primer pair comprises a first primer comprising a fragment of SEQ ID NO: 8 and a second primer comprising a fragment of SEQ ID NO: 9 or 10; or, alternatively, the primer pair comprises a first primer comprising a fragment of SEQ ID NO: 9 and the second primer comprises a fragment of SEQ ID NO: 8 or 10.
  • the primers can be of any length sufficient to amplify a DP061061-7 specific region including, for example, at least 6, 7, 8, 9, 10, 15, 20, 15 or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 nucleotides or longer. Additional primers are also set forth herein in Table 12.
  • any method to PCR amplify the DP061061-7 event or specific region can be employed, including for example, real time PCR. See, for example, Livak, et ah, (1995a). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system for detecting PCR product and nucleic acid hybridization. PCR methods and Applications. 4:357-362; US Patent Number 5,538,848; US Patent Number 5,723,591; Applied Biosystems User Bulletin No. 2, "Relative Quantitation of Gene Expression," P/N 4303859 and Applied Biosystems User Bulletin No. 5, “Multiplex PCR with Taqman VIC probes," P/N 4306236, each of which is herein incorporated by reference.
  • a method of detecting the presence of brassica event DP061061-7 or progeny thereof in a biological sample comprises (a) extracting a DNA sample from the biological sample; (b) providing a pair of DNA primer molecules targeting the insert and/or junction (c) providing DNA amplification reaction conditions; (d) performing the DNA amplification reaction, thereby producing a DNA amplicon molecule and (e) detecting the DNA amplicon molecule, wherein the detection of said DNA amplicon molecule in the DNA amplification reaction indicates the presence of Brassica event DP061061-7.
  • nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it needs only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. In hybridization techniques, all or part of a polynucleotide that selectively hybridizes to a target polynucleotide having a DP061061-7 specific event is employed.
  • stringent conditions or “stringent hybridization conditions” when referring to a polynucleotide probe conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background) are intended.
  • stringent conditions are conditions that permit the primer pair to hybridize to the target polynucleotide to which one primer having the corresponding wild-type sequence and another primer having the corresponding DP061061-7 inserted DNA sequence.
  • Stringent conditions are sequence- dependent and will be variable in different circumstances. 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, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of identity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length or less than 500 nucleotides in length.
  • a substantially identical or complementary sequence is a polynucleotide that will specifically hybridize to the complement of the nucleic acid molecule to which it is being compared under high stringency conditions.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6X sodium chloride/sodium citrate (SSC) at about 45° C, followed by a wash of 2XSSC at 50° C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na 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 may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 0.1X SSC at 60 to 65°C.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • a polynucleotide is said to be the "complement” of another polynucleotide if they exhibit complementarity.
  • molecules are said to exhibit "complete complementarity" when every nucleotide of one of the polynucleotide molecules is complementary to a nucleotide of the other.
  • Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency” conditions.
  • the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high-stringency” conditions.
  • the method comprises (a) contacting the biological sample with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from brassica event DP061061-7 and specifically detects the DP061061-7 event; (b) subjecting the sample and probe to stringent hybridization conditions and (c) detecting hybridization of the probe to the DNA, wherein detection of hybridization indicates the presence of the DP061061-7 event.
  • Various methods can be used to detect the DP061061-7 specific region or amplicon thereof, including, but not limited to, Genetic Bit Analysis (Nikiforov, et al, (1994) Nucleic Acid Res. 22:4167-4175) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate.
  • a single-stranded PCR product can be annealed to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base.
  • Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization and single base extension.
  • Another detection method is the Pyrosequencing technique as described by Winge, ((2000) Innov. Pharma. Tech. 00: 18-24).
  • an oligonucleotide is designed that overlaps the adjacent DNA and insert DNA junction.
  • the oligonucleotide is annealed to a single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin.
  • dNTPs are added individually and the incorporation results in a light signal which is measured.
  • a light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization and single or multi-base extension.
  • Fluorescence Polarization as described by Chen, et ah, ((1999) Genome Res. 9:492-498) is also a method that can be used to detect an amplicon of the invention.
  • an oligonucleotide is designed which overlaps the flanking and inserted DNA junction.
  • the oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert flanking sequence due to successful amplification, hybridization and single base extension.
  • Taqman® PE Applied Biosystems, Foster City, Calif.
  • a FRET oligonucleotide probe is designed which overlaps the flanking and insert DNA junction.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe.
  • a fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • Molecular Beacons have been described for use in sequence detection as described in Tyangi, et al, ((1996) Nature Biotech. 14:303-308). Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity.
  • the FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • a hybridization reaction using a probe specific to a sequence found within the amplicon is yet another method used to detect the amplicon produced by a PCR reaction.
  • kit refers to a set of reagents for the purpose of performing the method embodiments of the invention, more particularly, the identification and/or the detection of the DP061061-7 event in biological samples.
  • the kit of the invention can be used and its components can be specifically adjusted, for purposes of quality control (e.g. purity of seed lots), detection of event DP061061-7 in plant material or material comprising or derived from plant material, such as but not limited to food or feed products.
  • a kit for identifying event DP061061-7 in a biological sample comprises a first and a second primer, wherein the first and second primer amplify a polynucleotide comprising a DP061061-7 specific region.
  • the kit also comprises a polynucleotide for the detection of the DP061061-7 specific region.
  • the kit can comprise, for example, a first primer comprising a fragment of a polynucleotide of NO: 2, 8, 9, or 10, wherein the first or the second primer shares sufficient sequence homology or complementarity and specificity to the polynucleotide to amplify said DP061061-7 specific region.
  • the first primer comprises a fragment of a polynucleotide of SEQ ID NO: 8 and the second primer comprises a fragment of SEQ ID NO: 9, 10, or 1, wherein the first or the second primer shares sufficient sequence homology or complementarity to the polynucleotide to amplify the DP061061-7 specific region.
  • the first primer pair comprises SEQ ID NO: 9 or a variant or fragment thereof and the second primer comprises SEQ ID NO: 8, 10 or 1 or a variant or fragment thereof.
  • the primer pair can comprise a fragment of SEQ ID NO: 2 and a fragment of SEQ ID NO: 3.
  • the primers can be of any length sufficient to amplify the DP061061-7 region including, for example, at least 6, 7, 8, 9, 10, 15, 20, 15 or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 nucleotides or longer.
  • DNA detection kits comprising at least one polynucleotide that can specifically detect a DP061061-7 specific region or insert DNA, wherein said polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides homologous or complementary to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27
  • a kit for identifying event DP-061061-7 in a biological sample comprises a first and a second primer, wherein said first and said second primer amplify a polynucleotide comprising a DP-061061-7 specific region.
  • the kit further comprises a polynucleotide for the detection of the DP-061061-7 specific region.
  • the first primer comprises a first fragment of SEQ ID NO: 11 and the second primer comprises a second fragment of SEQ ID NO: 11, wherein the first and the second primer flank the DP-061061- 7 specific region and share sufficient sequence homology or complementarity to the polynucleotide to amplify said DP-061061-7 specific region.
  • a kit can therefore include a first primer comprising a fragment of SEQ ID NO: 8 and a second primer comprising a fragment of SEQ ID NO:9; or a first or a second primer comprising at least 8 consecutive polynucleotides of SEQ ID NO: 11; or a first or a second primer comprising at least 8 consecutive polynucleotides of SEQ ID NO: 8 or 9.
  • methods for detecting a GAT polypeptide comprising analyzing brassica plant tissues using an immunoassay comprising a GAT polypeptide-specific antibody or antibodies.
  • methods for detecting the presence of a polynucleotide that encodes a GAT polypeptide are provide and comprise assaying brassica plant tissue using PCR amplification. Kits for employing such methods are further provided.
  • any of the polynucleotides and fragments and variants thereof employed in the methods and compositions of the invention can share sequence identity to a region of the transgene insert of the DP061061-7 event, a junction sequence of the DP061061-7 event, or a region of the insert in combination with a region of the flanking sequence of the DP061061-7 event.
