US20030135879A1 - Methods and materials for making and using transgenic dicamba-degrading organisms - Google Patents

Methods and materials for making and using transgenic dicamba-degrading organisms Download PDF

Info

Publication number
US20030135879A1
US20030135879A1 US10/330,661 US33066102A US2003135879A1 US 20030135879 A1 US20030135879 A1 US 20030135879A1 US 33066102 A US33066102 A US 33066102A US 2003135879 A1 US2003135879 A1 US 2003135879A1
Authority
US
United States
Prior art keywords
dicamba
dna
degrading
seq
plant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/330,661
Inventor
Donald Weeks
Xiao-Zhuo Wang
Patricia Herman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Nebraska Lincoln
Original Assignee
University of Nebraska Lincoln
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/055,145 external-priority patent/US7022896B1/en
Application filed by University of Nebraska Lincoln filed Critical University of Nebraska Lincoln
Priority to US10/330,661 priority Critical patent/US20030135879A1/en
Publication of US20030135879A1 publication Critical patent/US20030135879A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8259—Phytoremediation
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • Dicamba is in the class of benzoic acid herbicides. It has been suggested that plants tolerant to benzoic acid herbicides, including dicamba, can be produced by incorporating a 1-aminocyclopropane-1-carboxylic acid (ACC) synthase antisense gene, an ACC oxidase antisense gene, an ACC deaminase gene, or combinations thereof, into the plants. See Canadian Patent Application 2,165,036 (published Jun. 16, 1996). However, no experimental data are presented in this application which demonstrate such tolerance.
  • ACC 1-aminocyclopropane-1-carboxylic acid
  • FIG. 1 A diagram of the proposed electron transport scheme for dicamba O-demethylase. Electrons from NADH are transferred sequentially from reductase DIC to ferredoxin DIC and then to oxygenase DIC . The reaction of oxygen with the substrate dicamba to form 3,6-dichlorosalicylic acid is catalyzed by oxygenase DIC . ox, oxidized; red, reduced.
  • the invention provides an isolated and at least partially purified dicamba-degrading reductase.
  • Dicamba-degrading reductase is defined herein to mean the reductases purified from P. maltophilia strain DI-6 and reductases which have an amino acid sequence which is at least about 65% homologous, preferably at least about 85% homologous, to that of one of the P. maltophilia reductases and which can participate in the degradation of dicamba.
  • “Dicamba-degrading reductases” include mutant reductases having the amino acid sequence of one of the P.
  • maltophilia reductases wherein one or more amino acids have been added to, deleted from, or substituted for, the amino acids of the P. maltophilia reductase sequence.
  • Activity of dicamba-degrading reductases can be determined as described in Examples 1 and 3.
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes, promoters from alcohol dehydrogenase genes, the TPI1 promoter, and the ADH2-4c promoter. See PCT WO 96/23898.
  • Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986).
  • Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988).
  • [ 14 C]dicamba and 14 C-labeled metabolites were separated by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the ether-extracted dicamba and its metabolites were redissolved in 50 ⁇ l of ether prior to being spotted onto a TLC plate.
  • the solvent system for running the TLC was chloroform-ethanol-acetic acid (85:10:5 [vol/vol/vol]).
  • the resolved reaction products were visualized and quantified by exposing the TLC plate to a phosphor screen for 24 hours and then scanning the screen in a PhosphorImager SF (Molecular Dynamics, Sunnyvale, Calif.).
  • Cell lysates Cells were grown to an optical density at 550 nm of 1.3 to 1.5 in liquid reduced chloride medium containing a mixture of glucose and Casamino Acids on a rotary shaker (250 rpm at 30° C.). The cells were harvested by centrifugation, washed twice with cold 100 mM MgCl 2 , and centrifuged again. Cell pastes were either used immediately or quickly frozen in liquid nitrogen and stored at ⁇ 80° C.
  • lysozyme After addition of 10 mg of lysozyme and 1 mg of DNase, cells were stirred for 10 min on ice and broken by sonication (model XL2020 sonicator; Heat Systems) on ice at a medium setting (setting 5) with twelve 20-second bursts and 40-second resting intervals. The resulting cell lysates were diluted to 90 ml with isolation buffer and centrifuged at 76,000 ⁇ g for 1 h at 4° C. The supernatant was used as the source of cleared cell lysate.
  • component I Purification of component I. Fractions containing component I activity (eluting from a Phenyl-Sepharose column at 0 M (NH 4 ) 2 SO 4 , fractions 128-145) were pooled to provide a total volume of 34 ml. The pooled samples were concentrated to 10 ml by centrifugation in a Centriprep-10 device (Amicon) and then applied to a Q-Sepharose (Fast Flow) FPLC column (Pharmacia) (2.5 by 6 cm) equilibrated with buffer A and washed with 80 ml of buffer A.
  • Proteins bound to the column were eluted with a 100 ml linear gradient of 0 to 0.6 M KCl in buffer A at a flow rate of 1 ml/min. Fractions were collected at 1.5 minute intervals. Those fractions exhibiting component I activity (fractions 29-37) were pooled, dialyzed against buffer A overnight at 4° C. and applied to a Mono Q HR 5/5 FPLC anion-exchange column in buffer A. Proteins were eluted at 1 ml/min by using a 50 ml gradient of increasing KCl concentration (0 to 0.5 M). Fractions showing component I activity (fractions 19 to 25) were pooled and concentrated to 0.4 ml by centrifugation in a Centricon-10 device.
  • the partially purified component I was diluted with cold 1% (w/v) glycine and concentrated by centrifugation in a Centricon-10 device three more times to desalt it in preparation for IEF electrophoresis.
  • the desalted and concentrated sample was then applied to a 6% (w/v) IEF (pH 4-6) gel and subjected to electrophoresis for 1.5 hours at 4° C. (see below).
  • the gel was washed with 25 mM cold phosphate buffer (pH 7.0) for 5 minutes and then each slice of the gel lane was diced into small (6 mm ⁇ 4 mm) pieces.
  • Fractions exhibiting component II activity were pooled, diluted with an equal volume of buffer A, and applied to a FPLC Mono Q column. Proteins were eluted from the column using the same KCl gradient as for component I. Fractions 20-25 showed component II activity. Partially purified component II was further purified by IEF (pH 4-6) electrophoresis using the same conditions as described for component I. The gel segment which showed component II activity was loaded onto a 12.5% (w/v) SDS-PAGE for further analysis.
  • component III Purification of component III.
  • Component III obtained by Phenyl-Sepharose column chromatography was dialyzed against buffer A overnight at 4° C. and applied to a FPLC Q-Sepharose column (2.5 by 6 cm). Conditions were identical to those described above for component I. Fractions exhibiting component III activity (fractions 26-38) were dialyzed against buffer B [10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 5% (v/v) glycerol, 1 mM dithiothreitol] and concentrated to 5 ml.
  • Blue dye affinity matrix [Cibacron Blue 3GA type 3000 (Sigma)] was packed into a FPLC column (1 ⁇ 5 cm) and pre-equilibrated with 20 ml of buffer B. Concentrated component III was loaded onto the blue dye column and washed with 20 ml of buffer B at a flow rate of 0.2 ml/min until the A 280 of the column effluent reached baseline levels. Bound protein was then eluted with 5 mM NADH in buffer B. Fractions containing reductase activity were detected by assaying for dicamba O-demethylase activity in the presence of an excess of components I and II and also by the ability of each fraction to reduce DCIP in the presence of NADH.
  • Fractions having strong reductase activity in both assays were pooled, dialyzed against buffer A containing 100 mM KCl, concentrated to 0.2 ml, and applied to a FPLC Superose 12 column. The same conditions were used for running the Superose column as described for component I.
  • Fractions containing proteins which catalyzed DCIP reduction were pooled, dialyzed against buffer A and applied to a FPLC Mono Q column. Proteins were eluted using the same conditions as for component I. Partially purified component III was further purified by IEF (pH 4-6) gel electrophoresis.
  • the reductase activity of proteins within the IEF gel was detected by assaying for DCIP reduction in an agarose gel overlay as described above.
  • the gel segment which showed component II activity was loaded onto a 12.5% (w/v) SDS-PAGE for further analysis.
  • Proteins were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in a mixture of isopropanol, water, and acetic acid at a ratio of 3:6:1 (v/v/v). Destaining was performed in a mixture of methanol, water, and acetic acid at a ratio of 7:83:10 (v/v/v).
  • M r The molecular weight of peptides under denaturing conditions was estimated using SDS-PAGE analysis.
  • bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B-12 (1.35 kDa).
  • the void volume of the Superose 12 column was calculated using Blue Dextran (M r 2,000,000, Sigma).
  • Dicamba O-demethylase activity was measured during incubations in vitro at temperatures ranging from 20° C. to 50° C. and at pH values from approximately 6 to 9. Activity peaked sharply at 30° C. and broadly at pH values between 6.5 and 7.5. Enzymatic activity was dependent on the type of pH buffer employed. At pH 7, for example, activity was approximately 40% lower in Tris-containing buffers than in phosphate-containing buffers.
  • Dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6 is a three component oxygenase (Wang, X -Z (1996) Ph.D. thesis, University of Kansas-Lincoln, Lincoln, Nebr.) responsible for the conversion of the herbicide, dicamba (2-methoxy-3,6-dichlorobenzoic acid), to 3,6-dichlorosalicylic acid (3,6-DCSA; 2-hydroxy-3,6-dichlorobenzoic acid).
  • Purification schemes have been devised which have allowed the isolation of each of the three components to a homogeneous or near-homogeneous state.
  • the oxygenase/hydroxylase component of a number of multicomponent systems is composed of an ( ⁇ ) n -type subunit arrangement in which the larger ⁇ subunit is approximately 50,000 Daltons in size and the smaller ⁇ subunit is approximately 20,000 Daltons in molecular mass (Harayama, et al. (1992) Annu. Rev. Microbiol. 46:565-601).
  • the oxygenase component of dicamba O-demethylase appears to possess a single subunit of approximately 40 kDa in molecular mass which may exist as a dimer in its native state.
  • Ferredoxin DIC Component II (ferredoxin DIC ) of dicamba O-demethylase was purified by several steps of column chromatography and IEF. Final purification by SDS-PAGE produced one major band of protein (M r ⁇ 28,000) and a smear of slightly smaller proteins.
  • FIG. 1 The reaction scheme for the reaction catalyzed by the three components of dicamba O-demethylase is presented in FIG. 1. Electrons from NADH are shuttled through a short electron chain consisting of the reductase and ferredoxin to the terminal oxygenase which catalyzes the oxidation of dicamba.
  • the similarities between dicamba O-demethylase and several multicomponent dioxygenases suggest that dicamba O-demethylase may potentially possess cryptic dioxygenase activity.
  • this enzyme is not in the class of dioxygenases which split O 2 and incorporate one atom of oxygen into the major substrate and the other into a small organic substrate such as ⁇ -ketoglutarate (Fukumori and Hausinger (1993) J. Biol. Chem. 268:24311-24317). Indeed, combinations of highly purified reductase DIC , ferredoxin DIC , and oxygenase DIC require only O 2 , NADH, Mg 2+ , Fe 2+ , and dicamba for activity.
  • the supernatant was retained and loaded at a flow rate of 5.0 ml/min onto a Pharmacia XK 26/20 column containing 25 ml of Phenyl-Sepharose 6 Fast Flow (high sub) that was equilibrated in buffer A containing 1.5 M (NH 4 ) 2 SO 4 .
  • Fractions that contained the reductase component were pooled, buffer exchanged into buffer B, and concentrated to approximately 2 ml using the Amicon concentrator with an YM30 membrane and a Centricon10 concentrator.
  • the 2 ml sample was applied to a pre-packed Pharmacia HiPrep 26/60 Sephacryl S-100 column equilibrated in buffer B and run at 0.5 ml/min.
  • the N-terminal sequence obtained from the purified ferredoxin protein was 29 amino acids in length (sequencing of proteins was performed by the Protein Core Facility at the University of Kansas-Lincoln using a standard Edman degradation procedure; see Example 1). A comparison of this sequence to the Genbank database showed that it was 35% identical in a 26 amino acid overlap to a terpredoxin from Pseudomonas sp., a bacterial [2Fe-2S] ferredoxin in the adrenodoxin family. The sequence information was used to design three degenerate oligonucleotide primers (two forward and one reverse).
  • the sequence of the two 17 mer forward primers was based on the N-terminal amino acid sequence obtained from the purified ferredoxin protein.
  • the sequence of the 17 mer reverse primer was based on a conserved sequence of six amino acids near the C-terminal end of six previously sequenced bacterial adrenodoxin-type ferredoxins.
  • the primers were used in a nested PCR reaction to amplify a 191 bp product from total P. maltophilia DNA.
  • the product was cloned into the pGEM-T Easy vector (Promega, Madison, Wis.) and sequenced. DNA sequencing was performed by the DNA Sequencing Core Facility at the University of Kansas-Lincoln using a standard dideoxy-mediated chain termination procedure.
  • This binary vector contains the peanut chlorotic virus full-length promoter (PCISV FLt36) with a duplicated enhancer domain for constitutive expression in plants and the pea rbcS 3′ sequence for efficient transcript termination (Maiti and Shepherd, Biochem. Biophys. Res. Commun., 244:440-444 (1998)). Constructs with all three genes on one binary vector can be produced using combinations of the three genes in a number of different orders and orientations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention provides isolated and at least partially-purified dicamba-degrading enzymes, isolated DNA molecules coding for dicamba-degrading enzymes, DNA constructs coding for dicamba-degrading enzymes, transgenic host cells comprising DNA coding for dicamba-degrading enzymes, and transgenic plants and plant parts comprising one or more cells comprising DNA coding for dicamba-degrading enzymes. Expression of the dicamba-degrading enzymes results in the production of dicamba-degrading organisms, including dicamba-tolerant plants. The invention further provides a method of controlling weeds in a field containing the transgenic dicamba-tolerant plants of the invention and a method of decontaminating a material containing dicamba comprising applying an effective amount of a transgenic microorganism or dicamba-degrading enzyme(s) of the invention to the material. Finally, the invention provides a method of selecting transformed plants and plant cells based on dicamba tolerance and a method of selecting or screening transformed host cells, intact organisms and parts of organisms based on the fluorescence of 3,6-dichlorosalicylic acid produced as a result of dicamba degradation.