  • Methods to determine the relationship of various sequences are known.
  • "reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides.
  • the comparison window is at least 20 contiguous nucleotides in length and optionally can be 30, 40, 50, 100 or longer.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-244 (1988); Higgins, et al, (1989) CABIOS 5: 151-153; Corpet, et al, (1988) Nucleic Acids Res. 16: 10881-90; Huang, et al, (1992) CABIOS 8: 155-65 and Pearson, et al, (1994) Meth. Mol. Biol. 24:307-331.
  • the ALIGN program is based on the algorithm of Myers and Miller, (1988) supra. A PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • the default parameters of the respective programs e.g., BLASTN for nucleotide sequences, BLASTX for proteins
  • Alignment may also be performed manually by inspection.
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2 and the BLOSUM62 scoring matrix or any equivalent program thereof.
  • equivalent program any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10 is intended.
  • GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443- 453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
  • gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
  • the default gap creation penalty is 50 while the default gap extension penalty is 3.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200.
  • the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
  • GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity.
  • the Quality is the metric maximized in order to align the sequences.
  • Ratio is the Quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915).
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the present invention provides methods for controlling weeds in an area of cultivation, preventing the development or the appearance of herbicide resistant weeds in an area of cultivation, producing a crop and increasing crop safety.
  • controlling and derivations thereof, for example, as in “controlling weeds” refers to one or more of inhibiting the growth, germination, reproduction and/or proliferation of and/or killing, removing, destroying or otherwise diminishing the occurrence and/or activity of a weed.
  • an "area of cultivation” comprises any region in which one desires to grow a plant.
  • Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc), a greenhouse, a growth chamber, etc.
  • the methods of the invention comprise planting the area of cultivation with the Brassica DP061061-7 seeds or plants, and in specific embodiments, applying to the crop, seed, weed or area of cultivation thereof an effective amount of a herbicide of interest. It is recognized that the herbicide can be applied before or after the crop is planted in the area of cultivation. Such herbicide applications can include an application of glyphosate.
  • the method of controlling weeds comprises planting the area with the DP061061-7 Brassica seeds or plants and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a herbicide, wherein said effective amount comprises an amount that is not tolerated by a second control crop when applied to the second crop, crop part, seed or the area of cultivation, wherein said second control crop does not express the GLYAT polynucleotide.
  • the method of controlling weeds comprises planting the area with a DP061061-7 Brassica crop seed or plant and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a glyphosate herbicide, wherein said effective amount comprises a level that is above the recommended label use rate for the crop, wherein said effective amount is tolerated when applied to the DP061061-7 Brassica crop, crop part, seed or the area of cultivation thereof.
  • a "control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell, and may be any suitable plant or plant cell.
  • a control plant or plant cell may comprise, for example: (a) a wild- type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell which is genetically identical to the subject plant or plant cell but which is not exposed to the same treatment (e.g., herbicide treatment) as the subject plant or plant cell; (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed or (f) the subject plant or plant cell itself, under conditions in which it has not been exposed to a particular treatment such as, for example, a
  • an appropriate control plant or control plant cell may have a different genotype from the subject plant or plant cell but may share the herbicide- sensitive characteristics of the starting material for the genetic alteration(s) which resulted in the subject plant or cell (see, e.g., Green, (1998) Weed Technology 12:474-477; Green and Ulrich, (1993) Weed Science 41:508-516.
  • the null segregant can be used as a control, as they are genetically identical to DP061061-7 with the exception of the transgenic insert DNA.
  • HRAC Herbicide Resistance Action Committee
  • WSSA the Weed Science Society of America
  • Herbicides can be classified by their mode of action and/or site of action and can also be classified by the time at which they are applied (e.g., preemergent or postemergent), by the method of application (e.g., foliar application or soil application) or by how they are taken up by or affect the plant. For example, thifensulfuron-methyl and tribenuron-methyl are applied to the foliage of a crop and are generally metabolized there, while rimsulfuron and chlorimuron-ethyl are generally taken up through both the roots and foliage of a plant.
  • Mode of action generally refers to the metabolic or physiological process within the plant that the herbicide inhibits or otherwise impairs
  • site of action generally refers to the physical location or biochemical site within the plant where the herbicide acts or directly interacts.
  • Herbicides can be classified in various ways, including by mode of action and/or site of action (see, e.g., Table 1).
  • a herbicide-tolerance gene that confers tolerance to a particular herbicide or other chemical on a plant expressing it will also confer tolerance to other herbicides or chemicals in the same class or subclass, for example, a class or subclass set forth in Table 1.
  • a transgenic plant of the invention is tolerant to more than one herbicide or chemical in the same class or subclass, such as, for example, an inhibitor of PPO, a sulfonylurea or a synthetic auxin.
  • the plants of the present invention can tolerate treatment with different types of herbicides (i.e., herbicides having different modes of action and/or different sites of action) as well as with higher amounts of herbicides than previously known plants, thereby permitting improved weed management strategies that are recommended in order to reduce the incidence and prevalence of herbicide-tolerant weeds.
  • Specific herbicide combinations can be employed to effectively control weeds.
  • the invention thereby provides a transgenic brassica plant which can be selected for use in crop production based on the prevalence of herbicide-tolerant weed species in the area where the transgenic crop is to be grown.
  • Methods are known in the art for assessing the herbicide tolerance of various weed species.
  • Weed management techniques are also known in the art, such as for example, crop rotation using a crop that is tolerant to a herbicide to which the local weed species are not tolerant.
  • HRAC Herbicide Resistance Action Committee
  • Weed Science Society of America and various state agencies (see, for example, herbicide tolerance scores for various broadleaf weeds from the 2004 Illinois Agricultural Pest Management Handbook) and one of skill in the art would be able to use this information to determine which crop and herbicide combinations should be used in a particular location.
  • N-phenylphthalimides a. Cinidon-ethyl b. Flumioxazin c. Flumiclorac-pentyl
  • Triketones a. Mesotrione
  • Phosphinic Acids a. Glufosinate-ammonium b. Bialaphos
  • Chloroacetamides a. Acetochlor b. Alachlor c. Butachlor d. Dimethachlor e. Dimethanamid f. Metazachlor g. Metolachlor h. Pethoxamid i. Pretilachlor j. Propachlor k. Propisochlor 1. Thenylchlor
  • glyphosate alone or in combination with another herbicide of interest, can be applied to the DP061061-7 Brassica plants or their area of cultivation.
  • Non-limiting examples of glyphosate formations are set forth in Table 2.
  • the glyphosate is in the form of a salt, such as, ammonium, isopropylammonium, potassium, sodium (including sesquisodium) or trimesium (alternatively named sulfosate).
  • a transgenic plant of the invention is used in a method of growing a DP061061-7 brassica crop by the application of herbicides to which the plant is tolerant.
  • herbicides which include, but are not limited to: acetochlor, acifluorfen and its sodium salt, aclonifen, acrolein (2-propenal), alachlor, alloxydim, ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos, asulam, atrazine, azimsulfuron, beflubutamid, benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap, bifenox, bilan
  • herbicides and agricultural chemicals are known in the art, such as, for example, those described in WO 2005/041654.
  • Other herbicides also include bioherbicides such as Alternaria destruens Simmons, Colletotrichum gloeosporiodes (Penz.) Penz. and Sacc, Drechsiera monoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz) Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Puccinia thlaspeos Schub.
  • bioherbicides such as Alternaria destruens Simmons, Colletotrichum gloeosporiodes (Penz.) Penz. and Sacc, Drechsiera monoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz) Ditmar: Fries, Phytophthora palmivora (Butl.) But
  • Combinations of various herbicides can result in a greater- than-additive (i.e., synergistic) effect on weeds and/or a less-than-additive effect (i.e., safening) on crops or other desirable plants.