Description

  • This application is a continuation-in-part of application Ser. No. 09/055,145, filed Apr. 3, 1998, and claims benefit of provisional applications 60/042,666 and 60/042,941, both filed Apr. 4, 1997, the complete disclosures of all three of which are incorporated herein by reference.[0001]
    • FIELD OF THE INVENTION
  • The present invention relates to transgenic organisms that are able to degrade the herbicide dicamba, including transgenic plants that have been made tolerant to dicamba. The invention also relates to dicamba-degrading enzymes and to DNA molecules and DNA constructs coding for dicamba-degrading enzymes. The invention further relates to a method of controlling weeds in fields of dicamba-tolerant transgenic plants and to a method of removing dicamba from materials contaminated with it (bioremediation). Finally, the invention relates to methods of selecting transformants based on dicamba tolerance or on detecting the fluorescence of 3,6-dichlorosalicylic acid which is generated as a result of dicamba degradation. [0002]
    • BACKGROUND
  • Herbicides are used routinely in agricultural production. Their effectiveness is often determined by their ability to kill weed growth in crop fields and the tolerance of the cash crop to the herbicide. If the cash crop is not tolerant to the herbicide, the herbicide will either diminish the productivity of the cash crop or kill it altogether. Conversely, if the herbicide is not strong enough, it may allow too much weed growth in the crop field which will, in turn, lessen the productivity of the cash crop. Therefore, it is desirable to produce economically important plants which are tolerant to herbicides. To protect the water and environmental quality of agricultural lands, it is also desirable to develop technologies to degrade herbicides in cases of accidental spills of the herbicide or in cases of unacceptably high levels of soil or water contamination. [0003]
  • Genes encoding enzymes which inactivate herbicides and other xenophobic compounds have previously been isolated from a variety of procaryotic and eucaryotic organisms. In some cases, these genes have been genetically engineered for successful expression in plants. Through this approach, plants have been developed which are tolerant to the [0004] herbicides 2,4-dichlorophenoxyacetic acid (Streber and Willmitzer (1989) Bio/Technology 7:811-816; 2,4-D), bromoxynil (Stalker et al. (1988) Science 242:419-423; tradename Buctril), glyphosate (Comai et al. (1985) Nature 317:741-744; tradename Round-Up) and phosphinothricin (De Block et al. (1987) EMBO J. 6:2513-2518; tradename Basta).
  • Dicamba (tradename Banvel) is used as a pre-emergent and post-emergent herbicide for the control of annual and perennial broadleaf weeds and several grassy weeds in corn, sorghum, small grains, pasture, hay, rangeland, sugarcane, asparagus, turf and grass seed crops. See [0005] Crop Protection Reference, pages 1803-1821 (Chemical & Pharmaceutical Press, Inc., New York, N.Y., 11th ed., 1995). Unfortunately, dicamba can injure many commercial crops (including beans, soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, and fruit trees), ornamental plants and trees, and other broadleaf plants when it comes into contact with them. Id. Dicamba is chemically stable and can sometimes be persistent in the environment.
  • Dicamba is in the class of benzoic acid herbicides. It has been suggested that plants tolerant to benzoic acid herbicides, including dicamba, can be produced by incorporating a 1-aminocyclopropane-1-carboxylic acid (ACC) synthase antisense gene, an ACC oxidase antisense gene, an ACC deaminase gene, or combinations thereof, into the plants. See Canadian Patent Application 2,165,036 (published Jun. 16, 1996). However, no experimental data are presented in this application which demonstrate such tolerance. [0006]
  • Bacteria that metabolize dicamba are known. See U.S. Pat. No. 5,445,962; Krueger et al., [0007] J. Agric. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbiol., 38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol., 40, 289-320 (1995). It has been suggested that the specific genes responsible for dicamba metabolism by these bacteria could be isolated and used to produce dicamba-resistant plants and other organisms. See id. and Yang et al., Anal. Biochem., 219:37-42 (1994). However, prior to the present invention, no such genes had been identified or isolated.
    • SUMMARY OF THE INVENTION
  • The invention provides an isolated and at least partially purified dicamba-degrading O-demethylase, an isolated and at least partially purified dicamba-degrading oxygenase, an isolated and at least partially purified dicamba-degrading ferredoxin, and an isolated and at least partially purified dicamba-degrading reductase, all as defined and described below. [0008]
  • The invention also provides an isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading oxygenase, an isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading ferredoxin, and an isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading reductase. The invention further provides a DNA construct comprising a DNA sequence coding for a dicamba-degrading oxygenase, a DNA sequence coding for a dicamba-degrading ferredoxin, or a DNA sequence coding for a dicamba-degrading reductase, each DNA coding sequence being operatively linked to expression control sequences. In addition, the invention provides a DNA construct comprising a DNA sequence coding for a dicamba-degrading oxygenase, a DNA sequence coding for a dicamba-degrading ferredoxin, and a DNA sequence coding for a dicamba-degrading reductase, each DNA coding sequence being operatively linked to expression control sequences. [0009]
  • The invention further provides a transgenic host cell comprising DNA coding for a dicamba-degrading oxygenase, DNA coding for a dicamba-degrading ferredoxin, or DNA coding for a dicamba-degrading reductase, each DNA being operatively linked to expression control sequences. In addition, the invention provides a transgenic host cell comprising DNA coding for a dicamba-degrading oxygenase and DNA coding for a dicamba-degrading ferredoxin, DNA coding for a dicamba-degrading reductase, or DNA coding for a dicamba-degrading ferredoxin and DNA coding for a dicamba-degrading reductase, each DNA being operatively linked to expression control sequences. The transgenic host cell may be a plant cell or a prokaryotic or eukaryotic microorganism. [0010]
  • The invention also provides a transgenic plant or plant part comprising one or more cells comprising DNA coding for a dicamba-degrading oxygenase, DNA coding for a dicamba-degrading ferredoxin, or DNA coding for a dicamba-degrading reductase, each DNA being operatively linked to expression control sequences. The invention further provides a transgenic plant or plant part comprising one or more cells comprising DNA coding for a dicamba-degrading oxygenase and DNA coding for a dicamba-degrading ferredoxin, DNA coding for a dicamba-degrading reductase, or DNA coding for a dicamba-degrading ferredoxin and DNA coding for a dicamba-degrading reductase, each DNA being operatively linked to expression control sequences. The transgenic plant or plant part is preferably tolerant to dicamba or has had its tolerance to dicamba increased as a result of the expression of the dicamba-degrading enzyme(s). [0011]
  • The invention also provides a method of controlling weeds in a field containing transgenic dicamba-tolerant plants. The method comprises applying an amount of dicamba to the field which is effective to control the weeds. [0012]
  • The invention further provides methods of decontaminating a material containing dicamba. In one embodiment, the method comprises applying an effective amount of a transgenic dicamba-degrading microorganism to the material. In another embodiment, the method comprises applying an effective amount of a dicamba-degrading O-demethylase or of a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase to the material. [0013]
  • The invention also provides a method of selecting transformed plant cells and transformed plants using dicamba tolerance as the selection marker. In one embodiment, the method comprises transforming at least some of the plant cells in a population of plant cells so that they are tolerant to dicamba and growing the resulting population of plant cells in a culture medium containing dicamba at a concentration selected so that transformed plant cells will grow and untransformed plant cells will not grow. In another embodiment, the method comprising applying dicamba to a population of plants suspected of comprising plants that have been transformed so that they are tolerant to dicamba, the dicamba being applied in an amount selected so that transformed plants will grow, and the growth of untransformed plants will be inhibited. [0014]
  • Finally, the invention provides a method of selecting, or screening for, transformed host cells, intact organisms, and parts of organisms. The method comprises providing a population of host cells, intact organisms, or parts of organisms suspected of comprising host cells, intact organisms, or parts of organisms that have been transformed so that they are able to degrade dicamba, contacting the population of host cells, intact organisms, or parts of organisms with dicamba, and ascertaining the presence or level of fluorescence due to 3,6-dichlorosalicylic acid. The 3,6-dichlorosalicyclic acid is generated in transformed host cells, intact organisms, or parts of organisms as a result of the degradation of dicamba, but will not be generated in untransformed host cells, intact organisms, or parts of organisms.[0015]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1. A diagram of the proposed electron transport scheme for dicamba O-demethylase. Electrons from NADH are transferred sequentially from reductase[0016] DIC to ferredoxinDIC and then to oxygenaseDIC. The reaction of oxygen with the substrate dicamba to form 3,6-dichlorosalicylic acid is catalyzed by oxygenaseDIC. ox, oxidized; red, reduced.
  • FIG. 2. Comparison of the derived amino acid sequence of the ferredoxin component of dicamba O-demethylase to the amino acid sequences of members of the adrenodoxin family of ferredoxins. In FIG. 2, ferr di6=ferredoxin component of dicamba O-demethylase from [0017] Pseudomonas maltophilia DI-6[SEQ ID NO: 5]; fer6 rhoca=ferredoxin from Rhodobacter capsulatus [SEQ ID NO: 10]; fer caucr=ferredoxin from Caulobacter crescentus [SEQ ID NO: 11]; thcc rhocr=ferredoxin from Rhodococcus erythropolis [SEQ ID NO: 12]; putx psepu=ferredoxin from Pseudomonas putida [SEQ ID NO: 13]; terp psesp=ferredoxin from Pseudomonas sp. [SEQ ID NO: 14]. Also in FIG. 2, the numbers 1-3 designate the three conserved motifs of the adrenodoxin family of bacterial ferredoxins.
  • FIG. 3. Comparison of the derived amino acid sequence of the two reductase components of dicamba O-demethylase to the amino acid sequences of members of the family of FAD-dependent pyridine nucleotide reductases. In FIG. 3, red1 di6=reductase component of dicamba O-demethylase from [0018] P. maltophilia DI-6[SEQ ID NO: 7]; red2 di6=reductase component of dicamba O-demethylase from P. maltophilia DI-6[SEQ ID NO: 9]; AJ002606=reductase from Sphingomonas sp. [SEQ ID NO: 15]; thcd rhoer=reductase from R. erythropolis [SEQ ID NO: 16]; cama psepu=reductase from P. putida [SEQ ID NO: 17]; tera pscsp=reductase from Pseudomonas sp. [SEQ ID NO: 18]. Also in FIG. 3, the numbers 1-5 designate the five conserved motifs of FAD-dependent pyridine nucleotide reductases.
    • DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS OF THE INVENTION
  • Prior studies (Cork and Kreuger, [0019] Advan. Appl. Microbiol. 36:1-56 and Yang et al. (1994) Anal. Biochem. 219:37-42) have shown that the soil bacterium, Pseudomonas maltophilia, strain DI-6, is capable of destroying the herbicidal activity of dicamba through a single step reaction in which dicamba (3,6-dichloro-2-methoxybenzoic acid) is converted to 3,6-dichlorosalicylic acid (3,6-DCSA). 3,6-DCSA has no herbicidal activity and has not been shown to have any detrimental effects on plants. In addition, 3,6-DCSA is readily degraded by the normal bacterial flora present in soil.
  • The experiments described herein confirm the hypothesis of Yang et al. (see id.) that an O-demethylase is responsible for the conversion of dicamba to 3,6-DCSA by [0020] P. maltophilia strain DI-6 and establish that the O-demethylase is a three-component enzyme system consisting of a reductase, a ferredoxin, and an oxygenase. See Examples 1 and 3 which describe in detail the isolation, purification and characterization of the P. maltophilia O-demethylase and its three components. The reaction scheme for the reaction catalyzed by the three components of dicamba O-demethylase is presented in FIG. 1. As illustrated in FIG. 1, electrons from NADH are shuttled through a short electron chain consisting of the reductase and ferredoxin to the terminal oxygenase which catalyzes the oxidation of dicamba to produce 3,6-DCSA.
  • In a first embodiment, the invention provides isolated and at least partially purified dicamba-degrading enzymes. “Isolated” is used herein to mean that the enzymes have at least been removed from the cells in which they are produced (i.e., they are contained in a cell lysate). “At least partially purified” is used herein to mean that they have been separated at least partially from the other components of the cell lysate. Preferably, the enzymes have been purified sufficiently so that the enzyme preparations are at least about 70% homogenous. [0021]
  • In particular, the invention provides an isolated and at least partially purified dicamba-degrading O-demethylase. “Dicamba-degrading O-demethylase” is defined herein to mean a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase, all as defined below. [0022]
  • The invention also provides an isolated and at least partially purified dicamba-degrading oxygenase. “Dicamba-degrading oxygenase” is defined herein to mean the oxygenase purified from [0023] P. maltophilia strain DI-6 and oxygenases which have an amino acid sequence which is at least about 65% homologous, preferably at least about 85% homologous, to that of the P. maltophilia oxygenase and which can participate in the degradation of dicamba. “Dicamba-degrading oxygenases” include mutant oxygenases having the amino acid sequence of the P. maltophilia oxygenase wherein one or more amino acids have been added to, deleted from, or substituted for, the amino acids of the P. maltophilia oxygenase sequence. Activity of dicamba-degrading oxygenases can be determined as described in Examples 1 and 3.
  • The invention further provides an isolated and at least partially purified dicamba-degrading ferredoxin. “Dicamba-degrading ferredoxin” is defined herein to mean the ferredoxin purified from [0024] P. maltophilia strain DI-6 and ferredoxins which have an amino acid sequence which is at least about 65% homologous, preferably at least about 85% homologous, to that of the P. maltophilia ferredoxin and which can participate in the degradation of dicamba. “Dicamba-degrading ferredoxins” include mutant ferredoxins having the amino acid sequence of the P. maltophilia ferredoxin wherein one or more amino acids have been added to, deleted from, or substituted for, the amino acids of the P. maltophilia ferredoxin sequence. Activity of dicamba-degrading ferredoxins can be determined as described in Examples 1 and 3.
  • Finally, the invention provides an isolated and at least partially purified dicamba-degrading reductase. “Dicamba-degrading reductase” is defined herein to mean the reductases purified from [0025] P. maltophilia strain DI-6 and reductases which have an amino acid sequence which is at least about 65% homologous, preferably at least about 85% homologous, to that of one of the P. maltophilia reductases and which can participate in the degradation of dicamba. “Dicamba-degrading reductases” include mutant reductases having the amino acid sequence of one of the P. maltophilia reductases wherein one or more amino acids have been added to, deleted from, or substituted for, the amino acids of the P. maltophilia reductase sequence. Activity of dicamba-degrading reductases can be determined as described in Examples 1 and 3.
  • Methods of determining the degree of homology of amino acid sequences are well known in the art. For instance, the FASTA program of the Genetics Computing Group (GCG) software package (University of Wisconsin, Madison, Wis.) can be used to compare sequences in various protein databases such as the Swiss Protein Database. [0026]
  • The dicamba-degrading enzymes of the invention can be isolated and purified as described in Examples 1 and 3 from [0027] P. maltophilia or other organisms. Suitable other organisms include bacteria other than P. maltophilia strain DI-6 that degrade dicamba. Several strains of such bacteria are known. See U.S. Pat. No. 5,445,962; Krueger et al., J. Agric. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbiol., 38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol., 40, 289-320 (1995). Other dicamba-degrading bacterial strains can be isolated as were these strains by methods well known in the art.
  • Preferably, however, the dicamba-degrading enzymes of the invention are prepared using recombinant DNA techniques (see below). In particular, mutant enzymes having the amino acid sequence of the [0028] P. maltophilia enzyme wherein one or more amino acids have been added to, deleted from, or substituted for, the amino acids of the P. maltophilia sequence are prepared in this manner using, for example, oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See Ausubel et al. (eds.), Current Protocols In Molecular Biology (Wiley Interscience 1990) and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991).
  • In a second embodiment, the invention provides isolated DNA molecules coding for dicamba-degrading enzymes of the invention. “Isolated” is used herein to mean that the DNA molecule has been removed from its natural environment or is not a naturally-occurring is DNA molecule. Methods of preparing these DNA molecules are well known in the art. See, e.g., Maniatis et al., [0029] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).
  • For instance, the DNA molecules of the invention may be isolated cDNA or genomic clones. The identification and isolation of clones coding for the dicamba-degrading enzymes of [0030] P. maltophilia strain DI-6 are described in Examples 2 and 4-5. Additional clones coding for dicamba-degrading enzymes can be obtained in a similar manner. The isolated clones, or portions of them, can be used as probes to identify and isolate additional clones from organisms other than the ones from which the clones were originally isolated. Suitable organisms include bacteria that degrade dicamba. As noted above, in addition to P. maltophilia strain DI-6, several strains of bacteria are known that degrade dicamba. See U.S. Pat. No. 5,445,962; Krueger et al., J. Agric. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbiol., 38, 1-66(1991); Cork and Khalil, Adv. Appl. Microbiol., 40, 289-320 (1995).
  • The DNA molecules of the invention can also be chemically synthesized using the sequences of isolated clones. Such techniques are well known in the art. For instance, DNA sequences may be synthesized by phosphoamidite chemistry in an automated DNA synthesizer. Chemical synthesis has a number of advantages. In particular, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence will be expressed may be used to optimize expression. Not all of the codons need to be altered to obtain improved expression, but preferably at least the codons rarely used in the host are changed to host-preferred codons. High levels of expression can be obtained by changing greater than about 50%, most preferably at least about 80%, of the codons to host-preferred codons. The codon preferences of many host cells are known. See, e.g., [0031] Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds., 1986), PCT WO 97/31115, PCT WO 97/11086, EP 646643, EP 553494, and U.S. Pat. Nos. 5,689,052, 5,567,862, 5,567,600, 5,552,299 and 5,017,692. The codon preferences of other host cells can be deduced by methods known in the art. Also, using chemical synthesis, the sequence of the DNA molecule or its encoded protein can be readily changed to, e.g., optimize expression (e.g., eliminate mRNA secondary structures that interfere with transcription or translation), add unique restriction sites at convenient points, delete protease cleavage sites, etc.
  • In a third embodiment, the present invention provides DNA constructs comprising DNA coding for a dicamba-degrading enzyme operatively linked to expression control sequences or a plurality of DNA coding sequences, each coding for a dicamba-degrading enzyme and each being operatively linked to expression control sequences. “DNA constructs” are defined herein to be constructed (non-naturally occurring) DNA molecules useful for introducing DNA into host cells, and the term includes chimeric genes, expression cassettes, and vectors. [0032]
  • As used herein “operatively linked” refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded proteins are expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g. Maniatis et al., [0033] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).
  • “Expression control sequences” are DNA sequences involved in any way in the control of transcription or translation in prokaryotes and eukaryotes. Suitable expression control sequences and methods of making and using them are well known in the art. [0034]
  • The expression control sequences must include a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the chosen host cell or organism. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occuring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, [0035] Nucleic Acids Res., 15, 2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts, et al., Proc. Natl Acad. Sci. USA, 76, 760-4 (1979). Many suitable promoters for use in prokaryotes and eukaryotes are well known in the art.
  • For instance, suitable constitutive promoters for use in plants include: the promoters from plant viruses, such as the 35S promoter from cauliflower mosaic virus (Odell et al., [0036] Nature 313:810-812 (1985), promoters of Chlorella virus methyltransferase genes (U.S. Pat. No. 5,563,328), and the full-length transcript promoter from figwort mosaic virus (U.S. Pat. No. 5,378,619); the promoters from such genes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)), ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)), pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)), MAS (Velten et al., EMBO J. 3:2723-2730 (1984)), maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231:276-285 (1992) and Atanassova et al., Plant Journal 2(3):291-300 (1992)), Brassica napus ALS3 (PCT application WO 97/41228); and promoters of various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002, 5,102,796, 5,182,200, 5,428,147).
  • Suitable inducible promoters for use in plants include: the promoter from the ACE1 system which responds to copper (Mett et al. [0037] PNAS 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen. Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)), and the promoter of the Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet. 227:229-237(1991). A particularly preferred inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. USA 88:10421 (1991). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269.
  • Suitable promoters for use in bacteria include the promoter of the [0038] Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gene, the Bacillus pumilus xylosidase gene, the phage lambda PR and PL promoters, and the Escherichia coli lac, trp and tac promoters. See PCT WO 96/23898 and PCT WO 97/42320.
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes, promoters from alcohol dehydrogenase genes, the TPI1 promoter, and the ADH2-4c promoter. See PCT WO 96/23898. [0039]
  • Suitable promoters for use in filamentous fungi include the ADH3 promoter, the tpiA promoter, the promoters of the genes encoding [0040] Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or Aspergillus awamori glucoamylase, R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, and Aspergillus nidulans acetamidase. See PCT WO 96/23898.
  • Suitable promoters for use in mammalian cells are the SV40 promoter, metallothionein gene promoter, murine mammary tumor virus promoter, [0041] Rous sarcoma virus promoter, and adenovirus 2 major late promoter. See PCT WO 96/23898 and PCT WO 97/42320.
  • Suitable promoters for use in insect cells include the polyhedrin promoter, P10 promoter, the [0042] Autographa californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter. See PCT WO 96/23898.
  • Finally, promoters composed of portions of other promoters and partially or totally synthetic promoters can be used. See, e.g., Ni et al., [0043] Plant J., 7:661-676(1995)and PCT WO 95/14098 describing such promoters for use in plants.
  • The promoter may include, or be modified to include, one or more enhancer elements. Preferably, the promoter will include a plurality of enhancer elements. Promoters containing enhancer elements provide for higher levels of transcription as compared to promoters which do not include them. Suitable enhancer elements for use in plants include the 35S enhancer element from cauliflower mosaic virus (U.S. Pat. Nos. 5,106,739 and 5,164,316) and the enhancer element from figwort mosaic virus (Maiti et al., [0044] Transgenic Res., 6, 143-156 (1997)). Other suitable enhancers for use in other cells are known. See PCT WO 96/23898 and Enhancers And Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1983).
  • For efficient expression, the coding sequences are preferably also operatively linked to a 3′ untranslated sequence. The 3′ untranslated sequence contains transcription and/or translation termination sequences. The 3′ untranslated regions can be obtained from the flanking regions of genes from bacterial, plant or other eukaryotic cells. For use in prokaryotes, the 3′ untranslated region will include a transcription termination sequence. For use in plants and other eukaryotes, the 3′ untranslated region will include a transcription termination sequence and a polyadenylation sequence. Suitable 3′ untranslated sequences for use in plants include those of the cauliflower mosaic virus 35S gene, the phaseolin seed storage protein gene, the pea ribulose biphosphate carboxylase small subunit E9 gene, the soybean 7S storage protein genes, the octopine synthase gene, and the nopaline synthase gene. [0045]
  • In plants and other eukaryotes, a 5′ untranslated sequence is also employed. The 5′ untranslated sequence is the portion of an mRNA which extends from the 5′ CAP site to the translation initiation codon. This region of the mRNA is necessary for translation initiation in eukaryotes and plays a role in the regulation of gene expression. Suitable 5′ untranslated regions for use in plants include those of alfalfa mosaic virus, cucumber mosaic virus coat protein gene, and tobacco mosaic virus. [0046]
  • As noted above, the DNA construct may be a vector. The vector may contain one or more replication systems which allow it to replicate in host cells. Self-replicating vectors include plasmids, cosmids and viral vectors. Alternatively, the vector may be an integrating vector which allows the integration into the host cell's chromosome of the sequences coding for the dicamba-degrading enzymes of the invention. The vector desirably also has unique restriction sites for the insertion of DNA sequences. If a vector does not have unique restriction sites, it may be modified to introduce or eliminate restriction sites to make it more suitable for further manipulations. [0047]
  • The DNA constructs of the invention can be used to transform a variety of host cells (see below). A genetic marker must be used for selecting transformed host cells (“a selection marker”). Selection markers typically allow transformed cells to be recovered by negative selection (i.e., inhibiting growth of cells that do not contain the selection marker) or by screening for a product encoded by the selection marker. [0048]
  • The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from Tn5, which, when placed under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al., [0049] Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).
  • Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., [0050] Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988).
  • Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., [0051] Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990).
  • Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, R. A., [0052] Plant Mol. Biol. Rep. 5:387 (1987)., Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131 (1987), De Block et al., EMBO J. 3:1681 (1984), green fluorescent protein (GFP) (Chalfie et al., Science 263:802 (1994), Haseloff et al., TIG 11:328-329 (1995) and PCT application WO 97/41228). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).
  • Suitable selection markers for use in prokaryotes and eukaryotes other than plants are also well known. See, e.g., PCT WO 96/23898 and PCT WO 97/42320. For instance, resistance to antibiotics (ampicillin, kanamycin, tetracyline, chloramphenicol, neomycin or hygromycin) may be used as the selection marker. [0053]
  • According to another aspect of the present invention, tolerance to dicamba can be used as a selection marker for plants and plant cells. “Tolerance” means that transformed plant cells are able to grow (survive and regenerate into plants) when placed in culture medium containing a level of dicamba that prevents untransformed cells from doing so. “Tolerance” also means that transformed plants are able to grow after application of an amount of dicamba that inhibits the growth of untransformed plants. [0054]
  • Methods of selecting transformed plant cells are well known in the art. Briefly, at least some of the plant cells in a population of plant cells (e.g., an explant or an embryonic suspension culture) are transformed with a DNA construct or combination of DNA constructs providing for dicamba degradation. The resulting population of plant cells is placed in culture medium containing dicamba at a concentration selected so that transformed plant cells will grow, whereas untransformed plant cells will not. Suitable concentrations of dicamba can be determined empirically as is known in the art. [0055]
  • Methods of selecting transformed plants are also known in the art. Briefly, dicamba is applied to a population of plants suspected of comprising a DNA construct or a combination of DNA constructs providing for dicamba degradation. The amount of dicamba is selected so that transformed plants will grow, and the growth of untransformed plants will be inhibited. The level of inhibition must be sufficient so that transformed and untransformed plants can be readily distinguished (i.e., inhibition must be statistically significant). Such amounts can be determined empirically as is known in the art. [0056]
  • Further, the generation of 3,6-DCSA as a result of the degradation of dicamba can be used for selection and screening. The generation of 3,6-DCSA can be readily ascertained by observing the fluorescence of this compound, allowing selection and screening of transformed host cells, intact organisms, and parts of organisms (e.g., microorganisms, plants, plant parts, and plant cells). In this regard, the invention allows for selection and screening of transformed host cells, intact organisms, and parts of organisms in the same manner as for green fluorescent protein (GFP). See U.S. Pat. Nos. 5,162,227 and 5,491,084 and PCT applications WO 96/27675, WO 97/11094, WO 97/41228 and WO 97/42320, all of which are incorporated herein by reference. In particular, 3,6-DCSA can be detected in transformed host cells, intact organisms, and parts of organisms using conventional spectrophotometric methods. For instance, microscopes can be fitted with appropriate filter combinations for fluorescence excitation and detection. A hand-held lamp may be used for benchtop work or field work (see Example 1). Fluorescence-activated cell sorting can also be employed. 3,6-DCSA is excited at a wavelength of 312-313 nm, with a maximum emission wavelength of 424 nm. [0057]
  • “Parts” of organisms include organs, tissues, or any other part. “Plant parts” include seeds, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc. [0058]
  • Selection based on dicamba tolerance or dicamba degradation can be used in the production of dicamba-tolerant plants or dicamba-degrading microorganisms, in which case the use of another selection marker may not be necessary. Selection based on dicamba tolerance or dicamba degradation can also be used in the production of transgenic cells or organisms that express other genes of interest. Many such genes are known and include genes coding for proteins of commercial value and genes that confer improved agronomic traits on plants (see, e.g., PCT WO 97/41228, the complete disclosure of which is incorporated herein by reference). [0059]
  • The DNA constructs of the invention can be used to transform a variety of host cells, including prokaryotes and eukaryotes. The DNA sequences coding for the dicamba-degrading enzyme(s) and the selection marker, if a separate selection marker is used, may be on the same or different DNA constructs. Preferably, they are arranged on a single DNA construct as a transcription unit so that all of the coding sequences are expressed together. Also, the gene(s) of interest and the DNA sequence(s) coding for the dicamba-degrading enzyme(s), when dicamba-tolerance or dicamba degradation is being used as a selection marker, may be on the same or different DNA constructs. Such constructs are prepared in the same manner as described above. [0060]
  • Suitable host cells include prokaryotic and eukaryotic microorganisms (e.g., bacteria (including [0061] Agrobacterium tumefaciens and Escherichia coli), yeast (including Saccharomyces cerevisiae) and other fungi (including Aspergillus sp.), plant cells, insect cells, and mammalian cells. Preferably, the host cell is one that does not normally degrade dicamba. However, the present invention can also be used to increase the level of dicamba degradation in host cells that normally degrade dicamba.
  • Thus, the “transgenic” cells and organisms of the invention include cells and organisms that do not normally degrade dicamba, but which have been transformed according to the invention so that they are able to degrade this herbicide. The “transgenic” cells and organisms of the invention also include cells and organisms that normally degrade dicamba, but which have been transformed according to the invention so that they are able to degrade more of this herbicide or to degrade the herbicide more efficiently. [0062]
  • Methods of transforming prokaryotic and eukaryotic host cells are well known in the art. See, e.g., Maniatis et al., [0063] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989); PCT WO 96/23898 and PCT WO 97/42320.
  • For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in [0064] Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.
  • The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., [0065] Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by numerous references, including Gruber et al., supra, Miki et al., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos. 4,940,838 and 5,464,763.
  • A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds sufficient to penetrate plant cell walls and membranes. Sanford et al., [0066] Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206 (1990), Klein et al., Biotechnology 10:268 (1992).
  • Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., [0067] Bio/Technology 9:996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).
  • Transgenic dicamba-tolerant plants of any type may be produced according to the invention. In particular, broadleaf plants (including beans, soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, fruit trees, and ornamental plants and trees) that are currently known to be injured by dicamba can be transformed so that they become tolerant to the herbicide. Other plants (such as corn, sorghum, small grains, sugarcane, asparagus, and grass) which are currently considered tolerant to dicamba can be transformed to increase their tolerance to the herbicide. “Tolerant” means that the transformed plants can grow in the presence of an amount of dicamba which inhibits the growth of untransformed plants [0068]
  • It is anticipated that the dicamba-degrading oxygenases of the invention can function with endogenous reductases and ferredoxins found in transgenic host cells and organisms to degrade dicamba. Plant chloroplasts are particularly rich in reductases and ferredoxins. Accordingly, a preferred embodiment for the production of transgenic dicamba-tolerant plants is the use of a sequence coding for a peptide that will direct the dicamba-degrading oxygenase into chloroplasts (“a chloroplast targeting sequence”). DNA coding for the chloroplast targeting sequence is preferably placed upstream (5′) of the sequence coding for the dicamba-degrading oxygenase, but may also be placed downstream (3′) of the coding sequence, or both upstream and downstream of the coding sequence. Exemplary chloroplast targeting sequences include the maize cab-m7 signal sequence (see Becker et al., [0069] Plant Mol. Biol. 20:49 (1992) and PCT WO 97/41228) and the pea glutathione reductase signal sequence (Creissen et al., Plant J. 2:129 (1991) and PCT WO 97/41228). An alternative preferred embodiment is the direct transformation of chloroplasts using a construct comprising a promoter functional in chloroplasts to obtain expression of the oxygenase in chloroplasts. See, e.g., PCT application WO 95/24492 and U.S. Pat. No. 5,545,818. Of course, if a selected transgenic host cell or organism does not produce sufficient endogenous reductase, ferredoxin, or both, the host cell or organism can be transformed so that it produces one or both of these enzymes as well as the oxygenase.
  • In yet another embodiment, the invention provides a method of controlling weeds in a field where transgenic dicamba-tolerant plants are growing. The method comprises applying an effective amount of dicamba to the field to control the weeds. Methods of applying dicamba and amounts of dicamba effective to control various types of weeds are known. See [0070] Crop Protection Reference, pages 1803-1821 (Chemical & Pharmaceutical Press, Inc., New York, N.Y., 11th ed., 1995).
  • In another embodiment, the invention provides a method of degrading dicamba present in a material, such as soil, water, or waste products of a dicamba manufacturing facility. Such degradation can be accomplished using the dicamba-degrading enzymes of the invention. The enzymes can be purified from microorganisms naturally expressing them (see Examples 1 and 3) or can be purified from transgenic host cells producing them. If the enzymes are used in such methods, then appropriate cofactors must also be provided (see Example 1). Effective amounts can be determined empirically as is known in the art (see Example 1). Alternatively, transgenic prokaryotic and eukaryotic microorganisms can be used to degrade dicamba in such materials. Transgenic prokaryotic and eukaryotic microorganisms can be produced as described above, and effective amounts can be determined empirically as is known in the art. [0071]
  • Dicamba is introduced into the environment in large quantities on a continuing basis. The elimination of dicamba is dependent in large part on the action of enzyme systems which are found in microorganisms inhabiting the soil and water of the planet. An understanding of these enzyme systems, including dicamba-degrading O-demethylases and their three components, is important in efforts to exploit natural and genetically modified microbes for bioremediation and the restoration of contaminated soil, water and other materials. Thus, the dicamba-degrading enzymes, DNA molecules, DNA constructs, etc., of the invention can be used as research tools for the study of dicamba degradation and bioremediation. [0072]
  • Finally, the dicamba-degrading enzymes of the invention can be used in an assay for dicamba. A sample suspected of containing dicamba is mixed with a dicamba-degrading O-demethylase or a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase. Suitable assays are described in Examples 1 and 3. In particular, detecting or quantitating the fluorescence due to the generation of 3,6-DCSA makes for a convenient assay. [0073]
    • EXAMPLES Example 1 Purification and Characterization of the Components of Dicamba O-Demethylase of Pseudomonas maltophilia DI-6 Methods and Materials
  • Bacterium and growth conditions. [0074] Pseudomonas maltophilia, strain DI-6 (Kreuger, et al., (1989) J. Agric. Food Chem., 37:534-538) was isolated from a soil site persistently contaminated with dicamba. The bacterium was provided by Dr. Douglas Cork of the Illinois Institute of Technology (Chicago, Ill.), and was maintained on reduced chloride medium (Kreuger, J. P., (1989) Ph.D. thesis, Illinois Institute of Technology, Chicago, Ill.), supplemented with either dicamba (2 mg/ml) or a mixture of glucose (2 mg/ml) and Casamino Acids (2 mg/ml). The carbon sources were filter-sterilized and added to the medium after it was autoclaved. Solid media were prepared by the addition of 1% (wt/vol) Gelrite (Scott Laboratories, West Warwick, R.I.).
  • Chemicals and reagents. Dicamba, 3,6-DCSA, and [[0075] 14C]dicamba (U-phenyl-14C, 42.4 mCi/mmol, radiochemical purity greater than 98%) were supplied by Sandoz Agro, Inc. (Des Plaines, Ill). To increase solubility, the dicamba and 3,6-DCSA stock solutions were prepared by titration with KOH to pH 7.0. All chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.), unless otherwise stated. Superose 12, Mono Q, Q-Sepharose (Fast Flow) and Phenyl-Sepharose (CL-4B) column packings for the FPLC (fast performance liquid chromatography) apparatus were obtained from Pharmacia (Milwaukee, Wis.). Ampholyte pH 4-6 and ampholyte pH 4-9 were purchased from Serva (Heidelberg, FRG). Acrylamide, β-mercaptoethanol, N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) were from Bio-Rad Laboratories (Hercules, Calif.). Thin layer chromatography (TLC) plates were silica gel (250 μm thickness) with UV 254 indicator, and were purchased from J. T. Baker Chemical Co. (Phillipsburg, N.J.).
  • Enzyme assays. Dicamba O-demethylase activity was assayed by measuring the formation of [[0076] 14C]3,6-DCSA from [14C]dicamba. Briefly, the activity in mixtures of enzyme components was measured at 30° C. in a standard 300 μl reaction mixture containing 25 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl2, 0.5 mM NADH (beta-nicotinamide adenine dinucleotide, reduced form), 0.5 mM ferrous sulfate, 50 μM cold dicamba, 2.5 μM [14C]dicamba (the final specific activity of the radioactive dicamba was 1.9 mCi/mmol), and different amounts of cell lysate or partially purified enzyme. All enzyme assays during the final purification steps were conducted in phosphate buffer because the pH optimum for dicamba O-demethylase activity was found to be in the mid range of phosphate buffers, and higher enzyme activity was observed with phosphate buffer compared to Tris-HCl [tris(hydroxymethyl)aminomethane hydrochloride] buffer at pH 7.0. Reactions were initiated by the addition of the substrate, dicamba. At specific times, the reactions were stopped by adding 50 μl of 5% (vol/vol) H2SO4. Dicamba and dicamba metabolites were then extracted twice with one volume of ether, and the extracts were evaporated to dryness. The efficiencies of recovery (means±standard deviations) for the extraction procedure were 87%±2% for dicamba and 85%±3% for 3,6-DCSA (Yang et al., Anal. Biochem. 219:37-42 (1994)).
  • [[0077] 14C]dicamba and 14C-labeled metabolites were separated by thin layer chromatography (TLC). The ether-extracted dicamba and its metabolites were redissolved in 50 μl of ether prior to being spotted onto a TLC plate. The solvent system for running the TLC was chloroform-ethanol-acetic acid (85:10:5 [vol/vol/vol]). The resolved reaction products were visualized and quantified by exposing the TLC plate to a phosphor screen for 24 hours and then scanning the screen in a PhosphorImager SF (Molecular Dynamics, Sunnyvale, Calif.). Estimates of the amount of radioactivity in a particular spot on the TLC plate were determined by comparing the total pixel count in that spot relative to a spot on the same plate containing a known amount of [14C]dicamba. One unit of activity was defined as the amount of enzyme that catalyzes the formation of 1 nmol of3,6-DCSA from dicamba per minute at 30° C. Specific activities were based on the total protein concentration of the assay mixture.
  • The activity of the reductase component of dicamba demethylase was assayed by measuring reduction of 2,6-dichlorophenolindophenol (DCIP) with a Hitachi U-2000 spectrophotometer. The reaction contained 0.5 mM NADH, 0.2 mM FAD (flavin adenine dinucleotide), 50 μM DCIP, 20 mM Tris buffer (pH 8.0), and 10-100 μl of enzyme sample in a total volume of 1 ml. The change in absorbance at 600 nm with time was measured at room temperature. Specific activity was calculated using an extinction coefficient at 600 nm of 21.0 mM[0078] −1 cm−1 for reduced DCIP. Specific activity was expressed as nmol DCIP reduced min−1 mg−1 of protein.
  • In addition, an in situ DCIP assay was used to detect and locate the reductase activity in protein preparations separated on isoelectric focusing (IEF) gels. After electrophoresis of the proteins on an IEF gel, lanes sliced from the gel were washed with 20 ml of cold 20 mM Tris-HCl buffer (pH 8.0). Low melting agarose was dissolved by heating in 10 ml of 20 mM Tris-HCl buffer (pH 8.0) at a final concentration of 1.5% (w/v). When the agarose cooled to near room temperature, it was supplemented with 0.2 mM FAD, 50 μM DCIP, and 0.5 mM NADH. The assay mixture was poured onto a glass plate and allowed to solidify. The gel piece was placed on top of the solidified reaction mixture and allowed to set at room temperature for 15 minutes. If the gel slice contained a protein with reductase activity, a colorless band of reduced DCIP was generated in the blue background of DCIP. [0079]
  • Cell lysates. Cells were grown to an optical density at 550 nm of 1.3 to 1.5 in liquid reduced chloride medium containing a mixture of glucose and Casamino Acids on a rotary shaker (250 rpm at 30° C.). The cells were harvested by centrifugation, washed twice with cold 100 mM MgCl[0080] 2, and centrifuged again. Cell pastes were either used immediately or quickly frozen in liquid nitrogen and stored at −80° C. At the time of enzyme purification, 25 g of frozen cells were thawed and resuspended in 50 ml of isolation buffer containing 25 mM Tris buffer (pH 7.0), 10 mM MgCl2, and 0.5 mM EDTA. Phenylmethylsulfonyl fluoride and dithiothreitol were added to final concentrations of 0.5 mM and 1 mM, respectively. After addition of 10 mg of lysozyme and 1 mg of DNase, cells were stirred for 10 min on ice and broken by sonication (model XL2020 sonicator; Heat Systems) on ice at a medium setting (setting 5) with twelve 20-second bursts and 40-second resting intervals. The resulting cell lysates were diluted to 90 ml with isolation buffer and centrifuged at 76,000×g for 1 h at 4° C. The supernatant was used as the source of cleared cell lysate.
  • Enzyme purification. All procedures were performed at 4° C., unless otherwise stated. Solid ammonium sulfate was slowly added to a 90-ml volume of cleared cell lysate to 40% (wt/vol) saturation, with constant stirring. After 15 minutes of stirring, the mixtures were centrifuged at 15,400×g for 15 minutes, and the precipitate was discarded. Additional solid ammonium sulfate was added to 70% (wt/vol) saturation, with constant stirring of the supernatant. After 15 min of stirring, the mixtures were centrifuged under the conditions described above. The supernatant was discarded, and the precipitate was resuspended in a minimal volume of buffer A (20 mM Tris [pH 8.0], 2.5 mM MgCl[0081] 2, 0.5 mM EDTA, 5% (vol/vol) glycerol, and 1 mM dithiothreitol).
  • The 40%-70% ammonium sulfate cut was then loaded onto a Phenyl-Sepharose column (2.5 by 10 cm) connected to a FPLC apparatus (Pharmacia) and eluted with a decreasing linear gradient of (NH[0082] 4)2SO4 from 10% (w/v) to 0% (w/v). The column was preequilibrated with buffer A containing 10% (wt/vol) armonium sulfate. The flow rate was 1 ml/min. Protein concentrations were continuously monitored at A280 during column elution. The column was washed with 120 ml of buffer A containing 10% (wt/vol) ammonium sulfate until baseline A280 readings were obtained. Bound proteins were eluted with a decreasing gradient of (NH4)2SO4 in buffer A [10 to 0% (wt/vol) (NH4)2SO4 in a total volume of 210 ml]. Fractions of 2 ml were collected. Aliquots of 10 μl were taken from each fraction and added to the standard dicamba O-demethylase assay mixture (see above), except that nonradioactive dicamba was used as the substrate. Dicamba O-demethylase activity was detected by monitoring the conversion of dicamba to the highly fluorescent reaction product 3,6-DCSA with a hand-held UV lamp (312 nm, Fotodyne) in a darkened room.
  • This procedure allowed resolution of dicamba O-demethylase into three pools containing the separated components (designated components I, II and III). Each component was essential for dicamba O-demethylase activity (see below). When a single component was assayed, the other two components were provided in excess. Fractions containing a single type of activity were pooled (component I, fractions 128-145; component II, unbound fractions 12-33; component III, fractions 62-92). [0083]
  • (i) Purification of component I. Fractions containing component I activity (eluting from a Phenyl-Sepharose column at 0 M (NH[0084] 4)2SO4, fractions 128-145) were pooled to provide a total volume of 34 ml. The pooled samples were concentrated to 10 ml by centrifugation in a Centriprep-10 device (Amicon) and then applied to a Q-Sepharose (Fast Flow) FPLC column (Pharmacia) (2.5 by 6 cm) equilibrated with buffer A and washed with 80 ml of buffer A. Proteins bound to the column were eluted with a 100 ml linear gradient of 0 to 0.6 M KCl in buffer A at a flow rate of 1 ml/min. Fractions were collected at 1.5 minute intervals. Those fractions exhibiting component I activity (fractions 29-37) were pooled, dialyzed against buffer A overnight at 4° C. and applied to a Mono Q HR 5/5 FPLC anion-exchange column in buffer A. Proteins were eluted at 1 ml/min by using a 50 ml gradient of increasing KCl concentration (0 to 0.5 M). Fractions showing component I activity (fractions 19 to 25) were pooled and concentrated to 0.4 ml by centrifugation in a Centricon-10 device. The concentrated sample was then subjected to chromatography on a Superose 12 FPLC column (1.6 by 50 cm) at a flow rate of 0.2 ml/min with buffer A containing 100 mM KCl. Fractions 7-10 showing component I activity were pooled and concentrated by centrifugation in a Centricon-10 device.
  • The partially purified component I was diluted with cold 1% (w/v) glycine and concentrated by centrifugation in a Centricon-10 device three more times to desalt it in preparation for IEF electrophoresis. The desalted and concentrated sample was then applied to a 6% (w/v) IEF (pH 4-6) gel and subjected to electrophoresis for 1.5 hours at 4° C. (see below). After electrophoresis, the gel was washed with 25 mM cold phosphate buffer (pH 7.0) for 5 minutes and then each slice of the gel lane was diced into small (6 mm×4 mm) pieces. Protein was eluted from the diced gel fragments by grinding them with a pipette tip in the presence of 10 μl of 25 mM phosphate buffer (pH 7.0). Protein from each segment was mixed with an excess of components II and III and assayed for dicamba O-demethylase activity. The gel segment which showed component I activity (which was also reddish brown in color) was loaded onto a 12.5% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) to check sample purity. [0085]
  • (ii) Purification of component II Component II obtained by Phenyl-Sepharose column chromatography was dialyzed against buffer A overnight at 4° C. and applied to a FPLC Q-Sepharose column (2.5 by 6 cm). Sample elution conditions were identical to those described above for component I except that the elution gradient was 0 to 1 M KCl in buffer A. Fractions exhibiting component II activity (fractions 30-37) were pooled, dialyzed against buffer A, concentrated to 0.4 ml and applied to a [0086] FPLC Superose 12 column (1.6 by 50 cm). The procedures for sample application and elution were identical to those described above for component I. Fractions exhibiting component II activity (fractions 3-6) were pooled, diluted with an equal volume of buffer A, and applied to a FPLC Mono Q column. Proteins were eluted from the column using the same KCl gradient as for component I. Fractions 20-25 showed component II activity. Partially purified component II was further purified by IEF (pH 4-6) electrophoresis using the same conditions as described for component I. The gel segment which showed component II activity was loaded onto a 12.5% (w/v) SDS-PAGE for further analysis.
  • (iii) Purification of component III. Component III obtained by Phenyl-Sepharose column chromatography was dialyzed against buffer A overnight at 4° C. and applied to a FPLC Q-Sepharose column (2.5 by 6 cm). Conditions were identical to those described above for component I. Fractions exhibiting component III activity (fractions 26-38) were dialyzed against buffer B [10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl[0087] 2, 5% (v/v) glycerol, 1 mM dithiothreitol] and concentrated to 5 ml. Blue dye affinity matrix [Cibacron Blue 3GA type 3000 (Sigma)] was packed into a FPLC column (1×5 cm) and pre-equilibrated with 20 ml of buffer B. Concentrated component III was loaded onto the blue dye column and washed with 20 ml of buffer B at a flow rate of 0.2 ml/min until the A280 of the column effluent reached baseline levels. Bound protein was then eluted with 5 mM NADH in buffer B. Fractions containing reductase activity were detected by assaying for dicamba O-demethylase activity in the presence of an excess of components I and II and also by the ability of each fraction to reduce DCIP in the presence of NADH. Fractions having strong reductase activity in both assays were pooled, dialyzed against buffer A containing 100 mM KCl, concentrated to 0.2 ml, and applied to a FPLC Superose 12 column. The same conditions were used for running the Superose column as described for component I. Fractions containing proteins which catalyzed DCIP reduction were pooled, dialyzed against buffer A and applied to a FPLC Mono Q column. Proteins were eluted using the same conditions as for component I. Partially purified component III was further purified by IEF (pH 4-6) gel electrophoresis. The reductase activity of proteins within the IEF gel was detected by assaying for DCIP reduction in an agarose gel overlay as described above. The gel segment which showed component II activity was loaded onto a 12.5% (w/v) SDS-PAGE for further analysis.
  • Determination of NH[0088] 2-terminal amino acid sequences. Protein bands were excised from IEF gels and placed in the wells of a 12.5% (w/v) SDS polyacrylamide gel. After electrophoresis, the gel slices containing the purified proteins were transblotted onto a PVDF (polyvinylidene difluoride) membrane (Millipore) in a Trans-Blot cell (Bio-Rad, Richmond, Calif.) at 25 volts for 16 hours. The blotting buffer was a solution of 20% (v/v) methanol with 10 mM CAPS [3-(cyclohexylamino)-1-propanesulfonic acid], pH 10.0. Sequencing was performed using an Applied Biosystems Inc. 420 H machine by Edman degradation (Edman and Henschen (1975) pages 232-279 in S. B. Needleman (ed.), Protein sequence determination, 2nd ed., Springer-Verlage, N.Y.).
  • Determination of protein concentration. Protein concentrations were determined by the method of Bradford (1976) [0089] Anal. Biochem. 72:248-254, with bovine serum albumin as the standard.
  • SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) was performed according to modified methods of Laemmli (Laemmli (1970) [0090] Nature, 227:680-685). 12.5% (w/v) SDS gels of 85×65×0.75 mm were made as follows: running gel: 2.5 ml 40% (w/v) acrylamide/bis solution (37:5:1), 1 ml running buffer solution [3M Tris-CHl (pH 8.8), 0.8% (w/v) SDS], 4.5 ml H2O, 5 μl TEMED, and 40 μl 10% (w/v) APS; stacking gel: 0.5 ml 40% (w/v) acrylamide/bis, 0.5 ml stacking buffer solution [1 M Tris-HCl (pH 6.8), 0.8% (w/v) SDS], 3 ml H2O, 5 μl TEMED, and 12.5 μl 10% (w/v) APS. The composition of the running buffer was 25 mM Tris-HCl (pH 8.3), 0.2 M glycine, and 0.1% (w/v) SDS. The sample buffer contained 0.25 ml stacking buffer, 0.6 ml 20% (w/v) SDS, 0.2 ml β-mercaptoethanol, and 0.95 ml 0.1% bromphenol blue (w/v) in 50% (v/v) glycerol. Electrophoresis was performed at 80 volts in a Bio-Rad Mini Gel apparatus until the tracking dye was 0.5 cm from the anode end of the gel. Proteins were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in a mixture of isopropanol, water, and acetic acid at a ratio of 3:6:1 (v/v/v). Destaining was performed in a mixture of methanol, water, and acetic acid at a ratio of 7:83:10 (v/v/v). Standard proteins (Gibco BRL) included: myosin (214.2 kDa), phosphorylase B (111.4 kDa), bovine serum albumin (74.25 kDa), ovalbumin (45.5 kDa), carbonic anhydrase (29.5 kDa), β-lactoglobulin (18.3 kDa), and lysozyme (15.4 kDa).
  • Determination of molecular weight. The molecular weight (M[0091] r) of peptides under denaturing conditions was estimated using SDS-PAGE analysis. The molecular weights of the native components I, II and III were estimated by gel filtration through a Superose 12 HR 10/30 FPLC column (Pharmacia) at a flow rate of 0.2 ml/min in buffer A containing 100 mM KCl. Calibration proteins were gel filtration standards from Bio-Rad. They were: bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B-12 (1.35 kDa). The void volume of the Superose 12 column was calculated using Blue Dextran (Mr 2,000,000, Sigma).
  • IEF. Isoelectric focusing (IEF) gel electrophoresis was performed in a vertical mini-gel apparatus (Model #MGV-100) from C.B.S. Scientific Co. (Del Mar, Calif.). IEF gels with 6% (w/v) polyacrylamide (70×90×1 mm) were made by mixing the following: 1.6 ml 30% (w/v) acrylamide/bis (37:5:1), 0.8 g glycerol, 0.32 ml ampholyte pH 4-6 (Serva), 0.08 ml ampholyte pH 4-9 (Serva), 5.2 ml H[0092] 2O, 10 μl TEMED, and 80 μl 10% (w/v) APS. The cathode buffer was 100 mM β-alanine and the anode buffer was 100 mM acetic acid. Protein samples in approximately 1 to 10 μl of 1% (w/v) glycine were mixed with an equal volume of sample buffer [50% (v/v) glycerol, 1.6% (v/v) ampholyte pH 4-9, 2.4% (v/v) ampholyte pH 4-6]. Samples were loaded at the cathode end of the gel and allowed to migrate at 200 volts for 1.5 hours and 400 volts for another 1.5 hours. Proteins were stained with Coomassie Brilliant Blue R-250 using the procedure described above for SDS polyacrylamide gels. IEF markers (Sigma) were: amyloglucosodase, pI 3.6; glucose oxidase, pI 4.2; trypsin inhibitor, pI 4.6; β-lactoglobulin A, pI 5.1; carbonic anhydrase II, pI 5.4; carbonic anhydrase II, pI 5.9 and carbonic anhydrase I, pI 6.6
  • Kinetic analysis. The kinetics of the demethylation reaction catalyzed by dicamba O-demethylase were studied by analyzing the initial rates of the reaction in the presence of a constant concentration of the enzyme and increasing concentrations of the substrate, dicamba. Reaction mixtures contained 25 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl[0093] 2, 0.5 mM NADH, 0.5 mM FeSO4, 25 μg of partially purified O-demethylase enzyme [the 40%-70% (w/v) (NH4)2SO4 fraction from a cleared cell lysate], various concentrations (0.5 to 50 μM) of dicamba and various concentrations (0.025 to 2.5 μM) of [14C]dicamba (U-phenyl-14C, 42.4 mCi/mmol) in a total volume of 300 μl. For assays with dicamba concentrations of 0.5 μM and 1 μM, the reaction volume was increased to 900 μl to ensure that sufficient amounts of radioactive dicamba and its metabolites were present to allow detection. In these reactions, the amounts of all other components in the reaction were tripled. The conversion of [14C]dicamba to [14C]3,6-DCSA was determined for different time points at each concentration of dicamba using a PhosphorImager SF to scan radioactivity on phosphor screens which had been exposed to TLC plates for 24 hours. One unit of activity was defined as the amount of enzyme that forms 1 nmol of 3,6-DCSA per minute at 30° C. The initial rates of each reaction were determined by plotting the reaction rate versus time at each substrate concentration. Data were modeled to Michaelis-Menten kinetics and values of Km and Vmax were determined by fitting to Lineweaver-Burk plots using SigmaPlot® (Jandel Scientific, Corte Madera, Calif.).
  • Oxygen requirement. Preliminary experiments using a Clark oxygen electrode indicated oxygen consumption during a standard dicamba O-demethylase assay with dicamba as a substrate. To verify a requirement for oxygen in the O demethylation of dicamba by dicamba O-demethylase, reactions were conducted in an anaerobic chamber which contained less than 1 ppm of oxygen. The procedures for displacement of oxygen from the reaction mixture were performed at 4° C. Reaction mixtures lacking enzyme were placed in a vial and sealed with a rubber stopper. For displacement of oxygen, the vial was evacuated twice by vacuum and flushed each time with nitrogen. After a third evacuation, the vial was flushed with 90% nitrogen plus 10% hydrogen. The enzyme solution was likewise purged of oxygen (with care taken not to bubble the enzyme solution). Both the reaction mixtures and enzyme solutions were transferred into an anaerobic chamber (95% N[0094] 2-5% H2 atmosphere). Two hundred forty microliters of cleared cell lysate was injected through the rubber stopper with a microsyringe and gently mixed with 960 μl of oxygen-free reaction mixture. Reactions were carried out at 30° C.
  • An examination of the reaction products on TLC plates showed that the rate of [[0095] 14C]3,6-DCSA production from [14C]dicamba under anaerobic conditions was significantly lower than the rate of reactions with the same amount of enzyme under aerobic conditions. Under anaerobic conditions, there was virtually no conversion of dicamba to 3,6-DCSA within 1 hour. However, when a parallel reaction mixture was taken from the anaerobic chamber after 30 min and incubated with air, a significant quantity of one of the components of the dicamba O-demethylase enzyme complex was an oxygenase.
  • It may be noted that the in vitro conversion of [[0096] 14C]dicamba to [14C]3,6-DCSA mimics the in vivo conversion pathway documented earlier (Cork and Kreuger, Adv. Appl. Microbiol. 36:1-66 (1991); Yang et al., Anal. Biochem. 219:37-42 (1994)). In these studies, DCSA was identified as a reaction product by both TLC and capillary electrophoresis. Stringent identification of the first major product of dicamba degradation as DCSA both in vivo and in vitro has been obtained by gas chromatography-mass spectrometry analyses.
  • Component and cofactor requirements. After the initial separation of the three components of dicamba O-demethylase by phenyl-Sepharose column chromatography, the partially purified preparations were taken individually through one additional purification on a Q-Sepharose column (2.5 by 6 cm). Samples were applied to a Q-Sepharose (Fast Flow) fast protein liquid chromatography column (Pharmacia) in buffer A and eluted with a 100-ml linear gradient of 0 to 0.6 M KCl (for the oxygenase component) or 0 to 1.0 M KCl (for the ferredoxin and reductase components) in 1.5-ml fractions. Appropriate pooled fractions from separate columns for oxygenase purification (fractions 29 to 37), for ferredoxin purification (fractions 30 to 37), or for reductase purification (fractions 26 to 38) were used for the determination of component and cofactor requirements. [0097]
  • The three components were assayed for O-demethylase activity in various combinations to determine component requirements. [0098]
  • To determine cofactor requirements, O-demethylase activity was assayed using a mixture of the three components with [[0099] 14C]dicamba for 30 minutes at 30° C. The amounts of partially purified protein (provided in an optimized ratio) in the reaction mixtures were 85 μg of oxygenase, 55 μg of ferredoxin and 50 μg of reductase. The concentration of cofactors used in the reaction mixtures were 0.5 mM NADH, 0.2 mM FAD, 0.5 mM FeSO4, 10 mM MgCl2, 0.5 mM NADPH, and 0.2 mM FMN.
    • Results
  • Component I. The component of dicamba O-demethylase which bound most tightly to the Phenyl-Sepharose column (designated initially as component I and subsequently identified as an oxygenase) was distinctly reddish brown in color. This indicated the potential presence of a protein(s) containing an iron-sulfur cluster(s) or a heme group(s). The fractions with component I activity from the Phenyl-Sepharose column were subjected to further purification by Q-Sepharose (Fast Flow) and Mono Q chromatography and then to separation on a Superose 12 size exclusion column. The component I protein was then further purified on an IEF gel. [0100]
  • Protein from the major band on the IEF gel (with an apparent pI of approximately 4.6) was excised and separated from any remaining minor contaminants by SDS-PAGE. The major component I protein obtained after purification by IEF was greater than 90% pure as judged by densitometric analysis of this SDS-polyacrylamide gel stained with Coomassie Blue. [0101]
  • The N-terminal amino acid sequence of the dominant protein with an apparent molecular mass of approximately 40,000 Daltons was determined. Results of amino acid sequencing indicated that the first 29 amino acids of the N-terminal region were present in the following sequence (residues in parentheses are best guesses): [0102]
    Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu Leu Ser Glu Lys Pro [SEQ ID NO:1]
    Leu Gly Arg Thr Ile Leu Asp (Asp or Thr) (Pro).
  • Comparison with amino acid sequences in various databases indicated little or no homology with NH[0103] 2-terminal sequences reported for other monoxygenases or dioxygenases.
  • Component II. The protein fraction which did not bind to a Phenyl-Sepharose column was designated as component II. Because this yellowish colored fraction could be replaced by ferredoxin from [0104] Clostridium pasteurianum (but with slower reaction rates) when assays were performed in combination with components I and III, it was tentatively designated as a ferredoxin-containing fraction. The Clostridium ferredoxin clearly functioned in place of component II, but given the highly impure nature of the component II used in these experiments, the efficiency of the Clostridium enzyme was significantly lower than that of the putative ferredoxin from strain DI-6. In particular, 55 μg of partially purified component II mixed with excess amounts of components I and III catalyzed the conversion of dicamba to 3,6-DCSA at a rate of approximately 5 nmol min−1 mg−1, while 100 μg of highly purified ferredoxin from Clostridium resulted in an activity of only 0.6 nmol min−1 mg−1.
  • Purification steps involving Q-Sepharose (Fast Flow) chromatography, [0105] Superose 12 gel filtration and Mono Q chromatography yielded approximately one milligram of purified protein from an initial 25 grams of cell paste. This fraction was purified in a similar manner to the oxygenase component by electrophoresis on an IEF gel and subsequent electrophoresis of the active IEF fraction on an SDS-polyacrylamide gel.
  • Analysis of component II activity in proteins eluted from segments of the IEF gel indicated that a fraction with a pI of approximately 3.0 contained the active protein in component II. Protein from this gel slice was eluted and subjected to SDS-PAGE. Staining of the gel with Coomassie Blue revealed one prominent band of protein (molecular weight of about 28,000 Daltons) along with a smear of lower molecular weight proteins. [0106]
  • Component III. Component III of dicamba O-demethylase was retained on a Phenyl-Sepharose column in high concentrations of (NH[0107] 4)2SO4 and eluted at approximately 4% (w/v) (NH4)2SO4. This light yellow fraction was tentatively identified as a reductase-containing fraction based on its ability to reduce oxidized cytochrome c and DCIP in the presence of NADH and because it could be replaced by cytochrome c reductase from porcine heart (Type 1, Sigma) in assays with components I and II. In this set of reactions, the use of 50 μg of partially purified component III produced a reaction rate of approximately 5 nmol min−1 mg−1 when mixed with an excess of components I and II. The highly purified cytochrome c reductase showed a specific activity of approximately 2.5 nmol min−1 mg−1 in the reaction, an activity well below that provided by component III when one considers the impurity of the crude component III used in these assays. In addition, component III exhibited reductase activity when incubated with cytochrome c or 2,6-dichlorophenol-indophenol (DCPIP) in the presence of NADH. Neither component I nor component II showed activity in either of these two reductase assays.
  • Additional purification of this fraction by chromatography on columns containing Q-Sepharose (Fast Flow), blue dye affinity matrix, [0108] Superose 12, and Mono Q packings resulted in low amounts of protein in the fractions with reductase activity. The component III protein was about 70% pure as judged by densitometric analysis of the active protein fraction after separation by SDS-PAGE and staining with Coomassie Blue.
  • To further exacerbate purification of component III, it was found that two different protein fractions from the Mono Q column step contained reductase activity when assayed with the ferredoxin and oxygenase components. Further purification of these two fractions by eletrophoresis on an IEF gel revealed that the reductase activities of the two fractions had distinctly different isoelectric points. This was demonstrated by excising lanes containing each of the two reductase fractions from the IEF gel and laying the slices on top of a pad of low melt agarose containing a DCIP reaction mixture. Reductase activity in both gel slices was identified by the NADH-dependent reduction of DCIP to its colorless, reduced form. The reductase in [0109] fraction 35 had an apparent pI of approximately 5.6 while the reductase in fraction 27 possessed an apparent pI of approximately 4.8.
  • Both reductase activities isolated from the IEF gel slices were unstable and present in low amounts. Indeed, only the reductase from [0110] fraction 35 from the Mono Q column fractionation retained sufficient protein concentration and activity to allow further purification and characterization. A slice from an IEF gel containing this reductase activity was eluted and separated from contaminating proteins by SDS-PAGE. The predominant protein in this gel was one with a mass of approximately 45,000 Daltons. Size exclusion chromatography had indicated an approximate molecular mass of 50,000 Daltons for component III in its native state.
  • Biochemical characteristics of dicamba O-demethylase. Dicamba O-demethylase activity was measured during incubations in vitro at temperatures ranging from 20° C. to 50° C. and at pH values from approximately 6 to 9. Activity peaked sharply at 30° C. and broadly at pH values between 6.5 and 7.5. Enzymatic activity was dependent on the type of pH buffer employed. At pH 7, for example, activity was approximately 40% lower in Tris-containing buffers than in phosphate-containing buffers. [0111]
  • Values for K[0112] m and Vmax for dicamba O-demethylase were estimated using SigmaPlot® to generate best fit curves from Michaelis-Menten and Lineweaver-Burk plots of data from duplicate experiments. The Km for dicamba was estimated to be approximately 9.9±3.9 μM and the Vmax for the reaction was estimated to be approximately 108±12 nmol/min/mg.
  • The three components were assayed for dicamba O-demethylase activity in various combinations. None of the components showed enzyme activity when assayed alone. Indeed, a significant amount of O-demethylase activity could be detected only when all three components were combined. A mixture of components I and II exhibited small amounts of enzyme activity, probably due to traces of component III contaminating the component I fractions. [0113]
  • Both NADH and NADPH supported enzyme activity, with NADH being markedly more effective than NADPH. Mg[0114] 2+ was necessary for enzyme activity. Fe2+, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) produced little or no stimulation of enzymatic activity with the partially purified protein preparations in these experiments. The highest activity was obtained using a combination of NADH, Fe2+, Mg2+, and FAD.
  • Discussion [0115]
  • Dicamba O-demethylase from [0116] Pseudomonas maltophilia, strain DI-6, is a three component oxygenase (Wang, X -Z (1996) Ph.D. thesis, University of Nebraska-Lincoln, Lincoln, Nebr.) responsible for the conversion of the herbicide, dicamba (2-methoxy-3,6-dichlorobenzoic acid), to 3,6-dichlorosalicylic acid (3,6-DCSA; 2-hydroxy-3,6-dichlorobenzoic acid). Purification schemes have been devised which have allowed the isolation of each of the three components to a homogeneous or near-homogeneous state.
  • Initial separation of the three components was achieved by chromatography on a Phenyl-Sepharose column. Enzymatic activities and other characteristics of the partially purified components allowed a tentative identification of the components as a reductase, a ferredoxin and an oxygenase—a composition similar to that found in a number of other previously studied heme-containing and nonheme-containing, multicomponent oxygenases (Batie, et al. (1992) pages 543-565, In F. Müller (ed.), [0117] Chemistry and biochemistry of flavoenzymes, vol. 111, CRC Press, Boca Raton; Harayama, et al. (1992) Annu. Rev. Microbiol. 46:565-601; Mason and Cammack (1992) Annu. Rev. Microbiol. 46:277-305; Rosche et al. (1995) Biochem. Biophys. Acta 1252:177-179). Component III isolated from the Phenyl-Sepharose column catalyzed the NADH-dependent reduction of both cytochrome c and the dye, DCIP. In addition, its ability to support conversion of dicamba to 3,6-DCSA when combined with components I and II could be replaced in part by cytochrome c reductase. Component II could be replaced by the addition of ferredoxin from Clostridium pasteurianum to reactions containing components I and III. The absolute need for molecular oxygen to support the O-demethylation reaction indicated that the remaining component was an oxygenase.
  • Oxygenase[0118] DIC. ComponentI of dicamba O-demethylase (designated as oxygenaseDIC) has been purified to homogeneity and subjected to N-terminal amino acid sequencing. The resulting sequence of twenty nine amino acid residues showed no significant homology to other protein sequences in the various data banks. However, the information obtained from this amino acid sequence permitted the design of degenerate oligonucleotide probes which have been successfully used to detect and clone the component I gene (see Example 2). Furthermore, a comparison of the amino acid sequence derived from the nucleotide sequence of this clone with that of the protein sequences in the data base showed strong homology to other oxygenases (see Example 2).
  • The apparent molecular mass of oxygenase[0119] DIC, estimated from its migration in SDS-polyacrylamide gels, is approximately 40,000 Daltons. Purified preparations of the oxygenase exhibited only one major band on SDS-polyacrylamide gels stained with Coomassie Blue and Edman degradation of the protein contained in that band indicated the presence of only one N-terminal species. Estimates derived from the behavior of native oxygenaseDIC on size exclusion columns suggests a molecular size of approximately 90,000 Daltons. All of these results suggest that the native oxygenase exists as a homodimer.
  • The oxygenase/hydroxylase component of a number of multicomponent systems is composed of an (αβ)[0120] n-type subunit arrangement in which the larger α subunit is approximately 50,000 Daltons in size and the smaller β subunit is approximately 20,000 Daltons in molecular mass (Harayama, et al. (1992) Annu. Rev. Microbiol. 46:565-601). In contrast, the oxygenase component of dicamba O-demethylase appears to possess a single subunit of approximately 40 kDa in molecular mass which may exist as a dimer in its native state. This (α)n-type subunit arrangement is similar to that found in other well characterized oxygenases such as 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. strain CBS (Markus, et al. (1986) J. Biol. Chem. 261:12883-12888), phthalate dioxygenase from Pseudomonas cepacia (Batie, et al. (1987) J. Biol. Chem. 262:1510-1518),4-sulphobenzoate 3,4-dioxygenase from Comamonas testosteroni (Locher, et al. (1991) Biochem. J., 274:833-842), 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 (Rosche et al. (1995) Biochem. Biophys. Acta 1252:177-179), 4-carboxydiphenyl ether dioxygenase from Pseudomonas pseudoalcaligenes (Dehmel, et al. (1995) Arch. Microbiol. 163:35-41), and 3-chlorobenzoate 3,4-dioxygenase from Pseudomonas putida, (Nakatsu, et al. (1995) Microbiology (Reading) 141:485-495).
  • Ferredoxin[0121] DIC. Component II (ferredoxinDIC) of dicamba O-demethylase was purified by several steps of column chromatography and IEF. Final purification by SDS-PAGE produced one major band of protein (Mr˜28,000) and a smear of slightly smaller proteins.
  • The N-terminal amino acid sequence of the protein with an apparent molecular weight of approximately 28,000 Daltons was determined. This amino acid sequence permitted the preparation of degenerate oligonucleotide probes, and these probes were used to isolate a genomic clone coding for this protein. Although the protein produced by this clone was a ferredoxin (ferrodoxin[0122] 28kDa), it was subsequently determined not to be active in the degradation of dicamba when combined with the other two components of dicamba O-demethylase (data not shown).
  • Other evidence supports the conclusion that ferrodoxin[0123] 28kDa is not the ferredoxin component of dicamba O-demethylase. First, the molecular mass of this protein(28 kDa) protein is significantly higher than that of the other ferredoxins found in multicomponent oxygenases from bacteria (i.e., 8-13 kDa) (Batie, et al. (1992) pages 543-565, In F. Müller (ed.), Chemistry and biochemistry of flavoenzymes, vol. III, CRC Press, Boca Raton; Harayama, et al. (1992) Annu. Rev. Microbiol. 46:565-601).
  • Second, a comparison of the N-terminal sequence of 20 amino acid residues obtained from ferredoxin[0124] 28kDa to other amino acid sequences in the various protein data banks using Genetics Computing Group (GCG) software package (University of Wisconsin, Madison, Wis.) revealed strong homology (80-85% identity compared to the most likely N-terminal sequence of ferredoxin28kDa) to a number of dicluster bacterial ferredoxins (those from Pseudomonas stutzeri, Pseudomonas putida, Rhodobacter capsulatus and Azotobacter vinelandii). The four dicluster ferredoxins which showed strong homology to ferredoxin28kDa have a [3Fe-4S] cluster followed by a [4Fe-4S] cluster at the N-terminus of the protein. This suggests that ferredoxin28kDa is distinctly different from the ferredoxin components with [2Fe-2S] clusters which are usually associated with non-heme multicomponent oxygenases (Harayama, et al. (1992) Annu. Rev. Microbiol. 46:565-601; Mason and Cammack (1992) Annu. Rev. Microbiol. 46:277-305; Rosche, et al. (1995) Biochem. Biophys. Acta 1252:177-179).
  • Reductase[0125] DIC. Component III of dicamba O-demethylase (designated as reductaseDIC) has been the most recalcitrant of the three components to purify. This is due in part to its apparent instability and low abundance in lysates of strain DI-6. Nonetheless, sufficient protein has been purified to assign a tentative molecular mass of 45,000 Daltons. This is similar to the molecular mass of approximately 50,000 Daltons obtained from size exclusion chromatography and suggests that reductaseDIC exists in its native form as a monomer. The purification of the reductase component has been further complicated by the fact that chromatography on a Mono Q column and IEF resolves purified reductase preparations into two activities with apparently distinct pI values. Both fractions from the Mono Q column functioned in combination with purified ferredoxinDIC and oxygenaseDIC to produce dicamba O-demethylase activity. The presence in Sphingomonas sp. strain RW1 of two similar flavoproteins which function equally well as reductase components in the three component dibenzofuran 4,4a-dioxygenase has recently been reported by Bünz and Cook (Bünz and Cook (1993) J. Bacteriol. 175:6467-6475). Interestingly, both reductases were 44,000 Daltons in molecular mass, quite similar to that of the 45,000 Dalton reductaseDIC. Multiple components of leghemoglobin reductase have also been observed in lupin root nodules using isoelectric focusing techniques (Topunov, et al. (1982) Biokhimiya (English edition) 162:378-379). In this case, IEF revealed four separate components with NADH-dependent reductase activity. The resolution of the question of whether there is only one reductaseDIC which exists in two forms or two distinct reductases in strain DI-6 will rely on the development of improved procedures for isolating larger quantities of the proteins and/or on the cloning and sequencing of the gene(s) involved (see Examples 3 and 5).
  • Dicamba O-demethlylase characteristics. In addition to the physical and biochemical properties of the individual components noted above, analyses of enzymatic activity have shown that the O-demethylase system has a strong affinity (K[0126] m=˜10 μM) for its substrate and a Vmax of approximately 100-110 nmol/min/mg. As expected for a soil bacterium collected in a temperate climatic zone, the maximal enzymatic activity was observed at temperatures near 30° C. While the pH optima for the enzyme system was in the range from pH 6.5 to pH 7.5, the activity measured with a given preparation of enzyme was strongly affected by the type of pH buffering system employed. Activity in the presence of Tris buffers was at least 40% lower than with phosphate buffers at the same pH.
  • The reaction scheme for the reaction catalyzed by the three components of dicamba O-demethylase is presented in FIG. 1. Electrons from NADH are shuttled through a short electron chain consisting of the reductase and ferredoxin to the terminal oxygenase which catalyzes the oxidation of dicamba. The similarities between dicamba O-demethylase and several multicomponent dioxygenases suggest that dicamba O-demethylase may potentially possess cryptic dioxygenase activity. It is clear, however, that this enzyme is not in the class of dioxygenases which split O[0127] 2 and incorporate one atom of oxygen into the major substrate and the other into a small organic substrate such as α-ketoglutarate (Fukumori and Hausinger (1993) J. Biol. Chem. 268:24311-24317). Indeed, combinations of highly purified reductaseDIC, ferredoxinDIC, and oxygenaseDIC require only O2, NADH, Mg2+, Fe2+, and dicamba for activity.
    • Example 2 Identification and Sequencing of a Clone Coding for the Oxygenase of Dicamba O-Demethylase of Pseudomonas maltophilia DI-6
  • As noted in Example 1, the first 29 amino acids of the N-terminal amino acid sequence of oxygenase[0128] DIC had been determined to be (residues in parentheses are best guesses):
    Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu Leu Ser Glu Lys [SEQ ID NO:1]
    Pro Leu Gly Arg Thr Ile Leu Asp (Asp or Thr) (Pro).
  • This sequence permitted the design of degenerate oligonucleotide probes which were synthesized by Operon, Alameda, Calif. In particular, a mixture of 32 probes, each of which was 17 nucleotides in length, and contained all of the possible nucleotide sequences which could encode the amino acid sequence highlighted in bold above, was used. The oligonucleotide probes were 3′-end-labeled with digoxigenin (DIG) according to instructions provided by Boehringer Mannheim, Indianapolis, Ind. [0129]
  • The DIG-labeled probes were first hybridized to [0130] P. maltophilia DI-6 genomic DNA which had been digested with various combinations of restriction enzymes, resolved on a 1% agarose gel, and blotted to a nylon filter. Based on these results, a size-fractionated genomic library was constructed in the pBluescript II KS+ vector and transformed into Escherichia coli DH5α competent cells. The genomic library contained 1-2 kb Xho I/Hind III fragments. The DIG-labeled oligonucleotide probes were hybridized to an array of bacterial colonies streaked on nylon filters. Plasmid DNA was isolated from positive colonies and subcloned. Both strands of each subclone were sequenced by the DNA Sequencing Facility at the University of Nebraska-Lincoln. Hybridization and detection of DIG-labeled probes were performed according to protocols provided by Boehringer Mannheim.
  • A genomic DNA clone coding for the oxygenase[0131] DIC was identified. The nucleotide sequence and the deduced amino acid sequence of the entire oxygenaseDIC are given in the Sequence Listing below as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • A comparison of the amino acid sequence derived from the nucleotide sequence of this clone with that of the protein sequences in the Swiss Protein Database showed homology to other oxygenases. Homology was determined using the FASTA program of the GCG software package. The strongest homology was with the oxygenase component of vanillate demethylase (from Pseudomonas sp., ATCC strain 19151) which showed 33.8% identity. [0132]
    • Example 3 Purification and Characterization of Components of Dicamba O-Demethylase of Pseudomonas maltophilia DI-6
  • Bacterial cultures and preparation of cleared cell lysates. [0133] Pseudomonas maltophilia, strain DI-6, was inoculated into six two-liter Erlenmeyer flasks containing one liter of reduced chloride medium (Kreuger, J. P. 1989. Ph.D. thesis. Illinois Institute of Technology, Chicago) supplemented with glucose (2.0 mg/ml) and Casamino acids (2.0 mg/ml) as the carbon sources. Cultures were incubated on an orbital shaker (225 rpm at 30° C.). Cells were harvested at an A600 ranging from 1.5 to 2.0 using a JLA-10.500 rotor in a Beckman Avanti J-251 Centrifuge at 4,000×g for 20 minutes. Pelleted cells were stored at −80° C. The frozen cells were resuspended in 40 ml of 100 mM MgCl2, pelleted again using the same conditions as above, and then resuspended in breaking buffer (100 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 7.2), 1 mM dithiothreitol, 5% glycerol) in a ratio of 2 ml breaking buffer per gram of cells wet weight. Lysozyme was added in a ratio of 80 μl per gram of cells along with a Protease Inhibitor Cocktail for bacterial extracts (Sigma, P 8465) in a ratio of 5 ml per 20 grams of cells wet weight. Finally, phenylmethylsulfonyl fluoride (0.1 M stock solution in 100% ethanol) was added in a ratio of 250 μl per 50 ml of breaking buffer. The cells were disrupted with a sonicator (Sonics and Materials Inc., Model VCX 600) in 9.0 second bursts, with 3.0 second resting periods, for 30 minutes at an amplitude of 50%. Lysed cells were centrifuged for 75 minutes at 56,000×g in a JA-25.50 rotor of a Beckman Avanti J-251 Centrifuge at 4° C. The supernatant (cleared cell lysate) was decanted and glycerol was added to a final concentration of 15% prior to storage at −80° C.
  • Initial purification of dicamba O-demethylase components. An aliquot of the cleared cell lysate containing approximately 2.7 grams of protein was applied to a Pharmacia XK 26/60 column containing 25 ml of DEAE-Sepharose Fast Flow equilibrated with 50 mM MOPS (pH7.2), 1 mM dithiothreitol, and 15% (v/v) glycerol (buffer A). The column was connected to a Bio-CAD Perfusion Chromatography Workstation (USDA NRICGP Grant #9504266) and run at a flow rate of 5.0 ml/min. After the column was loaded, it was washed with buffer A until the absorbance reading at 280 nm decreased to below 0.1. All three components of dicamba O-demethylase were bound to the DEAE column under these conditions. The column was developed with a linear gradient of 0 to 500 mM NaCl in buffer A. This resulted in the elution of the ferredoxin at 400 mM NaCl and the co-elution of the reductase and oxygenase components at 250 mM NaCl. [0134]
  • Purification of the ferredoxin[0135] DIC. Fractions containing the ferredoxinDIC eluted from the DEAE-Sepharose column were pooled and buffer exchanged into 50 mM MOPS (pH 7.2), 5% glycerol (v/v) and 200 mM NaCl (buffer B). They were then concentrated to approximately 2 ml using a Amicon Cell Concentrator with a YM10 membrane and a Centricon 10 concentrator. This sample was then applied to a pre-packed Pharmacia HiPrep 26/60 Sephacryl S-100 column equilibrated with buffer B and run at a flow rate of 0.5 ml/min on a Pharmacia FPLC apparatus. Fractions that showed activity were pooled and buffer exchanged into 50 mM MOPS (pH 7.2), 1 mM dithiothreitol and 5% glycerol (v/v) (buffer C). The fractions were concentrated to approximately 2 ml and then loaded onto a Pharmacia Mono Q HR 5/5 column equilibrated in buffer C. The column was developed with a linear gradient of 0-2.0 M NaCl in buffer C. Fractions that contained ferredoxin activity were assayed for protein content and stored at −80° C.
  • Purification of the reductase[0136] DIC. Fractions from the initial DEAE-Sepharose column containing the oxygenase/reductase components were pooled, and ammonium sulfate was added to a final concentration of 1.5 M. After incubating at 4° C. for 1.5 hours, the samples were centrifuged for 75 minutes at 56,000×g at 4° C. in a JA-25.50 rotor of a Beckman Avanti J-25I centrifuge. The supernatant was retained and loaded at a flow rate of 5.0 ml/min onto a Pharmacia XK 26/20 column containing 25 ml of Phenyl-Sepharose 6 Fast Flow (high sub) that was equilibrated in buffer A containing 1.5 M (NH4)2SO4. Fractions that contained the reductase component were pooled, buffer exchanged into buffer B, and concentrated to approximately 2 ml using the Amicon concentrator with an YM30 membrane and a Centricon10 concentrator. The 2 ml sample was applied to a pre-packed Pharmacia HiPrep 26/60 Sephacryl S-100 column equilibrated in buffer B and run at 0.5 ml/min. Fractions containing reductase activity were pooled, buffer exchanged into buffer C, and concentrated down to approximately 2 ml. The 2 ml sample was loaded onto a pre-packed Pharmacia Mono Q HR 5/5 column that was equilibrated in buffer C. The column was developed with a linear gradient of 0-2.0 M NaCl in buffer C at a flow rate of 0.5 ml/min. Fractions that showed reductase activity were assayed for protein content and stored at −80° C.
  • Rapid enzyme assays. Activity for each of the three individual components was monitored in reactions using [0137] 14C-labeled dicamba in a reaction containing an excess of the remaining two components. For each reaction, a buffer solution composed of 24 mM potassium phosphate (KPi) buffer (pH 7.0), 0.48 mM NADH, 0.48 mM FeSO4 and 9.6 mM MgCl2 was added to 200 μl of protein sample along with 30 μl of master mix (562.4 μl sterile water, 12.0 μl 50 mM dicamba and 25.6 μl 14C-dicamba stock solution [1.28 μCi]) for a total volume of 311 μl and a 14C-dicamba specific activity of 4.12 μCi/ml. After 60 min, each reaction was stopped by the addition of 50 μl 5% H2SO4 (v/v) and 500 μl ether. Reaction tubes were vortexed and centrifuged in a microfuge (Eppendorf, Model 5415C) at 14,000×g for 2 minutes. For a quick visual appraisal of enzymatic activity, each reaction tube was placed under a hand-held UV light (Fotodyne Inc., Model 3-6000) to detect fluorescence of the reaction product, DCSA. For more accurate, semi-quantitative measurements of enzymatic activities, reaction products were separated using thin layer chromatography as described in Example 1.
  • Protein concentration determinations. The fractions obtained at different stages of the purification protocol were-assayed for protein concentration using the Bradford assay (standard protocol; Bio-Rad; see Example 1). [0138]
    • Example 4 Identification and Sequencing of a Clone Coding for FerredoxinDIC
  • The N-terminal sequence obtained from the purified ferredoxin protein (purification described in Example 3) was 29 amino acids in length (sequencing of proteins was performed by the Protein Core Facility at the University of Nebraska-Lincoln using a standard Edman degradation procedure; see Example 1). A comparison of this sequence to the Genbank database showed that it was 35% identical in a 26 amino acid overlap to a terpredoxin from Pseudomonas sp., a bacterial [2Fe-2S] ferredoxin in the adrenodoxin family. The sequence information was used to design three degenerate oligonucleotide primers (two forward and one reverse). The sequence of the two 17 mer forward primers was based on the N-terminal amino acid sequence obtained from the purified ferredoxin protein. The sequence of the 17 mer reverse primer was based on a conserved sequence of six amino acids near the C-terminal end of six previously sequenced bacterial adrenodoxin-type ferredoxins. The primers were used in a nested PCR reaction to amplify a 191 bp product from total [0139] P. maltophilia DNA. The product was cloned into the pGEM-T Easy vector (Promega, Madison, Wis.) and sequenced. DNA sequencing was performed by the DNA Sequencing Core Facility at the University of Nebraska-Lincoln using a standard dideoxy-mediated chain termination procedure. An analysis of the predicted amino acid sequence of this clone confirmed that it matched the N-terminal and internal amino acid sequence previously obtained from the purified ferredoxin protein. Furthermore, the derived amino acid sequence had 48% identity over its entire length with a [2Fe-2S] ferredoxin from Rhodobacter capsulatus. The cloned fragment was labeled with digoxigenin (DIG) (Roche Diagnostics) using a standard PCR protocol (DIG/Genius System User's Guide) and hybridized by a Southern blot to total P. maltophilia DNA that had been digested with a number of restriction enzymes. A map of the restriction sites surrounding the gene was constructed based on the sizes of the restriction fragments that hybridized to the probe. This initial experiment showed that the gene was contained on a Xho I/Pst I fragment that was approximately 1 kb in length. Subsequently, total P. maltophilia DNA was digested with Xho I and Pst I, and the restriction fragments were resolved on a gel. Fragments between 0.5 and 1.5 kb in length were excised from the gel, ligated into the vector pBluescript II KS+ (Stratagene, Inc.) and transformed into DH5α cells (Gibco BRL, Inc.). Bacterial colonies containing the size-fractionated library were screened with the DIG-labeled probe and a 900 bp Xho I/Pst I fragment was identified. Sequence analysis showed that this clone contained a full-length 318 bp ferredoxin gene [SEQ ID NO: 4] that encoded an 11.4 kDa protein composed of 105 amino acid residues [SEQ ID NO: 5]. The amino acid sequence predicted by the cloned gene matched the N-terminal and internal amino acid sequence previously obtained from the purified ferredoxin protein. Furthermore, the predicted amino acid sequence was homologous over its entire length to five other members of the adrenodoxin family of [2Fe-2S] bacterial ferredoxins, ranging from 42% identity with a ferredoxin from Rhodobacter capsulatus to 35% identity with a ferredoxin from Pseudomonas (see FIG. 2). The other three ferredoxins were from Caulobacter crescentus, Rhodococcus erythropolis, and Pseudomonas putida. Proteins in this family bind a single 2Fe-2S iron-sulfur cluster and have three conserved motifs. Motif 1 includes three conserved cysteines which are 2Fe-2S ligands. Motif 2 contains a cluster of negatively charged residues. Motif 3 includes the fourth conserved cysteine of the 2Fe-2S cluster.
    • Example 5 Identification and Sequencing of Clones Coding ReductaseDIC
  • Two reductase genes were cloned by the same approach that was used in Example 4 to clone the ferredoxin gene. The N-terminal sequence obtained from the purified reductase protein (purification described in Example 3) was 25 amino acids in length. A comparison of this sequence to the Genbank database showed that it was 90% identical in a 20 amino acid overlap to a cytochrome P450-type reductase component of dioxin dioxygenase, a three component enzyme previously isolated from Sphingomonas sp. RW1. An internal sequence of 10 amino acid residues was also obtained from tryptic digests of the purified protein. The internal sequence had 87.5% identity with residues 62 through 69 of the same cytochrome P450-type reductase from Sphingomonas sp. RW1. This sequence information was used to design three degenerate oligonucleotide primers (two forward and one reverse). The sequence of the two 17 mer forward primers was based on the N-terminal amino acid sequence and the sequence of the 17 mer reverse primer was based on the internal amino acid sequence. The primers were used in a nested PCR reaction to amplify a 180 bp product from total [0140] P. maltophilia DNA. The product was cloned into the pGEM-T Easy vector and sequenced. An analysis of the predicted amino acid sequence of this clone confirmed that it encoded a protein that matched the N-terminal and internal amino acid sequence obtained from the purified reductase protein. Furthermore, the predicted sequence had 80% identity over its entire length with the cytochrome P-450 type reductase component of the dioxin dioxygenase from Sphingomonas sp. RW1.
  • The cloned fragment was labeled with DIG and hybridized by a Southern blot to total [0141] P. maltophilia DNA that had been digested with a number of restriction enzymes. The Southern blot showed that the DIG-labeled probe recognized two distinct loci in the various restriction digests of P. maltophilia total DNA. This observation suggested that there are two reductase genes located at different positions in the genome of P. maltophilia. It was possible that the two genes are identical duplications that encode identical reductase proteins. Alternatively, one of the genes could encode a truncated protein with no activity or a full-length protein with low activity in our dicamba O-demethylase assay. Because it was necessary to clone the gene that encodes a protein with optimal activity, the DIG-labeled probe was used to retrieve both reductase genes.
  • When total [0142] P. maltophilia DNA was digested with Kpn I and EcoR I, the DIG-labeled probe hybridized to one restriction fragment that was approximately 4.0 kb in length and to another larger fragment with a size of approximately 20 kb. A map of a number of restriction sites surrounding the gene located on the 4.0 kb Kpn I/EcoR I fragment was constructed based on the sizes of different restriction fragments that hybridized to the probe. The restriction map indicated that the entire gene should be located on this 4.0 kb fragment. Subsequently, total P. maltophilia DNA was digested with Kpn I and EcoR I, and the restriction fragments were resolved on a gel. Fragments between 3.0 and 5.0 kb in length were excised from the gel, ligated into the vector pBluescript II KS+, and transformed into DH5α cells. Bacterial colonies containing the size-fractionated library were screened with the DIG-labeled probe and a 4.3 kb Kpn I/EcoR I fragment was identified. Sequence analysis showed that this clone contained a full-length 1224 bp reductase gene [SEQ ID NO: 6] and encoded a 43.7 kDa protein consisting of 408 amino acids [SEQ ID NO: 7]. The amino acid sequence predicted by the cloned gene matched the N-terminal and internal amino acid sequence previously obtained from the purified reductase protein. Furthermore, the predicted amino acid sequence was homologous over its entire length to at least four other FAD-dependent pyridine nucleotide reductases, ranging from 69% identity with a cytochrome P450-type reductase component of dioxin dioxygenase from Sphingomonas sp. RW1 to 36% identity with the terpredoxin reductase from a Pseudomonas species (see FIG. 3). The two other FAD-dependent pyridine nucleotide reductases were from R. erythropolis and P. putida. Proteins in this family of FAD-dependent pyridine nucleotide reductases have five conserved motifs. Motifs 1 and 3 contain three conserved glycine residues and correspond to the ADP binding site for FAD and NAD(P) respectively. Motif 5 corresponds to the site binding the FAD flavin moiety.
  • To clone the second gene, total [0143] P. maltophilia DNA was digested with Kpn I and EcoR I and the resulting restriction fragments were resolved on an agarose gel. Fragments with a size of approximately 20 kb were excised from the gel, digested with a number of restriction enzymes, and then hybridized by Southern blot to the DIG-labeled probe. A map of the restriction sites surrounding the second gene was constructed based on the sizes of the restriction fragments that hybridized to the probe. These experiments showed that the full-length second reductase gene was contained on an Apa I fragment that was approximately 3.0 kb in length. Subsequently, total P. maltophilia DNA was digested with Apa I and the restriction fragments were resolved on a gel. Fragments between 2.0 and 4.0 kb in length were excised from the gel, ligated into the vector pBluescript II KS+, and transformed into DH5α cells. Bacterial colonies containing the size-fractionated library were screened with the DIG-labeled probe and a 3.0 kb Apa I fragment was identified. Sequence analysis showed that the 3.0 kb clone contained an open reading frame of 1227 bp [SEQ ID NO: 8] and encoded a 43.9 kDa protein consisting of 409 amino acids [SEQ ID NO: 9]. The amino acid sequence encoded by the second reductase gene is almost identical (98.8% identity) to the sequence of the first gene.
    • Example 6 Transformation of Plants
  • In order to place each of the three genes that encode the components of dicamba O-demethylase individually into cassettes suitable for expression in plants, the following steps were taken. Oligonucleotide primers were designed to generate a Nco I site at the 5′ end and an Xba I site at the 3′ end of each of the three genes by PCR amplification. The authenticity of the resulting PCR products was confirmed by sequencing, and each gene was then cloned individually into the pRTL2 vector (provided by Dr. Tom Clemente of the Plant Transformation Core Research Facility, University of Nebraska, Lincoln, Nebr.). This vector contains a 144 bp translation enhancer sequence from tobacco etch virus (TEV) (Carrington and Freed, [0144] J. Virology, 64:1590-1597(1990)) at the 5′ end of the polylinker. The oxygenase, reductase, and ferredoxin genes, each with a 5′ translation enhancer, were then cloned individually as Xho I/Xba I fragments into the plant expression vector pKLP36 (obtained from Indu Maiti, University of Kentucky, Lexington, Ky.) (Maiti and Shepard, Biochem. Biophys. Res. Commun., 244:440-444 (1998)). This binary vector contains the peanut chlorotic virus full-length promoter (PCISV FLt36) with a duplicated enhancer domain for constitutive expression in plants and the pea rbcS 3′ sequence for efficient transcript termination (Maiti and Shepherd, Biochem. Biophys. Res. Commun., 244:440-444 (1998)). Constructs with all three genes on one binary vector can be produced using combinations of the three genes in a number of different orders and orientations.
  • The following methods were employed to move the oxygenase, ferredoxin, and reductase constructs individually into [0145] Agrobacterium tumefaciens and to transform each construct into Arabidopsis and tobacco. The three constructs were moved into the A. tumefaciens strain C58C1 by a modified triparental mating procedure routinely used by the Plant Transformation Core Research Facility. This involved incubating Escherichia coli cells carrying each construct with a mixture of A. tumefaciens cells and E. coli cells carrying the helper plasmid pRK2013 (for plasmid mobilization). A. tumefaciens cells containing each of the constructs were then used to transform tobacco and Arabidopsis with the assistance of the Plant Transformation Core Research Facility. For tobacco, leaf explants were incubated with a suspension of A. tumefaciens cells containing each of the constructs, and shoots were regenerated on solid medium containing kanamycin (Horsch et al., Science, 227:1229-1231 (1985)). Ten shoots were selected from each of the three transformation experiments, placed on rooting medium for a few weeks, and then moved to pots in the greenhouse. For Arabidopsis, a pot of plants with flowers was incubated with a suspension of A. tumefaciens cells containing each of the constructs, and the plants were then allowed to set seed (Bechtold et al., C.R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 316:1194-1199 (1993); Clough and Bent, Plant J., 16(6):735-743 (1998)). The seed was collected and germinated on medium with kanamycin. After the seedlings had developed an adequate root system, several plants were selected from each transformation experiment and moved to pots in a growth chamber.
  • For evaluation of the expression of each gene in the transformed plants, Western blots of leaf lysates from several transformed plants were prepared and probed with polyclonal antibodies that detect the three components of dicamba O-demethylase. To determine enzyme activities for each enzyme in plant extracts, the approach was to combine leaf lysates from transformed plants expressing the oxygenase, ferredoxin or reductase proteins with excess amounts of the other O-demethylase components purified from [0146] P. maltophilia strain DI-6. These mixtures were tested for dicamba O-demethylase activity with both the standard 14C-labeled dicamba assay (see Examples 1 and 3) and an HPLC assay employing nonradioactive dicamba as substrate (dicamba and 3,6-DCSA elute at different points from the HPLC and can be quantitated).
  • To test expression of gene products in the chloroplast compartment, constructs were made with a transit peptide sequence at the 5′ end of the oxygenase, ferredoxin, and reductase genes. Such an approach was successful for introducing tolerance to the sulfonylurea herbicides into tobacco plants (O'Keefe et al., [0147] Plant Physiol., 105:473-478 (1994)).
  • To test the possibility that the codon usage of the bacterial ferredoxin gene was not fully optimal for efficient translation in a plant cell, a synthetic ferredoxin gene encoding the same amino acid sequence as the [0148] P. maltophilia strain DI-6 ferredoxin, but with optimized codon bias for dicot plants was synthesized by Entelechon, GmbH (Regensberg, Germany). It has been well documented that changes in codon usage are essential for optimal expression of the bacterial B.t. toxin genes in plant cells (see, e.g., Diehn et al., Genetic Engineering, 18:83-99 (1996)).
  • 1 18 1 29 PRT Pseudomonas maltophilia DI-6 unsure (28)..(28) Best guess for Xaa = Asp or Thr 1 Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu Leu 1 5 10 15 Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Xaa Xaa 20 25 2 1020 DNA Pseudomonas maltophilia DI-6 CDS (1)..(1020) 2 atg acc ttc gtc cgc aat gcc tgg tat gtg gcg gcg ctg ccc gag gaa 48 Met Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu 1 5 10 15 ctg tcc gaa aag ccg ctc ggc cgg acg att ctc gac aca ccg ctc gcg 96 Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu Ala 20 25 30 ctc tac cgc cag ccc gac ggt gtg gtc gcg gcg ctg ctc gac atc tgt 144 Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile Cys 35 40 45 ccg cac cgc ttc gcg ccg ctg agc gac ggc atc ctc gtc aac ggc cat 192 Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly His 50 55 60 ctc caa tgc ccc tat cac ggg ctg gaa ttc gat ggc ggc ggg cag tgc 240 Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys 65 70 75 80 gtc cat aac ccg cac ggc aat ggc gcc cgc ccg gct tcg ctc aac gtc 288 Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn Val 85 90 95 cgc tcc ttc ccg gtg gtg gag cgc gac gcg ctg atc tgg atc tgg ccc 336 Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp Pro 100 105 110 ggc gat ccg gcg ctg gcc gat cct ggg gcg atc ccc gac ttc ggc tgc 384 Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly Cys 115 120 125 cgc gtc gat ccc gcc tat cgg acc gtc ggc ggc tat ggg cat gtc gac 432 Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val Asp 130 135 140 tgc aac tac aag ctg ctg gtc gac aac ctg atg gac ctc ggc cac gcc 480 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His Ala 145 150 155 160 caa tat gtc cat cgc gcc aac gcc cag acc gac gcc ttc gac cgg ctg 528 Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg Leu 165 170 175 gag cgc gag gtg atc gtc ggc gac ggt gag ata cag gcg ctg atg aag 576 Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met Lys 180 185 190 att ccc ggc ggc acg ccg agc gtg ctg atg gcc aag ttc ctg cgc ggc 624 Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly 195 200 205 gcc aat acc ccc gtc gac gct tgg aac gac atc cgc tgg aac aag gtg 672 Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys Val 210 215 220 agc gcg atg ctc aac ttc atc gcg gtg gcg ccg gaa ggc acc ccg aag 720 Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro Lys 225 230 235 240 gag cag agc atc cac tcg cgc ggt acc cat atc ctg acc ccc gag acg 768 Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu Thr 245 250 255 gag gcg agc tgc cat tat ttc ttc ggc tcc tcg cgc aat ttc ggc atc 816 Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly Ile 260 265 270 gac gat ccg gag atg gac ggc gtg ctg cgc agc tgg cag gct cag gcg 864 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln Ala 275 280 285 ctg gtc aag gag gac aag gtc gtc gtc gag gcg atc gag cgc cgc cgc 912 Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg Arg 290 295 300 gcc tat gtc gag gcg aat ggc atc cgc ccg gcg atg ctg tcg tgc gac 960 Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys Asp 305 310 315 320 gaa gcc gca gtc cgt gtc agc cgc gag atc gag aag ctt gag cag ctc 1008 Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln Leu 325 330 335 gaa gcc gcc tga 1020 Glu Ala Ala 3 339 PRT Pseudomonas maltophilia DI-6 3 Met Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu 1 5 10 15 Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu Ala 20 25 30 Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile Cys 35 40 45 Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly His 50 55 60 Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys 65 70 75 80 Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn Val 85 90 95 Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp Pro 100 105 110 Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly Cys 115 120 125 Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val Asp 130 135 140 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His Ala 145 150 155 160 Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg Leu 165 170 175 Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met Lys 180 185 190 Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly 195 200 205 Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys Val 210 215 220 Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro Lys 225 230 235 240 Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu Thr 245 250 255 Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly Ile 260 265 270 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln Ala 275 280 285 Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg Arg 290 295 300 Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys Asp 305 310 315 320 Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln Leu 325 330 335 Glu Ala Ala 4 318 DNA Pseudomonas maltophilia DI-6 CDS (1)..(318) 4 atg ccg cag att acc gtc gtc aac cag tcg ggt gaa gaa tcc agc gtc 48 Met Pro Gln Ile Thr Val Val Asn Gln Ser Gly Glu Glu Ser Ser Val 1 5 10 15 gag gcg agt gaa ggc cgc acc ctg atg gaa gtc atc cgc gac agc ggt 96 Glu Ala Ser Glu Gly Arg Thr Leu Met Glu Val Ile Arg Asp Ser Gly 20 25 30 ttt gac gaa ctc ctg gcg ctt tgc ggc ggc tgc tgc tcg tgc gcg acc 144 Phe Asp Glu Leu Leu Ala Leu Cys Gly Gly Cys Cys Ser Cys Ala Thr 35 40 45 tgc cac gtc cac atc gac ccg gcc ttc atg gac aag ctg ccg gag atg 192 Cys His Val His Ile Asp Pro Ala Phe Met Asp Lys Leu Pro Glu Met 50 55 60 agc gaa gac gag aac gac ctg ctc gac agc tcg gac cac cgc aac gag 240 Ser Glu Asp Glu Asn Asp Leu Leu Asp Ser Ser Asp His Arg Asn Glu 65 70 75 80 tac tcg cgt ctc tcg tgc cag att ccg gtc acc ggc gcc ctc gaa ggc 288 Tyr Ser Arg Leu Ser Cys Gln Ile Pro Val Thr Gly Ala Leu Glu Gly 85 90 95 atc aag gtg acg atc gcg cag gaa gac tga 318 Ile Lys Val Thr Ile Ala Gln Glu Asp 100 105 5 105 PRT Pseudomonas maltophilia DI-6 5 Met Pro Gln Ile Thr Val Val Asn Gln Ser Gly Glu Glu Ser Ser Val 1 5 10 15 Glu Ala Ser Glu Gly Arg Thr Leu Met Glu Val Ile Arg Asp Ser Gly 20 25 30 Phe Asp Glu Leu Leu Ala Leu Cys Gly Gly Cys Cys Ser Cys Ala Thr 35 40 45 Cys His Val His Ile Asp Pro Ala Phe Met Asp Lys Leu Pro Glu Met 50 55 60 Ser Glu Asp Glu Asn Asp Leu Leu Asp Ser Ser Asp His Arg Asn Glu 65 70 75 80 Tyr Ser Arg Leu Ser Cys Gln Ile Pro Val Thr Gly Ala Leu Glu Gly 85 90 95 Ile Lys Val Thr Ile Ala Gln Glu Asp 100 105 6 1227 DNA Pseudomonas maltophilia DI-6 CDS (1)..(1227) 6 atg agc aag gca gac gtc gta atc gtg gga gcc ggg cat ggc ggc gca 48 Met Ser Lys Ala Asp Val Val Ile Val Gly Ala Gly His Gly Gly Ala 1 5 10 15 cag tgc gcg atc gcc ctt cgc cag aac ggc ttc gaa gga acc atc acc 96 Gln Cys Ala Ile Ala Leu Arg Gln Asn Gly Phe Glu Gly Thr Ile Thr 20 25 30 gtc atc ggt cgt gag ccg gaa tat ccc tat gag cgt ccg ccg ctc tcg 144 Val Ile Gly Arg Glu Pro Glu Tyr Pro Tyr Glu Arg Pro Pro Leu Ser 35 40 45 aag gaa tat ttc gcg cgc gag aag acc ttc gac cgc ctc tac atc cgt 192 Lys Glu Tyr Phe Ala Arg Glu Lys Thr Phe Asp Arg Leu Tyr Ile Arg 50 55 60 ccg ccg acg ttc tgg gcc gag aag aac atc gag ttc aag ctt ggc acc 240 Pro Pro Thr Phe Trp Ala Glu Lys Asn Ile Glu Phe Lys Leu Gly Thr 65 70 75 80 gaa gtc acc aag gtc gat ccc aag gcg cac gaa ctg acg ctc tcc aac 288 Glu Val Thr Lys Val Asp Pro Lys Ala His Glu Leu Thr Leu Ser Asn 85 90 95 ggc gag agc tac ggt tat ggc aag ctc gtc tgg gcc acc ggc ggc gat 336 Gly Glu Ser Tyr Gly Tyr Gly Lys Leu Val Trp Ala Thr Gly Gly Asp 100 105 110 ccg cgt cgc ctt tct tgc cag ggg gcc gac ctc acc ggc atc cac gcc 384 Pro Arg Arg Leu Ser Cys Gln Gly Ala Asp Leu Thr Gly Ile His Ala 115 120 125 gtg cgc acc cgc gag gac tgc gac acg ctg atg gcc gaa gtc gat gcg 432 Val Arg Thr Arg Glu Asp Cys Asp Thr Leu Met Ala Glu Val Asp Ala 130 135 140 ggc acg aag aac atc gtc gtc atc ggc ggc ggc tac atc ggt ctg gaa 480 Gly Thr Lys Asn Ile Val Val Ile Gly Gly Gly Tyr Ile Gly Leu Glu 145 150 155 160 gcc gct gcg gtg ctg tcc aag atg ggc ctc aag gtc acc ctg ctc gaa 528 Ala Ala Ala Val Leu Ser Lys Met Gly Leu Lys Val Thr Leu Leu Glu 165 170 175 gcg ctt ccg cgc gtg ctg gcg cgc gtt gcg ggt gaa gac ctc tcg acc 576 Ala Leu Pro Arg Val Leu Ala Arg Val Ala Gly Glu Asp Leu Ser Thr 180 185 190 ttc tac cag aag gaa cat gtc gat cac ggc gtc gac ctg cgc acc gaa 624 Phe Tyr Gln Lys Glu His Val Asp His Gly Val Asp Leu Arg Thr Glu 195 200 205 gtc atg gtc gac agc ctc gtc ggc gaa aac ggc aag gtc acc ggc gtg 672 Val Met Val Asp Ser Leu Val Gly Glu Asn Gly Lys Val Thr Gly Val 210 215 220 cag ctt gcc ggc ggc gaa gtg atc ccg gcc gaa ggc gtc atc gtc ggc 720 Gln Leu Ala Gly Gly Glu Val Ile Pro Ala Glu Gly Val Ile Val Gly 225 230 235 240 atc ggc atc gtg cct gcc gtc ggt ccg ctg atc gcg gcc ggc gcg gcc 768 Ile Gly Ile Val Pro Ala Val Gly Pro Leu Ile Ala Ala Gly Ala Ala 245 250 255 ggt gcc aac ggc gtc gac gtg gac gag tac tgc cgc acc tcg ctg ccc 816 Gly Ala Asn Gly Val Asp Val Asp Glu Tyr Cys Arg Thr Ser Leu Pro 260 265 270 gac atc tat gcg atc ggc gac tgt gcg gct ttc gcc tgc gac tac gcc 864 Asp Ile Tyr Ala Ile Gly Asp Cys Ala Ala Phe Ala Cys Asp Tyr Ala 275 280 285 ggc ggc aac gtg atg cgc gtg gaa tcg gtc cag aac gcc aac gac atg 912 Gly Gly Asn Val Met Arg Val Glu Ser Val Gln Asn Ala Asn Asp Met 290 295 300 ggc acc tgc gtg gcc aag gcg atc tgc ggc gac gag aag ccc tac aag 960 Gly Thr Cys Val Ala Lys Ala Ile Cys Gly Asp Glu Lys Pro Tyr Lys 305 310 315 320 gcg ttc ccg tgg ttc tgg tcc aac cag tac gac ctc aag ctg cag acc 1008 Ala Phe Pro Trp Phe Trp Ser Asn Gln Tyr Asp Leu Lys Leu Gln Thr 325 330 335 gcc ggc atc aac ctg ggc ttc gac aag acc gtg atc cgc ggc aat ccg 1056 Ala Gly Ile Asn Leu Gly Phe Asp Lys Thr Val Ile Arg Gly Asn Pro 340 345 350 gag gag cgc agc ttc tcg gtc gtc tat ctc aag gac ggc cgc gtg gtc 1104 Glu Glu Arg Ser Phe Ser Val Val Tyr Leu Lys Asp Gly Arg Val Val 355 360 365 gcg ctg gac tgc gtg aac atg gtc aag gat tac gtg cag ggc cgc aag 1152 Ala Leu Asp Cys Val Asn Met Val Lys Asp Tyr Val Gln Gly Arg Lys 370 375 380 ctg gtc gaa gcc ggg gcc acc ccc gac ctc gaa gcg ctg gcc gat gcc 1200 Leu Val Glu Ala Gly Ala Thr Pro Asp Leu Glu Ala Leu Ala Asp Ala 385 390 395 400 ggc aag ccg ctc aag gaa ctg ctc tag 1227 Gly Lys Pro Leu Lys Glu Leu Leu 405 7 408 PRT Pseudomonas maltophilia DI-6 7 Met Ser Lys Ala Asp Val Val Ile Val Gly Ala Gly His Gly Gly Ala 1 5 10 15 Gln Cys Ala Ile Ala Leu Arg Gln Asn Gly Phe Glu Gly Thr Ile Thr 20 25 30 Val Ile Gly Arg Glu Pro Glu Tyr Pro Tyr Glu Arg Pro Pro Leu Ser 35 40 45 Lys Glu Tyr Phe Ala Arg Glu Lys Thr Phe Asp Arg Leu Tyr Ile Arg 50 55 60 Pro Pro Thr Phe Trp Ala Glu Lys Asn Ile Glu Phe Lys Leu Gly Thr 65 70 75 80 Glu Val Thr Lys Val Asp Pro Lys Ala His Glu Leu Thr Leu Ser Asn 85 90 95 Gly Glu Ser Tyr Gly Tyr Gly Lys Leu Val Trp Ala Thr Gly Gly Asp 100 105 110 Pro Arg Arg Leu Ser Cys Gln Gly Ala Asp Leu Thr Gly Ile His Ala 115 120 125 Val Arg Thr Arg Glu Asp Cys Asp Thr Leu Met Ala Glu Val Asp Ala 130 135 140 Gly Thr Lys Asn Ile Val Val Ile Gly Gly Gly Tyr Ile Gly Leu Glu 145 150 155 160 Ala Ala Ala Val Leu Ser Lys Met Gly Leu Lys Val Thr Leu Leu Glu 165 170 175 Ala Leu Pro Arg Val Leu Ala Arg Val Ala Gly Glu Asp Leu Ser Thr 180 185 190 Phe Tyr Gln Lys Glu His Val Asp His Gly Val Asp Leu Arg Thr Glu 195 200 205 Val Met Val Asp Ser Leu Val Gly Glu Asn Gly Lys Val Thr Gly Val 210 215 220 Gln Leu Ala Gly Gly Glu Val Ile Pro Ala Glu Gly Val Ile Val Gly 225 230 235 240 Ile Gly Ile Val Pro Ala Val Gly Pro Leu Ile Ala Ala Gly Ala Ala 245 250 255 Gly Ala Asn Gly Val Asp Val Asp Glu Tyr Cys Arg Thr Ser Leu Pro 260 265 270 Asp Ile Tyr Ala Ile Gly Asp Cys Ala Ala Phe Ala Cys Asp Tyr Ala 275 280 285 Gly Gly Asn Val Met Arg Val Glu Ser Val Gln Asn Ala Asn Asp Met 290 295 300 Gly Thr Cys Val Ala Lys Ala Ile Cys Gly Asp Glu Lys Pro Tyr Lys 305 310 315 320 Ala Phe Pro Trp Phe Trp Ser Asn Gln Tyr Asp Leu Lys Leu Gln Thr 325 330 335 Ala Gly Ile Asn Leu Gly Phe Asp Lys Thr Val Ile Arg Gly Asn Pro 340 345 350 Glu Glu Arg Ser Phe Ser Val Val Tyr Leu Lys Asp Gly Arg Val Val 355 360 365 Ala Leu Asp Cys Val Asn Met Val Lys Asp Tyr Val Gln Gly Arg Lys 370 375 380 Leu Val Glu Ala Gly Ala Thr Pro Asp Leu Glu Ala Leu Ala Asp Ala 385 390 395 400 Gly Lys Pro Leu Lys Glu Leu Leu 405 8 1230 DNA Pseudomonas maltophilia DI-6 CDS (1)..(1230) 8 atg cag agg gca gac gtc gta atc gtg gga gcc ggg cat ggc ggt gca 48 Met Gln Arg Ala Asp Val Val Ile Val Gly Ala Gly His Gly Gly Ala 1 5 10 15 cag tgc gcg atc gcc ctt cgc cag aac ggc ttc gaa ggc acc atc acc 96 Gln Cys Ala Ile Ala Leu Arg Gln Asn Gly Phe Glu Gly Thr Ile Thr 20 25 30 gtc atc ggt cgt gag ccg gaa tat ccc tat gag cgt ccg ccg ctc tcg 144 Val Ile Gly Arg Glu Pro Glu Tyr Pro Tyr Glu Arg Pro Pro Leu Ser 35 40 45 aag gaa tat ttc gcg cgc gag aag acc ttc gac cgc ctc tac atc cgt 192 Lys Glu Tyr Phe Ala Arg Glu Lys Thr Phe Asp Arg Leu Tyr Ile Arg 50 55 60 ccg ccg acg ttc tgg gcc gag aag aac atc gag ttc aag ctt ggc acc 240 Pro Pro Thr Phe Trp Ala Glu Lys Asn Ile Glu Phe Lys Leu Gly Thr 65 70 75 80 gaa gtc acc aag gtc gat ccc aag gcg cac gaa ctg acg ctc tcc aac 288 Glu Val Thr Lys Val Asp Pro Lys Ala His Glu Leu Thr Leu Ser Asn 85 90 95 ggc gag agc tac ggt tat ggc aag ctc gtc tgg gcc acc ggc ggc gat 336 Gly Glu Ser Tyr Gly Tyr Gly Lys Leu Val Trp Ala Thr Gly Gly Asp 100 105 110 ccg cgt cgc ctt tct tgc cag ggg gcc gac ctc acc ggc atc cac gcc 384 Pro Arg Arg Leu Ser Cys Gln Gly Ala Asp Leu Thr Gly Ile His Ala 115 120 125 gtg cgc acc cgc gag gac tgc gac acg ctg atg gcc gaa gtc gat gcg 432 Val Arg Thr Arg Glu Asp Cys Asp Thr Leu Met Ala Glu Val Asp Ala 130 135 140 ggc acg aag aac atc gtc gtc atc ggc ggc ggc tac atc ggt ctg gaa 480 Gly Thr Lys Asn Ile Val Val Ile Gly Gly Gly Tyr Ile Gly Leu Glu 145 150 155 160 gcc gct gcg gtg ctg tcc aag atg ggc ctc aag gtc acc ctg ctc gaa 528 Ala Ala Ala Val Leu Ser Lys Met Gly Leu Lys Val Thr Leu Leu Glu 165 170 175 gcg ctt ccg cgc gtg ctg gcg cgc gtt gcg ggt gaa gac ctc tcg acc 576 Ala Leu Pro Arg Val Leu Ala Arg Val Ala Gly Glu Asp Leu Ser Thr 180 185 190 ttc tac cag aag gaa cat gtc gat cac ggc gtc gac ctg cgc acc gaa 624 Phe Tyr Gln Lys Glu His Val Asp His Gly Val Asp Leu Arg Thr Glu 195 200 205 gtc atg gtc gac agc ctc gtc ggc gaa aac ggc aag gtc acc ggc gtg 672 Val Met Val Asp Ser Leu Val Gly Glu Asn Gly Lys Val Thr Gly Val 210 215 220 cag ctt gcc ggc ggc gaa gtg atc ccg gcc gaa ggc gtc atc gtc ggc 720 Gln Leu Ala Gly Gly Glu Val Ile Pro Ala Glu Gly Val Ile Val Gly 225 230 235 240 atc ggc atc gtg cct gcc atc ggt ccg ctg atc gcg gcc ggc gcg gcc 768 Ile Gly Ile Val Pro Ala Ile Gly Pro Leu Ile Ala Ala Gly Ala Ala 245 250 255 ggc gcc aac ggc gtc gac gtg gac gag tac tgc cgc acc tcg ctg ccc 816 Gly Ala Asn Gly Val Asp Val Asp Glu Tyr Cys Arg Thr Ser Leu Pro 260 265 270 gac atc tat gcg atc ggc gac tgt gcg gct ttc gcc tgc gac tac gcc 864 Asp Ile Tyr Ala Ile Gly Asp Cys Ala Ala Phe Ala Cys Asp Tyr Ala 275 280 285 ggc ggc aac gtg atg cgc gtg gaa tcg gtc cag aac gcc aac gac atg 912 Gly Gly Asn Val Met Arg Val Glu Ser Val Gln Asn Ala Asn Asp Met 290 295 300 ggc acc tgc gtg gcc aag gcg atc tgc ggc gac gag aag ccc tac aag 960 Gly Thr Cys Val Ala Lys Ala Ile Cys Gly Asp Glu Lys Pro Tyr Lys 305 310 315 320 gcg ttc ccg tgg ttc tgg tcc aac cag tac gac ctc aag ctg cag acc 1008 Ala Phe Pro Trp Phe Trp Ser Asn Gln Tyr Asp Leu Lys Leu Gln Thr 325 330 335 gcc ggc atc aac ctg ggc ttc gac aag acc gtg atc cgc ggc aat ccg 1056 Ala Gly Ile Asn Leu Gly Phe Asp Lys Thr Val Ile Arg Gly Asn Pro 340 345 350 gag gag cgc agc ttc tcg gtc gtc tat ctc aag gac ggc cgc gtg gtc 1104 Glu Glu Arg Ser Phe Ser Val Val Tyr Leu Lys Asp Gly Arg Val Val 355 360 365 gcg ctg gac tgc gtg aac atg gtc aag gat tac gtg cag ggc cgc aag 1152 Ala Leu Asp Cys Val Asn Met Val Lys Asp Tyr Val Gln Gly Arg Lys 370 375 380 ctg gtc gaa gcc ggg gcc acc ccc gac ctc gaa gcg ctg gcc gat gcc 1200 Leu Val Glu Ala Gly Ala Thr Pro Asp Leu Glu Ala Leu Ala Asp Ala 385 390 395 400 ggc aag ccg ctc aag gaa ctg caa tac tag 1230 Gly Lys Pro Leu Lys Glu Leu Gln Tyr 405 9 409 PRT Pseudomonas maltophilia DI-6 9 Met Gln Arg Ala Asp Val Val Ile Val Gly Ala Gly His Gly Gly Ala 1 5 10 15 Gln Cys Ala Ile Ala Leu Arg Gln Asn Gly Phe Glu Gly Thr Ile Thr 20 25 30 Val Ile Gly Arg Glu Pro Glu Tyr Pro Tyr Glu Arg Pro Pro Leu Ser 35 40 45 Lys Glu Tyr Phe Ala Arg Glu Lys Thr Phe Asp Arg Leu Tyr Ile Arg 50 55 60 Pro Pro Thr Phe Trp Ala Glu Lys Asn Ile Glu Phe Lys Leu Gly Thr 65 70 75 80 Glu Val Thr Lys Val Asp Pro Lys Ala His Glu Leu Thr Leu Ser Asn 85 90 95 Gly Glu Ser Tyr Gly Tyr Gly Lys Leu Val Trp Ala Thr Gly Gly Asp 100 105 110 Pro Arg Arg Leu Ser Cys Gln Gly Ala Asp Leu Thr Gly Ile His Ala 115 120 125 Val Arg Thr Arg Glu Asp Cys Asp Thr Leu Met Ala Glu Val Asp Ala 130 135 140 Gly Thr Lys Asn Ile Val Val Ile Gly Gly Gly Tyr Ile Gly Leu Glu 145 150 155 160 Ala Ala Ala Val Leu Ser Lys Met Gly Leu Lys Val Thr Leu Leu Glu 165 170 175 Ala Leu Pro Arg Val Leu Ala Arg Val Ala Gly Glu Asp Leu Ser Thr 180 185 190 Phe Tyr Gln Lys Glu His Val Asp His Gly Val Asp Leu Arg Thr Glu 195 200 205 Val Met Val Asp Ser Leu Val Gly Glu Asn Gly Lys Val Thr Gly Val 210 215 220 Gln Leu Ala Gly Gly Glu Val Ile Pro Ala Glu Gly Val Ile Val Gly 225 230 235 240 Ile Gly Ile Val Pro Ala Ile Gly Pro Leu Ile Ala Ala Gly Ala Ala 245 250 255 Gly Ala Asn Gly Val Asp Val Asp Glu Tyr Cys Arg Thr Ser Leu Pro 260 265 270 Asp Ile Tyr Ala Ile Gly Asp Cys Ala Ala Phe Ala Cys Asp Tyr Ala 275 280 285 Gly Gly Asn Val Met Arg Val Glu Ser Val Gln Asn Ala Asn Asp Met 290 295 300 Gly Thr Cys Val Ala Lys Ala Ile Cys Gly Asp Glu Lys Pro Tyr Lys 305 310 315 320 Ala Phe Pro Trp Phe Trp Ser Asn Gln Tyr Asp Leu Lys Leu Gln Thr 325 330 335 Ala Gly Ile Asn Leu Gly Phe Asp Lys Thr Val Ile Arg Gly Asn Pro 340 345 350 Glu Glu Arg Ser Phe Ser Val Val Tyr Leu Lys Asp Gly Arg Val Val 355 360 365 Ala Leu Asp Cys Val Asn Met Val Lys Asp Tyr Val Gln Gly Arg Lys 370 375 380 Leu Val Glu Ala Gly Ala Thr Pro Asp Leu Glu Ala Leu Ala Asp Ala 385 390 395 400 Gly Lys Pro Leu Lys Glu Leu Gln Tyr 405 10 106 PRT Rhodobacter capsulatus 10 Ala Lys Ile Ile Phe Ile Glu His Asn Gly Thr Arg His Glu Val Glu 1 5 10 15 Ala Lys Pro Gly Leu Thr Val Met Glu Ala Ala Arg Asp Asn Gly Val 20 25 30 Pro Gly Ile Asp Ala Asp Cys Gly Gly Ala Cys Ala Cys Ser Thr Cys 35 40 45 His Ala Tyr Val Asp Pro Ala Trp Val Asp Lys Leu Pro Lys Ala Leu 50 55 60 Pro Thr Glu Thr Asp Met Ile Asp Phe Ala Tyr Glu Pro Asn Pro Ala 65 70 75 80 Thr Ser Arg Leu Thr Cys Gln Ile Lys Val Thr Ser Leu Leu Asp Gly 85 90 95 Leu Val Val His Leu Pro Glu Lys Gln Ile 100 105 11 106 PRT Caulobacter crescentus 11 Met Ala Lys Ile Thr Tyr Ile Gln His Asp Gly Ala Glu Gln Val Ile 1 5 10 15 Asp Val Lys Pro Gly Leu Thr Val Met Glu Gly Ala Val Lys Asn Asn 20 25 30 Val Pro Gly Ile Asp Ala Asp Cys Gly Gly Ala Cys Ala Cys Ala Thr 35 40 45 Cys His Val Tyr Val Asp Glu Ala Trp Leu Asp Lys Thr Gly Asp Lys 50 55 60 Ser Ala Met Glu Glu Ser Met Leu Asp Phe Ala Glu Asn Val Glu Pro 65 70 75 80 Asn Ser Arg Leu Ser Cys Gln Ile Lys Val Ser Asp Ala Leu Asp Gly 85 90 95 Leu Val Val Arg Leu Pro Glu Ser Gln His 100 105 12 106 PRT Rhodococcus erythropolis 12 Pro Thr Val Thr Tyr Val His Pro Asp Gly Thr Lys His Glu Val Glu 1 5 10 15 Val Pro Thr Gly Lys Arg Val Met Gln Ala Ala Ile Gly Ala Gly Ile 20 25 30 Asp Gly Ile Val Ala Glu Cys Gly Gly Gln Ala Met Cys Ala Thr Cys 35 40 45 His Val Tyr Val Glu Ser Pro Trp Ala Asp Lys Phe Pro Ser Ile Ser 50 55 60 Glu Glu Glu Asp Glu Met Leu Asp Asp Thr Val Ser Pro Arg Thr Glu 65 70 75 80 Ala Ser Arg Leu Ser Cys Gln Leu Val Val Ser Asp Asp Val Asp Gly 85 90 95 Leu Ile Val Arg Leu Pro Glu Glu Gln Val 100 105 13 106 PRT Pseudomonas putida 13 Ser Lys Val Val Tyr Val Ser His Asp Gly Thr Arg Arg Glu Leu Asp 1 5 10 15 Val Ala Asp Gly Val Ser Leu Met Gln Ala Ala Val Ser Asn Gly Ile 20 25 30 Tyr Asp Ile Val Gly Asp Cys Gly Gly Ser Ala Ser Cys Ala Thr Cys 35 40 45 His Val Tyr Val Asn Glu Ala Phe Thr Asp Lys Val Pro Ala Ala Asn 50 55 60 Glu Arg Glu Ile Gly Met Leu Glu Cys Val Thr Ala Glu Leu Lys Pro 65 70 75 80 Asn Ser Arg Leu Cys Cys Gln Ile Ile Met Thr Pro Glu Leu Asp Gly 85 90 95 Ile Val Val Asp Val Pro Asp Arg Gln Trp 100 105 14 105 PRT Pseudomonas sp. 14 Pro Arg Val Val Phe Ile Asp Glu Gln Ser Gly Glu Tyr Ala Val Asp 1 5 10 15 Ala Gln Asp Gly Gln Ser Leu Met Glu Val Ala Thr Gln Asn Gly Val 20 25 30 Pro Gly Ile Val Ala Glu Cys Gly Gly Ser Cys Val Cys Ala Thr Cys 35 40 45 Arg Ile Glu Ile Glu Asp Ala Trp Val Glu Ile Val Gly Glu Ala Asn 50 55 60 Pro Asp Glu Asn Asp Leu Leu Gln Ser Thr Gly Glu Pro Met Thr Ala 65 70 75 80 Gly Thr Arg Leu Ser Cys Gln Val Phe Ile Asp Pro Ser Met Asp Gly 85 90 95 Leu Ile Val Arg Val Pro Leu Pro Ala 100 105 15 409 PRT Sphingomonas sp. 15 Met Arg Ser Ala Asp Val Val Ile Val Gly Ala Gly His Ala Gly Ala 1 5 10 15 Gln Cys Ala Ile Ala Leu Arg Gln Ala Gly Tyr Glu Gly Ser Ile Ala 20 25 30 Leu Val Gly Arg Glu Asn Glu Val Pro Tyr Glu Arg Pro Pro Leu Ser 35 40 45 Lys Glu Tyr Phe Ser Arg Glu Lys Ser Phe Glu Arg Leu Tyr Ile Arg 50 55 60 Pro Pro Glu Phe Trp Arg Glu Lys Asp Ile His Leu Thr Leu Gly Ile 65 70 75 80 Glu Val Ser Ala Val Asp Pro Gly Ser Lys Val Leu Thr Leu Ser Asp 85 90 95 Gly Ser Ala Phe Ala Tyr Gly Gln Leu Val Trp Ala Thr Gly Gly Asp 100 105 110 Pro Arg Lys Leu Ala Cys Pro Gly Ala Glu Leu Ser Gly Val His Ala 115 120 125 Ile Arg Thr Arg Ala Asp Cys Asp Arg Leu Met Ala Glu Ile Asp Arg 130 135 140 Gly Leu Thr Gln Val Val Val Val Gly Gly Gly Tyr Ile Gly Leu Glu 145 150 155 160 Ala Ala Ala Val Leu Thr Lys Leu Asn Cys His Val Thr Leu Leu Glu 165 170 175 Ala Met Pro Arg Val Leu Ala Arg Val Ala Gly Thr Glu Leu Ser Thr 180 185 190 Phe Tyr Glu Asn Glu His Arg Gly His Gly Val Asp Leu Arg Thr Gly 195 200 205 Ile Thr Val Ala Ala Leu Glu Gly Gln Glu Ser Val Thr Gly Val Arg 210 215 220 Leu Gly Asp Gly Ser Val Leu Pro Ala Gln Ala Val Ile Val Gly Ile 225 230 235 240 Gly Ile Val Pro Ala Val Ala Pro Leu Ile Glu Ala Gly Ala Ala Gly 245 250 255 Asp Gly Gly Val Thr Val Asp Glu Tyr Cys Arg Thr Ser Leu Pro Asp 260 265 270 Val Phe Ala Ile Gly Asp Cys Ala Ser Phe Ser Cys Ser Phe Ala Asp 275 280 285 Gly Arg Val Leu Arg Val Glu Ser Val Gln Asn Ala Asn Asp Gln Ala 290 295 300 Ser Cys Val Ala Lys Thr Ile Cys Gly Asp Pro Gln Pro Tyr Arg Ala 305 310 315 320 Phe Pro Trp Phe Trp Ser Asn Gln Tyr Asp Leu Arg Leu Gln Thr Ala 325 330 335 Gly Leu Ser Leu Gly Tyr Asp Gln Thr Val Val Arg Gly Asp Pro Ala 340 345 350 Val Arg Ser Phe Ser Val Leu Tyr Leu Lys Gln Gly Arg Val Ile Ala 355 360 365 Leu Asp Cys Val Asn Met Val Lys Asp Tyr Val Gln Gly Arg Lys Leu 370 375 380 Val Glu Ala Asn Val Cys Val Ser Pro Glu Gln Leu Val Asp Thr Gly 385 390 395 400 Leu Ala Leu Lys Asp Leu Ile Pro Val 405 16 427 PRT Rhodococcus erythropolis 16 Ser Ile Val Ile Ile Gly Ser Gly Gln Ala Gly Phe Glu Ala Ala Val 1 5 10 15 Ser Leu Arg Ser His Gly Phe Ser Gly Thr Ile Thr Leu Val Gly Asp 20 25 30 Glu Pro Gly Val Pro Tyr Gln Arg Pro Pro Leu Ser Lys Ala Tyr Leu 35 40 45 His Ser Asp Pro Asp Arg Glu Ser Leu Ala Leu Arg Pro Ala Gln Tyr 50 55 60 Phe Asp Asp His Arg Ile Thr Leu Thr Cys Gly Lys Pro Val Val Arg 65 70 75 80 Ile Asp Arg Asp Ala Gln Arg Val Glu Leu Ile Asp Ala Thr Ala Ile 85 90 95 Glu Tyr Asp His Leu Ile Leu Ala Thr Gly Ala Arg Asn Arg Leu Leu 100 105 110 Pro Val Pro Gly Ala Asn Leu Pro Gly Val His Tyr Leu Arg Thr Ala 115 120 125 Gly Glu Ala Glu Ser Leu Thr Ser Ser Met Ala Ser Cys Ser Ser Leu 130 135 140 Val Val Ile Gly Ala Gly Phe Ile Gly Leu Glu Val Ala Ala Ala Ala 145 150 155 160 Arg Lys Lys Gly Leu Asp Val Thr Val Val Glu Ala Met Asp Arg Pro 165 170 175 Met Ala Arg Ala Leu Ser Ser Val Met Ser Gly Tyr Phe Ser Thr Ala 180 185 190 His Thr Glu His Gly Val His Met Arg Leu Ser Thr Gly Val Lys Thr 195 200 205 Ile Asn Ala Ala Asp Gly Arg Ala Ala Gly Val Thr Thr Asn Ser Gly 210 215 220 Asp Val Ile His Ala Asp Ala Val Val Val Gly Ile Gly Val Val Pro 225 230 235 240 Asn Ile Glu Leu Ala Ala Leu Thr Gly Leu Pro Val Asp Asn Gly Ile 245 250 255 Val Val Asp Glu Tyr Leu Arg Thr Pro Asp Glu Asn Ile Ser Ala Ile 260 265 270 Gly Asp Cys Ala Ala Tyr Pro Ile Pro Gly Lys Ala Gly Leu Val Arg 275 280 285 Leu Glu Ser Val Gln Asn Ala Val Asp Gln Ala Arg Cys Leu Ala Ala 290 295 300 Gln Leu Thr Gly Thr Ser Thr His Tyr Arg Ser Val Pro Trp Phe Trp 305 310 315 320 Ser Glu Gln Tyr Glu Ser Lys Leu Gln Met Ala Gly Leu Thr Ala Gly 325 330 335 Ala Asp Thr His Val Val Arg Gly Ser Val Asp Ser Gly Val Phe Ser 340 345 350 Ile Phe Cys Phe Leu Gly Thr Arg Leu Leu Gly Val Glu Ser Val Asn 355 360 365 Lys Pro Arg Asp His Met Ala Ala Arg Lys Ile Leu Ala Thr Glu Met 370 375 380 Pro Leu Thr Pro Glu Gln Ala Ala Asp Thr Asp Phe Asp Leu Lys Leu 385 390 395 400 Ala Ile Ala Arg His Lys Asp Thr His Glu Lys Asp Glu Val Ala Ser 405 410 415 Ala Asp Ile Gly Glu Arg Gln Val Val Ala Ser 420 425 17 422 PRT Pseudomonas putida 17 Met Asn Ala Asn Asp Asn Val Val Ile Val Gly Thr Gly Leu Ala Gly 1 5 10 15 Val Glu Val Ala Phe Gly Leu Arg Ala Ser Gly Trp Glu Gly Asn Ile 20 25 30 Arg Leu Val Gly Asp Ala Thr Val Ile Pro His His Leu Pro Pro Leu 35 40 45 Ser Lys Ala Tyr Leu Ala Gly Lys Ala Thr Ala Glu Ser Leu Tyr Leu 50 55 60 Arg Thr Pro Asp Ala Tyr Ala Ala Gln Asn Ile Gln Leu Leu Gly Gly 65 70 75 80 Thr Gln Val Thr Ala Ile Asn Arg Asp Arg Gln Gln Val Ile Leu Ser 85 90 95 Asp Gly Arg Ala Leu Asp Tyr Asp Arg Leu Val Leu Ala Thr Gly Gly 100 105 110 Arg Pro Arg Pro Leu Pro Val Ala Ser Gly Ala Val Gly Lys Ala Asn 115 120 125 Asn Phe Arg Tyr Leu Arg Thr Leu Glu Asp Ala Glu Cys Ile Arg Arg 130 135 140 Gln Leu Ile Ala Asp Asn Arg Leu Val Val Ile Gly Gly Gly Tyr Ile 145 150 155 160 Gly Leu Glu Val Ala Ala Thr Ala Ile Lys Ala Asn Met His Val Thr 165 170 175 Leu Leu Asp Thr Ala Ala Arg Val Leu Glu Arg Val Thr Ala Pro Pro 180 185 190 Val Ser Ala Phe Tyr Glu His Leu His Arg Glu Ala Gly Val Asp Ile 195 200 205 Arg Thr Gly Thr Gln Val Cys Gly Phe Glu Met Ser Thr Asp Gln Gln 210 215 220 Lys Val Thr Ala Val Leu Cys Glu Asp Gly Thr Arg Leu Pro Ala Asp 225 230 235 240 Leu Val Ile Ala Gly Ile Gly Leu Ile Pro Asn Cys Glu Leu Ala Ser 245 250 255 Ala Ala Gly Leu Gln Val Asp Asn Gly Ile Val Ile Asn Glu His Met 260 265 270 Gln Thr Ser Asp Pro Leu Ile Met Ala Val Gly Asp Cys Ala Arg Phe 275 280 285 His Ser Gln Leu Tyr Asp Arg Trp Val Arg Ile Glu Ser Val Pro Asn 290 295 300 Ala Leu Glu Gln Ala Arg Lys Ile Ala Ala Ile Leu Cys Gly Lys Val 305 310 315 320 Pro Arg Asp Glu Ala Ala Pro Trp Phe Trp Ser Asp Gln Tyr Glu Ile 325 330 335 Gly Leu Lys Met Val Gly Leu Ser Glu Gly Tyr Asp Arg Ile Ile Val 340 345 350 Arg Gly Ser Leu Ala Gln Pro Asp Phe Ser Val Phe Tyr Leu Gln Gly 355 360 365 Asp Arg Val Leu Ala Val Asp Thr Val Asn Arg Pro Val Glu Phe Asn 370 375 380 Gln Ser Lys Gln Ile Ile Thr Asp Arg Leu Pro Val Glu Pro Asn Leu 385 390 395 400 Leu Gly Asp Glu Ser Val Pro Leu Lys Glu Ile Ile Ala Ala Ala Lys 405 410 415 Ala Glu Leu Ser Ser Ala 420 18 409 PRT Pseudomonas sp. 18 Met Gly Glu Arg Arg Asp Thr Thr Val Ile Val Gly Ala Gly His Ala 1 5 10 15 Gly Thr Ala Ala Ala Phe Phe Leu Arg Glu Phe Gly Tyr His Gly Arg 20 25 30 Val Leu Leu Leu Ser Ala Glu Thr Gln His Pro Tyr Gln Arg Pro Pro 35 40 45 Leu Ser Lys Glu Tyr Leu Leu Ala Gln His Ser Thr Pro Ser Leu Leu 50 55 60 Lys Gly Lys Asp Ser Tyr Ala Arg Ala Asp Ile Glu Leu Cys Leu Gln 65 70 75 80 Asp Asp Val Leu Ser Ile Thr Pro Ala Ser Arg Gln Val Lys Ser Ser 85 90 95 Gln Gly Ser Tyr Thr Tyr Asp His Leu Ile Leu Ala Thr Gly Ser His 100 105 110 Pro Arg Phe Met Ala Thr Leu Gly Gln Ala Asp Asn Leu Cys Tyr Leu 115 120 125 Ser Asp Trp Asp Asp Ala Gly Arg Ile Arg Gln Gln Leu Gly Glu Ala 130 135 140 Ser Arg Ile Val Val Leu Gly Gly Gly Phe Ile Gly Leu Glu Ile Ala 145 150 155 160 Ser Ser Ala Cys Lys Met Gly Lys His Val Thr Val Ile Glu Arg Ala 165 170 175 Pro Arg Leu Leu Ser Arg Val Val Ser Glu Ala Phe Ala Thr Phe Ile 180 185 190 Gly Asp Ile His Leu Gly Asn Gly Ile Glu Leu Arg Leu Gly Glu Glu 195 200 205 Val Arg Glu Val Arg Arg Cys Thr Ser Gly Val Arg Val Asp Ala Val 210 215 220 Phe Leu Ser Asp Gly Gln Leu Leu Glu Cys Asp Met Leu Val Ile Gly 225 230 235 240 Val Gly Ser Glu Pro Arg Met Glu Leu Ala Thr Ala Ala Gly Leu Ala 245 250 255 Cys Ala Ser Gly Val Leu Val Asp Glu His Cys His Thr Ser Asp Pro 260 265 270 Phe Ile Ser Ala Ile Gly Asp Cys Val Ala Val Cys Pro Ser Pro Gly 275 280 285 His Gln Leu Pro Arg Arg Glu Ser Val Gln Asn Ala Thr Glu Gln Ala 290 295 300 Arg Leu Val Ala Ala Arg Leu Ser Gly Arg Pro Val Pro Pro Val Gln 305 310 315 320 Thr Pro Trp Phe Trp Ser Asp Gln Leu Gln Ala Arg Ile Asn Leu Ala 325 330 335 Gly Glu Arg Pro Ala Gln Gly Gln Val Ile Val Arg Arg Tyr Gly Gly 340 345 350 Asp Lys Val Ser Met Leu Tyr Leu Gln Asp Gln Gln Leu Val Ala Ile 355 360 365 Glu Ala Cys Asn Met Pro Gly Asp Cys Leu Leu Ala Arg Arg Ala Ile 370 375 380 Gly Gln Asn His Ser Leu Asp Leu Ala Arg Leu Val Asp Ala Asp Val 385 390 395 400 Pro Leu Lys Asp Ala Leu His Phe Ala 405