  • combinations of glyphosate with other herbicides having a similar spectrum of control but a different mode of action will be particularly advantageous for preventing the development of resistant weeds.
  • Herbicidally effective amounts of any particular herbicide can be easily determined by one skilled in the art through simple experimentation.
  • Herbicides may be classified into groups and/or subgroups as described herein above with reference to their mode of action, or they may be classified into groups and/or subgroups in accordance with their chemical structure.
  • Sulfonamide herbicides have as an essential molecular structure feature a sulfonamide moiety (-S(0) 2 NH-).
  • sulfonamide herbicides particularly comprise sulfonylurea herbicides, sulfonylaminocarbonyltriazolinone herbicides and triazolopyrimidine herbicides.
  • the sulfonamide moiety is a component in a sulfonylurea bridge (-S(0) 2 NHC(0)NH(R)-).
  • sulfonylurea herbicides the sulfonyl end of the sulfonylurea bridge is connected either directly or by way of an oxygen atom or an optionally substituted amino or methylene group to a typically substituted cyclic or acyclic group.
  • the amino group which may have a substituent such as methyl (R being CH 3 ) instead of hydrogen, is connected to a heterocyclic group, typically a symmetric pyrimidine or triazine ring, having one or two substituents such as methyl, ethyl, trifluoromethyl, methoxy, ethoxy, methylamino, dimethylamino, ethylamino and the halogens.
  • the sulfonamide moiety is a component of a sulfonylaminocarbonyl bridge (-S(0) 2 NHC(0)-).
  • sulfonylaminocarbonyltriazolinone herbicides the sulfonyl end of the sulfonylaminocarbonyl bridge is typically connected to substituted phenyl ring. At the opposite end of the sulfonylaminocarbonyl bridge, the carbonyl is connected to the 1- position of a triazolinone ring, which is typically substituted with groups such as alkyl and alkoxy.
  • the sulfonyl end of the sulfonamide moiety is connected to the 2-position of a substituted [ 1 ,2,4]triazolopyrimidine ring system and the amino end of the sulfonamide moiety is connected to a substituted aryl, typically phenyl, group or alternatively the amino end of the sulfonamide moiety is connected to the 2- position of a substituted [ 1 ,2,4]triazolopyrimidine ring system and the sulfonyl end of the sulfonamide moiety is connected to a substituted aryl, typically pyridinyl, group.
  • the methods further comprise applying to the crop and the weeds in a field a sufficient amount of at least one herbicide to which the crop seeds or plants are tolerant, such as, for example, glyphosate, a hydroxyphenylpyruvatedioxygenase inhibitor (e.g., mesotrione or sulcotrione), a phytoene desaturase inhibitor (e.g., diflufenican), a pigment synthesis inhibitor, sulfonamide, imidazolinone, bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate, glufosinate, triazolopyrimidine, pyrimidinyloxy(thio)benzoate or sulonylaminocarbonyltriazolinone, an acetyl Co-A carboxylase inhibitor such as quizalofop-P-ethyl, a synthetic auxin such as quinclorac,
  • the effective amount of herbicide applied to the field is sufficient to selectively control the weeds without significantly affecting the crop.
  • "Weed” as used herein refers to a plant which is not desirable in a particular area.
  • a “crop plant” as used herein refers to a plant which is desired in a particular area, such as, for example, a Brassica plant.
  • a weed is a non-crop plant or a non-crop species
  • a weed is a crop species which is sought to be eliminated from a particular area, such as, for example, an inferior and/or non- transgenic Brassica plant in a field planted with Brassica event DP061061-7 or a non- Brassica crop plant in a field planted with DP061061-7.
  • Weeds can be classified into two major groups: monocots and dicots.
  • Plant species can be controlled (i.e., killed or damaged) by the herbicides described herein. Accordingly, the methods of the invention are useful in controlling these plant species where they are undesirable (i.e., where they are weeds).
  • These plant species include crop plants as well as species commonly considered weeds, including but not limited to species such as: blackgrass (Alopecurus myosuroides), giant foxtail (Setaria faberi), large crabgrass (Digitaria sanguinalis), Surinam grass (Brachiaria decumbens), wild oat (Avena fatua), common cocklebur (Xanthium pensylvanicum), common lambsquarters (Chenopodium album), morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf (Abutilion theophrasti), common barnyardgrass (Echinochioa crus-galti), bermudagrass (Cynodon dactylon), downy brome
  • the weed comprises a herbicide-resistant ryegrass, for example, a glyphosate resistant ryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistant ryegrass and a non-selective herbicide resistant ryegrass.
  • the undesired plants are proximate the crop plants.
  • a method is considered to selectively control weeds when at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the weeds are significantly damaged or killed, while if crop plants are also present in the field, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the crop plants are significantly damaged or killed.
  • Representative sulfonylureas that can be applied at this level are set forth in Table 1.
  • glyphosate herbicides as a class contain the same active ingredient, but the active ingredient is present as one of a number of different salts and/or formulations.
  • herbicides known to inhibit ALS vary in their active ingredient as well as their chemical formulations.
  • One of skill in the art is familiar with the determination of the amount of active ingredient and/or acid equivalent present in a particular volume and/or weight of herbicide preparation.
  • an ALS inhibitor herbicide is employed. Rates at which the ALS inhibitor herbicide is applied to the crop, crop part, seed or area of cultivation can be any of the rates disclosed herein. In specific embodiments, the rate for the ALS inhibitor herbicide is about 0.1 to about 5000 g ai/hectare, about 0.5 to about 300 g ai/hectare or about 1 to about 150 g ai/hectare.
  • a particular herbicide is applied to a particular field (and any plants growing in it) no more than 1, 2, 3, 4, 5, 6, 7 or 8 times a year, or no more than 1, 2, 3, 4 or 5 times per growing season.
  • weeds which are susceptible to each of the herbicides exhibit damage from treatment with each of the herbicides which is additive or synergistic.
  • the application of each herbicide and/or chemical may be simultaneous or the applications may be at different times, so long as the desired effect is achieved. Furthermore, the application can occur prior to the planting of the crop.
  • the proportions of herbicides used in the methods of the invention with other herbicidal active ingredients in herbicidal compositions are generally in the ratio of 5000: 1 to 1 :5000, 1000: 1 to 1: 1000, 100: 1 to 1: 100, 10: 1 to 1 : 10 or 5: 1 to 1 :5 by weight.
  • the optimum ratios can be easily determined by those skilled in the art based on the weed control spectrum desired.
  • any combinations of ranges of the various herbicides disclosed in Table 1 can also be applied in the methods of the invention.
  • the invention provides improved methods for selectively controlling weeds in a field wherein the total herbicide application may be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of that used in other methods.
  • the amount of a particular herbicide used for selectively controlling weeds in a field may be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the amount of that particular herbicide that would be used in other methods, i.e., methods not utilizing a plant of the invention.
  • a DP061061-7 Brassica plant of the invention benefits from a synergistic effect, wherein the herbicide tolerance conferred by the GLYAT polypeptide and that conferred by a polypeptide providing tolerance to another herbicide is greater than expected from simply combining the herbicide tolerance conferred by each gene separately. See, e.g., McCutchen, et al, (1997) J. Econ. Entomol. 90: 1170-1180; Priesler, et al, (1999) J. Econ. Entomol. 92:598-603.
  • the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic herbicide combination” or a “synergistic herbicide composition” refer to circumstances under which the biological activity of a combination of herbicides, such as at least a first herbicide and a second herbicide, is greater than the sum of the biological activities of the individual herbicides.
  • Synergy expressed in terms of a "Synergy Index (SI),” generally can be determined by the method described by Kull, et al., (1961) Applied Microbiology 9:538. See also, Colby, (1967) Weeds 15:20-22.
  • the herbicide tolerance conferred on a DP061061-7 plant of the invention is additive; that is, the herbicide tolerance profile conferred by the herbicide tolerance genes is what would be expected from simply combining the herbicide tolerance conferred by each gene separately to a transgenic plant containing them individually.