Claims (108)

We claim:
1. An isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading oxygenase.
2. The DNA molecule of claim 1 comprising a DNA sequence coding for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
3. The DNA molecule of claim 2 comprising the nucleotide sequence of SEQ ID NO: 2.
4. A DNA construct comprising a DNA sequence coding for a dicamba-degrading oxygenase operatively linked to expression control sequences.
5. The DNA construct of claim 4 comprising a DNA sequence coding for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
6. The DNA construct of claim 5 comprising the nucleotide sequence of SEQ ID NO: 2.
7. The DNA construct of claim 4 which is a vector.
8. An isolated and at least partially purified dicamba-degrading oxygenase.
9. The dicamba-degrading oxygenase of claim 8 having the amino acid sequence of SEQ ID NO: 3.
10. An isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading ferredoxin.
11. The DNA molecule of claim 10 comprising a DNA sequence coding for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
12. The DNA molecule of claim 11 comprising the nucleotide sequence of SEQ ID NO: 4.
13. The DNA molecule of claim 11 comprising a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
14. A DNA construct comprising a DNA sequence coding for a dicamba-degrading ferredoxin operatively linked to expression control sequences.
15. The DNA construct of claim 14 comprising a DNA sequence coding for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
16. The DNA construct of claim 15 comprising the nucleotide sequence of SEQ ID NO: 4.
17. The DNA construct of claim 15 comprising a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
18. The DNA construct of claim 14 which is a vector.
19. An isolated and at least partially purified dicamba-degrading ferredoxin.
20. The dicamba-degrading ferredoxin of claim 19 which has the amino acid sequence of SEQ ID NO: 5.
21. An isolated DNA molecule comprising a DNA sequence coding for a dicamba-degrading reductase.
22. The DNA molecule of claim 21 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
23. The DNA molecule of claim 22 comprising the nucleotide sequence of SEQ ID NO: 6.
24. The DNA molecule of claim 21 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
25. The DNA molecule of claim 24 comprising the nucleotide sequence of SEQ ID NO: 8.
26. A DNA construct comprising a DNA sequence coding for a dicamba-degrading reductase operatively linked to expression control sequences.
27. The DNA construct of claim 26 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
28. The DNA construct of claim 27 comprising the nucleotide sequence of SEQ ID NO: 6.
29. The DNA construct of claim 26 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
30. The DNA construct of claim 29 comprising the nucleotide sequence of SEQ ID NO: 8.
31. The DNA construct of claim 26 which is a vector.
32. An isolated and at least partially purified dicamba-degrading reductase.
33. The dicamba-degrading reductase of claim 32 which has the amino acid sequence of SEQ ID NO: 7.
34. The dicamba-degrading reductase of claim 32 which has the amino acid sequence of SEQ ID NO: 9.
35. A DNA construct comprising a DNA sequence coding for a dicamba-degrading oxygenase, a DNA sequence coding for a dicamba-degrading ferredoxin, and a DNA sequence coding for a dicamba-degrading reductase, each DNA sequence being operatively linked to expression control sequences.
36. The DNA construct of claim 35 comprising a DNA sequence coding for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
37. The DNA construct of claim 36 comprising the nucleotide sequence of SEQ ID NO: 2.
38. The DNA construct of claim 35 comprising a DNA sequence coding for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
39. The DNA construct of claim 38 comprising the nucleotide sequence of SEQ ID NO: 4.
40. The DNA construct of claim 38 comprising a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
41. The DNA construct of claim 35 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
42. The DNA construct of claim 41 comprising the nucleotide sequence of SEQ ID NO: 6.
43. The DNA construct of claim 35 comprising a DNA sequence coding for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
44. The DNA construct of claim 43 comprising the nucleotide sequence of SEQ ID NO: 8.
45. The DNA construct of claim 35 which is a vector.
46. An isolated and at least partially purified dicamba-degrading O-demethylase.
47. A transgenic host cell comprising DNA coding for a dicamba-degrading oxygenase operatively linked to expression control sequences.
48. The transgenic host cell of claim 47 wherein the DNA codes for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
49. The transgenic host cell of claim 48 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 2.
50. The transgenic host cell of claim 47 which is a plant cell.
51. The transgenic host cell of claim 47 which is a microorganism.
52. A transgenic host cell comprising DNA coding for a dicamba-degrading ferredoxin operatively linked to expression control sequences.
53. The transgenic host cell of claim 52 wherein the DNA codes for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
54. The transgenic host cell of claim 53 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 4.
55. The transgenic host cell of claim 53 wherein the DNA comprises a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
56. The transgenic host cell of claim 52 which is a plant cell.
57. The transgenic host cell of claim 52 which is a microorganism.
58. A transgenic host cell comprising DNA coding for a dicamba-degrading reductase operatively linked to expression control sequences.
59. The transgenic host cell of claim 58 wherein the DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
60. The transgenic host cell of claim 59 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 6.
61. The transgenic host cell of claim 58 wherein the DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
62. The transgenic host cell of claim 61 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 8.
63. The transgenic host cell of claim 58 which is a plant cell.
64. The transgenic host cell of claim 58 which is a microorganism.
65. A transgenic host cell comprising a first DNA coding for a dicamba-degrading oxygenase and a second DNA coding for a dicamba-degrading ferredoxin, a third DNA coding for a dicamba-degrading reductase, or both the second DNA and the third DNA, each DNA being operatively linked to expression control sequences.
66. The transgenic host cell of claim 65 wherein the first DNA codes for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
67. The transgenic host cell of claim 66 wherein the first DNA comprises the nucleotide sequence of SEQ ID NO: 2.
68. The transgenic host cell of claim 65 wherein the second DNA codes for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
69. The transgenic host cell of claim 68 wherein the second DNA comprises the nucleotide sequence of SEQ ID NO: 4.
70. The transgenic host cell of claim 68 wherein the second DNA comprises a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
71. The transgenic host cell of claim 65 wherein the third DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
72. The transgenic host cell of claim 71 wherein the third DNA comprises the nucleotide sequence of SEQ ID NO: 6.
73. The transgenic host cell of claim 65 wherein the third DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
74. The transgenic host cell of claim 73 wherein the third DNA comprises the nucleotide sequence of SEQ ID NO: 8.
75. The transgenic host cell of claim 65 which is a plant cell.
76. The transgenic host cell of claim 65 which is a microorganism.
77. A transgenic plant or part of a plant comprising one or more cells comprising DNA coding for a dicamba-degrading oxygenase operatively linked to expression control sequences.
78. The transgenic plant or plant part of claim 77 wherein the DNA codes for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
79. The transgenic plant or plant part of claim 78 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 2.
80. The transgenic plant or plant part of claim 77 wherein the plant is a broadleaf plant which is tolerant to dicamba.
81. A transgenic plant or part of a plant comprising one or more cells comprising DNA coding for a dicamba-degrading ferredoxin operatively linked to expression control sequences.
82. The transgenic plant or plant part of claim 81 wherein the DNA codes for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
83. The trangenic plant or plant part of claim 82 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 4.
84. The trangenic plant or plant part of claim 82 wherein the DNA comprises a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
85. The transgenic plant or plant part of claim 81 wherein the plant is a broadleaf plant which is tolerant to dicamba.
86. A transgenic plant or part of a plant comprising one or more cells comprising DNA coding for a dicamba-degrading reductase operatively linked to expression control sequences.
87. The transgenic plant or plant part of claim 86 wherein the DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
88. The trangenic plant or plant part of claim 87 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 6.
89. The transgenic plant or plant part of claim 86 wherein the DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
90. The trangenic plant or plant part of claim 89 wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 8.
91. The transgenic plant or plant part of claim 86 wherein the plant is a broadleaf plant which is tolerant to dicamba.
92. A transgenic plant or part of a plant comprising one or more cells comprising a first DNA coding for a dicamba-degrading oxygenase and a second DNA coding for a dicamba-degrading ferredoxin, a third DNA coding for a dicamba-degrading reductase, or both the second DNA and the third DNA, each DNA being operatively linked to expression control sequences.
93. The transgenic plant or plant part of claim 92 wherein the first DNA codes for a dicamba-degrading oxygenase having the amino acid sequence of SEQ ID NO: 3.
94. The transgenic plant or plant part of claim 93 wherein the first DNA comprises the nucleotide sequence of SEQ ID NO: 2.
95. The transgenic plant or plant part of claim 92 wherein the second DNA codes for a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 5.
96. The trangenic plant or plant part of claim 95 wherein the second DNA comprises the nucleotide sequence of SEQ ID NO: 4.
97. The trangenic plant or plant part of claim 95 wherein the second DNA comprises a nucleotide sequence which has codons selected for optimized expression in dicotyledonous plants.
98. The transgenic plant or plant part of claim 92 wherein the third DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 7.
99. The transgenic plant or plant part of claim 98 wherein the third DNA comprises the nucleotide sequence of SEQ ID NO: 6.
100. The transgenic plant or plant part of claim 92 wherein the third DNA codes for a dicamba-degrading reductase having the amino acid sequence of SEQ ID NO: 9.
101. The transgenic plant or plant part of claim 100 wherein the third DNA comprises the nucleotide sequence of SEQ ID NO: 8.
102. The transgenic plant or plant part of claim 92 wherein the plant is a broadleaf plant which is tolerant to dicamba.
103. A method of controlling weeds in a field containing a dicamba-tolerant transgenic plant, the method comprising applying an amount of dicamba to the field effective to control the weeds in the field.
104. A method of decontaminating a material containing dicamba comprising applying an amount of a dicamba-degrading transgenic microorganism to the material, the amount being effective to degrade at least some of the dicamba in the material.
105. A method of decontaminating a material containing dicamba comprising applying an amount of a dicamba-degrading O-demethylase or of a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase to the material, the amount being effective to degrade at least some of the dicamba in the material.
106. A method of selecting transformed plant cells comprising:
providing a population of plant cells;
transforming at least some of the plant cells in the population of plant cells so that they are tolerant to dicamba; and
growing the resulting population of plant cells in a culture medium containing dicamba at a concentration selected so that transformed plant cells will grow and untransformed plant cells will not grow.
107. A method of selecting transformed plants comprising:
providing a population of plants suspected of comprising plants that have been transformed so that they are tolerant to dicamba; and
applying an amount of dicamba to the population of plants, the amount of dicamba being selected so that transformed plants will grow and growth of untransformed plants will be inhibited.
108. A method of selecting, or screening for, transformed host cells, intact organisms, and parts of organisms, the method comprising:
providing a population of host cells, intact organisms, or parts of organisms suspected of comprising host cells, intact organisms, or parts of organisms that have been transformed so that they are able to degrade dicamba;
contacting the population of host cells, intact organisms, or parts of organisms with dicamba; and
ascertaining the presence or level of fluorescence due to 3,6-dichlorosalicylic acid, the 3,6-dichlorosalicyclic acid being generated in the host cells, intact organisms, or parts of organisms as a result of the degradation of dicamba.
US10/330,661 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms Abandoned US20030135879A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/330,661 US20030135879A1 (en) 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US4294197P 1997-04-04 1997-04-04
US4266697P 1997-04-04 1997-04-04
US09/055,145 US7022896B1 (en) 1997-04-04 1998-04-03 Methods and materials for making and using transgenic dicamba-degrading organisms
US09/797,238 US7105724B2 (en) 1997-04-04 2001-02-28 Methods and materials for making and using transgenic dicamba-degrading organisms
US10/330,661 US20030135879A1 (en) 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/797,238 Division US7105724B2 (en) 1997-04-04 2001-02-28 Methods and materials for making and using transgenic dicamba-degrading organisms

Publications (1)

Publication Number Publication Date
US20030135879A1 true US20030135879A1 (en) 2003-07-17

Family

ID=27488810

Family Applications (5)

Application Number Title Priority Date Filing Date
US09/797,238 Expired - Lifetime US7105724B2 (en) 1997-04-04 2001-02-28 Methods and materials for making and using transgenic dicamba-degrading organisms
US10/330,662 Expired - Fee Related US7812224B2 (en) 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms
US10/330,661 Abandoned US20030135879A1 (en) 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms
US12/875,747 Expired - Lifetime US8119380B2 (en) 1997-04-04 2010-09-03 Methods and materials for making and using transgenic dicamba-degrading organisms
US13/348,900 Expired - Lifetime US8629323B2 (en) 1997-04-04 2012-01-12 Methods and materials for making and using transgenic dicamba-degrading organisms

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US09/797,238 Expired - Lifetime US7105724B2 (en) 1997-04-04 2001-02-28 Methods and materials for making and using transgenic dicamba-degrading organisms
US10/330,662 Expired - Fee Related US7812224B2 (en) 1997-04-04 2002-12-27 Methods and materials for making and using transgenic dicamba-degrading organisms

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/875,747 Expired - Lifetime US8119380B2 (en) 1997-04-04 2010-09-03 Methods and materials for making and using transgenic dicamba-degrading organisms
US13/348,900 Expired - Lifetime US8629323B2 (en) 1997-04-04 2012-01-12 Methods and materials for making and using transgenic dicamba-degrading organisms

Country Status (2)

Country Link
US (5) US7105724B2 (en)
WO (1) WO2002068607A2 (en)

Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005069986A2 (en) 2004-01-20 2005-08-04 Monsanto Technology Llc Chimeric promoters for use in plants
US20060057616A1 (en) * 2004-08-20 2006-03-16 Vironix Llc Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing
WO2006069017A2 (en) 2004-12-21 2006-06-29 Monsanto Technology, Llc Transgenic plants with enhanced agronomic traits
WO2007030432A2 (en) 2005-09-06 2007-03-15 Monsanto Technology Llc Vectors and methods for improved plant transformation efficiency
WO2007053482A2 (en) 2005-10-28 2007-05-10 Dow Agrosciences Llc Novel herbicide resistance genes
WO2007143690A2 (en) 2006-06-06 2007-12-13 Monsanto Technology Llc Methods for weed control
US20080015110A1 (en) * 2006-06-06 2008-01-17 Monsanto Technology Llc Modified dmo enzyme and methods of its use
WO2008021543A2 (en) 2006-08-17 2008-02-21 Monsanto Technology, Llc Transgenic plants with enhanced agronomic traits
US20080120739A1 (en) * 2006-06-06 2008-05-22 Monsanto Technology Llc Method for selection of transformed cells
WO2008105890A2 (en) 2007-02-26 2008-09-04 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
WO2008112633A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Method of meristem excision and transformation
US20080305952A1 (en) * 2006-10-25 2008-12-11 Arnevik Cindy L Cropping systems for managing weeds
US20090105077A1 (en) * 2006-10-16 2009-04-23 Monsanto Technology Llc Methods and compositions for improving plant health
US20100257631A1 (en) * 2009-04-07 2010-10-07 Boerma H Roger Soybean variety g00-3209
US20100279866A1 (en) * 2006-10-16 2010-11-04 Muhammad Bhatti Methods and Compositions for Improving Plant Health
US20100299773A1 (en) * 2009-05-20 2010-11-25 Monsanto Technology Llc Methods and compositions for selecting an improved plant
EP2298901A2 (en) 2004-04-30 2011-03-23 Dow AgroSciences LLC Novel herbicide resistance genes
WO2011066384A1 (en) 2009-11-24 2011-06-03 Dow Agrosciences Llc Aad-12 event 416, related transgenic soybean lines, and event-specific identification thereof
EP2336332A2 (en) 2008-04-29 2011-06-22 Monsanto Technology LLC Genes and uses for plant enhancement
WO2011088299A1 (en) 2010-01-14 2011-07-21 Monsanto Technology Llc Plant regulatory elements and uses thereof
EP2361986A1 (en) 2006-05-16 2011-08-31 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP2380988A2 (en) 2007-07-10 2011-10-26 Mosanto Technology LLC Transgenic plants with enhanced agronomic traits
WO2012030711A1 (en) 2010-08-30 2012-03-08 Agrigenetics, Inc. Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
EP2455491A2 (en) 2006-05-25 2012-05-23 Monsanto Technology LLC A method to identify disease resistant quantitative trait loci in soybean and compositions thereof
WO2012074868A2 (en) 2010-12-03 2012-06-07 Ms Technologies, Llc Optimized expression of glyphosate resistance encoding nucleic acid molecules in plant cells
EP2484769A2 (en) 2005-12-21 2012-08-08 Monsanto Technology LLC Transgenic plants with enhanced agronomic traits
WO2012128946A1 (en) 2011-03-18 2012-09-27 Ms Technologies Llc Regulatory regions preferentially expressing in non-pollen plant tissue
WO2012134921A2 (en) 2011-03-25 2012-10-04 Monsanto Technology Llc Plant regulatory elements and uses thereof
WO2012158535A1 (en) 2011-05-13 2012-11-22 Monsanto Technology Llc Plant regulatory elements and uses thereof
EP2543735A1 (en) 2007-06-06 2013-01-09 Monsanto Technology LLC Genes and uses for plant enhancement
EP2562259A2 (en) 2004-12-21 2013-02-27 Monsanto Technology LLC Transgenic plants with enhanced agronomic traits
WO2013040116A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040033A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040021A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040049A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040117A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040005A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040057A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
EP2607488A2 (en) 2008-04-07 2013-06-26 Monsanto Technology LLC Plant regulatory elements and uses thereof
WO2013112527A1 (en) 2012-01-23 2013-08-01 Dow Agrosciences Llc Herbicide tolerant cotton event pdab4468.19.10.3
WO2013116700A1 (en) 2012-02-01 2013-08-08 Dow Agrosciences Llc Glyphosate resistant plants and associated methods
WO2013130813A1 (en) 2012-02-29 2013-09-06 Dow Agrosciences Llc Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
WO2013158442A1 (en) 2012-04-20 2013-10-24 Monsanto Technology Llc Plant regulatory elements and uses thereof
WO2014153254A2 (en) 2013-03-14 2014-09-25 Pioneer Hi-Bred International Inc. Compositions and methods to control insect pests
WO2014150914A2 (en) 2013-03-15 2014-09-25 Pioneer Hi-Bred International, Inc. Phi-4 polypeptides and methods for their use
WO2014159306A1 (en) 2013-03-13 2014-10-02 Pioneer Hi-Bred International, Inc. Glyphosate application for weed control in brassica
WO2015023846A2 (en) 2013-08-16 2015-02-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015038734A2 (en) 2013-09-13 2015-03-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015066636A2 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal maize loci
WO2015066638A2 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal maize loci
WO2015066643A1 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal soybean loci
WO2015120276A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
WO2015120270A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International, Inc. Insecticidal proteins and methods for their use
WO2015130931A1 (en) 2014-02-28 2015-09-03 Dow Agrosciences Llc Root specific expression conferred by chimeric gene regulatory elements
WO2016044092A1 (en) 2014-09-17 2016-03-24 Pioneer Hi Bred International Inc Compositions and methods to control insect pests
WO2016061206A1 (en) 2014-10-16 2016-04-21 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3023499A1 (en) 2008-07-16 2016-05-25 Monsanto Technology LLC Methods and vectors for producing transgenic plants
WO2016099916A1 (en) 2014-12-19 2016-06-23 E. I. Du Pont De Nemours And Company Polylactic acid compositions with accelerated degradation rate and increased heat stability
WO2016114973A1 (en) 2015-01-15 2016-07-21 Pioneer Hi Bred International, Inc Insecticidal proteins and methods for their use
WO2016154631A1 (en) 2015-03-26 2016-09-29 The Texas A&M University System Conversion of lignin into bioplastics and lipid fuels
WO2016186986A1 (en) 2015-05-19 2016-11-24 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
WO2016205445A1 (en) 2015-06-16 2016-12-22 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2017023486A1 (en) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
WO2017040343A1 (en) 2015-08-28 2017-03-09 Pioneer Hi-Bred International, Inc. Ochrobactrum-mediated transformation of plants
WO2017059341A1 (en) 2015-10-02 2017-04-06 Monsanto Technology Llc Recombinant maize b chromosome sequence and uses thereof
WO2017105987A1 (en) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US9725730B2 (en) 2013-12-31 2017-08-08 Dow Agrosciences Llc Maize ubiquitin promoters
WO2017192560A1 (en) 2016-05-04 2017-11-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2017218207A1 (en) 2016-06-16 2017-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2018005411A1 (en) 2016-07-01 2018-01-04 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2018013333A1 (en) 2016-07-12 2018-01-18 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2018084936A1 (en) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3339441A1 (en) 2005-10-13 2018-06-27 Monsanto Technology LLC Methods for producing hybrid seed
US10030246B2 (en) 2013-12-31 2018-07-24 Dow Agrosciences Llc Maize ubiquitin promoters
US10030247B2 (en) 2013-12-31 2018-07-24 Dow Agrosciences Llc Maize ubiquitin promoters
US10036028B2 (en) 2013-12-31 2018-07-31 Dow Agrosciences Llc Maize ubiquitin promoters
EP3382028A1 (en) 2010-12-03 2018-10-03 Dow AgroSciences LLC Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof
US10113178B2 (en) 2014-03-20 2018-10-30 Monsanto Technology Llc Transgenic maize event MON 87419 and methods of use thereof
WO2019139616A1 (en) 2018-01-12 2019-07-18 The Texas A&M University System Increasing plant bioproduct yield
WO2019178042A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2019178038A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2022015619A2 (en) 2020-07-14 2022-01-20 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2023086765A2 (en) 2021-11-12 2023-05-19 Monsanto Technology Llc Compositions and methods for altering plant determinacy
US11732268B2 (en) 2016-06-28 2023-08-22 Monsanto Technology Llc Methods and compositions for use in genome modification in plants
US11920140B2 (en) 2017-08-22 2024-03-05 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease
US12004476B2 (en) 2022-01-26 2024-06-11 Monsanto Technology Llc. Cotton variety 20R750B3XF

Families Citing this family (1227)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105724B2 (en) 1997-04-04 2006-09-12 Board Of Regents Of University Of Nebraska Methods and materials for making and using transgenic dicamba-degrading organisms
CN101500421B (en) * 2006-06-06 2015-04-15 孟山都技术有限公司 Methods for weed control
CN101501196B (en) * 2006-06-06 2013-01-02 孟山都技术有限公司 Modified dicamba monooxygenases and methods of use thereof
CL2008000083A1 (en) * 2007-01-12 2008-07-18 Monsanto Technology Llc Soc Or CRYSTALLIZED POLYPEPTIDE OF DICAMBA MONOOXIGENASA (BMD); MOLECULA THAT INCLUDES A UNION SURFACE FOR DICAMBA; VEGETABLE CELL THAT UNDERSTANDS DMO.
AR061248A1 (en) * 2007-02-26 2008-08-13 Monsanto Technology Llc CHLOROPLASTIC TRANSIT PEPTIDES FOR THE EFFICIENT ADDRESS OF DMO AND ITS USES
AU2015203032B2 (en) * 2007-02-26 2017-03-02 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
BRPI0811294B8 (en) * 2007-05-09 2022-06-28 Dow Agrosciences Llc POLYNUCLEOTIDE ISOLATED, AND METHOD FOR WEED CONTROL IN AN AREA
US8097712B2 (en) 2007-11-07 2012-01-17 Beelogics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
US20120023622A1 (en) * 2007-12-18 2012-01-26 E.I. DuPont De Nemours and Company Pioneer Hi bred International Inc. Drought tolerant plants and related constructs and methods involving genes encoding ferredoxin family proteins
WO2010046422A2 (en) 2008-10-22 2010-04-29 Basf Se Use of auxin type herbicides on cultivated plants
WO2010046423A2 (en) 2008-10-22 2010-04-29 Basf Se Use of sulfonylurea herbicides on cultivated plants
UA116809C2 (en) 2009-06-25 2018-05-10 Басф Се APPLICATION OF AGROCHEMICAL MIXTURES FOR INCREASING THE VITALITY OF THE PLANT
EP3875592A3 (en) 2009-09-17 2021-11-03 Monsanto Technology LLC Soybean transgenic event mon 87708 and methods of use thereof
EA023099B1 (en) 2009-09-30 2016-04-29 Басф Се LOW-YET AMINE SALTS OF ANIONIC PESTICIDES
US8962584B2 (en) 2009-10-14 2015-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Compositions for controlling Varroa mites in bees
WO2011054741A2 (en) 2009-11-06 2011-05-12 Basf Se Crystalline complexes of 4-hydroxy benzoic acid and selected pesticides
JP2013512934A (en) 2009-12-08 2013-04-18 ビーエーエスエフ ソシエタス・ヨーロピア Pesticide mixture
US20120245031A1 (en) 2009-12-08 2012-09-27 Basf Se Pesticidal Mixtures
NZ627060A (en) 2010-03-08 2016-01-29 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
BR112012028132A2 (en) 2010-05-04 2015-09-15 Basf Se plants that have increased herbicide tolerance
US20130085066A1 (en) 2010-05-31 2013-04-04 Basf Se Method for Increasing the Health of a Plant
WO2011161131A1 (en) 2010-06-25 2011-12-29 Basf Se Herbicidal mixtures
WO2011161132A1 (en) 2010-06-25 2011-12-29 Basf Se Pesticidal mixtures
WO2012022729A2 (en) 2010-08-20 2012-02-23 Basf Se Method for improving the health of a plant
CA2805770A1 (en) 2010-08-24 2012-03-01 Basf Se Agrochemical mixtures for increasing the health of a plant
US9222100B2 (en) 2010-08-24 2015-12-29 Monsanto Technology Llc Methods and DNA constructs for autoregulating transgene silencing
CA2813112A1 (en) 2010-10-07 2012-04-12 Basf Se Use of strobilurins for increasing the gluten strength in winter cereals
EP2460404A1 (en) 2010-12-01 2012-06-06 Basf Se Compositions containing identical polyamine salts of mixed anionic pesticides
TWI667347B (en) 2010-12-15 2019-08-01 瑞士商先正達合夥公司 Soybean event syht0h2 and compositions and methods for detection thereof
WO2012080975A1 (en) 2010-12-16 2012-06-21 Basf Se Plants having increased tolerance to herbicides
US20130274104A1 (en) 2010-12-22 2013-10-17 Basf Se Agrochemical mixtures for increasing the health of a plant
US9198412B2 (en) 2011-05-02 2015-12-01 Basf Se Method for enhancing the performance of a pesticide with guanidines
US20140080706A1 (en) 2011-06-01 2014-03-20 Basf Se Method of preparing an aqueous tank mix comprising a base
US20140171319A1 (en) 2011-08-02 2014-06-19 Basf Se Aqueous Composition Comprising a Pesticide and a Base Selected from an Alkali Salt of Hydrogencarbonate
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US9840715B1 (en) 2011-09-13 2017-12-12 Monsanto Technology Llc Methods and compositions for delaying senescence and improving disease tolerance and yield in plants
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US9920326B1 (en) 2011-09-14 2018-03-20 Monsanto Technology Llc Methods and compositions for increasing invertase activity in plants
UY34687A (en) 2012-03-21 2013-09-30 Basf Se MIXING ASSISTANTS IN GLIFOSATO TANK THAT INCLUDE A SELECTED BASE OF A CARBONATE AND / OR A PHOSPHATE
CA2864957A1 (en) 2012-03-21 2013-09-26 Basf Se Tank mix adjuvant comprising an alkyl polyglucoside and a base
EP2827715A1 (en) 2012-03-21 2015-01-28 Basf Se Solid particulate tank mix adjuvant comprising a base selected from a carbonate and/or a phosphate
UY34688A (en) 2012-03-21 2013-09-30 Basf Se Mixing aid in liquid or particulate tank comprising a base selected from a mixture of carbonate and hydrogen carbonate
BR112014024012A8 (en) 2012-03-29 2017-07-25 Basf Se COCRYSTALS, PROCESS FOR THE PREPARATION OF COCRYSTALS, AGROCHEMICAL COMPOSITION, USE OF THE AGROCHEMICAL COMPOSITION AND METHOD FOR THE CONTROL OF UNDESIRABLE VEGETATION
AR091141A1 (en) 2012-05-23 2015-01-14 Monsanto Technology Llc HOPLOTIPOS ASSOCIATED WITH THE PERFORMANCE OF IMPROVED STACKED RISK IN TRANSGENIC PLANTS
CN104619843B (en) 2012-05-24 2020-03-06 A.B.种子有限公司 Compositions and methods for silencing gene expression
AR091489A1 (en) 2012-06-19 2015-02-11 Basf Se PLANTS THAT HAVE A GREATER TOLERANCE TO HERBICIDES INHIBITORS OF PROTOPORFIRINOGENO OXIDASA (PPO)
US10041087B2 (en) 2012-06-19 2018-08-07 BASF Agro B.V. Plants having increased tolerance to herbicides
EA201500022A1 (en) 2012-06-21 2015-06-30 Басф Се WATER COMPOSITION, CONTAINING DICAMBA AND MEANS, OBSTACLE TO DREAM
AU2013285521B2 (en) 2012-07-03 2016-11-03 Basf Se Highly concentrated aqueous formulation comprising an anionic pesticide and a base
KR20150036462A (en) 2012-07-09 2015-04-07 바스프 코포레이션 Drift control agent comprising polypropylene glycol and a triblock polymer
CN104768378A (en) 2012-10-01 2015-07-08 巴斯夫欧洲公司 Use of N-thio-anthranilamide compounds on cultivated plants
MX364070B (en) 2012-10-18 2019-04-10 Monsanto Technology Llc Methods and compositions for plant pest control.
WO2014079820A1 (en) 2012-11-22 2014-05-30 Basf Se Use of anthranilamide compounds for reducing insect-vectored viral infections
US20150307894A1 (en) 2012-11-28 2015-10-29 Monsanto Technology Llc Transgenic Plants With Enhanced Traits
CA2894213A1 (en) 2012-12-21 2014-06-26 Pioneer Hi-Bred International, Inc. Compositions and methods for auxin-analog conjugation
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
WO2014106837A2 (en) 2013-01-01 2014-07-10 A. B. Seeds Ltd. ISOLATED dsRNA MOLECULES AND METHODS OF USING SAME FOR SILENCING TARGET MOLECULES OF INTEREST
US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
KR20150128838A (en) 2013-03-07 2015-11-18 바스프 에스이 Co-crystals of pyrimethanil and selected dithiine tetracarboximide
CN105263329B (en) 2013-03-13 2020-09-18 孟山都技术公司 Methods and compositions for weed control
WO2014164761A1 (en) 2013-03-13 2014-10-09 Monsanto Technology Llc Methods and compositions for weed control
US20140283211A1 (en) 2013-03-14 2014-09-18 Monsanto Technology Llc Methods and Compositions for Plant Pest Control
CA2905595A1 (en) 2013-03-14 2014-09-25 Pioneer Hi-Bred International, Inc. Compositions having dicamba decarboxylase activity and methods of use
BR112015023286A2 (en) 2013-03-14 2018-03-06 Arzeda Corp recombinant polypeptide with dicamba decarboxylase activity, polynucleotide construct, cell, method of producing a host cell comprising a heterologous polynucleotide encoding a dicamba decarboxylase activity, method for decarboxylating dicamba, a dicamba derivative or a dicamba metabolite, method for detecting a polypeptide and method for detecting the presence of a polynucleotide encoding a polypeptide having dicamba decarboxylase activity
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US10443063B2 (en) 2013-06-11 2019-10-15 Syngenta Participations Ag Methods for generating transgenic plants
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
CN105980567B (en) 2013-07-19 2021-04-16 孟山都技术有限公司 Compositions and methods for controlling Beetle
AU2014307664A1 (en) 2013-08-12 2016-02-18 BASF Agro B.V. Plants having increased tolerance to herbicides (PPO)
US10087460B2 (en) 2013-08-12 2018-10-02 BASF Agro B.V. Transgenic or non-transgenic plants with mutated protoporphyrinogen oxidase having increased tolerance to herbicides
US9320233B1 (en) 2013-08-29 2016-04-26 Monsanto Technology Llc Soybean variety XB55B13
US9271463B1 (en) 2013-08-29 2016-03-01 Monsanto Technology Llc Soybean variety XB56V13
US9485932B1 (en) 2013-08-29 2016-11-08 Monsanto Technology Llc Soybean variety XB40U13
US9332708B2 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XBO9AE13
US9408359B1 (en) 2013-08-29 2016-08-09 Monsanto Technology Llc Soybean variety XB36G13
US9408357B1 (en) 2013-08-29 2016-08-09 Monsanto Technology Llc Soybean variety XB35X13
US9433175B2 (en) 2013-08-29 2016-09-06 Monsanto Technology Llc Soybean variety XB33Z13
US9332710B2 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XB22A13
US9485930B1 (en) 2013-08-29 2016-11-08 Monsanto Technology Llc Soybean variety XB35W13
US9332713B1 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XB44K13
US9485931B1 (en) 2013-08-29 2016-11-08 Monsanto Technology Llc Soybean variety XB39C13
US9271464B1 (en) 2013-08-29 2016-03-01 Monsanto Technology Llc Soybean variety XB35Y13
US9332712B2 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XB28T13
US9408358B1 (en) 2013-08-29 2016-08-09 Monsanto Technology Llc Soybean variety XB34Y13
CA2861225A1 (en) 2013-08-29 2015-02-28 Monsanto Technology Llc Soybean variety xb04f13
US9332709B2 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XB21Z13
CA2861217C (en) 2013-08-29 2022-07-19 Monsanto Technology Llc Soybean variety xb007g13
US9445567B1 (en) 2013-08-29 2016-09-20 Monsanto Technology Llc Soybean variety XB39D13
US9332711B2 (en) 2013-08-29 2016-05-10 Monsanto Technology Llc Soybean variety XB23AB13
US9271462B1 (en) 2013-08-29 2016-03-01 Monsanto Technology Llc Soybean variety XB49AE13
US9480226B1 (en) 2013-08-29 2016-11-01 Monsanto Technology Llc Soybean variety XB34Z13
WO2015054106A1 (en) 2013-10-07 2015-04-16 Monsanto Technology Llc Transgenic plants with enhanced traits
CN105849266A (en) 2013-11-04 2016-08-10 孟山都技术公司 Compositions and methods for controlling arthropod parasite and pest infestations
UA119253C2 (en) 2013-12-10 2019-05-27 Біолоджикс, Інк. METHOD FOR VARROA TREATMENT AND VEGETABLES
US9497914B2 (en) 2013-12-12 2016-11-22 Monsanto Technology Llc Soybean variety 01045730
US9554528B2 (en) 2013-12-13 2017-01-31 Monsanto Technology Llc Soybean variety 01045954
US9554529B2 (en) 2013-12-13 2017-01-31 Monsanto Technology Llc Soybean variety 01046035
US9357731B2 (en) 2013-12-13 2016-06-07 Monsanto Technology Llc Soybean variety 01046948
US9370155B2 (en) 2013-12-16 2016-06-21 Monsanto Technology Llc Soybean variety 01046182
US9445568B1 (en) 2013-12-17 2016-09-20 Monsanto Technology Llc Soybean variety 01046626
CA2875106A1 (en) 2013-12-17 2015-06-17 Monsanto Technology Llc Soybean variety 01046426
US9370157B2 (en) 2013-12-17 2016-06-21 Monsanto Technology Llc Soybean variety 01046703
US9345217B2 (en) 2013-12-17 2016-05-24 Monsanto Technology Llc Soybean variety 01046814
US9386754B2 (en) 2013-12-17 2016-07-12 Monsanto Technology, Llc Soybean variety 01046551
US9357732B2 (en) 2013-12-18 2016-06-07 Monsanto Technology Llc Soybean variety 01046898
US9363967B2 (en) 2013-12-18 2016-06-14 Monsanto Technology Llc Soybean variety 01046942
US9370156B2 (en) 2013-12-18 2016-06-21 Monsanto Technology Llc Soybean variety 01046863
MX368629B (en) 2014-01-15 2019-10-08 Monsanto Technology Llc Methods and compositions for weed control using epsps polynucleotides.
CN110506752B (en) 2014-04-01 2022-02-18 孟山都技术公司 Compositions and methods for controlling insect pests
WO2015150465A2 (en) 2014-04-03 2015-10-08 Basf Se Plants having increased tolerance to herbicides
US9363974B2 (en) 2014-05-08 2016-06-14 Monsanto Technology Llc Soybean variety 01051545
US9363973B2 (en) 2014-05-08 2016-06-14 Monsanto Technology Llc Soybean variety 01051540
US9521816B2 (en) 2014-05-08 2016-12-20 Monsanto Technology Llc Soybean variety 01051547
US9345224B2 (en) 2014-05-08 2016-05-24 Monsanto Technology Llc Soybean variety 01051076
US9510544B2 (en) 2014-05-08 2016-12-06 Monsanto Technology Llc Soybean variety 01050893
US9510545B2 (en) 2014-05-08 2016-12-06 Monsanto Technology Llc Soybean variety 01050923
CA2890604A1 (en) 2014-05-08 2015-11-08 Monsanto Technology Llc Soybean variety 01050938
US9655321B2 (en) 2014-05-09 2017-05-23 Monsanto Technology Llc Soybean variety 01051585
US9655320B2 (en) 2014-05-09 2017-05-23 Monsanto Technology Llc Soybean variety 01051581
US9655322B2 (en) 2014-05-09 2017-05-23 Monsanto Technology Llc Soybean variety 01051603
US9345225B2 (en) 2014-05-09 2016-05-24 Monsanto Technology Llc Soybean variety 01051559
AU2015280252A1 (en) 2014-06-23 2017-01-12 Monsanto Technology Llc Compositions and methods for regulating gene expression via RNA interference
US11807857B2 (en) 2014-06-25 2023-11-07 Monsanto Technology Llc Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
RS60016B1 (en) 2014-06-26 2020-04-30 Basf Agrochemical Products Bv Seed treatment with acetolactate synthase (als) inhibitors
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
EP2979549A1 (en) 2014-07-31 2016-02-03 Basf Se Method for improving the health of a plant
US9585347B2 (en) 2014-09-29 2017-03-07 Monsanto Technology Llc Soybean variety 01046668
US10219515B2 (en) 2014-11-07 2019-03-05 Basf Se Agrochemical adjuvant containing 2-oxo-1,3-dioxolan-4 carboxylates
US9320241B1 (en) 2014-11-19 2016-04-26 Monsanto Technology Llc Soybean variety XR37Z14RX
CA2883599C (en) 2014-11-19 2017-09-05 Monsanto Technology Llc Soybean variety xr27ak14rx
US9320240B1 (en) 2014-11-19 2016-04-26 Monsanto Technology Llc Soybean variety XR35AS14RX
US9585321B1 (en) 2014-11-19 2017-03-07 Monsanto Technology Llc Soybean variety XR34AT14RX
US9320242B1 (en) 2014-11-19 2016-04-26 Monsanto Technology Llc Soybean variety XR43A14RX
US9480228B2 (en) 2014-11-19 2016-11-01 Monsanto Technology Llc Soybean variety XR30AR14RX
CA2883685C (en) 2014-11-19 2017-08-29 Monsanto Technology Llc Soybean variety xr30at14rx
US9675026B1 (en) 2014-11-19 2017-06-13 Monsanto Technology Llc Soybean variety XR35AP14RX
US9402362B1 (en) 2014-11-19 2016-08-02 Monsanto Technology Llc Soybean variety XR47AB14RX
US9648819B1 (en) 2014-11-19 2017-05-16 Monsanto Technology Llc Soybean variety XR57F14RX
US9332717B1 (en) 2014-11-19 2016-05-10 Monsanto Technology Llc Soybean variety XR68D14RX
US9320239B1 (en) 2014-11-19 2016-04-26 Monsanto Technology Llc Soybean variety XR35AR14RX
US9320243B1 (en) 2014-11-19 2016-04-26 Monsanto Technology Llc Soybean variety XR45Z14RX
CA2883608C (en) 2014-11-24 2017-08-29 Monsanto Technology Llc Soybean variety xb15r14r2
US9332716B1 (en) 2014-11-24 2016-05-10 Monsanto Technology LC Soybean variety XB42A14R2
US9433180B1 (en) 2014-11-24 2016-09-06 Monsanto Technology Llc Soybean variety XB35AL14R2
US9648818B1 (en) 2014-11-24 2017-05-16 Monsanto Technology Llc Soybean variety XB37X14R2
US9635819B1 (en) 2014-11-24 2017-05-02 Monsanto Technology Llc Soybean variety XB57E14R2
US9320238B1 (en) 2014-11-24 2016-04-26 Monsanto Technology Llc Soybean variety XB20AA14R2
US9414562B1 (en) 2014-11-24 2016-08-16 Monsanto Technology Llc Soybean variety XB47AA14R2
US9271470B1 (en) 2014-11-24 2016-03-01 Monsanto Technology Llc Soybean variety XB69F14R2
CA2883610C (en) 2014-11-24 2017-09-05 Monsanto Technology Llc Soybean variety xb07a14r2
EP3028573A1 (en) 2014-12-05 2016-06-08 Basf Se Use of a triazole fungicide on transgenic plants
WO2016091674A1 (en) 2014-12-12 2016-06-16 Basf Se Use of cyclaniliprole on cultivated plants
WO2016091675A1 (en) 2014-12-12 2016-06-16 Basf Se Method for improving the health of a plant
US9743602B2 (en) 2014-12-22 2017-08-29 Monsanto Technology Llc Cotton variety 14R911B2XF
US9615530B2 (en) 2014-12-22 2017-04-11 Monsanto Technology Llc Cotton variety 14R938B2XF
US9723801B2 (en) 2014-12-22 2017-08-08 Monsanto Technology Llc Cotton variety 14R952B2XF
US9820452B2 (en) 2014-12-22 2017-11-21 Monsanto Technology Llc Cotton variety 14R915B2XF
US9743605B2 (en) 2014-12-22 2017-08-29 Monsanto Technology Llc Cotton variety 14R950B2XF
US9743604B2 (en) 2014-12-22 2017-08-29 Monsanto Technology Llc Cotton variety 14R949B2XF
US9826691B2 (en) 2014-12-22 2017-11-28 Monsanto Technology Llc Cotton variety 14R914B2XF
US9788507B2 (en) 2014-12-22 2017-10-17 Monsanto Technology Llc Cotton variety 14R955B2XF
US9743603B2 (en) 2014-12-22 2017-08-29 Monsanto Technology Llc Cotton variety 14R925B2XF
US9924654B2 (en) 2014-12-22 2018-03-27 Monsanto Technology Llc Cotton variety 14R922B2XF
US9668447B2 (en) 2014-12-22 2017-06-06 Monsanto Technology Llc Cotton variety 14R941B2XF
US9717208B2 (en) 2014-12-22 2017-08-01 Monsanto Technology Llc Cotton variety 14R953B2XF
US9820451B2 (en) 2014-12-22 2017-11-21 Monsanto Technology Llc Cotton variety 14R948B2XF
US9820450B2 (en) 2014-12-22 2017-11-21 Monsanto Technology Llc Cotton variety 14R942B2XF
EA201791613A1 (en) 2015-01-21 2017-12-29 Басф Се PLANTS WITH ENHANCED STABILITY TO HERBICIDES
MX395326B (en) 2015-01-22 2025-03-25 Monsanto Technology Llc COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA.
WO2016130813A1 (en) 2015-02-11 2016-08-18 Adjuvants Plus Usa, Inc. An agrochemical formulation aid for micronutrient uptake in plants, plant health benefits and herbicide performance
WO2016128470A1 (en) 2015-02-11 2016-08-18 Basf Se Herbicide-resistant hydroxyphenylpyruvate dioxygenases
US9854765B2 (en) 2015-02-26 2018-01-02 Monsanto Technology Llc Cotton variety 14R934B2XF
US9351467B1 (en) 2015-03-02 2016-05-31 Monsanto Technology Llc Soybean variety XB32AQ13R2
CA2980505A1 (en) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Use of an insecticidal carboxamide compound against pests on cultivated plants
US9615525B2 (en) 2015-05-20 2017-04-11 Monsanto Technology Llc Soybean variety 01051642
US9609826B2 (en) 2015-05-20 2017-04-04 Monsanto Technology Llc Soybean variety 01050982
US9585335B2 (en) 2015-05-20 2017-03-07 Monsanto Technology Llc Soybean variety 01050939
US9585336B2 (en) 2015-05-20 2017-03-07 Monsanto Technology Llc Soybean variety 01059528
WO2016196738A1 (en) 2015-06-02 2016-12-08 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
WO2016196782A1 (en) 2015-06-03 2016-12-08 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
AU2016280215B2 (en) 2015-06-17 2022-07-28 BASF Agro B.V. Plants having increased tolerance to herbicides
US9788516B2 (en) 2015-06-25 2017-10-17 Monsanto Technology Llc Soybean variety 01058627
US9894860B2 (en) 2015-06-25 2018-02-20 Monsanto Technology Llc Soybean variety 01058780
US9814205B2 (en) 2015-06-25 2017-11-14 Monsanto Technology Llc Soybean variety 01058730
US9807961B2 (en) 2015-06-25 2017-11-07 Monsanto Technology Llc Soybean variety 01058472
US9781898B2 (en) 2015-06-25 2017-10-10 Monsanto Technology Llc Soybean variety 01058771
US9814206B2 (en) 2015-06-25 2017-11-14 Monsanto Technology Llc Soybean variety 01051877
US9781897B2 (en) 2015-06-25 2017-10-10 Monsanto Technology Llc Soybean variety 01058741
US10076093B2 (en) 2015-06-25 2018-09-18 Monsanto Technology Llc Soybean variety 01058524
US9974268B2 (en) 2015-06-25 2018-05-22 Monsanto Technology Llc Soybean variety 01051074
US9743631B2 (en) 2015-06-25 2017-08-29 Monsanto Technology Llc Soybean variety 01058733
US9832964B2 (en) 2015-06-25 2017-12-05 Monsanto Technology Llc Soybean variety 01051705
US9986704B2 (en) 2015-06-25 2018-06-05 Monsanto Technology Llc Soybean variety 01057162
US9743630B2 (en) 2015-06-25 2017-08-29 Monsanto Technology Llc Soybean variety 01058664
US9775320B2 (en) 2015-06-25 2017-10-03 Monsanto Technology Llc Soybean variety 01058744
US9907262B2 (en) 2015-06-25 2018-03-06 Monsanto Technology Llc Soybean variety 01051117
US9826701B2 (en) 2015-06-25 2017-11-28 Monsanto Technology Llc Soybean variety 01050769
US9807962B2 (en) 2015-06-25 2017-11-07 Monsanto Technology Llc Soybean variety 01058613
US10104848B2 (en) 2015-06-25 2018-10-23 Monsanto Technology Llc Soybean variety 01051773
US9832963B2 (en) 2015-06-25 2017-12-05 Monsanto Technology Llc Soybean variety 01058403
US9807963B2 (en) 2015-06-28 2017-11-07 Monsanto Technology Llc Soybean variety 01052094
US9844199B2 (en) 2015-06-28 2017-12-19 Monsanto Technology Llc Soybean variety 01058475
US9844200B2 (en) 2015-06-28 2017-12-19 Monsanto Technology Llc Soybean variety 01058749
US9848569B2 (en) 2015-06-28 2017-12-26 Monsanto Technology Llc Soybean variety 01057209
US9924677B2 (en) 2015-06-28 2018-03-27 Monsanto Technology Llc Soybean variety 01052136
US9907265B2 (en) 2015-06-28 2018-03-06 Monsanto Technology Llc Soybean variety 01058380
US9974269B2 (en) 2015-06-28 2018-05-22 Monsanto Technology Llc Soybean variety 01058766
US9820458B2 (en) 2015-06-28 2017-11-21 Monsanto Technology Llc Soybean variety 01052110
US9924676B2 (en) 2015-06-28 2018-03-27 Monsanto Technology Llc Soybean variety 01057057
US9826703B2 (en) 2015-06-28 2017-11-28 Monsanto Technology Llc Soybean variety 01058683
US10051807B2 (en) 2015-06-28 2018-08-21 Monsanto Technology Llc Soybean variety 01058772
US9907266B2 (en) 2015-06-28 2018-03-06 Monsanto Technology Llc Soybean variety 01057255
US9907267B2 (en) 2015-06-29 2018-03-06 Monsanto Technology Llc Soybean variety 01057383
US9901056B2 (en) 2015-06-29 2018-02-27 Monsanto Technology Llc Soybean variety 01057377
US9907268B2 (en) 2015-06-29 2018-03-06 Monsanto Technology Llc Soybean variety 01057333
US9968057B2 (en) 2015-06-29 2018-05-15 Monsanto Technology Llc Soybean variety 01058336
US10034445B2 (en) 2015-06-29 2018-07-31 Monsanto Technology Llc Soybean variety 01050611
US9924679B2 (en) 2015-06-29 2018-03-27 Monsanto Technology Llc Soybean variety 01057010
US9901057B2 (en) 2015-06-29 2018-02-27 Monsanto Technology Llc Soybean variety 01050566
US9844201B2 (en) 2015-06-29 2017-12-19 Monsanto Technology Llc Soybean variety 01050745
US9907269B2 (en) 2015-06-29 2018-03-06 Monsanto Technology Llc Soybean variety 01050536
US9924678B2 (en) 2015-06-29 2018-03-27 Monsanto Technology Llc Soybean variety 01057325
US9907271B2 (en) 2015-06-29 2018-03-06 Monsanto Technology Llc Soybean variety 01056960
US9907270B2 (en) 2015-06-29 2018-03-06 Monsanto Technology Llc Soybean variety 01057497
US10034444B2 (en) 2015-06-30 2018-07-31 Monsanto Technology Llc Soybean variety 01050609
US9907263B2 (en) 2015-06-30 2018-03-06 Monsanto Technology Llc Soybean variety 01050612
US9907264B2 (en) 2015-06-30 2018-03-06 Monsanto Technology Llc Soybean variety 01052085
US9814207B2 (en) 2015-06-30 2017-11-14 Monsanto Technology Llc Soybean variety 01058565
US9826702B2 (en) 2015-06-30 2017-11-28 Monsanto Technology Llc Soybean variety 01051835
US9901055B2 (en) 2015-06-30 2018-02-27 Monsanto Technology Llc Soybean variety 01050596
US9820459B2 (en) 2015-06-30 2017-11-21 Monsanto Technology Llc Soybean variety 01058808
US10004197B2 (en) 2015-06-30 2018-06-26 Monsanto Technology Llc Soybean variety 01050595
US10070606B2 (en) 2015-06-30 2018-09-11 Monsanto Technology Llc Soybean variety 01058789
US9832965B2 (en) 2015-06-30 2017-12-05 Monsanto Technology Llc Soybean variety 01051604
WO2017011879A1 (en) * 2015-07-23 2017-01-26 Commonwealth Scientific And Industrial Research Organisation Monooxygenases and method for production of hydroxylated cineole
US9907273B2 (en) 2015-09-24 2018-03-06 Monsanto Technology Llc Soybean variety 01051571
US9901060B2 (en) 2015-09-24 2018-02-27 Monsanto Technology Llc Soybean variety 01051946
US9901061B2 (en) 2015-09-24 2018-02-27 Monsanto Technology Llc Soybean variety 01052008
US9901059B2 (en) 2015-09-24 2018-02-27 Monsanto Technology Llc Soybean variety 01051956
US9901062B2 (en) 2015-09-24 2018-02-27 Monsanto Technology Llc Soybean variety 01052029
US9907274B2 (en) 2015-09-24 2018-03-06 Monsanto Technology Llc Soybean variety 01052030
US9894862B2 (en) 2015-09-24 2018-02-20 Monsanto Technology Llc Soybean variety 01058666
US12203089B2 (en) 2015-10-22 2025-01-21 Basf Se Plants having increased tolerance to herbicides
CA3001979A1 (en) 2015-10-22 2017-04-27 Basf Se Plants having increased tolerance to herbicides
US9629325B1 (en) 2015-12-01 2017-04-25 Monsanto Technology Llc Soybean variety 01057518
UA128078C2 (en) 2015-12-16 2024-04-03 Сінгента Партісіпейшнс Аг Genetic regions & genes associated with increased yield in plants
US9980448B2 (en) 2015-12-18 2018-05-29 Monsanto Technology Llc Cotton variety 15R515B2XF
US9854761B2 (en) 2015-12-18 2018-01-02 Monsanto Technology Llc Cotton variety 15R510B2XF
US9854762B2 (en) 2015-12-18 2018-01-02 Monsanto Technology Llc Cotton variety 15R509B2XF
US9961845B2 (en) 2015-12-18 2018-05-08 Monsanto Technology Llc Cotton variety 15R551B2XF
AU2017212500B9 (en) 2016-01-26 2023-05-11 Monsanto Technology Llc Compositions and methods for controlling insect pests
US9596818B1 (en) 2016-03-21 2017-03-21 Monsanto Technology Llc Soybean variety XR30AX15X
US9560827B1 (en) 2016-03-21 2017-02-07 Monsanto Technology Llc Soybean variety XR45C15X
US9861053B1 (en) 2016-03-21 2018-01-09 Monsanto Technology Llc Soybean variety XR36AC15X
US9585354B1 (en) 2016-03-21 2017-03-07 Monsanto Technology Llc Soybean variety XR48C15X
US9861055B1 (en) 2016-03-21 2018-01-09 Monsanto Technology Llc Soybean variety XR39U15X
US9565821B1 (en) 2016-03-21 2017-02-14 Monsanto Technology Llc Soybean variety XR47AF15X
US9788508B1 (en) 2016-03-21 2017-10-17 Monsanto Technology Llc Soybean variety XR20AH15X
US9560828B1 (en) 2016-03-21 2017-02-07 Monsanto Technology Llc Soybean variety XR46Z15X
US9603331B1 (en) 2016-03-21 2017-03-28 Monsanto Technology Llc Soybean variety XR39V15X
US9596819B1 (en) 2016-03-21 2017-03-21 Monsanto Technology Llc Soybean variety XR33AU15X
US9585355B1 (en) 2016-03-21 2017-03-07 Monsanto Technology Llc Soybean variety XR40AA15X
US9861056B1 (en) 2016-03-21 2018-01-09 Monsanto Technology Llc Soybean variety XR56J15X
US9763413B1 (en) 2016-03-21 2017-09-19 Monsanto Technology Llc Soybean variety XR23AQ15X
US9730415B1 (en) 2016-03-21 2017-08-15 Monsanto Technology Llc Soybean variety XR08A15X
US9763414B1 (en) 2016-03-21 2017-09-19 Monsanto Technology Llc Soybean variety XR25AL15X
US9699999B1 (en) 2016-03-21 2017-07-11 Monsanto Technology Llc Soybean variety XR22K15X
US9763412B1 (en) 2016-03-21 2017-09-19 Monsanto Technology Llc Soybean variety XR09AP15X
US9565822B1 (en) 2016-03-21 2017-02-14 Monsanto Technology Llc Soybean variety XR37AE15X
US9763411B1 (en) 2016-03-21 2017-09-19 Monsanto Technology Llc Soybean variety XR09AL15X
US9700000B1 (en) 2016-03-21 2017-07-11 Monsanto Technology Llc Soybean variety XR38W15X
US9560829B1 (en) 2016-03-21 2017-02-07 Monsanto Technology Llc Soybean variety XR47AG15X
US10010040B1 (en) 2016-03-21 2018-07-03 Monsanto Technology Llc Soybean variety XR50E15X
US9861054B1 (en) 2016-03-21 2018-01-09 Monsanto Technology Llc Soybean variety XR36Z15X
US9585357B1 (en) 2016-03-22 2017-03-07 Monsanto Technology Llc Soybean cultivar 53146341
US9655336B1 (en) 2016-03-22 2017-05-23 Monsanto Technology Llc Soybean cultivar 58150703
US9770000B1 (en) 2016-03-22 2017-09-26 Monsanto Technology Llc Soybean cultivar 56042428
US9609832B1 (en) 2016-03-22 2017-04-04 Monsanto Technology Llc Soybean cultivar 53032202
US9609833B1 (en) 2016-03-22 2017-04-04 Monsanto Technology Llc Soybean cultivar 58111502
US9609831B1 (en) 2016-03-22 2017-04-04 Monsanto Technology Llc Soybean cultivar 56052100
US9585356B1 (en) 2016-03-22 2017-03-07 Monsanto Technology Llc Soybean cultivar 52232331
US9648835B1 (en) 2016-03-22 2017-05-16 Monsanto Technology Llc Soybean cultivar 53030521
US9591827B1 (en) 2016-03-22 2017-03-14 Monsanto Technology Llc Soybean cultivar 57160653
US9578849B1 (en) 2016-03-22 2017-02-28 Monsanto Technology Llc Soybean cultivar 55021012
US9706729B1 (en) 2016-03-22 2017-07-18 Monsanto Technology Llc Soybean cultivar 51240902
US9648836B1 (en) 2016-03-22 2017-05-16 Monsanto Technology Llc Soybean cultivar 50262411
US9648834B1 (en) 2016-03-22 2017-05-16 Monsanto Technology Llc Soybean cultivar 51201110
US9609834B1 (en) 2016-03-22 2017-04-04 Monsanto Technology Llc Soybean cultivar 55030014
US10219463B2 (en) 2016-03-25 2019-03-05 Monsanto Technology Llc Cotton variety 15R513B2XF
EP3054014A3 (en) 2016-05-10 2016-11-23 BASF Plant Science Company GmbH Use of a fungicide on transgenic plants
MX393332B (en) 2016-05-20 2025-03-24 Basf Agro Bv DUAL TRANSIT PEPTIDES FOR TARGETING POLYPEPTIDES.
US9924672B2 (en) 2016-05-31 2018-03-27 Monsanto Technology Llc Soybean variety 01056984
US9867352B2 (en) 2016-05-31 2018-01-16 Monsanto Technology Llc Soybean variety 01057081
US9867351B2 (en) 2016-05-31 2018-01-16 Monsanto Technology Llc Soybean variety 01057076
US10226001B2 (en) 2016-05-31 2019-03-12 Monsanto Technology Llc Soybean variety 01056938
US9888648B2 (en) 2016-05-31 2018-02-13 Monsanto Technology Llc Soybean variety 01057115
US9756823B1 (en) 2016-05-31 2017-09-12 Monsanto Technology Llc Soybean variety 01056961
US9888647B2 (en) 2016-05-31 2018-02-13 Monsanto Technology Llc Soybean variety 01057072
US9901051B2 (en) 2016-05-31 2018-02-27 Monsanto Technology Llc Soybean variety 01057075
US9907247B2 (en) 2016-05-31 2018-03-06 Monsanto Technology Llc Soybean variety 01056929
US9888646B2 (en) 2016-05-31 2018-02-13 Monsanto Technology Llc Soybean variety 01057021
US9872469B2 (en) 2016-05-31 2018-01-23 Monsanto Technology Llc Soybean variety 01056991
US9877450B2 (en) 2016-05-31 2018-01-30 Monsanto Technology Llc Soybean variety 01056979
US9901052B2 (en) 2016-05-31 2018-02-27 Monsanto Technology Llc Soybean variety 01057176
US9750218B1 (en) 2016-05-31 2017-09-05 Monsanto Technology Llc Soybean variety 01056968
US9907246B2 (en) 2016-05-31 2018-03-06 Monsanto Technology Llc Soybean variety 01056904
US10051806B2 (en) 2016-05-31 2018-08-21 Monsanto Technology Llc Soybean variety 01057079
US9713311B1 (en) 2016-05-31 2017-07-25 Monsanto Technology Llc Soybean variety 01057181
US9743624B1 (en) 2016-05-31 2017-08-29 Monsanto Technology Llc Soybean variety 01056994
US9907245B2 (en) 2016-05-31 2018-03-06 Monsanto Technology Llc Soybean variety 01056902
US9743623B1 (en) 2016-05-31 2017-08-29 Monsanto Technology Llc Soybean variety 01056977
US9788515B1 (en) 2016-06-01 2017-10-17 Monsanto Technology Llc Soybean variety 01057347
US9706738B1 (en) 2016-06-01 2017-07-18 Monsanto Technology Llc Soybean variety 01057282
US9832953B1 (en) 2016-06-01 2017-12-05 Monsanto Technology Llc Soybean variety 01057348
US9848563B1 (en) 2016-06-01 2017-12-26 Monsanto Technology Llc Soybean variety 01058353
US9907251B2 (en) 2016-06-01 2018-03-06 Monsanto Technology Llc Soybean variety 01058313
US9907250B2 (en) 2016-06-01 2018-03-06 Monsanto Technology Llc Soybean variety 01057493
US9706739B1 (en) 2016-06-01 2017-07-18 Monsanto Technology Llc Soybean variety 01057290
US9907249B2 (en) 2016-06-01 2018-03-06 Monsanto Technology Llc Soybean variety 01057459
US9788514B1 (en) 2016-06-01 2017-10-17 Monsanto Technology Llc Soybean variety 01057345
US9848562B1 (en) 2016-06-01 2017-12-26 Monsanto Technology Llc Soybean variety 01058349
US10104847B2 (en) 2016-06-01 2018-10-23 Monsanto Technology Llc Soybean variety 01057454
US10226002B2 (en) 2016-06-01 2019-03-12 Monsanto Technology Llc Soybean variety 01057408
US9968054B2 (en) 2016-06-01 2018-05-15 Monsanto Technology Llc Soybean variety 01057207
US9661823B1 (en) 2016-06-01 2017-05-30 Monsanto Technology Llc Soybean variety 01057424
US9700008B1 (en) 2016-06-01 2017-07-11 Monsanto Technology Llc Soybean variety 01057341
US10231398B2 (en) 2016-06-01 2019-03-19 Monsanto Technology Llc Soybean variety 01057400
US9974264B2 (en) 2016-06-01 2018-05-22 Monsanto Technology Llc Soybean variety 01057212
US9907248B2 (en) 2016-06-01 2018-03-06 Monsanto Technology Llc Soybean variety 01057390
US9854772B2 (en) 2016-06-01 2018-01-02 Monsanto Technology Llc Soybean variety 01058360
US9907252B2 (en) 2016-06-01 2018-03-06 Monsanto Technology Llc Soybean variety 01058320
US9848564B1 (en) 2016-06-02 2017-12-26 Monsanto Technology Llc Soybean variety 01058864
US9901053B2 (en) 2016-06-02 2018-02-27 Monsanto Technology Llc Soybean variety 01058606
US9832956B1 (en) 2016-06-02 2017-12-05 Monsanto Technology Llc Soybean variety 01063886
US9907254B2 (en) 2016-06-02 2018-03-06 Monsanto Technology Llc Soybean variety 01058698
US9907253B2 (en) 2016-06-02 2018-03-06 Monsanto Technology Llc Soybean variety 01058513
US9832957B1 (en) 2016-06-02 2017-12-05 Monsanto Technology Llc Soybean variety 01058863
US9807960B1 (en) 2016-06-02 2017-11-07 Monsanto Technology Llc Soybean variety 01063888
US9700009B1 (en) 2016-06-02 2017-07-11 Monsanto Technology Llc Soybean variety 01058861
US9854773B2 (en) 2016-06-02 2018-01-02 Monsanto Technology Llc Soybean variety 01058444
US9867354B2 (en) 2016-06-02 2018-01-16 Monsanto Technology Llc Soybean variety 01058529
US9867353B2 (en) 2016-06-02 2018-01-16 Monsanto Technology Llc Soybean variety 01058528
US9872470B2 (en) 2016-06-02 2018-01-23 Monsanto Technology Llc Soybean variety 01058469
US9693530B1 (en) 2016-06-02 2017-07-04 Monsanto Technology Llc Soybean variety 01063884
US9693529B1 (en) 2016-06-02 2017-07-04 Monsanto Technology Llc Soybean variety 01063878
US9700010B1 (en) 2016-06-02 2017-07-11 Monsanto Technology Llc Soybean variety 01063871
US9832955B1 (en) 2016-06-02 2017-12-05 Monsanto Technology Llc Soybean variety 01058718
US9907256B2 (en) 2016-06-02 2018-03-06 Monsanto Technology Llc Soybean variety 01058822
US9907255B2 (en) 2016-06-02 2018-03-06 Monsanto Technology Llc Soybean variety 01058767
US9832954B1 (en) 2016-06-02 2017-12-05 Monsanto Technology Llc Soybean variety 01058711
US9717213B1 (en) 2016-06-03 2017-08-01 Monsanto Technology Llc Soybean variety 01064120
US9737037B1 (en) 2016-06-03 2017-08-22 Monsanto Technology Llc Soybean variety 01063921
US9907260B2 (en) 2016-06-03 2018-03-06 Monsanto Technology Llc Soybean variety 01063906
US9907258B2 (en) 2016-06-03 2018-03-06 Monsanto Technology Llc Soybean variety 01063898
US9730419B1 (en) 2016-06-03 2017-08-15 Monsanto Technology Llc Soybean variety 01064108
US9717211B1 (en) 2016-06-03 2017-08-01 Monsanto Technology Llc Soybean variety 01063940
US9955645B2 (en) 2016-06-03 2018-05-01 Monsanto Technology Llc Soybean variety 01063902
US9743625B1 (en) 2016-06-03 2017-08-29 Monsanto Technology Llc Soybean variety 01063903
US10219462B2 (en) 2016-06-03 2019-03-05 Monsanto Technology Llc Soybean variety 01063907
US9848565B1 (en) 2016-06-03 2017-12-26 Monsanto Technology Llc Soybean variety 01063939
US9907261B2 (en) 2016-06-03 2018-03-06 Monsanto Technology Llc Soybean variety 01063928
US9750221B1 (en) 2016-06-03 2017-09-05 Monsanto Technology Llc Soybean variety 01063935
US9907259B2 (en) 2016-06-03 2018-03-06 Monsanto Technology Llc Soybean variety 01063916
US9832958B1 (en) 2016-06-03 2017-12-05 Monsanto Technology Llc Soybean variety 01064122
US9907257B2 (en) 2016-06-03 2018-03-06 Monsanto Technology Llc Soybean variety 01063892
US9717212B1 (en) 2016-06-03 2017-08-01 Monsanto Technology Llc Soybean variety 01064113
US9901054B2 (en) 2016-06-03 2018-02-27 Monsanto Technology Llc Soybean variety 01063900
US10058047B2 (en) 2016-06-03 2018-08-28 Monsanto Technology Llc Soybean variety 01063918
US9730418B1 (en) 2016-06-03 2017-08-15 Monsanto Technology Llc Soybean variety 01063920
US9839187B1 (en) 2016-06-06 2017-12-12 Monsanto Technology Llc Soybean variety 01064165
US10064356B2 (en) 2016-06-06 2018-09-04 Monsanto Technology Llc Soybean variety 01064137
US9730420B1 (en) 2016-06-06 2017-08-15 Monsanto Technology Llc Soybean variety 01064123
US9848566B1 (en) 2016-06-06 2017-12-26 Monsanto Technology Llc Soybean variety 01064124
US9743627B1 (en) 2016-06-06 2017-08-29 Monsanto Technology Llc Soybean variety 01064182
US9832961B1 (en) 2016-06-06 2017-12-05 Monsanto Technology Llc Soybean variety 01064167
US9872471B2 (en) 2016-06-06 2018-01-23 Monsanto Technology Llc Soybean variety 01064177
US9848567B1 (en) 2016-06-06 2017-12-26 Monsanto Technology Llc Soybean variety 01064142
US9844197B1 (en) 2016-06-06 2017-12-19 Monsanto Technology Llc Soybean variety 01064151
US9832959B1 (en) 2016-06-06 2017-12-05 Monsanto Technology Llc Soybean variety 01064131
US9924673B2 (en) 2016-06-06 2018-03-27 Monsanto Technology Llc Soybean variety 01064169
US9867356B2 (en) 2016-06-06 2018-01-16 Monsanto Technology Llc Soybean variety 01064143
US9832962B1 (en) 2016-06-06 2017-12-05 Monsanto Technology Llc Soybean variety 01064172
US9743626B1 (en) 2016-06-06 2017-08-29 Monsanto Technology Llc Soybean variety 01064148
US9867355B2 (en) 2016-06-06 2018-01-16 Monsanto Technology Llc Soybean variety 01064176
US9795106B1 (en) 2016-06-06 2017-10-24 Monsanto Technology Llc Soybean variety 01064150
US9832960B1 (en) 2016-06-06 2017-12-05 Monsanto Technology Llc Soybean variety 01064152
US9844198B1 (en) 2016-06-07 2017-12-19 Monsanto Technology Llc Soybean variety 01063894
US9743629B1 (en) 2016-06-07 2017-08-29 Monsanto Technology Llc Soybean variety 01058714
US9848568B1 (en) 2016-06-07 2017-12-26 Monsanto Technology Llc Soybean variety 01064130
US10136599B2 (en) 2016-06-29 2018-11-27 Monsanto Technology Llc Cotton variety 15R535B2XF
CA3030803A1 (en) 2016-07-15 2018-01-18 Basf Se Plants having increased tolerance to herbicides
BR112019001483A2 (en) 2016-07-27 2019-09-10 Basf Agro Bv method for controlling unwanted vegetation at a plantation site
CA3042939A1 (en) 2016-12-08 2018-06-14 Syngenta Participations Ag Methods for improving transformation frequency
CN110382702A (en) 2016-12-20 2019-10-25 巴斯夫农业公司 Plants with increased herbicide tolerance
EP3338552A1 (en) 2016-12-21 2018-06-27 Basf Se Use of a tetrazolinone fungicide on transgenic plants
US9963714B1 (en) 2016-12-30 2018-05-08 Monsanto Technology Llc Cotton variety 16R247NRB2XF
US10499605B2 (en) 2017-03-03 2019-12-10 Monsanto Technology Llc Cotton variety 16R251NRB2XF
US9930858B1 (en) 2017-03-29 2018-04-03 Monsanto Technology Llc Soybean cultivar 62232543
US9999193B1 (en) 2017-03-29 2018-06-19 Monsanto Technology Llc Soybean cultivar 65090203
US9930857B1 (en) 2017-03-29 2018-04-03 Monsato Technology Llc Soybean cultivar 61311131
US9913448B1 (en) 2017-03-29 2018-03-13 Monsanto Technology Llc Soybean cultivar 63281310
US9907283B1 (en) 2017-03-29 2018-03-06 Monsanto Technology Llc Soybean cultivar 62103227
US9888659B1 (en) 2017-03-29 2018-02-13 Monsanto Technology Llc Soybean cultivar 61040705
US10039249B1 (en) 2017-03-29 2018-08-07 Monsanto Technology Llc Soybean cultivar 61162227
US9913449B1 (en) 2017-03-29 2018-03-13 Monsanto Technology Llc Soybean cultivar 67272133
US9930856B1 (en) 2017-03-29 2018-04-03 Monsanto Technology Llc Soybean cultivar 62030513
US9907282B1 (en) 2017-03-29 2018-03-06 Monsanto Technology Llc Soybean cultivar 60371632
US9907280B1 (en) 2017-03-29 2018-03-06 Monsanto Technology Llc Soybean cultivar 64492916
US9907279B1 (en) 2017-03-29 2018-03-06 Monsanto Technology Llc Soybean cultivar 63282422
US9961868B1 (en) 2017-03-29 2018-05-08 Monsanto Technology Llc Soybean cultivar 60111034
US9907281B1 (en) 2017-03-29 2018-03-06 Monsanto Technology Llc Soybean cultivar 64291310
US9913450B1 (en) 2017-03-29 2018-03-13 Monsanto Technology Llc Soybean cultivar 68071303
US9894869B1 (en) 2017-03-29 2018-02-20 Monsanto Technology Llc Soybean cultivar 64091728
US11109591B2 (en) 2017-04-24 2021-09-07 Taminco Bvba Single phase liquids of alkanolamine salts of dicamba
US10314273B2 (en) 2017-06-26 2019-06-11 Monsanto Technology Llc Soybean variety 01068110
US10342198B2 (en) 2017-06-26 2019-07-09 Monsanto Technology Llc Soybean variety 01064381
US10383304B2 (en) 2017-06-26 2019-08-20 Monsanto Technology Llc Soybean variety 01063944
US10010046B1 (en) 2017-06-26 2018-07-03 Monsanto Technology Llc Soybean variety 01064401
US10368518B2 (en) 2017-06-26 2019-08-06 Monsanto Technology Llc Soybean variety 01068117
US10034447B1 (en) 2017-06-26 2018-07-31 Monsanto Technology Llc Soybean variety 01064421
US10342197B2 (en) 2017-06-26 2019-07-09 Monsanto Technology Llc Soybean variety 01064349
US10039255B1 (en) 2017-06-26 2018-08-07 Monsanto Technology Llc Soybean variety 01064252
US10314274B2 (en) 2017-06-26 2019-06-11 Monsanto Technology Llc Soybean variety 01068109
US10455788B2 (en) 2017-06-26 2019-10-29 Monsanto Technology Llc Soybean variety 01068888
US10034448B1 (en) 2017-06-26 2018-07-31 Monsanto Technology Llc Soybean variety 01064516
US10342199B2 (en) 2017-06-26 2019-07-09 Monsanto Technology Llc Soybean variety 01068883
US10405511B2 (en) 2017-06-26 2019-09-10 Monsanto Technology Llc Soybean variety 01064350
US10039254B1 (en) 2017-06-26 2018-08-07 Monsanto Technology Llc Soybean variety 01064117
US10201138B2 (en) 2017-06-28 2019-02-12 Monsanto Technology Llc Soybean variety 01064519
US10178838B1 (en) 2017-06-28 2019-01-15 Monsanto Technology Llc Soybean variety 5PQNG18
US10368511B2 (en) 2017-06-28 2019-08-06 Monsanto Technology Llc Soybean variety 01068085
US10036027B1 (en) 2017-06-28 2018-07-31 Monsanto Technology Llc Soybean variety 01064536
US10182539B1 (en) 2017-06-28 2019-01-22 Monsanto Technology Llc Soybean variety 5PDZB74
US10206350B1 (en) 2017-06-28 2019-02-19 Monsanto Technology Llc Soybean variety 5PDSD70
US10172310B1 (en) 2017-06-28 2019-01-08 Monsanto Technology Llc Soybean variety 5PYFS21
US10244704B2 (en) 2017-06-28 2019-04-02 Monsanto Technologies Llc Soybean variety 01068084
US10244705B2 (en) 2017-06-28 2019-04-02 Monsanto Technology Llc Soybean variety 01064265
US10271493B2 (en) 2017-06-28 2019-04-30 Monsanto Technology Llc Soybean variety 01064280
US10292358B2 (en) 2017-06-28 2019-05-21 Monsanto Technology Llc Soybean variety 01064327
US10011845B1 (en) 2017-06-28 2018-07-03 Monsanto Technology Llc Soybean variety 01068893
US10178837B1 (en) 2017-06-28 2019-01-15 Monsanto Technology Llc Soybean variety 5PHQP31
US10188059B1 (en) 2017-06-28 2019-01-29 Monsanto Technology Llc Soybean variety 5PUWT62
US10206349B1 (en) 2017-06-28 2019-02-19 Monsanto Technology Llc Soybean variety 5PPHU37
US10053699B1 (en) 2017-06-28 2018-08-21 Monsanto Technology Llc Soybean variety 01068892
US10194612B2 (en) 2017-06-28 2019-02-05 Monsanto Technology Llc Soybean variety 01068093
US10053700B1 (en) 2017-06-28 2018-08-21 Monsanto Technology Llc Soybean variety 01064288
US10196646B2 (en) 2017-06-28 2019-02-05 Monsanto Technology Llc Soybean variety 01064344
US10368512B2 (en) 2017-06-28 2019-08-06 Monsanto Technology Llc Soybean variety 01068083
US10349598B2 (en) 2017-06-28 2019-07-16 Monsanto Technology Llc Soybean variety 01068088
US10194613B2 (en) 2017-06-28 2019-02-05 Monsanto Technology Llc Soybean variety 01064363
US10231403B2 (en) 2017-07-06 2019-03-19 Monsanto Technology Llc Soybean variety 01068141
US10064367B1 (en) 2017-07-06 2018-09-04 Monsanto Technology Llc Soybean variety 01067515
US10238057B2 (en) 2017-07-06 2019-03-26 Monsanto Technology Llc Soybean variety 01064461
US10194614B2 (en) 2017-07-06 2019-02-05 Monsanto Technology Llc Soybean variety 01064567
US10064382B1 (en) 2017-07-06 2018-09-04 Monsanto Technology Llc Soybean variety 01064035
US10064381B1 (en) 2017-07-06 2018-09-04 Monsanto Technology Llc Soybean variety 01067510
US10070616B1 (en) 2017-07-06 2018-09-11 Monsanto Technology Llc Soybean variety 01067507
US10194616B2 (en) 2017-07-06 2019-02-05 Monsanto Technology Llc Soybean variety 01064568
US10098305B1 (en) 2017-07-06 2018-10-16 Monsanto Technology Llc Soybean variety 01064403
US10064379B1 (en) 2017-07-06 2018-09-04 Monsanto Technology Llc Soybean variety 01064450
US10201139B2 (en) 2017-07-06 2019-02-12 Monsanto Technology Llc Soybean variety 01068144
US10244706B2 (en) 2017-07-06 2019-04-02 Monsanto Technology Llc Soybean variety 01064571
US10070617B1 (en) 2017-07-06 2018-09-11 Monsanto Technology Llc Soybean variety 01063975
US10064369B1 (en) 2017-07-19 2018-09-04 Monsanto Technology Llc Soybean variety 01068131
US10440908B2 (en) 2017-07-19 2019-10-15 Monsanto Technology Llc Soybean variety 01064245
US10238076B2 (en) 2017-07-19 2019-03-26 Monsanto Technology, Llc Soybean variety 01068130
US10231422B2 (en) 2017-07-19 2019-03-19 Monsanto Technology Llc Soybean variety 01068134
US10398105B2 (en) 2017-07-19 2019-09-03 Monsanto Technology Llc Soybean variety 01064198
US10314275B2 (en) 2017-07-19 2019-06-11 Monsanto Technology Llc Soybean variety 01064238
US10231405B2 (en) 2017-07-19 2019-03-19 Monsanto Technology Llc Soybean variety 01068135
US10356997B2 (en) 2017-07-19 2019-07-23 Monsanto Technology Llc Soybean variety 01068891
US10058061B1 (en) 2017-07-19 2018-08-28 Monsanto Technology Llc Soybean variety 01068112
US10212901B2 (en) 2017-07-19 2019-02-26 Monsanto Technology Llc Soybean variety 01068895
US10064368B1 (en) 2017-07-19 2018-09-04 Monsanto Technology Llc Soybean variety 01068133
US10194617B1 (en) 2017-07-19 2019-02-05 Monsanto Technology Llc Soybean variety 01068066
US10231421B2 (en) 2017-07-19 2019-03-19 Monsanto Technology Llc Soybean variety 01064231
US10206352B2 (en) 2017-07-19 2019-02-19 Monsanto Technology Llc Soybean variety 01068065
US10136602B1 (en) 2017-07-19 2018-11-27 Monsanto Technology Llc Soybean variety 01068087
US10231404B2 (en) 2017-07-19 2019-03-19 Monsanto Technology Llc Soybean variety 01064187
US10206351B2 (en) 2017-07-19 2019-02-19 Monsanto Technology Llc Soybean variety 01068060
US10098306B1 (en) 2017-07-19 2018-10-16 Monsanto Technology Llc Soybean variety 01068070
US10219466B2 (en) 2017-07-19 2019-03-05 Monsanto Technology Llc Soybean variety 01064243
US10426116B2 (en) 2017-07-19 2019-10-01 Monsanto Technology Llc Soybean variety 01068124
US10058060B1 (en) 2017-07-19 2018-08-28 Monsanto Technology Llc Soybean variety 01068082
US10098308B1 (en) 2017-07-20 2018-10-16 Monsanto Technology Llc Soybean variety 01068054
US10051823B1 (en) 2017-07-20 2018-08-21 Monsanto Technology Llc Soybean variety 01064868
US10285367B2 (en) 2017-07-20 2019-05-14 Monsanto Technology Llc Soybean variety 01064508
US10117400B1 (en) 2017-07-20 2018-11-06 Monsanto Technology Llc Soybean variety 01064185
US10117401B1 (en) 2017-07-20 2018-11-06 Monsanto Technology Llc Soybean variety 01064488
US10098307B1 (en) 2017-07-20 2018-10-16 Monsanto Technology Llc Soybean variety 01064838
US10231406B2 (en) 2017-07-20 2019-03-19 Monsanto Technology Llc Soybean variety 01068831
US10244723B2 (en) 2017-07-20 2019-04-02 Monsanto Technology Llc Soybean variety 01068058
US10064370B1 (en) 2017-07-20 2018-09-04 Monsanto Technology Llc Soybean variety 01064806
US10143167B1 (en) 2017-07-20 2018-12-04 Monsanto Technology Llc Soybean variety 01064871
US10058062B1 (en) 2017-07-20 2018-08-28 Monsanto Technology Llc Soybean variety 01064840
US10206353B2 (en) 2017-07-20 2019-02-19 Monsanto Technology Llc Soybean variety 01064485
US10428347B2 (en) 2017-07-24 2019-10-01 Spogen Biotech Inc. Herbicide-detoxifying enzymes and uses thereof
US10117402B1 (en) 2017-07-31 2018-11-06 Monsanto Technology Llc Soybean variety 01068884
US10368513B2 (en) 2017-07-31 2019-08-06 Monsanto Technology Llc Soybean variety 01064206
US10231408B1 (en) 2017-08-23 2019-03-19 Monsanto Technology Llc Soybean variety 01064013
US10257994B2 (en) 2017-08-23 2019-04-16 Monsanto Technology Llc Soybean variety 01068143
US10271494B2 (en) 2017-08-23 2019-04-30 Monsanto Technology Llc Soybean variety 01068122
US10231407B1 (en) 2017-08-23 2019-03-19 Monsanto Technology Llc Soybean variety 01063959
US10251354B2 (en) 2017-08-23 2019-04-09 Monsanto Technology Llc Soybean variety 01064596
US10285368B2 (en) 2017-08-23 2019-05-14 Monsanto Technology Llc Soybean variety 01064056
US10117404B1 (en) 2017-08-23 2018-11-06 Monsanto Technology Llc Soybean variety 01064613
US10264748B2 (en) 2017-08-23 2019-04-23 Monsanto Technology Llc Soybean variety 01064212
US10238059B2 (en) 2017-08-23 2019-03-26 Monsanto Technology Llc Soybean variety 01064460
US10117403B1 (en) 2017-08-23 2018-11-06 Monsanto Technology Llc Soybean variety 01064099
US10244708B2 (en) 2017-08-23 2019-04-02 Monsanto Technology Llc Soybean variety 01064594
US10130060B1 (en) 2017-08-24 2018-11-20 Monsanto Technology Llc Soybean variety 01068804
US10045506B1 (en) 2017-08-24 2018-08-14 Monsanto Technology Llc Soybean variety 01064415
US10136603B1 (en) 2017-08-24 2018-11-27 Monsanto Technology Llc Soybean variety 01067535
US10039256B1 (en) 2017-08-24 2018-08-07 Monsanto Technology Llc Soybean variety 01064446
US10231423B1 (en) 2017-08-24 2019-03-19 Monsanto Technology Llc Soybean variety 01064404
US10130058B1 (en) 2017-08-24 2018-11-20 Monsanto Technology Llc Soybean variety 01064723
US10051824B1 (en) 2017-08-24 2018-08-21 Monsanto Technology Llc Soybean variety 01064393
US10130059B1 (en) 2017-08-24 2018-11-20 Monsanto Technology Llc Soybean variety 01068801
US10123501B1 (en) 2017-08-24 2018-11-13 Monsanto Technology Llc Soybean variety 01064722
US10314276B2 (en) 2017-08-24 2019-06-11 Monsanto Technology Llc Soybean variety 01064370
US10051825B1 (en) 2017-08-24 2018-08-21 Monsanto Technology Llc Soybean variety 01064400
US10045507B1 (en) 2017-08-24 2018-08-14 Monsanto Technology Llc Soybean variety 01064406
US10342200B2 (en) 2017-08-24 2019-07-09 Monsanto Technology Llc Soybean variety 01067525
US10271495B2 (en) 2017-08-29 2019-04-30 Monsanto Technology Llc Soybean variety 01064036
US10244724B2 (en) 2017-08-29 2019-04-02 Monsanto Technology Llc Soybean variety 01064667
US10271497B2 (en) 2017-08-29 2019-04-30 Monsanto Technology Llc Soybean variety 01064054
US10314265B2 (en) 2017-08-29 2019-06-11 Monsanto Technology Llc Soybean variety 01064669
US10433504B2 (en) 2017-08-29 2019-10-08 Monsanto Technology Llc Soybean variety 01064105
US10342201B2 (en) 2017-08-29 2019-07-09 Monsanto Technology Llc Soybean variety 01067531
US10159206B1 (en) 2017-08-29 2018-12-25 Monsanto Technology Llc Soybean variety 01067519
US10271496B2 (en) 2017-08-29 2019-04-30 Monsanto Technology Llc Soybean variety 01064102
US10238077B2 (en) 2017-08-29 2019-03-26 Monsanto Technology Llc Soybean variety 01064104
US10485197B2 (en) 2017-08-29 2019-11-26 Monsanto Technology Llc Soybean variety 01064625
US10257995B2 (en) 2017-08-29 2019-04-16 Monsanto Technology Llc Soybean variety 01064083
US10231409B1 (en) 2017-08-29 2019-03-19 Monsanto Technology Llc Soybean variety 01064026
US10314277B2 (en) 2017-08-29 2019-06-11 Monsanto Technology Llc Soybean variety 01064636
US10159205B1 (en) 2017-08-29 2018-12-25 Monsanto Technology Llc Soybean variety 01064633
US10357007B2 (en) 2017-08-30 2019-07-23 Monsanto Technology Llc Soybean variety 01064743
US10212902B1 (en) 2017-08-30 2019-02-26 Monsanto Technology Llc Soybean variety 01067523
US10231425B1 (en) 2017-08-30 2019-03-19 Monsanto Technology Llc Soybean variety 01067534
US10188072B1 (en) 2017-08-30 2019-01-29 Monsanto Technology Llc Soybean variety 01064694
US10117409B1 (en) 2017-08-30 2018-11-06 Monsanto Technology Llc Soybean variety 01064756
US10342202B2 (en) 2017-08-30 2019-07-09 Monsanto Technology Llc Soybean variety 01064775
US10188061B1 (en) 2017-08-30 2019-01-29 Monsanto Technology Llc Soybean variety 01064486
US10117405B1 (en) 2017-08-30 2018-11-06 Monsanto Technology Llc Soybean variety 01064511
US10051827B1 (en) 2017-08-30 2018-08-21 Monsanto Technology Llc Soybean variety 01064164
US10231424B1 (en) 2017-08-30 2019-03-19 Monsanto Technology Llc Soybean variety 01067526
US10219484B1 (en) 2017-08-30 2019-03-05 Monsanto Technology Llc Soybean variety 01064735
US10278350B2 (en) 2017-08-30 2019-05-07 Monsanto Technology Llc Soybean variety 01064672
US10117408B1 (en) 2017-08-30 2018-11-06 Monsanto Technology Llc Soybean variety 01064491
US10314278B2 (en) 2017-08-30 2019-06-11 Monsanto Technology Llc Soybean variety 01064635
US10117406B1 (en) 2017-08-30 2018-11-06 Monsanto Technology Llc Soybean variety 01064166
US10219485B1 (en) 2017-08-30 2019-03-05 Monsanto Technology Llc Soybean variety 01069661
US10064371B1 (en) 2017-08-30 2018-09-04 Monsanto Technology Llc Soybean variety 01064809
US10231426B1 (en) 2017-08-30 2019-03-19 Monsanto Technology Llc Soybean variety 01064748
US10219467B1 (en) 2017-08-30 2019-03-05 Monsanto Technology Llc Soybean variety 01064677
US10271498B2 (en) 2017-08-30 2019-04-30 Monsanto Technology Llc Soybean variety 01064676
US10117407B1 (en) 2017-08-30 2018-11-06 Monsanto Technology Llc Soybean variety 01064490
US10051826B1 (en) 2017-08-30 2018-08-21 Monsanto Technology Llc Soybean variety 01064189
US10194618B1 (en) 2017-08-30 2019-02-05 Monsanto Technology Llc Soybean variety 01064184
CN107828803B (en) * 2017-11-03 2020-12-22 南京农业大学 3,6-Dichlorosalicylic acid 5-hydroxylase gene dsmABC and its application
CN111801012A (en) 2017-11-29 2020-10-20 巴斯夫欧洲公司 Plants with increased herbicide tolerance
US10856512B2 (en) 2017-12-28 2020-12-08 Monsanto Technology Llc Cotton variety 16R336B3XF
US10842122B2 (en) 2017-12-28 2020-11-24 Monsanto Technology Llc Cotton variety 16R123XF
US10863713B2 (en) 2017-12-28 2020-12-15 Monsanto Technology Llc Cotton variety 16R341B3XF
US10863714B2 (en) 2017-12-28 2020-12-15 Monsanto Technology Llc Cotton variety 16R330B3XF
US10863715B2 (en) 2017-12-28 2020-12-15 Monsanto Technology Llc Cotton variety 16R343B3XF
US10849302B2 (en) 2017-12-28 2020-12-01 Monsanto Technology Llc Cotton variety 16R335B3XF
US10842121B2 (en) 2017-12-28 2020-11-24 Monsanto Technology Llc Cotton variety 16R141XF
US10842120B2 (en) 2017-12-28 2020-11-24 Monsanto Technology Llc Cotton variety 16R125XF
US10842123B2 (en) 2017-12-28 2020-11-24 Monsanto Technology Llc Cotton variety 16R324B3XF
US10925248B2 (en) 2017-12-28 2021-02-23 Monsanto Technology Llc Cotton variety 16R225NRB2XF
US10869455B2 (en) 2017-12-28 2020-12-22 Monsanto Technology Llc Cotton variety 16R345B3XF
US10856513B2 (en) 2017-12-28 2020-12-08 Monsanto Technology Llc Cotton variety 16R338B3XF
US10939656B2 (en) 2017-12-28 2021-03-09 Monsanto Technology Llc Cotton variety 16R228NRB2XF
US10874079B2 (en) 2017-12-28 2020-12-29 Monsanto Technology Llc Cotton variety 16R351B3XF
AU2019210093B2 (en) 2018-01-17 2025-03-20 Basf Se Plants having increased tolerance to herbicides
US10368519B1 (en) 2018-05-24 2019-08-06 Monsanto Technology Llc Soybean variety 5PYGS18
US10480005B1 (en) 2018-05-24 2019-11-19 Monsanto Technology Llc Soybean variety 5PEKB42
US10368520B1 (en) 2018-05-24 2019-08-06 Monsanto Technology Llc Soybean variety 5PCDJ10
US10420313B1 (en) 2018-05-24 2019-09-24 Monsanto Technology Llc Soybean variety 5PKVF24
US10412921B1 (en) 2018-05-24 2019-09-17 Monsanto Technology Llc Soybean variety 5PYUP46
US10477818B1 (en) 2018-05-24 2019-11-19 Monsanto Technology Llc Soybean variety 5PTJB40
US10420312B1 (en) 2018-05-24 2019-09-24 Monsanto Technology Llc Soybean variety 5PKGL32
US10517255B1 (en) 2018-08-06 2019-12-31 Monsanto Technology Llc Soybean cultivar 62391708
US10258014B1 (en) 2018-08-06 2019-04-16 Monsanto Technology Llc Soybean cultivar 76051047
US10342204B1 (en) 2018-08-06 2019-07-09 Monsanto Technology Llc Soybean cultivar 65382631
US10368521B1 (en) 2018-08-06 2019-08-06 Monsanto Technology Llc Soybean cultivar 70490447
US10342206B1 (en) 2018-08-06 2019-07-09 Monsanto Technology Llc Soybean cultivar 60390131
US10238079B1 (en) 2018-08-06 2019-03-26 Monsanto Technology Llc Soybean cultivar 77301427
US10238080B1 (en) 2018-08-06 2019-03-26 Monsanto Technology Llc Soybean cultivar 79072143
US10264760B1 (en) 2018-08-06 2019-04-23 Monsanto Technology Llc Soybean cultivar 79380729
US10349607B1 (en) 2018-08-06 2019-07-16 Monsanto Technology Llc Soybean cultivar 78091047
US10342207B1 (en) 2018-08-06 2019-07-09 Monsanto Technology Llc Soybean cultivar 68381333
US10349608B1 (en) 2018-08-06 2019-07-16 Monsanto Technology Llc Soybean cultivar 70520427
US10321654B1 (en) 2018-08-06 2019-06-18 Monsanto Technology Llc Soybean cultivar 76222418
US10398122B1 (en) 2018-08-06 2019-09-03 Monsanto Technology Llc Soybean cultivar 77252418
US10357010B1 (en) 2018-08-06 2019-07-23 Monsanto Technology Llc Soybean cultivar 68352516