  • Additive and/or synergistic activity for two or more herbicides against key weed species will increase the overall effectiveness and/or reduce the actual amount of active ingredient(s) needed to control said weeds.
  • the plant of the invention may display tolerance to a higher dose or rate of herbicide and/or the plant may display tolerance to additional herbicides or other chemicals beyond those to which it would be expected to display tolerance.
  • a DP061061-7 Brassica plant may show tolerance to organophosphate compounds such as insecticides and/or inhibitors of 4- hydroxyphenylpyruvate dioxygenase.
  • the DP061061-7 Brassica plants of the invention when further comprising genes conferring tolerance to other herbicides, can exhibit greater than expected tolerance to various herbicides, including but not limited to glyphosate, ALS inhibitor chemistries and sulfonylurea herbicides.
  • the DP061061-7 Brassica plant plants of the invention may show tolerance to a particular herbicide or herbicide combination that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400% or 500% or more higher than the tolerance of an appropriate control plant that contains only a single herbicide tolerance gene which confers tolerance to the same herbicide or herbicide combination.
  • DP061061-7 Brassica plants may show decreased damage from the same dose of herbicide in comparison to an appropriate control plant, or they may show the same degree of damage in response to a much higher dose of herbicide than the control plant.
  • a particular herbicide used for selectively containing weeds in a field is more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or greater than the amount of that particular herbicide that would be used in other methods, i.e., methods not utilizing a plant of the invention.
  • a DP061061-7 Brassica plant of the invention shows improved tolerance to a particular formulation of a herbicide active ingredient in comparison to an appropriate control plant.
  • Herbicides are sold commercially as formulations which typically include other ingredients in addition to the herbicide active ingredient; these ingredients are often intended to enhance the efficacy of the active ingredient.
  • Such other ingredients can include, for example, safeners and adjuvants (see, e.g., Green and Foy, (2003) "Adjuvants: Tools for Enhancing Herbicide Performance," in Weed Biology and Management, ed. Inderjit (Kluwer Academic Publishers, The Netherlands)).
  • a DP061061-7 Brassica plant of the invention can show tolerance to a particular formulation of a herbicide (e.g., a particular commercially available herbicide product) that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000% or more higher than the tolerance of an appropriate control plant that contains only a single herbicide tolerance gene which confers tolerance to the same herbicide formulation.
  • a herbicide e.g., a particular commercially available herbicide product
  • a DP061061-7 Brassica plant of the invention shows improved tolerance to a herbicide or herbicide class to which at least one other herbicide tolerance gene confers tolerance as well as improved tolerance to at least one other herbicide or chemical which has a different mechanism or basis of action than either glyphosate or the herbicide corresponding to said at least one other herbicide tolerance gene.
  • This surprising benefit of the invention finds use in methods of growing crops that comprise treatment with various combinations of chemicals, including, for example, other chemicals used for growing crops.
  • a DP061061-7 Brassica plant may also show improved tolerance to chlorpyrifos, a systemic organophosphate insecticide.
  • the invention also provides a DP061061-7 Brassica plant that confers tolerance to glyphosate (i.e., a GLYAT gene) which shows improved tolerance to chemicals which affect the cytochrome P450 gene, and methods of use thereof.
  • glyphosate i.e., a GLYAT gene
  • the DP061061-7 Brassica plants also show improved tolerance to dicamba.
  • the improved tolerance to dicamba may be evident in the presence of glyphosate and a sulfonylurea herbicide.
  • a herbicide combination is applied over a DP061061-7 Brassica plant, where the herbicide combination produces either an additive or a synergistic effect for controlling weeds.
  • Such combinations of herbicides can allow the application rate to be reduced, a broader spectrum of undesired vegetation to be controlled, improved control of the undesired vegetation with fewer applications, more rapid onset of the herbicidal activity or more prolonged herbicidal activity.
  • An “additive herbicidal composition” has a herbicidal activity that is about equal to the observed activities of the individual components.
  • a “synergistic herbicidal combination” has a herbicidal activity higher than what can be expected based on the observed activities of the individual components when used alone. Accordingly, the presently disclosed subject matter provides a synergistic herbicide combination, wherein the degree of weed control of the mixture exceeds the sum of control of the individual herbicides.
  • the degree of weed control of the mixture exceeds the sum of control of the individual herbicides by any statistically significant amount including, for example, about 1% to 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to 120% or greater.
  • a "synergistically effective amount" of a herbicide refers to the amount of one herbicide necessary to elicit a synergistic effect in another herbicide present in the herbicide composition.
  • the term "synergist,” and derivations thereof, refer to a substance that enhances the activity of an active ingredient (ai), i.e., a substance in a formulation from which a biological effect is obtained, for example, a herbicide.
  • the presently disclosed subject matter provides a method for controlling weeds in an area of cultivation.
  • the method comprises: (a) planting the area with a DP061061-7 crop seeds or crop plants which also comprise polynucleotides conferring ALS-inhibitor tolerance; and (b) applying to the weed, the crop plants, a crop part, the area of cultivation or a combination thereof, an effective amount of a herbicide composition comprising at least one of a synergistically effective amount of glyphosate and a synergistically effective amount of an ALS inhibitor (for example, but not limited to, a sulfonylurea herbicide) or agriculturally suitable salts thereof, wherein at least one of: (i) the synergistically effective amount of the glyphosate is lower than an amount of glyphosate required to control the weeds in the absence of the sulfonylurea herbicide; (ii) the synergistically effective
  • the herbicide composition used in the presently disclosed method for controlling weeds comprises a synergistically effective amount of glyphosate and a sulfonylurea herbicide.
  • the presently disclosed synergistic herbicide composition comprises glyphosate and a sulfonylurea herbicide selected from the group consisting of metsulfuron-methyl, chlorsulfuron and triasulfuron.
  • the synergistic herbicide combination further comprises an adjuvant such as, for example, an ammonium sulfate-based adjuvant, e.g., ADD-UP ® (Wenkem., Halfway House, Midrand, South Africa).
  • the presently disclosed synergistic herbicide compositions comprise an additional herbicide, for example, an effective amount of a pyrimidinyloxy(thio)benzoate herbicide.
  • the pyrimidinyloxy(thio)benzoate herbicide comprises bispyribac, e.g., (VELOCITY ® , Valent U.S.A. Corp., Walnut Creek, California, United States of America) or an agriculturally suitable salt thereof.
  • the glyphosate is applied pre-emergence, post-emergence or pre- and post- emergence to the undesired plants or plant crops and/or the ALS inhibitor herbicide (i.e., the sulfonylurea herbicide) is applied pre-emergence, post-emergence or pre- and post- emergence to the undesired plants or plant crops.
  • the glyphosate and/or the ALS inhibitor herbicide i.e., the sulfonylurea herbicide
  • the synergistic herbicide composition is applied, e.g., step (b) above, at least once prior to planting the crop(s) of interest, e.g., step (a) above.
  • Weeds that can be difficult to control with glyphosate alone in fields where a crop is grown include but are not limited to the following: horseweed (e.g., Conyza canadensis); rigid ryegrass (e.g., Lolium rigidum); goosegrass (e.g., Eleusine indica); Italian ryegrass (e.g., Lolium multiflorum); hairy fleabane (e.g., Conyza bonariensis); buckhorn plantain (e.g., Plantago lanceolata); common ragweed (e.g., Ambrosia artemisifolia); morning glory (e.g., Ipomoea spp.); waterhemp (e.g., Amaranthus spp.); field bindweed (e.g., Convolvulus arvensis); yellow nutsedge (e.g., Cyperus esculentus); common lambsquarters (
  • Brassica plants comprising the DP061061-7 event and tolerance to another herbicide are particularly useful in allowing the treatment of a field (and therefore any crop growing in the field) with combinations of herbicides that would cause unacceptable damage to crop plants that did not contain both of these polynucleotides.
  • Plants of the invention that are tolerant to glyphosate and other herbicides such as, for example, sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate and/or sulfonylaminocarbonyltriazolinone herbicides in addition to being tolerant to at least one other herbicide with a different mode of action or site of action are particularly useful in situations where weeds are tolerant to at least two of the same herbicides to which the plants are tolerant. In this manner, plants of the invention make possible improved control of weeds that are tolerant to more than one herbicide.
  • herbicides such as, for example, sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate and/or sulfonylaminocarbonyltriazolinone herbicides in addition to being tolerant to at least one other herbicide with a
  • weed control in fields where current commercial crops include glyphosate and, optionally, 2,4-D; this combination, however, has some disadvantages.
  • glyphosate and, optionally, 2,4-D
  • Another commonly used treatment for weed control in brassica fields is the sulfonylurea herbicide chlorimuron-ethyl, which has significant residual activity in the soil and thus maintains selective pressure on all later- emerging weed species, creating a favorable environment for the growth and spread of sulfonylurea-resistant weeds.
  • Fields may be treated with sulfonylurea, imidazolinone, triazolopyrimi dines, pyrimidiny(thio)benzoates and/or sulfonylaminocarbonyltriazonlinone such as the sulfonylurea chlorimuron-ethyl, either alone or in combination with other herbicides, such as a combination of glyphosate and tribenuron-methyl (available commercially as Express®).
  • This combination has several advantages for weed control under some circumstances, including the use of herbicides with different modes of action and the use of herbicides having a relatively short period of residual activity in the soil.
  • a herbicide having a relatively short period of residual activity is desirable, for example, in situations where it is important to reduce selective pressure that would favor the growth of herbicide-tolerant weeds.
  • other considerations may be more important, such as, for example, the need to prevent the development of and/or appearance of weeds in a field prior to planting a crop by using a herbicide with a relatively long period of residual activity. Treatments that include both tribenuron-methyl and thifensulfuron-methyl may be particularly useful.
  • thifensulfuron-methyl available commercially as Harmony GT®.
  • thifensulfuron-methyl available commercially as Harmony GT®.
  • thifensulfuron-methyl is that the higher application rates required for consistent weed control often cause injury to a crop growing in the same field.
  • DP061061-7 Brassica plants comprising additional tolerance can be treated with a combination of glyphosate and thifensulfuron-methyl, which has the advantage of using herbicides with different modes of action.
  • weeds that are resistant to either herbicide alone are controlled by the combination of the two herbicides, and the improved DP061061-7 Brassica plants would not be significantly damaged by the treatment.
  • Other herbicides which are used for weed control in fields where current commercial varieties of crops (including, for example, Brassicas) are grown are the triazolopyrimidine herbicide cloransulam-methyl (available commercially as FirstRate®) and the imidazolinone herbicide imazaquin (available commercially as Sceptor®). When these herbicides are used individually they may provide only marginal control of weeds.
  • glyphosate e.g., Roundup® (glyphosate isopropylamine salt)
  • imazapyr currently available commercially as Arsenal®
  • chlorimuron-ethyl currently available commercially as Classic®
  • quizalofop- P-ethyl currently available commercially as Assure II®
  • fomesafen currently available commercially as Flexstar®
  • Fields containing the DP061061-7 Brassica plants with additional herbicide tolerance may also be treated, for example, with a combination of herbicides including glyphosate, rimsulfuron, and dicamba or mesotrione. This combination may be particularly useful in controlling weeds which have developed some tolerance to herbicides which inhibit ALS.
  • Another combination of herbicides which may be particularly useful for weed control includes glyphosate and at least one of the following: metsulfuron-methyl (commercially available as Ally®), imazapyr (commercially available as Arsenal®), imazethapyr, imazaquin and sulfentrazone. It is understood that any of the combinations discussed above or elsewhere herein may also be used to treat areas in combination with any other herbicide or agricultural chemical.
  • Some commonly-used treatments for weed control in fields where current commercial crops (including, for example, Brassica) are grown include glyphosate (currently available commercially as Roundup®), rimsulfuron (currently available commercially as Resolve® or Matrix®), dicamba (commercially available as Clarity®), atrazine and mesotrione (commercially available as Callisto®).
  • glyphosate currently available commercially as Roundup®
  • rimsulfuron currently available commercially as Resolve® or Matrix®
  • dicamba commercially available as Clarity®
  • atrazine and mesotrione commercially available as Callisto®
  • glyphosate currently available commercially as Roundup®
  • chlorimuron-ethyl chlorimuron-ethyl, tribenuron-methyl
  • rimsulfuron currently available commercially as Resolve® or Matrix®
  • imazethapyr imazapyr and imazaquin.
  • DP061061-7 Brassica with an additional herbicide tolerance trait can be treated with a combination of herbicides that would cause unacceptable damage to standard plant varieties, including combinations of herbicides that include at least one of those mentioned above.
  • a herbicide may be formulated and applied to an area of interest such as, for example, a field or area of cultivation, in any suitable manner.
  • a herbicide may be applied to a field in any form, such as, for example, in a liquid spray or as solid powder or granules.
  • the herbicide or combination of herbicides that are employed in the methods comprise a tankmix or a premix.
  • a herbicide may also be formulated, for example, as a "homogenous granule blend" produced using blends technology (see, e.g., US Patent Number 6,022,552, entitled "Uniform Mixtures of Pesticide Granules").
  • the blends technology of US Patent Number 6,022,552 produces a nonsegregating blend (i.e., a "homogenous granule blend") of formulated crop protection chemicals in a dry granule form that enables delivery of customized mixtures designed to solve specific problems.
  • a homogenous granule blend can be shipped, handled, subsampled and applied in the same manner as traditional premix products where multiple active ingredients are formulated into the same granule.
  • a "homogenous granule blend” is prepared by mixing together at least two extruded formulated granule products.
  • each granule product comprises a registered formulation containing a single active ingredient which is, for example, a herbicide, a fungicide and/or an insecticide.
  • the uniformity (homogeneity) of a "homogenous granule blend” can be optimized by controlling the relative sizes and size distributions of the granules used in the blend.
  • the diameter of extruded granules is controlled by the size of the holes in the extruder die and a centrifugal sifting process may be used to obtain a population of extruded granules with a desired length distribution (see, e.g., US Patent Number 6,270,025).
  • a homogenous granule blend is considered to be "homogenous" when it can be subsampled into appropriately sized aliquots and the composition of each aliquot will meet the required assay specifications.
  • a large sample of the homogenous granule blend is prepared and is then subsampled into aliquots of greater than the minimum statistical sample size.Blends also afford the ability to add other agrochemicals at normal, labeled use rates such as additional herbicides (a 3 rd /4 th mechanism of action), fungicides, insecticides, plant growth regulators and the like thereby saving costs associated with additional applications.
  • any herbicide formulation applied over the DP061061-7 Brassica plant can be prepared as a "tank-mix" composition.
  • each ingredient or a combination of ingredients can be stored separately from one another. The ingredients can then be mixed with one another prior to application. Typically, such mixing occurs shortly before application.
  • each ingredient, before mixing typically is present in water or a suitable organic solvent.
  • Woods "The Formulator's Toolbox— Product Forms for Modern Agriculture” Pesticide Chemistry and Bioscience, The Food-Environment Challenge, Brooks and Roberts, Eds., Proceedings of the 9th International Congress on Pesticide Chemistry, The Royal Society of Chemistry, Cambridge, 1999, pp. 120-133.
  • the methods of the invention further allow for the development of herbicide combinations to be used with the DP061061-7 Brassica plants.
  • the environmental conditions in an area of cultivation are evaluated.
  • Environmental conditions that can be evaluated include, but are not limited to, ground and surface water pollution concerns, intended use of the crop, crop tolerance, soil residuals, weeds present in area of cultivation, soil texture, pH of soil, amount of organic matter in soil, application equipment and tillage practices.