US10362758B1 (en) 2018-08-06 2019-07-30 Monsanto Technology Llc Soybean cultivar 64272401
US10258013B1 (en) 2018-08-06 2019-04-16 Monsanto Technology Llc Soybean cultivar 64442216
US10238066B1 (en) 2018-08-06 2019-03-26 Monsanto Technology Llc Soybean cultivar 73301323
US10602704B2 (en) 2018-08-22 2020-03-31 Monsanto Technology Llc Soybean variety 01068313
US10602689B2 (en) 2018-08-22 2020-03-31 Monsanto Technology Llc Soybean variety 01068908
US10555481B1 (en) 2018-08-22 2020-02-11 Monsanto Technology Llc Soybean variety 01068364
US10575489B1 (en) 2018-08-22 2020-03-03 Monsanto Technology Llc Soybean variety 01067545
US10897868B2 (en) 2018-08-22 2021-01-26 Monsanto Technology Llc Soybean variety 01072327
US10905080B2 (en) 2018-08-22 2021-02-02 Monsanto Technology Llc Soybean variety 01072355
US10999998B2 (en) 2018-08-22 2021-05-11 Monsanto Technology Llc Soybean variety 01072346
US10582686B1 (en) 2018-08-22 2020-03-10 Monsanto Technology Llc Soybean variety 01064580
US10624285B2 (en) 2018-08-22 2020-04-21 Monsanto Technology Llc Soybean variety 01068376
US10624303B2 (en) 2018-08-22 2020-04-21 Monsanto Technology Llc Soybean variety 01072279
US11044876B2 (en) 2018-08-22 2021-06-29 Monsanto Technology Llc Soybean variety 01072345
US10973200B2 (en) 2018-08-22 2021-04-13 Monsanto Technology Llc Soybean variety 01072342
US10897869B2 (en) 2018-08-22 2021-01-26 Monsanto Technology Llc Soybean variety 01072333
US11206791B2 (en) 2018-08-22 2021-12-28 Monsanto Technology Llc Soybean variety 01072337
US10561099B1 (en) 2018-08-22 2020-02-18 Monsanto Technology Llc Soybean variety 01068352
US10555484B1 (en) 2018-08-22 2020-02-11 Monsanto Technology Llc Soybean variety 01068903
US10993404B2 (en) 2018-08-22 2021-05-04 Monsanto Technology Llc Soybean variety 01072356
US10973199B2 (en) 2018-08-22 2021-04-13 Monsanto Technology Llc Soybean variety 01072341
US11096363B2 (en) 2018-08-22 2021-08-24 Monsanto Technology Llc Soybean variety 01072331
US11109552B2 (en) 2018-08-22 2021-09-07 Monsanto Technology Llc Soybean variety 01072332
US10555480B1 (en) 2018-08-22 2020-02-11 Monsanto Technology Llc Soybean variety 01068350
US10555483B1 (en) 2018-08-22 2020-02-11 Monsanto Technology Llc Soybean variety 01067598
US10555482B1 (en) 2018-08-22 2020-02-11 Monsanto Technology Llc Soybean variety 01068427
US10624302B2 (en) 2018-08-22 2020-04-21 Monsanto Technology Llc Soybean variety 01072280
US10973198B2 (en) 2018-08-22 2021-04-13 Monsanto Technology Llc Soybean variety 01072340
US10561098B1 (en) 2018-08-22 2020-02-18 Monsanto Technology Llc Soybean variety 01067649
US10939652B2 (en) 2018-08-22 2021-03-09 Monsanto Technology Llc Soybean variety 01072350
US10905081B2 (en) 2018-08-22 2021-02-02 Monsanto Technology Llc Soybean variety 01072329
US11026389B2 (en) 2018-08-22 2021-06-08 Monsanto Technology Llc Soybean variety 01072367
US11013197B2 (en) 2018-08-22 2021-05-25 Monsanto Technology Llc Soybean variety 01072368
US10973201B2 (en) 2018-08-22 2021-04-13 Monsanto Technology Llc Soybean variety 01072339
US11109553B2 (en) 2018-08-22 2021-09-07 Monsanto Technology Llc Soybean variety 01072328
US10897870B2 (en) 2018-08-22 2021-01-26 Monsanto Technology Llc Soybean variety 01072322
US10918065B2 (en) 2018-08-22 2021-02-16 Monsanto Technology Llc Soybean variety 01072343
US10617089B2 (en) 2018-08-22 2020-04-14 Monsanto Technology Llc Soybean variety 01067827
US10575490B1 (en) 2018-08-22 2020-03-03 Monsanto Technology Llc Soybean variety 01067547
US10524446B1 (en) 2018-08-23 2020-01-07 Monsanto Technology Llc Soybean variety 01072245
US10499601B1 (en) 2018-08-23 2019-12-10 Monsanto Technology Llc Soybean variety 01068356
US10448609B1 (en) 2018-08-23 2019-10-22 Monsanto Technology Llc Soybean variety 01072293
US10506786B1 (en) 2018-08-23 2019-12-17 Monsanto Technology Llc Soybean variety 01067789
US10555486B1 (en) 2018-08-23 2020-02-11 Monsanto Technology Llc Soybean variety 01068910
US10548284B1 (en) 2018-08-23 2020-02-04 Monsanto Technology Llc Soybean variety 01072294
US10448608B1 (en) 2018-08-23 2019-10-22 Monsanto Technology Llc Soybean variety 01067812
US10463000B1 (en) 2018-08-23 2019-11-05 Monsanto Technology Llc Soybean variety 01072224
US10492450B1 (en) 2018-08-23 2019-12-03 Monsanto Technology Llc Soybean variety 01064752
US10631499B2 (en) 2018-08-23 2020-04-28 Monsanto Technology Llc Soybean variety 01072291
US10555485B1 (en) 2018-08-23 2020-02-11 Monsanto Technology Llc Soybean variety 01073532
US10687501B2 (en) 2018-08-23 2020-06-23 Monsanto Technology Llc Soybean variety 01068438
US10638707B2 (en) 2018-08-23 2020-05-05 Monsanto Technology Llc Soybean variety 01067639
US10631486B2 (en) 2018-08-23 2020-04-28 Monsanto Technology Llc Soybean variety 01068187
US10638708B2 (en) 2018-08-23 2020-05-05 Monsanto Technology Llc Soybean variety 01068325
US10687502B2 (en) 2018-08-23 2020-06-23 Monsanto Technology Llc Soybean variety 01068433
US10609888B2 (en) 2018-08-23 2020-04-07 Monsanto Technology Llc Soybean variety 01068346
US10624287B2 (en) 2018-08-23 2020-04-21 Monsanto Technology Llc Soybean variety 01072275
US10512239B1 (en) 2018-08-23 2019-12-24 Monsanto Technology Llc Soybean variety 01072273
US10624286B2 (en) 2018-08-23 2020-04-21 Monsanto Technology Llc Soybean variety 01072276
US10631500B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01067788
US10548285B1 (en) 2018-08-24 2020-02-04 Monsanto Technology Llc Soybean variety 01067703
US10512241B1 (en) 2018-08-24 2019-12-24 Monsanto Technology Llc Soybean variety 01072232
US10561106B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01068466
US10638710B2 (en) 2018-08-24 2020-05-05 Monsanto Technology Llc Soybean variety 01068195
US10631505B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01069676
US10561102B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01068182
US10660292B2 (en) 2018-08-24 2020-05-26 Monsanto Technology Llc Soybean variety 01068157
US10631507B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01068485
US10582689B1 (en) 2018-08-24 2020-03-10 Monsanto Technology Llc Soybean variety 01067573
US10561104B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01072234
US10506787B1 (en) 2018-08-24 2019-12-17 Monsanto Technology Llc Soybean variety 01072220
US10561101B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01068184
US10638709B2 (en) 2018-08-24 2020-05-05 Monsanto Technology Llc Soybean variety 01068191
US10582688B1 (en) 2018-08-24 2020-03-10 Monsanto Technology Llc Soybean variety 01067543
US10631503B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01067814
US10617090B2 (en) 2018-08-24 2020-04-14 Monsanto Technology Llc Soybean variety 01068163
US10631506B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01068838
US10582687B1 (en) 2018-08-24 2020-03-10 Monsanto Technology Llc Soybean variety 01068359
US10542721B1 (en) 2018-08-24 2020-01-28 Monsanto Technology Llc Soybean variety 01072231
US10582690B1 (en) 2018-08-24 2020-03-10 Monsanto Technology Llc Soybean variety 01067790
US10555487B1 (en) 2018-08-24 2020-02-11 Monsanto Technology Llc Soybean variety 01067694
US10602705B2 (en) 2018-08-24 2020-03-31 Monsanto Technology Llc Soybean variety 01068821
US10485213B1 (en) 2018-08-24 2019-11-26 Monsanto Technology Llc Soybean variety 01068968
US10517259B1 (en) 2018-08-24 2019-12-31 Monsanto Technology Llc Soybean variety 01068824
US10463010B1 (en) 2018-08-24 2019-11-05 Monsanto Technology Llc Soybean variety 01068189
US10631501B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01067793
US10448610B1 (en) 2018-08-24 2019-10-22 Monsanto Technology Llc Soybean variety 01067781
US10631502B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01067840
US10631504B2 (en) 2018-08-24 2020-04-28 Monsanto Technology Llc Soybean variety 01072295
US10512240B1 (en) 2018-08-24 2019-12-24 Monsanto Technology Llc Soybean variety 01072228
US10645898B2 (en) 2018-08-24 2020-05-12 Monsanto Technology Llc Soybean variety 01068192
US10470395B1 (en) 2018-08-24 2019-11-12 Monsanto Technology Llc Soybean variety 01067663
US10531629B1 (en) 2018-08-24 2020-01-14 Monsanto Technology Llc Soybean variety 01072223
US10561105B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01072235
US10477821B1 (en) 2018-08-24 2019-11-19 Monsanto Technology Llc Soybean variety 01067769
US10561100B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01067742
US10492451B1 (en) 2018-08-24 2019-12-03 Monsanto Technology Llc Soybean variety 0107226
US10470425B1 (en) 2018-08-24 2019-11-12 Monsanto Technology Llc Soybean variety 01067783
US10624304B2 (en) 2018-08-24 2020-04-21 Monsanto Technology Llc Soybean variety 01068381
US10575491B1 (en) 2018-08-24 2020-03-03 Monsanto Technology Llc Soybean variety 01072289
US10561103B1 (en) 2018-08-24 2020-02-18 Monsanto Technology Llc Soybean variety 01073501
US10555488B1 (en) 2018-08-24 2020-02-11 Monsanto Technology Llc Soybean variety 01067714
US10582678B1 (en) 2018-08-27 2020-03-10 Monsanto Technology Llc Soybean variety 01072239
US10602692B2 (en) 2018-08-27 2020-03-31 Monsanto Technology Llc Soybean variety 01072252
US10743493B2 (en) 2018-08-27 2020-08-18 Monsanto Technology Llc Soybean variety 01077438
US10499602B1 (en) 2018-08-27 2019-12-10 Monsanto Technology Llc Soybean variety 01067553
US10555489B1 (en) 2018-08-27 2020-02-11 Monsanto Technology Llc Soybean variety 01068972
US10631508B2 (en) 2018-08-27 2020-04-28 Monsanto Technology Llc Soybean variety 01068228
US10477822B1 (en) 2018-08-27 2019-11-19 Monsanto Technology Llc Soybean variety 01069686
US10499603B1 (en) 2018-08-27 2019-12-10 Monsanto Technology Llc Soybean variety 01068207
US10548286B1 (en) 2018-08-27 2020-02-04 Monsanto Technology Llc Soybean variety 01068171
US10575493B1 (en) 2018-08-27 2020-03-03 Monsanto Technology Llc Soybean variety 01067819
US10568291B1 (en) 2018-08-27 2020-02-25 Monsanto Technology Llc Soybean variety 01067741
US10477823B1 (en) 2018-08-27 2019-11-19 Monsanto Technology Llc Soybean variety 01069720
US10660293B2 (en) 2018-08-27 2020-05-26 Monsanto Technology Llc Soybean variety 01077434
US10463011B1 (en) 2018-08-27 2019-11-05 Monsanto Technology Llc Soybean variety 01068162
US10743492B2 (en) 2018-08-27 2020-08-18 Monsanto Technology Llc Soybean variety 01077433
US10687503B2 (en) 2018-08-27 2020-06-23 Monsanto Technology Llc Soybean variety 01068213
US10602706B2 (en) 2018-08-27 2020-03-31 Monsanto Technology Llc Soybean variety 01077437
US10492453B1 (en) 2018-08-27 2019-12-03 Monsanto Technology Llc Soybean variety 01072254
US10512242B1 (en) 2018-08-27 2019-12-24 Monsanto Technology Llc Soybean variety 01077436
US10631509B2 (en) 2018-08-27 2020-04-28 Monsanto Technology Llc Soybean variety 01067807
US10743491B2 (en) 2018-08-27 2020-08-18 Monsanto Technology Llc Soybean variety 01077432
US10492452B1 (en) 2018-08-27 2019-12-03 Monsanto Technology Llc Soybean variety 01067646
US10463013B1 (en) 2018-08-27 2019-11-05 Monsanto Technology Llc Soybean variety 01069709
US10561107B1 (en) 2018-08-27 2020-02-18 Monsanto Technology Llc Soybean variety 01068807
US10463012B1 (en) 2018-08-27 2019-11-05 Monsanto Technology Llc Soybean variety 01069688
US10602691B2 (en) 2018-08-27 2020-03-31 Monsanto Technology Llc Soybean variety 01072253
US10582679B1 (en) 2018-08-27 2020-03-10 Monsanto Technology Llc Soybean variety 01072243
US10568290B1 (en) 2018-08-27 2020-02-25 Monsanto Technology Llc Soybean variety 01068219
US10660294B2 (en) 2018-08-27 2020-05-26 Monsanto Technology Llc Soybean variety 01077435
US10492454B1 (en) 2018-08-27 2019-12-03 Monsanto Technology Llc Soybean variety 01072256
US10506788B1 (en) 2018-08-27 2019-12-17 Monsanto Technology Llc Soybean variety 01072292
US10575492B1 (en) 2018-08-27 2020-03-03 Monsanto Technology Llc Soybean variety 01068915
US10602690B2 (en) 2018-08-27 2020-03-31 Monsanto Technology Llc Soybean variety 01068227
US10506789B1 (en) 2018-12-04 2019-12-17 Monsanto Technology Llc Soybean cultivar 61381330
US10918071B2 (en) 2018-12-19 2021-02-16 Monsanto Technology Llc Cotton variety 16R346B3XF
US10905085B2 (en) 2018-12-19 2021-02-02 Monsanto Technology Llc Cotton variety 17R808B3XF
US10905086B2 (en) 2018-12-19 2021-02-02 Monsanto Technology Llc Cotton variety 17R820B3XF
US10918070B2 (en) 2018-12-19 2021-02-16 Monsanto Technology Llc Cotton variety 17R806B3XF
US10874081B2 (en) 2018-12-19 2020-12-29 Monsanto Technology Llc Cotton variety 17R709XF
US10918075B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R845B3XF
US10918077B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R827B3XF
US10918076B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R817B3XF
US10939660B2 (en) 2019-01-09 2021-03-09 Monsanto Technology Llc Cotton variety 17R740XF
US10939658B2 (en) 2019-01-09 2021-03-09 Monsanto Technology Llc Cotton variety 16R232B2XF
US10918074B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R844B3XF
US10925250B2 (en) 2019-01-09 2021-02-23 Monsanto Technology Llc Cotton variety 17R816B3XF
US10939661B2 (en) 2019-01-09 2021-03-09 Monsanto Technology Llc Cotton variety 17R815B3XF
US10905087B2 (en) 2019-01-09 2021-02-02 Monsanto Technology Llc Cotton variety 16R246NRB2XF
US10918073B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R819B3XF
US10918072B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R814B3XF
US10939659B2 (en) 2019-01-09 2021-03-09 Monsanto Technology Llc Cotton variety 16R353B3XF
US10925249B2 (en) 2019-01-09 2021-02-23 Monsanto Technology Llc Cotton variety 17R933NRB3XF
US10918034B2 (en) 2019-01-09 2021-02-16 Monsanto Technology Llc Cotton variety 17R737XF
US10602693B1 (en) 2019-06-04 2020-03-31 Monsanto Technology Llc Soybean cultivar 81302732
US10617080B1 (en) 2019-06-04 2020-04-14 Monsanto Technology Llc Soybean cultivar 87352527
US10631512B1 (en) 2019-06-04 2020-04-28 Monsanto Technology Llc Soybean cultivar 85322536
US10631513B1 (en) 2019-06-04 2020-04-28 Monsanto Technology Llc Soybean cultivar 87181312
US10624305B1 (en) 2019-06-04 2020-04-21 Monsanto Technology Llc Soybean cultivar 85161422
US10617091B1 (en) 2019-06-04 2020-04-14 Monsanto Technology Llc Soybean cultivar 86331141
US10667484B1 (en) 2019-06-04 2020-06-02 Monsanto Technology Llc Soybean cultivar 82322805
US10638713B1 (en) 2019-06-05 2020-05-05 Monsanto Technology Llc Soybean cultivar 85220103
US10638686B1 (en) 2019-06-05 2020-05-05 Monsanto Technology Llc Soybean cultivar 84161023
US10631514B1 (en) 2019-06-05 2020-04-28 Monsanto Technology Llc Soybean cultivar 80131402
US10624288B1 (en) 2019-06-05 2020-04-21 Monsanto Technology Llc Soybean cultivar 85061635
US10660295B1 (en) 2019-06-05 2020-05-26 Monsanto Technology Llc Soybean cultivar 86072029
US10548287B1 (en) 2019-06-05 2020-02-04 Monsanto Technology Llc Soybean cultivar 86411138
US10602708B1 (en) 2019-06-05 2020-03-31 Monsanto Technology Llc Soybean cultivar 82370721
US10602709B1 (en) 2019-06-05 2020-03-31 Monsanto Technology Llc Soybean cultivar 86360847
US10602707B1 (en) 2019-06-05 2020-03-31 Monsanto Technology Llc Soybean cultivar 85442619
US10609883B1 (en) 2019-06-05 2020-04-07 Monsanto Technology Llc Soybean cultivar 82340103
US10631487B1 (en) 2019-06-06 2020-04-28 Monsanto Technology Llc Soybean cultivar 83240828
US10813335B1 (en) 2019-06-06 2020-10-27 Monsanto Technology Llc Soybean cultivar 80261309
US10561109B1 (en) 2019-06-06 2020-02-18 Monsanto Technology Llc Soybean cultivar 82042815
US10617092B1 (en) 2019-06-06 2020-04-14 Monsanto Technology Llc Soybean cultivar 80411617
US10548289B1 (en) 2019-06-06 2020-02-04 Monsanto Technology Llc Soybean cultivar 82290447
US10638687B1 (en) 2019-06-06 2020-05-05 Monsanto Technology Llc Soybean cultivar 88103087
US10609889B1 (en) 2019-06-06 2020-04-07 Monsanto Technology Llc Soybean cultivar 89001711
US10638714B1 (en) 2019-06-06 2020-05-05 Monsanto Technology Llc Soybean cultivar 84062517
US10548273B1 (en) 2019-06-06 2020-02-04 Monsanto Technology Llc Soybean cultivar 87120510
US10785950B1 (en) 2019-06-06 2020-09-29 Stine Seed Farm, Inc. Soybean cultivar 86190345
US10561108B1 (en) 2019-06-06 2020-02-18 Monsanto Technology Llc Soybean cultivar 80431932
US10548288B1 (en) 2019-06-06 2020-02-04 Mansanto Technology Llc Soybean cultivar 80372800
US10568292B1 (en) 2019-06-06 2020-02-25 Monsanto Technology Llc Soybean cultivar 80181322
US10568284B1 (en) 2019-06-06 2020-02-25 Monsanto Technology Llc Soybean cultivar 87161804
US10561089B1 (en) 2019-06-07 2020-02-18 Monsanto Technology Llc Soybean cultivar 84060345
US10555491B1 (en) 2019-06-07 2020-02-11 Monsanto Technology Llc Soybean cultivar 83072718
US10548274B1 (en) 2019-06-07 2020-02-04 Monsanto Technology Llc Soybean cultivar 83262718
US10660296B1 (en) 2019-06-07 2020-05-26 Monsanto Technology Llc Soybean cultivar 84241941
US10555492B1 (en) 2019-06-07 2020-02-11 Monsanto Technology Llc Soybean cultivar 81430038
US10555490B1 (en) 2019-06-07 2020-02-11 Monsanto Technology Llc Soybean cultivar 85370909
US10548290B1 (en) 2019-06-07 2020-02-04 Monsanto Technology Llc Soybean cultivar 89031941
US10602710B1 (en) 2019-06-07 2020-03-31 Monsanto Technology Llc Soybean cultivar 89282206
US10568294B1 (en) 2019-06-07 2020-02-25 Monsanto Technology Llc Soybean cultivar 82440038
US10568293B1 (en) 2019-06-07 2020-02-25 Monsanto Technology Llc Soybean cultivar AR 86112649
US10561090B1 (en) 2019-06-07 2020-02-18 Monsanto Technology Llc Soybean cultivar 86370345
US10939643B2 (en) 2019-07-29 2021-03-09 Monsanto Technology Llc Soybean variety 01072768
US10912270B1 (en) 2019-07-29 2021-02-09 Monsanto Technology Llc Soybean variety 01072737
US10932428B2 (en) 2019-07-29 2021-03-02 Monsanto Technology Llc Soybean variety 01072749
US10925235B1 (en) 2019-07-29 2021-02-23 Monsanto Technology Llc Soybean variety 01072810
US10925237B1 (en) 2019-07-29 2021-02-23 Monsanto Technology Llc Soybean variety 01073288
US11096357B2 (en) 2019-07-29 2021-08-24 Monsanto Technology Llc Soybean variety 01073351
US10849300B1 (en) 2019-07-29 2020-12-01 Monsanto Technology Llc Soybean variety 01073359
US10925238B1 (en) 2019-07-29 2021-02-23 Monsanto Technology Llc Soybean variety 01073320
US10925236B1 (en) 2019-07-29 2021-02-23 Monsanto Technology Llc Soybean variety 01072823
US10863709B1 (en) 2019-07-29 2020-12-15 Monsanto Technology Llc Soybean variety 01072789
US10993401B2 (en) 2019-07-29 2021-05-04 Monsanto Technology Llc Soybean variety 01073256
US10918051B1 (en) 2019-07-29 2021-02-16 Monsanto Technology Llc Soybean variety 01072804
US10905071B1 (en) 2019-07-29 2021-02-02 Monsanto Technology Llc Soybean variety 01072744
US10918053B1 (en) 2019-07-29 2021-02-16 Monsanto Technology Llc Soybean variety 01073354
US10918052B1 (en) 2019-07-29 2021-02-16 Monsanto Technology Llc Soybean variety 01073365
US10863708B1 (en) 2019-07-29 2020-12-15 Monsanto Technology Llc Soybean variety 01073439
US10918054B1 (en) 2019-07-29 2021-02-16 Monsanto Technology Llc Soybean variety 01073353
US10918050B1 (en) 2019-07-29 2021-02-16 Monsanto Technology Llc Soybean variety 01072791
US10905072B1 (en) 2019-07-29 2021-02-02 Monsanto Technology Llc Soybean variety 01073330
US10869452B1 (en) 2019-07-29 2020-12-22 Monsanto Technology Llc Soybean variety 01072767
US10932429B2 (en) 2019-07-29 2021-03-02 Monsanto Technology Llc Soybean variety 01073265
US10849301B1 (en) 2019-07-30 2020-12-01 Monsanto Technology Llc Soybean variety 01072784
US10897864B1 (en) 2019-07-30 2021-01-26 Monsanto Technology Llc Soybean variety 01073453
US10925239B1 (en) 2019-07-30 2021-02-23 Monsanto Technology Llc Soybean variety 01073452
US10939646B2 (en) 2019-07-30 2021-03-09 Monsanto Technology Llc Soybean variety 01073479
US10881073B1 (en) 2019-07-30 2021-01-05 Monsanto Technology Llc Soybean variety 01072782
US10939645B2 (en) 2019-07-30 2021-03-09 Monsanto Technology Llc Soybean variety 01073480
US10881074B1 (en) 2019-07-30 2021-01-05 Monsanto Technology Llc Soybean variety 01072802
US11266100B2 (en) 2019-07-30 2022-03-08 Monsanto Technology Llc Soybean variety 01073441
US10918055B1 (en) 2019-07-30 2021-02-16 Monsanto Technology Llc Soybean variety 01073472
US10939644B2 (en) 2019-07-30 2021-03-09 Monsanto Technology Llc Soybean variety 01073461
US10856508B1 (en) 2019-07-30 2020-12-08 Monsanto Technology Llc Soybean variety 01073445
US10888063B1 (en) 2019-07-30 2021-01-12 Monsanto Technology Llc Soybean variety 01072800
US10925240B1 (en) 2019-07-30 2021-02-23 Monsanto Technology Llc Soybean variety 01073455
US10856507B1 (en) 2019-07-30 2020-12-08 Monsanto Technology Llc Soybean variety 01073444
US10827714B1 (en) 2019-07-30 2020-11-10 Monsanto Technology Llc Soybean variety 01073443
US10842116B1 (en) 2019-07-31 2020-11-24 Monsanto Technology Llc Soybean variety 01072832
US10874078B1 (en) 2019-07-31 2020-12-29 Monsanto Technology Llc Soybean variety 01072817
US10939648B2 (en) 2019-07-31 2021-03-09 Monsanto Technology Llc Soybean variety 01072899
US11122759B2 (en) 2019-07-31 2021-09-21 Monsanto Technology Llc Soybean variety 01072870
US10939647B2 (en) 2019-07-31 2021-03-09 Monsanto Technology Llc Soybean variety 01072860
US10918056B1 (en) 2019-07-31 2021-02-16 Monsanto Technology Llc Soybean variety 01077815
US10973194B2 (en) 2019-07-31 2021-04-13 Monsanto Technology Llc Soybean variety 01072867
US10842117B1 (en) 2019-07-31 2020-11-24 Monsanto Technology Llc Soybean variety 01072842
US10973193B2 (en) 2019-07-31 2021-04-13 Monsanto Technology Llc Soybean variety 01072864
US10869453B1 (en) 2019-07-31 2020-12-22 Monsanto Technology Llc Soybean variety 01072813
US10945397B2 (en) 2019-07-31 2021-03-16 Monsanto Technology Llc Soybean variety 01073506
US10827715B1 (en) 2019-07-31 2020-11-10 Monsanto Technology Llc Soybean variety 01077799
US10912271B1 (en) 2019-07-31 2021-02-09 Monsanto Technology Llc Soybean variety 01072822
US10959389B2 (en) 2019-07-31 2021-03-30 Monsanto Technology Llc Soybean variety 01073503
US10863710B1 (en) 2019-07-31 2020-12-15 Monsanto Technology Llc Soybean variety 01072819
US11026387B2 (en) 2019-08-01 2021-06-08 Monsanto Technology Llc Soybean variety 01073016
US11019787B2 (en) 2019-08-01 2021-06-01 Monsanto Technology Llc Soybean variety 01072951
US11129346B2 (en) 2019-08-01 2021-09-28 Monsanto Technology Llc Soybean variety 01073533
US10925241B1 (en) 2019-08-01 2021-02-23 Monsanto Technology Llc Soybean variety 01072974
US10905074B1 (en) 2019-08-01 2021-02-02 Monsanto Technology Llc Soybean variety 01072906
US11006602B2 (en) 2019-08-01 2021-05-18 Monsanto Technology Llc Soybean variety 01072946
US11019788B2 (en) 2019-08-01 2021-06-01 Monsanto Technology Llc Soybean variety 01072980
US10905073B1 (en) 2019-08-01 2021-02-02 Monsanto Technology Llc Soybean variety 01072883
US11026388B2 (en) 2019-08-01 2021-06-08 Monsanto Technology Llc Soybean variety 01073060
US10905075B1 (en) 2019-08-01 2021-02-02 Monsanto Technology Llc Soybean variety 01073059
US11019789B2 (en) 2019-08-01 2021-06-01 Monsanto Technology Llc Soybean variety 01073534
US11096359B2 (en) 2019-08-07 2021-08-24 Monsanto Technology Llc Soybean variety 01077377
US11019790B2 (en) 2019-08-07 2021-06-01 Monsanto Technology Llc Soybean variety 01073304
US11076552B2 (en) 2019-08-07 2021-08-03 Monsanto Technology Llc Soybean variety 01073356
US10973195B2 (en) 2019-08-07 2021-04-13 Monsanto Technology Llc Soybean variety 01073325
US10980203B2 (en) 2019-08-07 2021-04-20 Monsanto Technology Llc Soybean variety 01073305
US10912273B1 (en) 2019-08-07 2021-02-09 Monsanto Technology Llc Soybean variety 01073355
US11044867B2 (en) 2019-08-15 2021-06-29 Monsanto Technology Llc Soybean variety 01073133
US11071271B2 (en) 2019-08-15 2021-07-27 Monsanto Technology Llc Soybean variety 01073065
US10993407B2 (en) 2019-08-15 2021-05-04 Monsanto Technology Llc Cotton variety 17R818B3XF
US10952392B2 (en) 2019-08-15 2021-03-23 Monsanto Technology Llc Soybean variety 01073151
US11096361B2 (en) 2019-08-15 2021-08-24 Monsanto Technology Llc Soybean variety 01073072
US11109550B2 (en) 2019-08-15 2021-09-07 Monsanto Technology Llc Soybean variety 01073116
US11013196B2 (en) 2019-08-15 2021-05-25 Monsanto Technology Llc Soybean variety 01073094
US10945398B2 (en) 2019-08-15 2021-03-16 Monsanto Technology Llc Soybean variety 01073160
US10952391B2 (en) 2019-08-15 2021-03-23 Monsanto Technology Llc Soybean variety 01073150
US11044865B2 (en) 2019-08-15 2021-06-29 Monsanto Technology Llc Soybean variety 01073029
US10952393B2 (en) 2019-08-15 2021-03-23 Monsanto Technology Llc Soybean variety 01073169
US10959390B2 (en) 2019-08-15 2021-03-30 Monsanto Technology Llc Soybean variety 01073069
US11096360B2 (en) 2019-08-15 2021-08-24 Monsanto Technology Llc Soybean variety 01067560
US11044866B2 (en) 2019-08-15 2021-06-29 Monsanto Technology Llc Soybean variety 01073114
US10973196B2 (en) 2019-08-16 2021-04-13 Monsanto Technology Llc Soybean variety 01073237
US11044869B2 (en) 2019-08-16 2021-06-29 Monsanto Technology Llc Soybean variety 01073270
US11044868B2 (en) 2019-08-16 2021-06-29 Monsanto Technology Llc Soybean variety 01073261
US11129347B2 (en) 2019-08-16 2021-09-28 Monsanto Technology Llc Soybean variety 01073210
US11071272B2 (en) 2019-08-16 2021-07-27 Monsanto Technology Llc Soybean variety 01073195
US10863711B1 (en) 2019-08-16 2020-12-15 Monsanto Technology Llc Soybean variety 01073279
US11096362B2 (en) 2019-08-16 2021-08-24 Monsanto Technology Llc Soybean variety 01073204
US11109551B2 (en) 2019-08-16 2021-09-07 Monsanto Technology Llc Soybean variety 01073253
US11160237B2 (en) 2019-08-16 2021-11-02 Monsanto Technology Llc Soybean variety 01073183
US10863712B1 (en) 2019-08-16 2020-12-15 Monsanto Technology Llc Soybean variety 01073281
US10856509B1 (en) 2019-08-16 2020-12-08 Monsanto Technology Llc Soybean variety 01073199
US11006603B2 (en) 2019-08-16 2021-05-18 Monsanto Technology Llc Soybean variety 01072287
US10869454B1 (en) 2019-08-16 2020-12-22 Monsanto Technology Llc Soybean variety 01073329
US10932430B1 (en) 2019-08-16 2021-03-02 Monsanto Technology Llc Soybean variety 01073203
US11039590B2 (en) 2019-08-16 2021-06-22 Monsanto Technology Llc Soybean variety 01073272
US11039591B2 (en) 2019-08-16 2021-06-22 Monsanto Technology Llc Soybean variety 01073340
US10905079B1 (en) 2019-08-16 2021-02-02 Monsanto Technology Llc Soybean variety 01073335
US11129349B2 (en) 2019-08-16 2021-09-28 Monsanto Technology Llc Soybean variety 01073350
US11129348B2 (en) 2019-08-16 2021-09-28 Monsanto Technology Llc Soybean variety 01073219
CA3145101A1 (en) 2019-09-03 2021-03-11 Timothy H Anderson Polymers for spray drift control of pesticide spray
US11284587B2 (en) 2019-09-16 2022-03-29 Monsanto Technology Llc Soybean variety 01077381
US11259493B2 (en) 2019-09-16 2022-03-01 Monsanto Technology Llc Soybean variety 01077341
US11259494B2 (en) 2019-09-16 2022-03-01 Monsanto Technology Llc Soybean variety 01077345
US11266109B2 (en) 2019-09-16 2022-03-08 Monsanto Technology Llc Soybean variety 01077385
US11266110B2 (en) 2019-09-16 2022-03-08 Monsanto Technology Llc Soybean variety 01077337
US11350595B2 (en) 2019-09-16 2022-06-07 Monsanto Technology, Llc Soybean variety 01077372
US11185043B2 (en) 2019-09-16 2021-11-30 Monsanto Technology Llc Soybean variety 01077334
US11266105B2 (en) 2019-09-16 2022-03-08 Monsanto Technology Llc Soybean variety 01077338
US11178841B2 (en) 2019-09-16 2021-11-23 Monsanto Technology Llc Soybean variety 01077386
US11266106B2 (en) 2019-09-16 2022-03-08 Monsanto Technology Llc Soybean variety 01081407
US11229182B2 (en) 2019-09-16 2022-01-25 Monsanto Technology Llc Soybean variety 01077387
US11266107B2 (en) 2019-09-16 2022-03-08 Monsanto Technology Llc Soybean variety 01081408
US11185044B2 (en) 2019-09-16 2021-11-30 Monsanto Technology Llc Soybean variety 01077343
US11337400B2 (en) 2019-09-16 2022-05-24 Monsanto Technology, Llc Soybean variety 01077373
US11576340B2 (en) 2019-09-17 2023-02-14 Monsanto Technology, Llc Soybean variety 01081414
US11266108B2 (en) 2019-09-17 2022-03-08 Monsanto Technology Llc Soybean variety 01081409
US11297792B2 (en) 2019-09-17 2022-04-12 Monsanto Technology Llc Soybean variety 01077344
US11576341B2 (en) 2019-09-17 2023-02-14 Monsanto Technology, Llc Soybean variety 01072361
US11266111B2 (en) 2019-09-17 2022-03-08 Monsanto Technology Llc Soybean variety 01077383
CN111118037B (en) * 2019-10-14 2021-07-13 中国农业科学院生物技术研究所 Gene DICX4 with Degradation Function of Herbicides Dicamba and Its Application
CN111139238B (en) * 2019-10-14 2021-08-03 中国农业科学院生物技术研究所 Degradation gene dicX3 of herbicide dicamba and its application
US11272679B2 (en) 2019-10-18 2022-03-15 Monsanto Technology Llc Soybean variety 01077330
US11330783B2 (en) 2019-10-18 2022-05-17 Monsanto Technology, Llc Soybean variety 01077336
US11330777B2 (en) 2019-10-18 2022-05-17 Monsanto Technology, Llc Soybean variety 01077390
US11324181B2 (en) 2019-10-18 2022-05-10 Monsanto Technology Llc Soybean variety 01077335
US11252921B2 (en) 2019-10-18 2022-02-22 Monsanto Technology Llc Soybean variety 01077393
US11252922B2 (en) 2019-10-18 2022-02-22 Monsanto Technology Llc Soybean variety 01077391
US20220386602A1 (en) 2019-11-15 2022-12-08 Basf Corporation Methods of using a composition comprising an anionic pesticide and a buffer
US11051483B1 (en) 2019-12-16 2021-07-06 Monsanto Technology Llc Cotton variety 18R448B3XF
US11154029B2 (en) 2019-12-16 2021-10-26 Monsanto Technology Llc Cotton variety 18R445B3XF
US11064672B2 (en) 2019-12-16 2021-07-20 Monsanto Technology Llc Cotton variety 18R438B3XF
US11026392B1 (en) 2019-12-16 2021-06-08 Monsanto Technology Llc Cotton variety 18R421B3XF
US11039594B1 (en) 2019-12-16 2021-06-22 Monsanto Technology Llc Cotton variety 18R423B3XF
CN115698282A (en) 2020-01-13 2023-02-03 福路伦特生物科学公司 Single cell sequencing
US11310991B2 (en) 2020-01-24 2022-04-26 Monsanto Technology Llc Cotton variety 18R540B3XF
US11197454B2 (en) 2020-01-24 2021-12-14 Monsanto Technology Llc Cotton variety 18R062
US11134645B2 (en) 2020-01-24 2021-10-05 Monsanto Technology Llc Cotton variety 18R441B3XF
US11129354B2 (en) 2020-01-24 2021-09-28 Monsanto Technology Llc Cotton variety 18R435B3XF
US11129352B2 (en) 2020-01-24 2021-09-28 Monsanto Technology Llc Cotton variety 18R418B3XF
US11134646B2 (en) 2020-01-24 2021-10-05 Monsanto Technology Llc Cotton variety 18R459B3XF
US11185046B2 (en) 2020-01-24 2021-11-30 Monsanto Technology Llc Cotton variety 18R409B3XF
US11129353B2 (en) 2020-01-24 2021-09-28 Monsanto Technology Llc Cotton variety 18R419B3XF
US11206800B2 (en) 2020-01-24 2021-12-28 Monsanto Technology Llc Cotton variety 18R067
US11154030B2 (en) 2020-02-12 2021-10-26 Monsanto Technology Llc Cotton variety 16R020
US11197455B2 (en) 2020-02-12 2021-12-14 Monsanto Technology Llc Cotton variety 18R410B3XF
US11330787B2 (en) 2020-02-12 2022-05-17 Monsanto Technology Llc Cotton variety 18R420B3XF
US11866782B2 (en) 2020-03-16 2024-01-09 Fluent Biosciences Inc. Multi-omic analysis in monodisperse droplets
US12428685B2 (en) 2020-03-24 2025-09-30 Illumina, Inc. Viral detection using template emulsification
EP4164382A1 (en) 2020-06-15 2023-04-19 Basf Se A stable, solvent-free, self-emulsifiable concentrate
JP2023534028A (en) 2020-07-15 2023-08-07 フルーエント バイオサイエンシーズ インコーポレイテッド Oligos for stepwise ligation
US11234405B1 (en) 2020-09-09 2022-02-01 Monsanto Technology Llc Soybean cultivar 90110208
US11206797B1 (en) 2020-09-09 2021-12-28 Monsanto Technology Llc Soybean cultivar 97320218
US11259495B1 (en) 2020-09-09 2022-03-01 Monsanto Technology Llc Soybean cultivar 90381408
US11252923B1 (en) 2020-09-09 2022-02-22 Monsanto Technology Llc Soybean cultivar 99003018
US11234406B1 (en) 2020-09-09 2022-02-01 Monsanto Technology Llc Soybean cultivar 98010818
US11277996B1 (en) 2020-09-11 2022-03-22 Monsanto Technology Llc Soybean cultivar 97051600
US11229183B1 (en) 2020-09-11 2022-01-25 Monsanto Technology Llc Soybean cultivar 93070703
US11310988B2 (en) 2020-09-11 2022-04-26 Monsanto Technology Llc Soybean cultivar 92340030
US11317587B2 (en) 2020-09-11 2022-05-03 Stine Seed Farm, Inc. Soybean cultivar 96030600
US20220079070A1 (en) 2020-09-11 2022-03-17 Monsanto Technology Llc Soybean cultivar 94112440
US11357200B2 (en) 2020-09-11 2022-06-14 Monsanto Technology, Llc Soybean cultivar 92131600
US11330784B2 (en) 2020-09-11 2022-05-17 Monsanto Technology, Llc Soybean cultivar 95322700
US11284592B1 (en) 2020-09-11 2022-03-29 Monsanto Technology Llc Soybean cultivar 91030603
US11317588B2 (en) 2020-09-11 2022-05-03 Stine Seed Farm, Inc. Soybean cultivar 97080600
US11350596B2 (en) 2020-09-11 2022-06-07 Monsanto Technology, Llc Soybean cultivar 96050600
US11439111B2 (en) 2020-09-11 2022-09-13 Monsanto Technology Llc Soybean cultivar 99354100
US11369074B2 (en) 2020-09-15 2022-06-28 Monsanto Technology Llc Soybean cultivar 99122620
US11363778B2 (en) 2020-09-15 2022-06-21 Monsanto Technology Llc Soybean cultivar 90221620
US11406080B2 (en) 2020-09-15 2022-08-09 Stine Seed Farm, Inc. Soybean cultivar 92420140
US11425879B2 (en) 2020-09-15 2022-08-30 Monsanto Technology, Llc Soybean cultivar 93054930
US11399497B2 (en) 2020-09-15 2022-08-02 Monsanto Technology, Llc Soybean cultivar 93333110
US11337401B2 (en) 2020-09-15 2022-05-24 Monsanto Technology, Llc Soybean cultivar 90370640
US11399496B2 (en) 2020-09-15 2022-08-02 Monsanto Technology, Llc Soybean cultivar 90116100
US11324182B2 (en) 2020-09-15 2022-05-10 Stine Seed Farm, Inc. Soybean cultivar 95060020
US11412692B2 (en) 2020-09-15 2022-08-16 Monsanto Technology Llc Soybean cultivar 90290600
US11744211B2 (en) 2020-09-15 2023-09-05 Monsanto Technology, Llc Soybean cultivar 92310017
US11343989B2 (en) 2020-09-15 2022-05-31 Monsanto Technology, Llc Soybean cultivar 92350300
US11324183B2 (en) 2020-09-15 2022-05-10 Stine Seed Farm, Inc. Soybean cultivar 90250820
US11419299B2 (en) 2020-09-15 2022-08-23 Monsanto Technology Llc Soybean cultivar 91361201
US11363779B2 (en) 2020-09-15 2022-06-21 Monsanto Technology Llc Soybean cultivar 90301201
US11357201B2 (en) 2020-09-15 2022-06-14 Monsanto Technology, Llc Soybean cultivar 94411660
US11343988B2 (en) 2020-09-15 2022-05-31 Stine Seed Farm, Inc. Soybean cultivar 91041800
US11324184B2 (en) 2020-09-15 2022-05-10 Stine Seed Farm, Inc. Soybean cultivar 97061850
US11317589B2 (en) 2020-09-15 2022-05-03 Monsanto Technology Llc Soybean cultivar 95430820
US11343987B2 (en) 2020-09-15 2022-05-31 Stine Seed Farm, Inc. Soybean cultivar 90401320
US11350597B2 (en) 2020-09-15 2022-06-07 Monsanto Technology, Llc Soybean cultivar 94111580
US11310989B2 (en) 2020-09-15 2022-04-26 Monsanto Technology Llc Soybean cultivar 94071660
US11363780B2 (en) 2020-09-15 2022-06-21 Monsanto Technology Llc Soybean cultivar 93272026
US11606926B2 (en) 2020-09-15 2023-03-21 Monsanto Technology Llc Soybean cultivar 94030201
US11477960B2 (en) 2020-11-02 2022-10-25 Monsanto Technology Llc Soybean variety 01077912
US11653612B2 (en) 2020-11-02 2023-05-23 Monsanto Technology Llc Soybean variety 01077826
US11596122B2 (en) 2020-11-02 2023-03-07 Monsanto Technology Llc Soybean variety 01077891
US11510382B2 (en) 2020-11-02 2022-11-29 Monsanto Technology Llc Soybean variety 01078845
US11653613B2 (en) 2020-11-02 2023-05-23 Monsanto Technology Llc Soybean variety 01077829
US11547075B2 (en) 2020-11-02 2023-01-10 Monsanto Technology Llc Soybean variety 01078822
US11612128B2 (en) 2020-11-02 2023-03-28 Monsanto Technology Llc Soybean variety 01083666
US11357205B2 (en) 2020-11-05 2022-06-14 Monsanto Technology Llc Soybean variety 01083631
US11445688B2 (en) 2020-11-05 2022-09-20 Monsanto Technology, Llc Soybean variety 01077870
US11284594B1 (en) 2020-11-05 2022-03-29 Monsanto Technology Llc Soybean variety 01077848
US11284593B1 (en) 2020-11-05 2022-03-29 Monsanto Technology Llc Soybean variety 01083687
US11277997B1 (en) 2020-11-05 2022-03-22 Monsanto Technology Llc Soybean variety 01077853
US11457591B2 (en) 2020-11-05 2022-10-04 Monsanto Technology, Llc Soybean variety 01077893
US11533883B2 (en) 2020-11-05 2022-12-27 Monsanto Technology, Llc Soybean variety 01077890
US11445689B2 (en) 2020-11-05 2022-09-20 Monsanto Technology, Llc Soybean variety 01078100
US11343990B2 (en) 2020-11-05 2022-05-31 Monsanto Technology, Llc Soybean variety 01083439
US11343991B2 (en) 2020-11-05 2022-05-31 Monsanto Technology, Llc Soybean variety 01078115
US11343992B2 (en) 2020-11-05 2022-05-31 Monsanto Technology, Llc Soybean variety 01078550
US11343993B2 (en) 2020-11-05 2022-05-31 Monsanto Technology, Llc Soybean variety 01083650
US11412694B2 (en) 2020-11-05 2022-08-16 Monsanto Technology Llc Soybean variety 01077905
US11350600B2 (en) 2020-11-05 2022-06-07 Monsanto Technology, Llc Soybean variety 01083699
US11464195B2 (en) 2020-11-06 2022-10-11 Monsanto Technology Llc Soybean variety 01083654
US11343994B1 (en) 2020-11-06 2022-05-31 Monsanto Technology, Llc Soybean variety 01083655
US11343996B1 (en) 2020-11-06 2022-05-31 Monsanto Technology, Llc Soybean variety 01077875
US11439114B2 (en) 2020-11-06 2022-09-13 Monsanto Technology, Llc Soybean variety 01078032
US11547076B2 (en) 2020-11-06 2023-01-10 Monsanto Technology, Llc Soybean variety 01077931
US11510384B2 (en) 2020-11-06 2022-11-29 Monsanto Technology, Llc Soybean variety 01078675
US11350601B2 (en) 2020-11-06 2022-06-07 Monsanto Technology, Llc Soybean variety 01077876
US11528879B2 (en) 2020-11-06 2022-12-20 Monsanto Technology, Llc Soybean variety 01083659
US11382301B2 (en) 2020-11-06 2022-07-12 Monsanto Technology, Llc Soybean variety 01077881
US11369076B2 (en) 2020-11-06 2022-06-28 Monsanto Technology, Llc Soybean variety 01078749
US11484000B2 (en) 2020-11-06 2022-11-01 Monsanto Technology, Llc Soybean variety 01083661
US11445690B2 (en) 2020-11-06 2022-09-20 Monsanto Technology Llc Soybean variety 01083639
US11553671B2 (en) 2020-11-06 2023-01-17 Monsanto Technology, Llc Soybean variety 01083653
US11457601B2 (en) 2020-11-06 2022-10-04 Monsanto Technology Llc Soybean variety 01083638
US11343995B1 (en) 2020-11-06 2022-05-31 Monsanto Technology, Llc Soybean variety 01083647
US11477961B2 (en) 2020-11-06 2022-10-25 Monsanto Technology, Llc Soybean variety 01077940
US11330785B1 (en) 2020-11-06 2022-05-17 Monsanto Technology, Llc Soybean variety 01077941
US11432518B2 (en) 2020-11-06 2022-09-06 Monsanto Technology, Llc Soybean variety 01078759
US11357206B2 (en) 2020-11-06 2022-06-14 Monsanto Technology Llc Soybean variety 01077974
US11533884B2 (en) 2020-11-06 2022-12-27 Monsanto Technology, Llc Soybean variety 01083660
US11547077B2 (en) 2020-11-06 2023-01-10 Monsanto Technology, Llc Soybean variety 01083688
US11457600B2 (en) 2020-11-06 2022-10-04 Monsanto Technology, Llc Soybean variety 01083646
US11432520B2 (en) 2020-12-22 2022-09-06 Monsanto Technology Llc Cotton variety 19R228B3XF
US11432521B2 (en) 2020-12-22 2022-09-06 Monsanto Technology Llc Cotton variety 19R242NRB3XF
US11432519B2 (en) 2020-12-22 2022-09-06 Monsanto Technology Llc Cotton variety 19R227B3XF
US11317592B1 (en) 2020-12-22 2022-05-03 Monsanto Technology Llc Cotton variety 19R238NRB3XF
US11344000B1 (en) 2020-12-22 2022-05-31 Monsanto Technology Llc Cotton variety 19R113B3XF
US11653614B2 (en) 2021-02-11 2023-05-23 Eagle Research LLC Soybean cultivar P-17
US11606927B2 (en) 2021-02-24 2023-03-21 Monsanto Technology Llc Cotton variety 19R130B3XF
US11602114B2 (en) 2021-02-24 2023-03-14 Monsanto Technology Llc Cotton variety 19R107B3XF
US11589544B2 (en) 2021-02-24 2023-02-28 Monsanto Technology Llc Cotton variety 19R229B3XF
US11589545B2 (en) 2021-02-24 2023-02-28 Monsanto Technology Llc Cotton variety 19R240B3XF
US11737420B2 (en) 2021-02-24 2023-08-29 Monsanto Technology Llc Cotton variety 19R241NRB3XF
US11602116B2 (en) 2021-02-24 2023-03-14 Monsanto Technology Llc Cotton variety 19R236B3XF
US11606928B2 (en) 2021-02-24 2023-03-21 Monsanto Technology Llc Cotton variety 19R231B3XF
US11432522B1 (en) 2021-02-24 2022-09-06 Monsanto Technology Llc Cotton variety 19R244B3XF
US11602115B2 (en) 2021-02-24 2023-03-14 Monsanto Technology Cotton variety 19R233B3XF
US11617336B2 (en) 2021-02-26 2023-04-04 Monsanto Technology Llc Cotton variety 19R254B3XF
US11432523B1 (en) 2021-02-26 2022-09-06 Monsanto Technology Llc Cotton variety 19R125B3XF
US11617335B2 (en) 2021-02-26 2023-04-04 Monsanto Technology Llc Cotton variety 19R249B3XF
US11602117B2 (en) 2021-02-26 2023-03-14 Monsanto Technology Llc Cotton variety 19R245B3XF
US11497188B2 (en) 2021-02-26 2022-11-15 Monsanto Technology Llc Cotton variety 19R132B3XF
US20230019117A1 (en) * 2021-07-15 2023-01-19 Fluent Biosciences Inc. Decentralized workflows for single cell analysis
US11647721B2 (en) 2021-08-24 2023-05-16 Monsanto Technology Llc Soybean cultivar 02330517
US11647723B2 (en) 2021-08-24 2023-05-16 Monsanto Technology Llc Soybean cultivar 07060434
US11641838B2 (en) 2021-08-24 2023-05-09 Monsanto Technology Llc Soybean cultivar 04130514
US11666031B2 (en) 2021-08-24 2023-06-06 Monsanto Technology Llc Soybean cultivar 05140102
US11678636B2 (en) 2021-08-24 2023-06-20 Monsanto Technology, Llc Soybean cultivar 04070428
US11641837B2 (en) 2021-08-24 2023-05-09 Monsanto Technology Llc Soybean cultivar 02340134
US11696556B2 (en) 2021-08-24 2023-07-11 Monsanto Technology, Llc Soybean cultivar 07080746
US11647722B2 (en) 2021-08-24 2023-05-16 Monsanto Technology Llc Soybean cultivar 02140434
US11647724B2 (en) 2021-08-24 2023-05-16 Monsanto Technology Llc Soybean cultivar 03230124
US11678635B2 (en) 2021-08-24 2023-06-20 Stine Seed Farm, Inc. Soybean cultivar 01160922
US11647720B2 (en) 2021-08-24 2023-05-16 Monsanto Technology Llc Soybean cultivar 01130157
US12029189B2 (en) 2021-08-30 2024-07-09 Monsanto Technology Llc Soybean cultivar 02170813
US11856914B2 (en) 2021-08-31 2024-01-02 Monsanto Technology, Llc Soybean cultivar 01140108
US11700826B2 (en) 2021-08-31 2023-07-18 Monsanto Technology, Llc Soybean cultivar 02410315
US11696557B2 (en) 2021-08-31 2023-07-11 Monsanto Technology, Llc Soybean cultivar 04060955
US11700825B2 (en) 2021-08-31 2023-07-18 Monsanto Technology, Llc Soybean cultivar 06170324
US11716947B2 (en) 2021-08-31 2023-08-08 Monsanto Technology, Llc Soybean cultivar 07030013
US11647725B2 (en) 2021-08-31 2023-05-16 Monsanto Technology Llc Soybean cultivar 07160416
US11700827B2 (en) 2021-08-31 2023-07-18 Monsanto Technology, Llc Soybean cultivar 07220837
US11647726B2 (en) 2021-08-31 2023-05-16 Monsanto Technology Llc Soybean cultivar 04070902
US11696558B2 (en) 2021-08-31 2023-07-11 Monsanto Technology, Llc Soybean cultivar 02021411
US11800847B2 (en) 2021-08-31 2023-10-31 Monsanto Technology, Llc Soybean cultivar 05050112
US11647727B2 (en) 2021-08-31 2023-05-16 Monsanto Technology Llc Soybean cultivar 04120446
US11672230B2 (en) 2021-08-31 2023-06-13 Monsanto Technology, Llc Soybean cultivar 03260108
US11672229B2 (en) 2021-08-31 2023-06-13 Monsanto Technology, Llc Soybean cultivar 08180710
CN117897378A (en) 2021-09-03 2024-04-16 巴斯夫农业种子解决方案美国有限责任公司 Plants with increased tolerance to herbicides
WO2023044364A1 (en) 2021-09-15 2023-03-23 Enko Chem, Inc. Protoporphyrinogen oxidase inhibitors
US11917969B2 (en) 2021-09-28 2024-03-05 Monsanto Technology Llc Soybean variety 01083899
US11917968B2 (en) 2021-09-28 2024-03-05 Monsanto Technology Llc Soybean variety 01083684
US11825805B2 (en) 2021-09-28 2023-11-28 Monsanto Technology, Llc Soybean variety 01083845
US11985949B2 (en) 2021-09-28 2024-05-21 Monsanto Technology Llc Soybean variety 01091555
US11856918B2 (en) 2021-09-28 2024-01-02 Monsanto Technology, Llc Soybean variety 01084034
US11730132B2 (en) 2021-09-28 2023-08-22 Monsanto Technology, Llc Soybean variety 01083701
US11997978B2 (en) 2021-09-28 2024-06-04 Monsanto Technology Llc Soybean variety 01083711
US11825804B2 (en) 2021-09-28 2023-11-28 Monsanto Technology, Llc Soybean variety 01083877
US11825806B2 (en) 2021-09-28 2023-11-28 Monsanto Technology, Llc Soybean variety 01083849
US11819003B2 (en) 2021-09-28 2023-11-21 Monsanto Technology, Llc Soybean variety 01083835
US11744215B2 (en) 2021-09-28 2023-09-05 Monsanto Technology, Llc Soybean variety 01084068
US11903357B2 (en) 2021-09-28 2024-02-20 Monsanto Technology Llc Soybean variety 01083737
US11707044B2 (en) 2021-09-28 2023-07-25 Monsanto Technology, Llc Soybean variety 01083999
US11839192B2 (en) 2021-09-29 2023-12-12 Monsanto Technology, Llc Soybean variety 01091705
US11744217B2 (en) 2021-09-29 2023-09-05 Monsanto Technology, Llc Soybean variety 01091711
US11917970B2 (en) 2021-09-29 2024-03-05 Monsanto Technology Llc Soybean variety 01091697
US11930768B2 (en) 2021-09-29 2024-03-19 Monsanto Technology Llc Soybean variety 01091702
US11917971B2 (en) 2021-09-29 2024-03-05 Monsanto Technology Llc Soybean variety 01091703
US11716957B2 (en) 2021-09-29 2023-08-08 Monsanto Technology, Llc Soybean variety 01091574
US11925161B2 (en) 2021-09-29 2024-03-12 Monsanto Technology Llc Soybean variety 01091628
US11716958B2 (en) 2021-09-29 2023-08-08 Monsanto Technology, Llc Soybean variety 01091691
US11744216B2 (en) 2021-09-29 2023-09-05 Monsanto Technology, Llc Soybean variety 01091693
US11758869B2 (en) 2021-09-29 2023-09-19 M.S. Technologies, L.L.C. Soybean variety 01091607
US12089555B2 (en) 2021-09-30 2024-09-17 Monsanto Technology Llc Soybean variety 01091725
US12004474B2 (en) 2021-09-30 2024-06-11 Monsanto Technology Llc Soybean variety 01091721
US11751529B2 (en) 2021-09-30 2023-09-12 M.S. Technologies, L.L.C. Soybean variety 01091734
US12004475B2 (en) 2021-09-30 2024-06-11 Monsanto Technology Llc Soybean variety 01091767
US11930769B2 (en) 2021-09-30 2024-03-19 Monsanto Technology Llc Soybean variety 01091763
US11744218B2 (en) 2021-09-30 2023-09-05 Monsanto Technology, Llc Soybean variety 01091749
US11917972B2 (en) 2021-10-01 2024-03-05 Monsanto Technology Llc Soybean variety 01091777
US11963509B2 (en) 2021-10-01 2024-04-23 Monsanto Technology Llc Soybean variety 01091775
US11930770B2 (en) 2021-10-01 2024-03-19 Monsanto Technology Llc Soybean variety 01091765
US11917973B2 (en) 2021-10-01 2024-03-05 Monsanto Technology Llc Soybean variety 01091785
US11832578B2 (en) 2021-10-04 2023-12-05 Monsanto Technology, Llc Soybean variety 01091795
US11917974B2 (en) 2021-10-04 2024-03-05 Monsanto Technology, Llc Soybean variety 01091792
US12082547B2 (en) 2021-10-04 2024-09-10 Monsanto Technology Llc Soybean variety 01091811
US11839193B2 (en) 2021-10-04 2023-12-12 Monsanto Technology, Llc Soybean variety 01091798
US11903358B2 (en) 2021-10-04 2024-02-20 Monsanto Technology Llc Soybean variety 01091805
US12193386B2 (en) 2021-11-30 2025-01-14 Monsanto Technology Llc Soybean variety 01091764
US12075745B2 (en) 2021-12-23 2024-09-03 Monsanto Technology Llc Cotton variety 19R325B3TXF
US12356950B2 (en) 2021-12-23 2025-07-15 Monsanto Technology Llc Cotton variety 20R816B3TXF
CN118525002A (en) 2022-01-14 2024-08-20 恩科化学公司 Protoporphyrinogen oxidase inhibitors
US12102052B2 (en) 2022-01-26 2024-10-01 Monsanto Technology Llc Cotton variety 20R827B3TXF
US12193404B2 (en) 2022-01-26 2025-01-14 Monsanto Technology Llc Cotton variety 20R828B3TXF
US12070008B2 (en) 2022-01-26 2024-08-27 Monsanto Technology Llc Cotton variety 19R239B3XF
US12369556B2 (en) 2022-01-26 2025-07-29 Monsanto Technology Llc Cotton variety 19R311B3TXF
US12108730B2 (en) 2022-01-26 2024-10-08 Monsanto Technology Llc Cotton variety 19R316B3TXF
US12108731B2 (en) 2022-01-26 2024-10-08 Monsanto Technology Llc Cotton variety 20R824B3TXF
US11985950B2 (en) 2022-01-26 2024-05-21 Monsanto Technology Llc Cotton variety 19R114B3XF
US12245568B2 (en) 2022-01-26 2025-03-11 Monsanto Technology Llc Cotton variety 19R331B3TXF
US12108732B2 (en) 2022-01-26 2024-10-08 Monsanto Technology Llc Cotton variety 20R914XF
US12382917B2 (en) 2022-01-26 2025-08-12 Monsanto Technology Llc Cotton variety 20R821B3TXF
US12295332B2 (en) 2022-09-21 2025-05-13 Monsanto Technology Llc Soybean cultivar 19221717
US12207615B2 (en) 2022-09-21 2025-01-28 Monsanto Technology Llc Soybean cultivar 15301811
US12207614B2 (en) 2022-09-21 2025-01-28 Monsanto Technology Llc Soybean cultivar 15280717
US12284968B2 (en) 2022-09-21 2025-04-29 Monsanto Technology Llc Soybean cultivar 16150010
US12207612B2 (en) 2022-09-21 2025-01-28 Monsanto Technology Llc Soybean cultivar 18181400
US12402594B2 (en) 2022-09-21 2025-09-02 Monsanto Technology Llc Soybean cultivar 15202010
US12336490B2 (en) 2022-09-21 2025-06-24 Monsanto Technology Llc Soybean cultivar 11251200
US12268167B2 (en) 2022-09-21 2025-04-08 Monsanto Technology Llc Soybean cultivar 15040915
US12239087B2 (en) 2022-09-21 2025-03-04 Monsanto Technology Llc Soybean cultivar 17111743
US12284966B2 (en) 2022-09-21 2025-04-29 Monsanto Technology Llc Soybean cultivar 19241023
US12284965B2 (en) 2022-09-21 2025-04-29 Monsanto Technology Llc Soybean cultivar 12350011
US12414529B2 (en) 2022-09-21 2025-09-16 Monsanto Technology Llc Soybean cultivar 11232411
US12207613B2 (en) 2022-09-21 2025-01-28 Monsanto Technology Llc Soybean cultivar 17101336
US12268166B2 (en) 2022-09-21 2025-04-08 Monsanto Technology Llc Soybean cultivar 14030941
US12274225B2 (en) 2022-09-21 2025-04-15 Monsanto Technology Llc Soybean cultivar 12340924
US12268168B2 (en) 2022-09-21 2025-04-08 Monsanto Technology Llc Soybean cultivar 17041010
US12396426B2 (en) 2022-09-21 2025-08-26 Monsanto Technology Llc Soybean cultivar 18071201
US12284967B2 (en) 2022-09-21 2025-04-29 Monsanto Technology Llc Soybean cultivar 13323023
US12219915B2 (en) 2022-09-28 2025-02-11 Monsanto Technology Llc Soybean cultivar 15161227
US12284971B2 (en) 2022-09-28 2025-04-29 Monsanto Technology Llc Soybean cultivar 13380227
US12302845B2 (en) 2022-09-28 2025-05-20 Monsanto Technology Llc Soybean cultivar 15091010
US12274227B2 (en) 2022-09-28 2025-04-15 Monsanto Technology Llc Soybean cultivar 11021540
US12268172B2 (en) 2022-09-28 2025-04-08 Monsanto Technology Llc Soybean cultivar 13011855
US12268174B2 (en) 2022-09-28 2025-04-08 Monsanto Technology Llc Soybean cultivar 15410048
US12207616B2 (en) 2022-09-28 2025-01-28 Monsanto Technology Llc Soybean cultivar 17370111
US12213442B2 (en) 2022-09-28 2025-02-04 Monsanto Technology Llc Soybean cultivar 10220111
US12225871B2 (en) 2022-09-28 2025-02-18 Monsanto Technology Llc Soybean cultivar 15341968
US12274226B2 (en) 2022-09-28 2025-04-15 Monsanto Technology Llc Soybean cultivar 10411540
US12274228B2 (en) 2022-09-28 2025-04-15 Mansanto Technology Llc Soybean cultivar 14102235
US12268173B2 (en) 2022-09-28 2025-04-08 Monsanto Technology Llc Soybean cultivar 17050534
US12284970B2 (en) 2022-09-28 2025-04-29 Monsanto Technology Llc Soybean cultivar 12070855
US12302848B2 (en) 2022-11-01 2025-05-20 Monsanto Technology Llc Soybean variety 01091599
US12295333B2 (en) 2022-11-02 2025-05-13 Monsanto Technology Llc Soybean variety 01094697
US12284973B2 (en) 2022-11-02 2025-04-29 Monsanto Technology Llc Soybean variety 01094700
US12389862B2 (en) 2022-11-02 2025-08-19 Monsanto Technology Llc Soybean variety 01091626
US12396427B2 (en) 2022-11-02 2025-08-26 Monsanto Technoloy LLC Soybean variety 01091614
US12295334B2 (en) 2022-11-02 2025-05-13 Monsanto Technology Llc Soybean variety 01093524
US12295335B2 (en) 2022-11-03 2025-05-13 Monsanto Technology Llc Soybean variety 01094667
US12284974B2 (en) 2022-11-03 2025-04-29 Monsanto Technology Llc Soybean variety 01094189
US12329115B2 (en) 2022-11-04 2025-06-17 Monsanto Technology Llc Soybean variety 01094676
US12324397B2 (en) 2022-11-07 2025-06-10 Monsanto Technology Llc Soybean variety 01094715
US12295336B2 (en) 2022-11-10 2025-05-13 Monsanto Technology Llc Soybean variety 01094682
US12356946B2 (en) 2022-11-15 2025-07-15 Monsanto Technology Llc Soybean variety 01094720
US12317850B2 (en) 2022-11-15 2025-06-03 Monsanto Technology Llc Soybean variety 01094668
US12219916B2 (en) 2022-11-15 2025-02-11 Monsanto Technology Llc Soybean variety 01094718
US12310318B2 (en) 2022-11-15 2025-05-27 Monsanto Technology Llc Soybean variety 01094669
US12295337B2 (en) 2022-11-16 2025-05-13 Monsanto Technology Llc Soybean variety 01094726
US12225872B2 (en) 2022-11-17 2025-02-18 Monsanto Technology Llc Soybean variety 01094758
US12310319B2 (en) 2022-11-17 2025-05-27 Monsanto Technology Llc Soybean variety 01094752
US12369554B2 (en) 2022-11-17 2025-07-29 Monsanto Technology Llc Soybean variety 01094759
US12295338B2 (en) 2022-11-17 2025-05-13 Monsanto Technology Llc Soybean variety 01094762
US12310320B2 (en) 2022-11-17 2025-05-27 Monsanto Technology Llc Soybean variety 01094766
US12356947B2 (en) 2022-11-17 2025-07-15 Monsanto Technology Llc Soybean variety 01094755
US12389863B2 (en) 2022-11-20 2025-08-19 Monsanto Technology Llc Soybean variety 01094793
US12310321B2 (en) 2022-11-20 2025-05-27 Monsanto Technology Llc Soybean variety 01094775
US12310322B2 (en) 2022-11-20 2025-05-27 Monsanto Technology Llc Soybean variety 01094785
US12356948B2 (en) 2022-11-20 2025-07-15 Monsanto Technology Llc Soybean variety 01094790
US12376549B2 (en) 2022-11-20 2025-08-05 Monsanto Technology Llc Soybean variety 01094788
US12245566B2 (en) 2022-11-20 2025-03-11 Monsanto Technology Llc Soybean variety 01094772
US12329116B2 (en) 2022-11-21 2025-06-17 Monsanto Technology Llc Soybean variety 01094813
US12310323B2 (en) 2022-11-21 2025-05-27 Monsanto Technology Llc Soybean variety 01094797
US12402595B2 (en) 2022-11-21 2025-09-02 Monsanto Technology Llc Soybean variety 01094810
US12295339B2 (en) 2022-11-21 2025-05-13 Monsanto Technology Llc Soybean variety 01094816
US12302849B2 (en) 2022-11-21 2025-05-20 Monsanto Technology Llc Soybean variety 01094817
US12396428B2 (en) 2022-11-22 2025-08-26 Monsanto Technology Llc Soybean variety 01094840
US12342786B2 (en) 2022-11-22 2025-07-01 Monsanto Technology Llc Soybean variety 01094835
US12317852B2 (en) 2022-11-22 2025-06-03 Monsanto Technology Llc Soybean variety 01098165
US12382914B2 (en) 2022-11-22 2025-08-12 Monsanto Technology Llc Soybean variety 01098202
US12396429B2 (en) 2022-11-22 2025-08-26 Monsanto Technology Llc Soybean variety 01098164
US12295340B2 (en) 2022-11-22 2025-05-13 Monsanto Technology Llc Soybean variety 01094839
US12364241B2 (en) 2022-11-23 2025-07-22 Monsanto Technology Llc Soybean variety 01098236
US12302850B2 (en) 2022-11-24 2025-05-20 Monsanto Technology Llc Soybean variety 01098267
US12389864B2 (en) 2022-11-24 2025-08-19 Monsanto Technology Llc Soybean variety 01098296
US12382915B2 (en) 2022-11-24 2025-08-12 Monsanto Technology Llc Soybean variety 01098300
US12376550B2 (en) 2022-11-24 2025-08-05 Monsanto Technology Llc Soybean variety 01098249
US12376551B2 (en) 2022-11-24 2025-08-05 Monsanto Technology Llc Soybean variety 01098301
US12295342B2 (en) 2022-11-26 2025-05-13 Monsanto Technology Llc Soybean variety 01098302
US12369555B2 (en) 2022-11-26 2025-07-29 Monsanto Technology Llc Soybean variety 01098305
US12295343B2 (en) 2022-11-26 2025-05-13 Monsanto Technology Llc Soybean variety 01098326
US12382916B2 (en) 2022-11-26 2025-08-12 Monsanto Technology Llc Soybean variety 01098337
CN120529828A (en) 2022-12-12 2025-08-22 巴斯夫农业解决方案美国有限责任公司 Plants with increased tolerance to herbicides
US12389867B2 (en) 2022-12-30 2025-08-19 Monsanto Technology Llc Cotton variety 21R4127B3TXF
US12389868B2 (en) 2022-12-30 2025-08-19 Monsanto Technology Llc Cotton variety 20R733B3XF
US12284976B2 (en) 2022-12-30 2025-04-29 Monsanto Technology Llc Cotton variety 21R4132B3TXF
US12356951B2 (en) 2023-01-25 2025-07-15 Monsanto Technology Llc Cotton variety 21R523XF
US12414532B2 (en) 2023-01-25 2025-09-16 Monsanto Technology Llc Cotton variety 20R731B3XF
US12426566B2 (en) 2023-01-25 2025-09-30 Monsanto Technology Llc Cotton variety 20R751B3XF
US12414533B2 (en) 2023-01-25 2025-09-16 Monsanto Technology Llc Cotton variety 20R752B3XF
US12419262B2 (en) 2023-01-26 2025-09-23 Monsanto Technology Llc Cotton variety 21R4221B3TXF
US12433235B2 (en) 2023-01-26 2025-10-07 Monsanto Technology Llc Cotton variety 21R4432B3TXF
US12419263B2 (en) 2023-01-26 2025-09-23 Monsanto Technology Llc Cotton variety 21R4126B3TXF
US12419261B2 (en) 2023-01-26 2025-09-23 Monsanto Technology Llc Cotton variety 21R626B3XF
US11917976B1 (en) 2023-06-20 2024-03-05 Kg Agri Products, Inc. Soybean cultivar GL2876
WO2025038723A1 (en) 2023-08-14 2025-02-20 The Texas A&M University System Corn endotype-derived polypeptides capable of metabolizing fumonisin
WO2025076411A1 (en) * 2023-10-05 2025-04-10 The Board Of Trustees Of The University Of Illinois Pantaphos compounds and methods
WO2025153657A2 (en) 2024-01-17 2025-07-24 Basf Se Plants having increased tolerance to herbicides
WO2025153595A1 (en) 2024-01-17 2025-07-24 Basf Se Plants having increased tolerance to herbicides