  • an effective amount of a combination of herbicides can be applied to the crop, crop part, seed of the crop or area of cultivation.
  • the herbicide applied to the DP061061-7 Brassica plants of the invention serves to prevent the initiation of growth of susceptible weeds and/or serve to cause damage to weeds that are growing in the area of interest.
  • the herbicide or herbicide mixture exert these effects on weeds affecting crops that are subsequently planted in the area of interest (i.e., field or area of cultivation).
  • the application of the herbicide combination need not occur at the same time. So long as the field in which the crop is planted contains detectable amounts of the first herbicide and the second herbicide is applied at some time during the period in which the crop is in the area of cultivation, the crop is considered to have been treated with a mixture of herbicides according to the invention.
  • methods of the invention encompass applications of herbicide which are "preemergent,” "postemergent,” “preplant incorporation” and/or which involve seed treatment prior to planting.
  • methods are provided for coating seeds.
  • the methods comprise coating a seed with an effective amount of a herbicide or a combination of herbicides (as disclosed elsewhere herein).
  • the seeds can then be planted in an area of cultivation.
  • seeds having a coating comprising an effective amount of a herbicide or a combination of herbicides (as disclosed elsewhere herein).
  • Preemergent refers to a herbicide which is applied to an area of interest (e.g., a field or area of cultivation) before a plant emerges visibly from the soil.
  • Postemergent refers to a herbicide which is applied to an area after a plant emerges visibly from the soil.
  • preemergent and postemergent are used with reference to a weed in an area of interest, and in some instances these terms are used with reference to a crop plant in an area of interest. When used with reference to a weed, these terms may apply to only a particular type of weed or species of weed that is present or believed to be present in the area of interest.
  • rimsulfuron has both preemergence and postemergence activity, while other herbicides have predominately preemergence (metolachlor) or postemergence (glyphosate) activity.
  • the invention provides improved methods of growing a crop and/or controlling weeds such as, for example, "pre-planting burn down," wherein an area is treated with herbicides prior to planting the crop of interest in order to better control weeds.
  • the invention also provides methods of growing a crop and/or controlling weeds which are "no-till” or “low-till” (also referred to as “reduced tillage”). In such methods, the soil is not cultivated or is cultivated less frequently during the growing cycle in comparison to traditional methods; these methods can save costs that would otherwise be incurred due to additional cultivation, including labor and fuel costs.
  • the methods of the invention encompass the use of simultaneous and/or sequential applications of multiple classes of herbicides.
  • the methods of the invention involve treating a plant of the invention and/or an area of interest (e.g., a field or area of cultivation) and/or weed with just one herbicide or other chemical such as, for example, a sulfonylurea herbicide.
  • the time at which a herbicide is applied to an area of interest may be important in optimizing weed control.
  • the time at which a herbicide is applied may be determined with reference to the size of plants and/or the stage of growth and/or development of plants in the area of interest, e.g., crop plants or weeds growing in the area.
  • the stages of growth and/or development of plants are known in the art.
  • the time at which a herbicide or other chemical is applied to an area of interest in which plants are growing may be the time at which some or all of the plants in a particular area have reached at least a particular size and/or stage of growth and/or development, or the time at which some or all of the plants in a particular area have not yet reached a particular size and/or stage of growth and/or development.
  • the DP061061-7 Brassica plants of the invention show improved tolerance to postemergence herbicide treatments.
  • plants of the invention may be tolerant to higher doses of herbicide, tolerant to a broader range of herbicides, and/or may be tolerant to doses of herbicide applied at earlier or later times of development in comparison to an appropriate control plant.
  • the DP061061-7 Brassica plants of the invention show an increased resistance to morphological defects that are known to result from treatment at particular stages of development.
  • the glyphosate-tolerant plants of the invention find use in methods involving herbicide treatments at later stages of development than were previously feasible.
  • plants of the invention may be treated with a particular herbicide that causes morphological defects in a control plant treated at the same stage of development, but the glyphosate-tolerant plants of the invention will not be significantly damaged by the same treatment.
  • a crop rotation scheme may be chosen based on residual effects from treatments that will be used for each crop and their effect on the crop that will subsequently be grown in the same area.
  • One of skill in the art is familiar with techniques that can be used to evaluate the residual effect of a herbicide; for example, generally, glyphosate has very little or no soil residual activity, while herbicides that act to inhibit ALS vary in their residual activity levels. Residual activities for various herbicides are known in the art, and are also known to vary with various environmental factors such as, for example, soil moisture levels, temperature, pH and soil composition (texture and organic matter).
  • the transgenic plants of the invention provide improved tolerance to treatment with additional chemicals commonly used on crops in conjunction with herbicide treatments, such as safeners, adjuvants such as ammonium sulfonate and crop oil concentrate, and the like.
  • herbicide treatments such as safeners, adjuvants such as ammonium sulfonate and crop oil concentrate, and the like.
  • safener refers to a substance that when added to a herbicide formulation eliminates or reduces the phytotoxic effects of the herbicide to certain crops.
  • safener depends, in part, on the crop plant of interest and the particular herbicide or combination of herbicides included in the synergistic herbicide composition.
  • Exemplary safeners suitable for use with the presently disclosed herbicide compositions include, but are not limited to, those disclosed in US Patent Numbers 4,808,208; 5,502,025; 6,124,240 and US Patent Application Publication Numbers 2006/0148647; 2006/0030485; 2005/0233904; 2005/0049145; 2004/0224849; 2004/0224848; 2004/0224844; 2004/0157737; 2004/0018940; 2003/0171220; 2003/0130120; 2003/0078167, the disclosures of which are incorporated herein by reference in their entirety.
  • the methods of the invention can involve the use of herbicides in combination with herbicide safeners such as benoxacor, BCS (l-bromo-4-[(chloromethyl) sulfonyl]benzene), cloquintocet-mexyl, cyometrinil, dichlormid, 2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG 191), fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen-ethyl, mefenpyr-diethyl, methoxyphenone ((4-methoxy-3 -methylphenyl)(3 -methylphenyl)-methanone), naphthalic anhydride (1,8-naphthalic anhydride) and oxabetrinil to increase crop safety.
  • herbicide safeners such as benoxacor, BCS (l-bromo
  • Antidotally effective amounts of the herbicide safeners can be applied at the same time as the compounds of this invention, or applied as seed treatments. Therefore an aspect of the present invention relates to the use of a mixture comprising glyphosate, at least one other herbicide and an antidotally effective amount of a herbicide safener.
  • Seed treatment is particularly useful for selective weed control, because it physically restricts antidoting to the crop plants. Therefore a particularly useful embodiment of the present invention is a method for selectively controlling the growth of weeds in a field comprising treating the seed from which the crop is grown with an antidotally effective amount of safener and treating the field with an effective amount of herbicide to control weeds. Antidotally effective amounts of safeners can be easily determined by one skilled in the art through simple experimentation.
  • An antidotally effective amount of a safener is present where a desired plant is treated with the safener so that the effect of a herbicide on the plant is decreased in comparison to the effect of the herbicide on a plant that was not treated with the safener; generally, an antidotally effective amount of safener prevents damage or severe damage to the plant treated with the safener.
  • One of skill in the art is capable of determining whether the use of a safener is appropriate and determining the dose at which a safener should be administered to a crop.
  • the herbicide or herbicide combination applied to the plant of the invention acts as a safener.
  • a first herbicide or a herbicide mixture is applied at an antidotally effect amount to the plant.
  • a method for controlling weeds in an area of cultivation comprises planting the area with crop seeds or plants which comprise a first polynucleotide encoding a polypeptide that can confer tolerance to glyphosate operably linked to a promoter active in a plant; and, a second polynucleotide encoding an ALS inhibitor-tolerant polypeptide operably linked to a promoter active in a plant.
  • a combination of herbicides comprising at least an effective amount of a first and a second herbicide is applied to the crop, crop part, weed or area of cultivation thereof.
  • the effective amount of the herbicide combination controls weeds; and, the effective amount of the first herbicide is not tolerated by the crop when applied alone when compared to a control crop that has not been exposed to the first herbicide; and, the effective amount of the second herbicide is sufficient to produce a safening effect, wherein the safening effect provides an increase in crop tolerance upon the application of the first and the second herbicide when compared to the crop tolerance when the first herbicide is applied alone.
  • the combination of safening herbicides comprises a first ALS inhibitor and a second ALS inhibitor.
  • the safening effect is achieved by applying an effective amount of a combination of glyphosate and at least one ALS inhibitor chemistry.
  • Such mixtures provides increased crop tolerance (i.e., a decrease in herbicidal injury). This method allows for increased application rates of the chemistries post or pre-treatment. Such methods find use for increased control of unwanted or undesired vegetation.
  • a safening affect is achieved when the DP061061-7 brassica crops, crop part, crop seed, weed or area of cultivation is treated with at least one herbicide from the sulfonylurea family of chemistry in combination with at least one herbicide from the imidazolinone family.
  • This method provides increased crop tolerance (i.e., a decrease in herbicidal injury).
  • the sulfonylurea comprises rimsulfuron and the imidazolinone comprises imazethapyr.
  • glyphosate is also applied to the crop, crop part or area of cultivation.
  • an “adjuvant” is any material added to a spray solution or formulation to modify the action of an agricultural chemical or the physical properties of the spray solution. See, for example, Green and Foy, (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” in Weed Biology and Management, ed. Inderjit (Kluwer Academic Publishers, The Netherlands).
  • Adjuvants can be categorized or subclassified as activators, acidifiers, buffers, additives, adherents, antiflocculants, antifoamers, defoamers, antifreezes, attractants, basic blends, chelating agents, cleaners, colorants or dyes, compatibility agents, cosolvents, couplers, crop oil concentrates, deposition agents, detergents, dispersants, drift control agents, emulsifiers, evaporation reducers, extenders, fertilizers, foam markers, formulants, inerts, humectants, methylated seed oils, high load COCs, polymers, modified vegetable oils, penetrators, repellants, petroleum oil concentrates, preservatives, rainfast agents, retention aids, solubilizers, surfactants, spreaders, stickers, spreader stickers, synergists, thickeners, translocation aids, uv protectants, vegetable oils, water conditioners and wetting agents.
  • methods of the invention can comprise the use of a herbicide or a mixture of herbicides, as well as, one or more other insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants or other biologically active compounds or entomopathogenic bacteria, virus or fungi to form a multi-component mixture giving an even broader spectrum of agricultural protection.
  • Examples of such agricultural protectants which can be used in methods of the invention include: insecticides such as abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, cartap, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin, dimethoate, dinotefur
  • Bacillus thuringiensis subsp. Kurstaki and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g., Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardine fungus; and entomopathogenic virus including baculovirus, nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus (GV) such as CpGV.
  • NPV nucleopolyhedro virus
  • GV granulosis virus
  • the weight ratios of these various mixing partners to other compositions (e.g., herbicides) used in the methods of the invention typically are between 100: 1 and 1: 100, or between 30: 1 and 1 :30, between 10: 1 and 1 : 10, or between 4: 1 and 1 :4.
  • the present invention also pertains to a composition
  • a composition comprising a biologically effective amount of a herbicide of interest or a mixture of herbicides, and an effective amount of at least one additional biologically active compound or agent and can further comprise at least one of a surfactant, a solid diluent or a liquid diluent.
  • Such biologically active compounds or agents are: insecticides such as abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethi
  • Bacillus thuringiensis subsp. Kurstaki and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g., Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardine fungus; and entomopathogenic virus including baculovirus, nucleopolyhedro virus (NPV) such as HzNPV, AfNPV and granulosis virus (GV) such as CpGV.
  • Methods of the invention may also comprise the use of plants genetically transformed to express proteins toxic to invertebrate pests (such as Bacillus thuringiensis delta-endotoxins).
  • the effect of exogenously applied invertebrate pest control compounds may be synergistic with the expressed toxin proteins.
  • compositions of the present invention can further comprise a biologically effective amount of at least one additional invertebrate pest control compound or agent having a similar spectrum of control but a different mode of action.
  • a plant protection compound e.g., protein
  • a biologically effective amount of a compound of this invention can also provide a broader spectrum of plant protection and be advantageous for resistance management.
  • methods of the invention employ a herbicide or herbicide combination and may further comprise the use of insecticides and/or fungicides, and/or other agricultural chemicals such as fertilizers.
  • the use of such combined treatments of the invention can broaden the spectrum of activity against additional weed species and suppress the proliferation of any resistant biotypes.
  • Methods of the invention can further comprise the use of plant growth regulators such as aviglycine, N-(phenylmethyl)-lH-purin-6-amine, ethephon, epocholeone, gibberellic acid, gibberellin A4 and A7, harpin protein, mepiquat chloride, prohexadione calcium, prohydrojasmon, sodium nitrophenolate and trinexapac -methyl and plant growth modifying organisms such as Bacillus cereus strain BP01.
  • plant growth regulators such as aviglycine, N-(phenylmethyl)-lH-purin-6-amine, ethephon, epocholeone, gibberellic acid, gibberellin A4 and A7, harpin protein, mepiquat chloride, prohexadione calcium, prohydrojasmon, sodium nitrophenolate and trinexapac -methyl
  • plant growth regulators such as aviglycine, N-(phenylmethyl)-
  • GLYAT4621 glyphosate acetyltransferase protein
  • Brassica Brassica napus L.
  • glyphosate acetyltransferase gene Glyat4621
  • Plasmid PHP28181 contains an expression cassette as further described hereafter.
  • DNA construct PHP28181 was made by cloning the GAT4621:PrNII TERM fragment excised from DNA construct pZSL149 with BamHI and Mfel double digestion downstream to the AT-UBQ10 promoter of DNA construct QC272 in the same BamHI and Mfel sites using T4 DNA ligase (New England Lab).
  • the resulting PHP28181 DNA contains the expression cassette: AT-UBQ10 (DUPONT) PRO:GAT4621:PINII TERM. See, Figure 1 and Figure 2.
  • the 2112 bp PHP28181A DNA fragment was prepared from plasmid PHP28181 with Hindlll and Notl restriction enzyme double digestion.
  • the digested plasmid DNA was resolved in a 1% agarose gel by electrophoresis.
  • the DNA band of the correct size was excised and DNA fragment was extracted using a Qiagen DNA fragment extraction kit (Qiagen).
  • DNA fragment purity was checked by PCR with a series of dilutions of amp+ positive control DNA since the PHP28181 plasmid contains an amp+ gene in its backbone. DNA fragment concentration was measured spectrophotometrically and confirmed by comparing to DNA low mass markers (InVitrogen) in an agarose gel.
  • Gold particles coated with the PHP28181A DNA fragment were used for transformation.
  • Biolistic transformation was carried out using the PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA) as described by Klein, et al., (1987) Nature 327:70-73.
  • Transformed embryogenic microspores were cultured in fresh medium in dark conditions for 10-12 days, then under dim light for 1-3 weeks. Green embryos were transferred to fresh medium and cultured for two weeks to select for glyphosate tolerance. Germinated shoots and/or plants were transferred to growth medium supplemented with glyphosate.
  • the glyat4621 gene was derived from the soil bacterium Bacillus licheniformis and was synthesized by a gene shuffling process to optimize the acetyltransferase activity of the GLYAT4621 enzyme (Castle, et al, (2004) Science 304: 1151-1154).
  • the inserted fragment ( Figure 3) from this plasmid contains the glyat4621 gene cassette.
  • the expression of the glyat4621 gene is controlled by the UBQ10 regulatory region from Arabidopsis and the pinll terminator (see, Table 4).
  • a summary of the transformation fragment of plasmid PHP28181 is shown in Table 4.
  • the genetic elements of plasmid PHP28181 used in the creation of DP-061061-7 are shown in Table 3.
  • PCR polymerase chain reaction
  • genomic DNA from the test substance (plant material of event DP- 061061-7) and the control substance (plant material of a non-genetically modified Brassica with a genetic background representative of the event background) is isolated and subjected to qualitative PCR amplification using a construct-specific primer pair.
  • the PCR products are separated on 1.5% or 2% agarose gels to confirm the presence of the inserted construct in the genomic DNA generated from the test substance, and absence in the genomic DNA generated from the control substance.
  • a reference standard 100 base pair DNA Ladder; Invitrogen Corporation Catalog # 10380-012
  • Test and control samples are harvested from plants. Genomic DNA extraction from the test and control tissues is performed using a standard urea extraction protocol, if leaf tissue.
  • Genomic DNA from the test and control samples is isolated using Wizard® Magnetic 96 DNA Plant System (Promega Corporation Catalog # FF3760), if seed tissue. Genomic DNA is quantified on a spectrofluorometer using PicoGreen® reagent (Molecular Probes, Inc., Eugene, OR) and/or visualized on an agarose gel to confirm quantitation values and to determine the DNA quality.
  • Genomic DNA isolated from plant material of event DP-061061-7 and control samples is subjected to PCR amplification (PCR Master Mix Catalog #7505 from Promega Corporation) utilizing a construct-specific primer pair which spans at least a portion of the glyat4621 coding region, and allows for the unique identification of brassica event DP-061061-7.
  • a second primer set is used to amplify an endogenous gene as a positive control for PCR amplification.
  • the PCR target site and size of the expected PCR product for each primer set are compared to the observed results.
  • Southern blot analyses (Southern, 1975) are performed to investigate the number of sites of insertion of the transforming DNA, the copy number and functional integrity of the genetic elements and the absence of plasmid backbone sequences.
  • Genomic DNA is extracted from lyophilized tissue sampled from DP-061061-7 Brassica and non-genetically- modified control plants. Genomic DNA is digested with restriction endonuclease enzymes and size- separated on an agarose gel. A molecular weight marker is run alongside samples for size estimation purposes. DNA fragments separated on agarose gel are depurinated, denatured and neutralized in situ and transferred to a nylon membrane. Following transfer to the membrane, the DNA is bound to the membrane by UV crosslinking. Fragments homologous to the glyat4621 gene are generated by PCR from plasmid PHP28181, separated on an agarose gel by size, exsized and purified using a gel extraction kit.
  • Labeled probe is hybridized to the target DNA on the nylon membranes for detection of the specific fragments. Washes after hybridization are carried out at high stringency. Blots are exposed to X-ray film for one or more time points to detect hybridizing fragments and visualize molecular weight markers.
  • samples of genomic DNA from plants containing the event DP- 061061-7 insert (lanes 2, 4, 6, 8, 10, 12, 14 and 16) and negative segregants from the same T1F2 generation (lanes 3, 5, 7, 9, 11, 13, 15 and 17) were subjected to restriction endonuclease digestion with Ncol (lanes 2-13) or Sspl (lanes 14-17) followed by alkaline agarose gel electrophoresis and transfer onto nylon membrane.
  • GLYAT4621 protein Expression of the GLYAT4621 protein is evaluated using leaf tissue collected from transgenic plants. For example, four fresh leaf punches may be collected and ground in sample extraction buffer using a GenoGrinder (Spex Certiprep). Total Extractable Protein (TEP) can be determined using the Bio-Rad Protein assay, which is based on the Bradford dye-binding procedure. Sample extracts may be diluted in sample extraction buffer for ELISA analysis.
  • the levels of expression of the GAT4621 protein in DP-061061-7 Brassica can be determined by quantitative enzyme linked immunosorbent assay (ELISA) of samples obtained from multiple field trial locations. Replicate seed samples (three replicates) may be obtained from DP-061061-7 plants treated with the maximum recommended label rate of Touchdown® Total glyphosate herbicide (500 g/1 glyphosate as potassium salt; 0.60- 1.35 1/ha), applied at the cotyledon to 6-leaf stage, as this represents a likely commercial cultivation scenario.
  • ELISA quantitative enzyme linked immunosorbent assay
  • Another way to verify the expression of the insert in DP-061061-7 Brassica plants is to evaluate the transformed plants' tolerance to glyphosate. Multigenerational stability and within-generation segregation of the herbicide tolerant trait conferred by expression of the GAT4621 enzyme will be confirmed using a functional assay for herbicide tolerance. Tests are conducted on at least three generations of plant material. Herbicide injury may be scored as described in Table 6.
  • Table 5 Segregation of glyphosate tolerance trait in different generations derived from event DP-061061-7.
  • Genomic DNA isolated from leaf of DP-061061-7 canola (T2F2 generation) and control canola (non-genetically modified) was subjected to PCR amplification (Roche High Fidelity PCR Master Kit, Roche Catalog # 12140314001) utilizing the construct- specific primer pair (09-0-3290/09-0-3288) which spans the ubiquitin promoter and the gatA62 ⁇ gene cassette ( Figure 5).
  • a second primer set (09-0-2812/09-0-2813) was used to amplify the endogenous canola FatA gene as a positive control for PCR amplification.
  • the PCR target site and size of the expected PCR product for each primer sets are shown in Table 9.
  • PCR reagents and reaction conditions are shown in Table 10.
  • the primer sequences used in this study are listed in Table 11. In this study, 100 ng of leaf genomic DNA was used in all PCR reactions.
  • a PCR product of approximately 600 bp in size amplified by the construct-specific primer set 09-0-3290/09-0-3288 was observed in PCR reactions using plasmid PHP28181 (10 ng) as a template and three DP-061061-7 canola plants, but absent in three control canola plants and the no-template control (Figure 6). Samples were loaded as shown in Table 7.
  • flanking genomic DNA border regions of the DP-061061-7 event were determined. Flanking genomic sequence of DP-061061-7 is included within SEQ ID NO: 2. PCR amplification from the insert and border sequences confirms that the border regions are of Brassica origin and that the junction regions can be used for identification of DP-061061- 7 Brassica. Overall, characterization of the insert and genomic border sequences, along with Southern blot data, indicate a single insertion of the DNA fragment present in the Brassica genome. Various molecular techniques are then used to specifically characterize the integration site.
  • flanking genomic border regions are cloned and sequenced using the GenomeWalker and inverse PCR methods. Using information from the flanking border sequence, PCR is performed on DP-061061-7 genomic DNA and unmodified control genomic DNA. Those skilled in the art will also include a control PCR using an endogenous gene to verify that the isolated genomic DNA is suitable for PCR amplification.

Abstract

L'invention concerne des compositions et des procédés associés à des plantes transgéniques Brassica résistantes au glyphosate. Plus précisément, la présente invention concerne des plantes Brassica présentant un événement DP-061061-7 qui confère une résistance au glyphosate. La plante Brassica porteuse de l'événement DP-061061-7 à l'emplacement chromosomique indiqué comprend les jonctions génomiques/transgéniques de SEQ ID NO: 2 ou les jonctions génomiques/transgéniques représentées dans SEQ ID NO: 12 et/ou 13. La caractérisation du site d'insertion génomique des événements assure une efficacité reproductive accrue et permet l'utilisation de marqueurs moléculaires pour retrouver le transgène inséré dans les populations se reproduisant et dans leur descendance. En outre, l'invention concerne divers procédés et diverses compositions permettant l'identification, la détection et l'utilisation des événements.
PCT/US2010/058002 2010-11-24 2010-11-24 Événement dp-061061-7 de brassica gat et compositions et procédés pour l'identifier et/ou le détecter WO2012071039A1 (fr)

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