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567600A (en) 1983-09-26 1996-10-22 Mycogen Plant Sciences, Inc. Synthetic insecticidal crystal protein gene
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US4816403A (en) 1986-08-04 1989-03-28 Louisiana State University Detoxification of chlorinated aromatic compounds by organism NRRL B-18086
US5017692A (en) 1986-09-04 1991-05-21 Schering Corporation Truncated human interleukin-a alpha
US5263992A (en) 1986-10-17 1993-11-23 Bio-Metric Systems, Inc. Biocompatible device with covalently bonded biocompatible agent
US5445962A (en) 1988-08-05 1995-08-29 Atallah; Yousef H. Microorganisms
FR2655048A1 (en) 1989-11-27 1991-05-31 Inst Nat Sante Rech Med PROCESS FOR OBTAINING BIOACTIVE COPOLYMERS BY GRAFTING VINYL PATTERNS AND RESULTING GRAFT COPOLYMERS.
AU3124793A (en) 1991-10-29 1993-06-07 Clover Consolidated, Limited Crosslinkable polysaccharides, polycations and lipids useful for encapsulation and drug release
AU648214B2 (en) 1991-12-31 1994-04-14 Lucky Limited Recombinant gene coding for human alpha interferon and expression vector thereof, etc.
WO1993016176A1 (en) 1992-02-13 1993-08-19 Bio-Metric Systems, Inc. Immobilization of chemical species in crosslinked matrices
HU214698B (en) 1992-04-09 1998-06-29 Gyógyszerkutató Intézet Kft. Process for producing recombinant desulphatohirudin hv-1 peptides
US5563328A (en) 1992-08-19 1996-10-08 Board Of Regents, University Of Nebraska-Lincoln Promoters from chlorella virus genes providing for expression of genes in prokaryotic and eukaryotic hosts
EP0646643B1 (en) 1993-06-30 2001-12-05 Lucky Ltd. Modified human granulocyte macrophage-colony stimulating factor gene and expression thereof in yeast
US5362865A (en) 1993-09-02 1994-11-08 Monsanto Company Enhanced expression in plants using non-translated leader sequences
US5689052A (en) 1993-12-22 1997-11-18 Monsanto Company Synthetic DNA sequences having enhanced expression in monocotyledonous plants and method for preparation thereof
US5608147A (en) * 1994-01-11 1997-03-04 Kaphammer; Bryan J. tfdA gene selectable markers in plants and the use thereof
US5545818A (en) 1994-03-11 1996-08-13 Calgene Inc. Expression of Bacillus thuringiensis cry proteins in plant plastids
US5795737A (en) 1994-09-19 1998-08-18 The General Hospital Corporation High level expression of proteins
US5656422A (en) 1994-10-05 1997-08-12 Idaho Research Foundation, Inc. Compositions and methods for detection of 2,4-dichlorophenoxyacetic acid and related compounds
DE4444708A1 (en) 1994-12-15 1996-06-20 Basf Ag Use of auxin-type herbicides for the treatment of transgenic crop plants
DE19518247A1 (en) 1995-05-18 1996-11-21 Stoess & Co Gelatine Water resistant barrier material
DE19533682A1 (en) 1995-09-12 1997-03-13 Biotronik Mess & Therapieg Process for depositing and immobilizing heparin on inorganic substrate surfaces of cardiovascular implants
PL328730A1 (en) 1996-02-22 1999-02-15 Merck & Co Inc Synthetic hiv genes
AR006928A1 (en) 1996-05-01 1999-09-29 Pioneer Hi Bred Int AN ISOLATED DNA MOLECULA CODING A GREEN FLUORESCENT PROTEIN AS A TRACEABLE MARKER FOR TRANSFORMATION OF PLANTS, A METHOD FOR THE PRODUCTION OF TRANSGENIC PLANTS, A VECTOR OF EXPRESSION, A TRANSGENIC PLANT AND CELLS OF SUCH PLANTS.
KR100558838B1 (en) 1997-04-04 2006-03-10 보드 오브 리젠츠 오브 디 유니버시티 오브 네브라스카 Methods and Materials for the Preparation and Use of Transgenic Dicamba-Degrading Organisms
US7105724B2 (en) * 1997-04-04 2006-09-12 Board Of Regents Of University Of Nebraska Methods and materials for making and using transgenic dicamba-degrading organisms
US7022896B1 (en) * 1997-04-04 2006-04-04 Board Of Regents Of University Of Nebraska Methods and materials for making and using transgenic dicamba-degrading organisms
US7884262B2 (en) * 2006-06-06 2011-02-08 Monsanto Technology Llc Modified DMO enzyme and methods of its use

Cited By (177)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2333079A1 (en) 2004-01-20 2011-06-15 Monsanto Technology LLC Chimeric promoters for use in plants
WO2005069986A2 (en) 2004-01-20 2005-08-04 Monsanto Technology Llc Chimeric promoters for use in plants
EP2868749A1 (en) 2004-01-20 2015-05-06 Monsanto Technology LLC Chimeric promoters for use in plants
EP2308976A2 (en) 2004-04-30 2011-04-13 Dow AgroSciences LLC Novel herbicide resistance gene
US11149283B2 (en) 2004-04-30 2021-10-19 Dow Agrosciences Llc Herbicide resistance genes
EP2308977A2 (en) 2004-04-30 2011-04-13 Dow AgroSciences LLC Novel herbicide resistance gene
EP2298901A2 (en) 2004-04-30 2011-03-23 Dow AgroSciences LLC Novel herbicide resistance genes
US10947555B2 (en) 2004-04-30 2021-03-16 Dow Agrosciences Llc Herbicide resistance genes
EP2319932A2 (en) 2004-04-30 2011-05-11 Dow AgroSciences LLC Novel herbicide resistance gene
US11299745B1 (en) 2004-04-30 2022-04-12 Dow Agrosciences Llc Herbicide resistance genes
US7309589B2 (en) 2004-08-20 2007-12-18 Vironix Llc Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing
US20060057616A1 (en) * 2004-08-20 2006-03-16 Vironix Llc Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing
WO2006069017A2 (en) 2004-12-21 2006-06-29 Monsanto Technology, Llc Transgenic plants with enhanced agronomic traits
EP2562259A2 (en) 2004-12-21 2013-02-27 Monsanto Technology LLC Transgenic plants with enhanced agronomic traits
WO2007030432A2 (en) 2005-09-06 2007-03-15 Monsanto Technology Llc Vectors and methods for improved plant transformation efficiency
EP3536793A1 (en) 2005-09-06 2019-09-11 Monsanto Technology LLC Vectors and methods for improved plant transformation efficiency
EP3339441A1 (en) 2005-10-13 2018-06-27 Monsanto Technology LLC Methods for producing hybrid seed
WO2007053482A2 (en) 2005-10-28 2007-05-10 Dow Agrosciences Llc Novel herbicide resistance genes
US11371055B2 (en) 2005-10-28 2022-06-28 Corteva Agriscience Llc Herbicide resistance genes
EP3241430A1 (en) 2005-10-28 2017-11-08 Dow AgroSciences LLC Novel herbicide resistance genes
EP2484202A1 (en) 2005-10-28 2012-08-08 Dow AgroSciences LLC Novel herbicide resistance genes
EP2484767A1 (en) 2005-10-28 2012-08-08 Dow AgroSciences LLC Novel Herbicide resistance genes
EP2484769A2 (en) 2005-12-21 2012-08-08 Monsanto Technology LLC Transgenic plants with enhanced agronomic traits
EP2371964A1 (en) 2006-05-16 2011-10-05 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP2361986A1 (en) 2006-05-16 2011-08-31 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP2803728A1 (en) 2006-05-16 2014-11-19 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP3608413A1 (en) 2006-05-16 2020-02-12 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP3196311A1 (en) 2006-05-16 2017-07-26 Monsanto Technology LLC Use of non-agrobacterium bacterial species for plant transformation
EP2455490A2 (en) 2006-05-25 2012-05-23 Monsanto Technology LLC A method to identify disease resistant quantitative trait loci in soybean and compositions thereof
EP2455491A2 (en) 2006-05-25 2012-05-23 Monsanto Technology LLC A method to identify disease resistant quantitative trait loci in soybean and compositions thereof
EP2455489A2 (en) 2006-05-25 2012-05-23 Monsanto Technology LLC A method to identify disease resistant quantitative trait loci in soybean and compositions thereof
USRE44971E1 (en) 2006-06-06 2014-06-24 Monsanto Technology Llc Method for selection of transformed cells
US20110152096A1 (en) * 2006-06-06 2011-06-23 Feng Paul C C Methods for weed control
US7884262B2 (en) 2006-06-06 2011-02-08 Monsanto Technology Llc Modified DMO enzyme and methods of its use
US7855326B2 (en) 2006-06-06 2010-12-21 Monsanto Technology Llc Methods for weed control using plants having dicamba-degrading enzymatic activity
US7851670B2 (en) 2006-06-06 2010-12-14 Monsanto Technology Llc Method for selection of transformed cells
WO2007143690A2 (en) 2006-06-06 2007-12-13 Monsanto Technology Llc Methods for weed control
US20080015110A1 (en) * 2006-06-06 2008-01-17 Monsanto Technology Llc Modified dmo enzyme and methods of its use
USRE45048E1 (en) 2006-06-06 2014-07-22 Monsanto Technology Llc Methods for weed control using plants having dicamba-degrading enzymatic activity
US20080119361A1 (en) * 2006-06-06 2008-05-22 Feng Paul C C Methods for weed control
US20080120739A1 (en) * 2006-06-06 2008-05-22 Monsanto Technology Llc Method for selection of transformed cells
US8629328B2 (en) 2006-06-06 2014-01-14 Monsanto Technology Llc Methods for weed control using plants transformed with dicamba monooxygenase
WO2008021543A2 (en) 2006-08-17 2008-02-21 Monsanto Technology, Llc Transgenic plants with enhanced agronomic traits
EP2540831A2 (en) 2006-08-17 2013-01-02 Monsanto Technology, LLC Transgenic plants with enhanced agronomic traits
US8207092B2 (en) 2006-10-16 2012-06-26 Monsanto Technology Llc Methods and compositions for improving plant health
US9155300B2 (en) 2006-10-16 2015-10-13 Monsanto Technology Llc Methods and compositions for improving plant health
US8754011B2 (en) 2006-10-16 2014-06-17 Monsanto Technology Llc Methods and compositions for improving plant health
US20090105077A1 (en) * 2006-10-16 2009-04-23 Monsanto Technology Llc Methods and compositions for improving plant health
US20130217130A1 (en) * 2006-10-16 2013-08-22 Paul C. C. Feng Methods and compositions for improving plant health
US20100279866A1 (en) * 2006-10-16 2010-11-04 Muhammad Bhatti Methods and Compositions for Improving Plant Health
US20080305952A1 (en) * 2006-10-25 2008-12-11 Arnevik Cindy L Cropping systems for managing weeds
EP2454940A2 (en) 2006-10-25 2012-05-23 Monsanto Technology LLC Cropping systems for managing weeds
US7939721B2 (en) 2006-10-25 2011-05-10 Monsanto Technology Llc Cropping systems for managing weeds
US8791325B2 (en) 2007-02-26 2014-07-29 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US20090029861A1 (en) * 2007-02-26 2009-01-29 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
EP2527453A2 (en) 2007-02-26 2012-11-28 Monsanto Technology LLC Chloroplast transit peptides for efficient targeting of DMO and uses thereof
EP2527452A2 (en) 2007-02-26 2012-11-28 Monsanto Technology LLC Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US20110126307A1 (en) * 2007-02-26 2011-05-26 Feng Paul C C Chloroplast transit peptides for efficient targeting of dmo and uses thereof
US8084666B2 (en) 2007-02-26 2011-12-27 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US7838729B2 (en) 2007-02-26 2010-11-23 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US8420888B2 (en) 2007-02-26 2013-04-16 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
WO2008105890A2 (en) 2007-02-26 2008-09-04 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
EP3290520A1 (en) 2007-03-09 2018-03-07 Monsanto Technology LLC Preparation and use of plant embryo explants for transformation
EP2425709A1 (en) 2007-03-09 2012-03-07 Monsanto Technology, LLC Preparation and use of plant embryo explants for transformation
WO2008112645A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
WO2008112633A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Method of meristem excision and transformation
EP3916097A1 (en) 2007-03-09 2021-12-01 Monsanto Technology LLC Preparation and use of plant embryo explants for transformation
EP2811026A2 (en) 2007-03-09 2014-12-10 Monsanto Technology LLC Methods for plant transformation using spectinomycin selection
EP2450448A1 (en) 2007-03-09 2012-05-09 Monsanto Technology LLC Methods for plant transformation using spectinomycin selection
EP3567113A1 (en) 2007-06-06 2019-11-13 Monsanto Technology LLC Genes and uses for plant enhancement
EP2698433A1 (en) 2007-06-06 2014-02-19 Monsanto Technology LLC Genes and uses for plant enhancement
EP2543735A1 (en) 2007-06-06 2013-01-09 Monsanto Technology LLC Genes and uses for plant enhancement
EP2840142A1 (en) 2007-06-06 2015-02-25 Monsanto Technology LLC Genes and uses for plant enhancement
EP2380988A2 (en) 2007-07-10 2011-10-26 Mosanto Technology LLC Transgenic plants with enhanced agronomic traits
EP2573178A2 (en) 2007-07-10 2013-03-27 Monsanto Technology LLC Transgenic plants with enhanced agronomic traits
EP2607488A2 (en) 2008-04-07 2013-06-26 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2607489A2 (en) 2008-04-07 2013-06-26 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2716763A2 (en) 2008-04-29 2014-04-09 Monsanto Technology LLC Genes and uses for plant enhancement
EP2537937A2 (en) 2008-04-29 2012-12-26 Monsanto Technology LLC Genes and uses for plant enhancement
EP2336332A2 (en) 2008-04-29 2011-06-22 Monsanto Technology LLC Genes and uses for plant enhancement
EP3023499A1 (en) 2008-07-16 2016-05-25 Monsanto Technology LLC Methods and vectors for producing transgenic plants
US8304616B2 (en) 2009-04-07 2012-11-06 University Of Georgia Research Foundation, Inc. Soybean variety G00-3209
US20100257631A1 (en) * 2009-04-07 2010-10-07 Boerma H Roger Soybean variety g00-3209
US20100299773A1 (en) * 2009-05-20 2010-11-25 Monsanto Technology Llc Methods and compositions for selecting an improved plant
US9944944B2 (en) 2009-11-24 2018-04-17 Dow Agrosciences Llc AAD-12 event 416, related transgenic soybean lines, and event-specific identification thereof
WO2011066384A1 (en) 2009-11-24 2011-06-03 Dow Agrosciences Llc Aad-12 event 416, related transgenic soybean lines, and event-specific identification thereof
EP2982757A1 (en) 2010-01-14 2016-02-10 Monsanto Technology LLC Plant regulatory elements and uses thereof
WO2011088299A1 (en) 2010-01-14 2011-07-21 Monsanto Technology Llc Plant regulatory elements and uses thereof
EP3473721A1 (en) 2010-01-14 2019-04-24 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP3760726A1 (en) 2010-01-14 2021-01-06 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP3473722A1 (en) 2010-01-14 2019-04-24 Monsanto Technology LLC Plant regulatory elements and uses thereof
WO2012030711A1 (en) 2010-08-30 2012-03-08 Agrigenetics, Inc. Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
EP3382028A1 (en) 2010-12-03 2018-10-03 Dow AgroSciences LLC Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof
WO2012074868A2 (en) 2010-12-03 2012-06-07 Ms Technologies, Llc Optimized expression of glyphosate resistance encoding nucleic acid molecules in plant cells
WO2012128946A1 (en) 2011-03-18 2012-09-27 Ms Technologies Llc Regulatory regions preferentially expressing in non-pollen plant tissue
WO2012134921A2 (en) 2011-03-25 2012-10-04 Monsanto Technology Llc Plant regulatory elements and uses thereof
EP2733152A1 (en) 2011-03-25 2014-05-21 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2733151A1 (en) 2011-03-25 2014-05-21 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2752491A2 (en) 2011-05-13 2014-07-09 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2762570A2 (en) 2011-05-13 2014-08-06 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2752489A2 (en) 2011-05-13 2014-07-09 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP3321366A1 (en) 2011-05-13 2018-05-16 Monsanto Technology LLC Plant regulatory elements and uses thereof
WO2012158535A1 (en) 2011-05-13 2012-11-22 Monsanto Technology Llc Plant regulatory elements and uses thereof
EP4186977A1 (en) 2011-05-13 2023-05-31 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2752490A2 (en) 2011-05-13 2014-07-09 Monsanto Technology LLC Plant regulatory elements and uses thereof
EP2752492A2 (en) 2011-05-13 2014-07-09 Monsanto Technology LLC Plant regulatory elements and uses thereof
WO2013040057A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
EP3382027A2 (en) 2011-09-13 2018-10-03 Monsanto Technology LLC Methods and compositions for weed control
WO2013040033A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040021A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
EP3296402A2 (en) 2011-09-13 2018-03-21 Monsanto Technology LLC Methods and compositions for weed control
WO2013040049A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040116A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040117A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
WO2013040005A1 (en) 2011-09-13 2013-03-21 Monsanto Technology Llc Methods and compositions for weed control
EP3434780A1 (en) 2011-09-13 2019-01-30 Monsanto Technology LLC Methods and compositions for weed control
EP3434779A1 (en) 2011-09-13 2019-01-30 Monsanto Technology LLC Methods and compositions for weed control
WO2013112527A1 (en) 2012-01-23 2013-08-01 Dow Agrosciences Llc Herbicide tolerant cotton event pdab4468.19.10.3
EP3219200A1 (en) 2012-02-01 2017-09-20 Dow Agrosciences Llc Glyphosate resistant plants and associated methods
WO2013116700A1 (en) 2012-02-01 2013-08-08 Dow Agrosciences Llc Glyphosate resistant plants and associated methods
EP3470522A2 (en) 2012-02-01 2019-04-17 Dow AgroSciences LLC Novel class of glyphosate resistance genes
WO2013130813A1 (en) 2012-02-29 2013-09-06 Dow Agrosciences Llc Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
WO2013158442A1 (en) 2012-04-20 2013-10-24 Monsanto Technology Llc Plant regulatory elements and uses thereof
WO2014159306A1 (en) 2013-03-13 2014-10-02 Pioneer Hi-Bred International, Inc. Glyphosate application for weed control in brassica
WO2014153254A2 (en) 2013-03-14 2014-09-25 Pioneer Hi-Bred International Inc. Compositions and methods to control insect pests
EP3744727A1 (en) 2013-03-14 2020-12-02 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2014150914A2 (en) 2013-03-15 2014-09-25 Pioneer Hi-Bred International, Inc. Phi-4 polypeptides and methods for their use
WO2015023846A2 (en) 2013-08-16 2015-02-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015038734A2 (en) 2013-09-13 2015-03-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3692786A1 (en) 2013-09-13 2020-08-12 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP4159028A1 (en) 2013-09-13 2023-04-05 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3862434A1 (en) 2013-11-04 2021-08-11 Dow AgroSciences LLC Optimal soybean loci
WO2015066643A1 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal soybean loci
WO2015066638A2 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal maize loci
WO2015066636A2 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal maize loci
US10030247B2 (en) 2013-12-31 2018-07-24 Dow Agrosciences Llc Maize ubiquitin promoters
US9725730B2 (en) 2013-12-31 2017-08-08 Dow Agrosciences Llc Maize ubiquitin promoters
US10036028B2 (en) 2013-12-31 2018-07-31 Dow Agrosciences Llc Maize ubiquitin promoters
US10030246B2 (en) 2013-12-31 2018-07-24 Dow Agrosciences Llc Maize ubiquitin promoters
US9885054B2 (en) 2013-12-31 2018-02-06 Dow Agrosciences Llc Maize ubiquitin promoters
WO2015120276A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
WO2015120270A1 (en) 2014-02-07 2015-08-13 Pioneer Hi Bred International, Inc. Insecticidal proteins and methods for their use
EP3705489A1 (en) 2014-02-07 2020-09-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015130931A1 (en) 2014-02-28 2015-09-03 Dow Agrosciences Llc Root specific expression conferred by chimeric gene regulatory elements
US10113178B2 (en) 2014-03-20 2018-10-30 Monsanto Technology Llc Transgenic maize event MON 87419 and methods of use thereof
US11098321B2 (en) 2014-03-20 2021-08-24 Monsanto Technology Llc Transgenic maize event MON 87419 and methods of use thereof
US11987798B2 (en) 2014-03-20 2024-05-21 Monsanto Technology Llc Transgenic maize event MON 87419 and methods of use thereof
WO2016044092A1 (en) 2014-09-17 2016-03-24 Pioneer Hi Bred International Inc Compositions and methods to control insect pests
WO2016061206A1 (en) 2014-10-16 2016-04-21 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2016099916A1 (en) 2014-12-19 2016-06-23 E. I. Du Pont De Nemours And Company Polylactic acid compositions with accelerated degradation rate and increased heat stability
WO2016114973A1 (en) 2015-01-15 2016-07-21 Pioneer Hi Bred International, Inc Insecticidal proteins and methods for their use
WO2016154631A1 (en) 2015-03-26 2016-09-29 The Texas A&M University System Conversion of lignin into bioplastics and lipid fuels
WO2016186986A1 (en) 2015-05-19 2016-11-24 Pioneer Hi Bred International Inc Insecticidal proteins and methods for their use
WO2016205445A1 (en) 2015-06-16 2016-12-22 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
WO2017023486A1 (en) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
EP3943602A1 (en) 2015-08-06 2022-01-26 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
WO2017040343A1 (en) 2015-08-28 2017-03-09 Pioneer Hi-Bred International, Inc. Ochrobactrum-mediated transformation of plants
WO2017059341A1 (en) 2015-10-02 2017-04-06 Monsanto Technology Llc Recombinant maize b chromosome sequence and uses thereof
US11732269B2 (en) 2015-10-02 2023-08-22 Monsanto Technology Llc Recombinant maize B chromosome sequence and uses thereof
WO2017105987A1 (en) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3960863A1 (en) 2016-05-04 2022-03-02 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2017192560A1 (en) 2016-05-04 2017-11-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2017218207A1 (en) 2016-06-16 2017-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
US11732268B2 (en) 2016-06-28 2023-08-22 Monsanto Technology Llc Methods and compositions for use in genome modification in plants
EP3954202A1 (en) 2016-07-01 2022-02-16 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2018005411A1 (en) 2016-07-01 2018-01-04 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2018013333A1 (en) 2016-07-12 2018-01-18 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
EP4050021A1 (en) 2016-11-01 2022-08-31 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2018084936A1 (en) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US11920140B2 (en) 2017-08-22 2024-03-05 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease
US12173295B2 (en) 2017-08-22 2024-12-24 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease
WO2019139616A1 (en) 2018-01-12 2019-07-18 The Texas A&M University System Increasing plant bioproduct yield
WO2019178042A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2019178038A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2022015619A2 (en) 2020-07-14 2022-01-20 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2023086765A2 (en) 2021-11-12 2023-05-19 Monsanto Technology Llc Compositions and methods for altering plant determinacy
US12004476B2 (en) 2022-01-26 2024-06-11 Monsanto Technology Llc. Cotton variety 20R750B3XF

Also Published As

Publication number Publication date
US7812224B2 (en) 2010-10-12
US20030115626A1 (en) 2003-06-19
WO2002068607A3 (en) 2003-02-20
US20110061137A1 (en) 2011-03-10
US8629323B2 (en) 2014-01-14
US7105724B2 (en) 2006-09-12
US20120151630A1 (en) 2012-06-14
WO2002068607A2 (en) 2002-09-06
US8119380B2 (en) 2012-02-21
US20060168700A1 (en) 2006-07-27

Similar Documents

Publication Publication Date Title
US7812224B2 (en) Methods and materials for making and using transgenic dicamba-degrading organisms
US7022896B1 (en) Methods and materials for making and using transgenic dicamba-degrading organisms
CA2285676C (en) Methods and materials for making and using transgenic dicamba-degrading organisms
EP1947926B1 (en) Novel herbicide resistance genes
EP1379539B1 (en) Methods and materials for making and using transgenic dicamba-degrading organisms
AU2002252165A1 (en) Methods and materials for making and using transgenic dicamba-degrading organisms
AU2005203471B2 (en) Methods and Materials for Making and Using Transgenic Dicamba-Degrading Organisms
AU781125B2 (en) Methods and materials for making and using transgenic dicamba-degrading organisms
Weeks et al. Methods and materials for making and using transgenic dicamba-degrading organisms
HK1152077A (en) Methods and materials for making and using transgenic dicamba-degrading organisms
MXPA99009086A (en) Methods and materials for making and using transgenic dicamba-degrading organisms
AU2012261523A1 (en) Novel herbicide resistance genes

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION