WO2002092856A1 - Procede de selection de constructions d'adn pouvant conferer une tolerance aux herbicides chez des plantes - Google Patents

Procede de selection de constructions d'adn pouvant conferer une tolerance aux herbicides chez des plantes Download PDF

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WO2002092856A1
WO2002092856A1 PCT/US2002/014793 US0214793W WO02092856A1 WO 2002092856 A1 WO2002092856 A1 WO 2002092856A1 US 0214793 W US0214793 W US 0214793W WO 02092856 A1 WO02092856 A1 WO 02092856A1
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plant
crop
tolerance
glyphosate
dna
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PCT/US2002/014793
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English (en)
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R. Eric Cerny
Yun-Chia Sophia Chen
Jeanne G. Layton
Bernard Sammons
R. Douglas Sammons
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Monsanto Technology Llc
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Priority to US10/332,494 priority Critical patent/US7977046B2/en
Publication of WO2002092856A1 publication Critical patent/WO2002092856A1/fr
Priority to US13/104,854 priority patent/US20110271399A1/en

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

Definitions

  • This invention relates in general to plant molecular biology and plant genetic engineering for herbicide tolerance and, more particularly, to a method for selecting DNA constructs with enhanced herbicide tolerance performance in transgenic crop plants from a plurality of DNA constructs.
  • the invention more particularly is related to the use of transgenic model plants and the measurement of herbicide tolerance phenotypes to select DNA constructs that are useful in crop plants.
  • One of the goals of plant genetic engineering is to produce plants with agronomically important characteristics or traits.
  • Recent advances in genetic engineering have provided the requisite tools to produce transgenic plants that contain and express foreign genes (Kahl et al, World J. of Microbiol. Biotech. 11:449-460, 1995).
  • Particularly desirable traits or qualities of interest for plant genetic engineering would include but are not limited to resistance to insects, fungal diseases, and other pests and disease-causing agents, tolerances to herbicides, enhanced stability or shelf-life, yield, environmental tolerances, and nutritional enhancements.
  • the technological advances in plant transformation and regeneration have enabled researchers to take exogenous DNA, such as a gene or genes from a heterologous or a native source, and incorporate the exogenous DNA into the plant's genome.
  • expression of a novel gene that is not normally expressed in a particular plant or plant tissue may confer a desired phenotypic effect.
  • transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.
  • a DNA construct that includes a heterologous gene sequence is introduced into a crop plant cell.
  • the plant cell is then regenerated to produce a transgenic crop plant.
  • the DNA construct includes a plant promoter that is operably linked to the heterologous gene sequence, often a promoter not normally associated with the heterologous gene.
  • the promoter controls expression of the introduced DNA sequence to which the promoter is operably linked and thus affects the desired characteristic conferred by the DNA sequence.
  • the exact level of expression of the new phenotype and its subsequent commercial utility in the crop plant is generally not known until the DNA construct has been successfully introduced into the target crop.
  • Transgenic crop plants are usually difficult, time consuming and expensive to transform, with cotton and soybeans being leading examples.
  • promoters and other regulatory elements that affect the transgene expression such that a gene or genes is transcribed efficiently at the right time during plant growth and development, in the optimal location in the plant, and in the amount necessary to produce the desired effect.
  • the level and rate of transcription of the transgene of interest is controlled by the various genetic elements of the transgene expression cassette. Promoters, introns, and leaders affect the expression level of the gene of interest, as well as the tissue localization, which is especially important for herbicide tolerance genes. It has been particularly difficult to predict the performance of DNA constructs for herbicide tolerance at a whole crop plant level.
  • Arabidopsis thaliana has a long history of use as a model plant to test the expression pattern of individual promoters, usually by placing them 5' to a reporter gene such as GUS, and the expression of transgenes. Also, many useful genes have been isolated from Arabidopsis and transferred to crop plant species. However, even though Arabidopsis genes have been isolated and transferred to other plants and DNA constructs having heterologous DNA sequences have been transformed into Arabidopsis, there has not been an effort to develop a system where Arabidopsis is used for the purpose of selecting from a number of possibly efficacious DNA constructs comprising a herbicide tolerance gene, a smaller number that would then be transformed into a crop plant of interest.
  • the method of the present invention provides a process for selecting DNA constructs that have the greatest potential for producing herbicide tolerance in crop plants.
  • the present invention provides a method of selecting a DNA construct effective for conferring herbicide tolerance in crop plants from a plurality of DNA constructs by screening the constructs for efficacy in a transgenic model plant.
  • the method includes the steps of: transforming a plant cell of a model plant with DNA constructs, each comprising a herbicide tolerance gene, regenerating the plant cell into a whole plant, treating the plant with an effective dose of the herbicide prior to flower formation, and scoring the plant for vegetative and reproductive fertility. If one or more of the DNA construct provides a high level of vegetative and reproductive herbicide tolerance in the model plant, then the construct or constructs are transformed into a crop plant cell, the crop plant cell is regenerated into a whole crop plant.
  • the crop plant is treated with an effective dose of the herbicide and further propagated to provide seeds and plants to cross with crop plants of the same species.
  • the transgenic model plant provides a high throughput testing system from which efficacious DNA constructs can be selected prior to transformation into crop plants.
  • One embodiment of the invention uses an Arabidopsis species as the model plant, preferably A. thaliana.
  • Another embodiment of the invention uses micro-tomato as the model plant.
  • Other easily transformable, non-crop plants may also be used.
  • DNA constructs comprising a herbicide tolerance gene coding sequence can be any coding sequence that confers transgenic plant cell tolerance to herbicides.
  • herbicides are glyphosate, glufosinate, sulfonylureas, arylphenoxyproprionates, imidazolinones, bromozynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, or isoxaslutole herbicides.
  • the coding sequences would encode for herbicide resistant enzymes or enzymes that degrade or detoxify the herbicide when expressed in plant cells.
  • coding sequences include, but are not limited to, native and modified plant EPSP synthases, native and modified bacterial EPSP synthases, glyphosate oxidoreductase, glutamine synthetase, phosphinothricin acetyltransferase, modified plant acetolactate synthase, modified acetyl coenzyme A carboxylase. More preferably, the herbicide tolerance gene coding sequence encodes an EPSP synthase conferring high tolerance to glyphosate herbicide while maintaining good enzymatic characteristics, such as the CP4 EPSP synthase disclosed in U.S. Patent 5,633,435, incorporated herein by reference.
  • the DNA constructs selected by the method of the present invention provide complete vegetative and reproductive herbicide tolerance to the crop plant.
  • the DNA constructs selected by the method of the present invention provide complete vegetative tolerance to the herbicide and incomplete reproductive tolerance to the crop plant, thereby providing a conditional sterility trait to the crop plant that is useful for hybrid seed production.
  • the crop species is selected from the dicot crops that include, but are not limited to, cotton, soybean, canola, tomato, and alfalfa.
  • the crop is selected from monocot crops that include, but are not limited to, corn, wheat, rice, barley, lettuce, and turf grasses.
  • Nucleic acid (sequence) or “polynucleotide (sequence)” refers to single- or double- stranded DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5' (upstream) end to the 3' (downstream) end.
  • the nucleic acid can represent the sense or complementary (antisense) strand.
  • “Native” refers to a naturally occurring (“wild-type”) nucleic acid sequence.
  • Heterologous sequence refers to a sequence which originates from a foreign source or species or, if from the same source, is modified from its original form.
  • nucleic acid sequence is substantially separated or purified away from other nucleic acid sequences with which the nucleic acid is normally associated in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal or extrachromosomal DNA.
  • the term embraces nucleic acids that are biochemically purified so as to substantially remove contaminating nucleic acids and other cellular components.
  • the term also embraces recombinant nucleic acids and chemically synthesized nucleic acids.
  • substantially purified refers to a molecule separated from other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free from the other molecules (exclusive of solvent) present in the natural mixture. The term “substantially purified” is not intended to encompass molecules present in their native state.
  • a first nucleic acid sequence displays "substantially identity" to a reference nucleic acid sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions totaling less than 20 percent of the reference sequence over the window of comparison) with the other nucleic acid (or its complementary strand), there is at least about 75% nucleotide sequence identity, preferably at least about 80% identity, more preferably at least about 85% identity, and most preferably at least about 90% identity over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of the first nucleic acid.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; preferably by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI.
  • the reference nucleic acid may be a full-length molecule or a portion of a longer molecule. Alternatively, two nucleic acids are have substantial identity if one hybridizes to the other under stringent conditions, as defined below.
  • a first nucleic acid sequence is "operably linked" with a second nucleic acid sequence when the sequences are so arranged that the first nucleic acid sequence affects the function of the second nucleic-acid sequence.
  • the two sequences are part of a single contiguous nucleic acid molecule and more preferably are adjacent.
  • a promoter is operably linked to a gene if the promoter regulates or mediates transcription of the gene in a cell.
  • a "recombinant" nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • Techniques for nucleic-acid manipulation are well-known (see for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Mailga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995; Birren et al, Genome Analysis: volume 1, Analyzing DNA, (1997), volume 2, Detecting Genes, (1998), volume 3, Cloning Systems, (1999) volume 4, Mapping Genomes, (1999), Cold Spring Harbor, New York).
  • nucleic acids Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al, J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.
  • a "synthetic nucleic acid sequence” can be designed and chemically synthesized for enhanced expression in particular host cells and for the purposes of cloning into appropriate constructs. Synthetic DNAs designed to enhance expression in a particular host should therefore reflect the pattern of codon usage in the host cell.
  • Computer programs are available for these purposes including but not limited to the "BestFit” or “Gap” programs of the Sequence Analysis Software Package, Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, WI 53711.
  • amplification of nucleic acids or “nucleic acid reproduction” refers to the production of additional copies of a nucleic acid sequence and is carried out using polymerase chain reaction (PCR) technologies.
  • PCR polymerase chain reaction
  • a variety of amplification methods are known in the art and are described, z ' wter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al, Academic Press, San Diego, 1990.
  • a primer refers to a short oligonucleotide of defined sequence which is annealed to a DNA template to initiate the polymerase chain reaction.
  • Transformed refers to a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid, such as a recombinant construct.
  • the introduced nucleic acid is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced nucleic acid is inherited by subsequent progeny.
  • a “transgenic” or “transformed” cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a "transgenic" plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant construct or construct.
  • gene refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.
  • Some genes can be transcribed into mRNA and translated into polypeptides (structural genes); other genes can be transcribed into RNA (e.g. rRNA, tRNA); and other types of gene function as regulators of expression (regulator genes).
  • “Expression” of a gene refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein. Gene expression is controlled or modulated by regulatory elements including 5' regulatory elements such as promoters.
  • Genetic component refers to any nucleic acid sequence or genetic element which may also be a component or part of an expression construct.
  • Examples of genetic components include, but are not limited to promoter regions, 5' untranslated leaders, introns, genes, 3' untranslated regions, and other regulatory sequences or sequences which affect transcription or translation of one or more nucleic acid sequences.
  • DNA construct refers to a DNA molecule capable of plant genomic integration, comprising one or more transgene DNA sequences that have been linked in a functionally operative manner using well-known recombinant DNA techniques.
  • a "plurality" of DNA constructs refers to two or more.
  • Complementary refers to the natural association of nucleic acid sequences by base- pairing (A-G-T pairs with the complementary sequence T-C-A). Complementarity between two single-stranded molecules may be partial, if only some of the nucleic acids pair are complementary; or complete, if all bases pair are complementary. The degree of complementarity affects the efficiency and strength of hybridization and amplification reactions.
  • “Homology” refers to the level of similarity between nucleic acid or amino acid sequences in terms of percent nucleotide or amino acid positional identity, respectively, i.e., sequence similarity or identity. Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • Promoter refers to a nucleic acid sequence located upstream or 5' to a translational start codon of an open reading frame (or protein-coding region) of a gene and that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.
  • a "plant promoter” is a native or non-native promoter that is functional in plant cells. Constitutive promoters are functional in most or all tissues of a plant throughout plant development. Tissue-, organ- or cell-specific promoters are expressed only or predominantly in a particular tissue, organ, or cell type, respectively.
  • a promoter may display "enhanced" expression, i.e., a higher level of expression, in one part (e.g., cell type, tissue, or organ) of the plant compared to other parts of the plant.
  • Temporally regulated promoters are functional only or predominantly during certain periods of plant development or at certain times of day, as in the case of genes associated with circadian rhythm, for example.
  • Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.
  • Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
  • Suitable expression constructs for introducing exogenous DNA into plant cells are well known to those of skill in the art. They would include, but are not limited to, disarmed Ti- plasmids for Agj'obacterium-mediated methods. These constructs can contain a resistance marker (although usually not necessary to the practice of the present invention in addition to the herbicide resistance coding sequence), one or more T-DNA borders, and origins of replication for E. coli and Agrobacterium along with one or more genes of interest and associated regulatory regions.
  • Those of skill in the art are aware that for Agrobacterium- e ⁇ iat ⁇ d - transformation a number of strains and methods are available. Such strains would include but are not limited to Agrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularly preferred strains are Agrobacterium tumefaciens strains.
  • Suitable methods include but are not limited to bacterial infection (e.g., with Agrobacterium as described above), binary bacterial artificial chromosome constructs, direct delivery of DNA (e.g. via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers), and acceleration of DNA coated particles (reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991).
  • transgenic plants reported include but are not limited to cotton (U. S. Patent No. 5,004,863; U. S. Patent No. 5,159,135; U. S. Patent No. 5,518,908, WO 97/43430), soybean (U. S. Patent No. 5,569,834; U. S. Patent No. 5,416,011; McCabe et al, Bio/Technology, 6:923, 1988; Christou et al, Plant Physiol., 87:671, 1988); Brassica (U. S. Patent No. 5,463,174), and peanut (Cheng et al, Plant Cell Rep., 15: 653, 1996).
  • Exemplary nucleic acids which may be introduced by the methods encompassed by the present invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques.
  • exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, or structure as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express.
  • exogenous gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell.
  • the type of DNA included in the exogenous DNA can include DNA which is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
  • N-phosphonomethylglycine also known as glyphosate
  • Glyphosate is a well known herbicide that has activity on a broad spectrum of plant species.
  • Glyphosate is the active ingredient of Roundup® (Monsanto Co.), a safe herbicide having a desirably short half life in the environment. When applied onto a plant surface, glyphosate moves systemically through the plant. Glyphosate is toxic to plants by inhibiting the shikimic acid pathway that provides a precursor for the synthesis of aromatic amino acids.
  • glyphosate affects the conversion of phosphoenolpyruvate and 3-phosphoshikimic acid to 5-enolpyruvyl-3- phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid (hereinafter referred to as EPSP synthase or EPSPS).
  • EPSP synthase EPSPS
  • the term "glyphosate” should be considered to include any herbicidally effective form of N- phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in planta.
  • glyphosate tolerant plants by inserting into the plant genome a DNA molecule that causes the production of higher levels of wild-type EPSPS (Shah et al, Science 233:478-481 (1986).
  • Glyphosate tolerance can also be achieved by the expression of EPSPS variants that have lower affinity for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (U.S. Patent No.4,535,060) or the expression of EPSPS genes isolated from organisms resistant to glyphosate activity (U.S. Patent No. 5,633,435).
  • Variants of the wild-type EPSPS enzyme have been isolated that are glyphosate-resistant as a result of alterations in the EPSPS amino acid coding sequence (Kishore et al, Annu. Rev. Biochem. 57:627-663 (1988); Schulz et al, Arch. Microbiol. 137:121-123 (1984); Sost et al, FEBS Lett. 173:238-241 (1984); Kishore et al, In "Biotechnology for Crop Protection” ACS Symposium Series No. 379. Eds. Hedlin et al, 37-48 (1988). Enzymes that degrade glyphosate in the plant tissues (U.S. Patent No.
  • glyphosate tolerance has been genetically engineered into corn (U.S. Patent No. 5,554,798), wheat (Zhou et al. Plant Cell Rep. 15:159-163, (1995), soybean (WO 9200377) and canola (WO 9204449).
  • glyphosate is used herein to refer collectively to the parent herbicide N-phosphonomethylglycine (otherwise known as glyphosate acid), to a salt or ester thereof, or to a compound which is converted to N-phosphonomethylglycine in plant tissues or which otherwise provides N-phosphonomethylglycine in ionic form (otherwise known as glyphosate ion).
  • N-phosphonomethylglycine otherwise known as glyphosate acid
  • salt or ester thereof or to a compound which is converted to N-phosphonomethylglycine in plant tissues or which otherwise provides N-phosphonomethylglycine in ionic form
  • water-soluble glyphosate salts useful herein are disclosed in U.S. Patents No. 3,799,758 and No. 4,405,531 to Franz, the disclosure of which is incorporated herein by reference.
  • Glyphosate salts that can be used according to the present invention include but are not restricted to alkali metal, for example sodium and potassium, salts; ammonium salt; C,. 16 alkylammonium, for example dimethylammonium and isopropylammonium, salts; C 6 alkanolammonium, for example monoethanolammonium, salt; C I-16 alkylsulfonium, for example trimethylsulfonium, salts; mixtures thereof and the like.
  • the glyphosate acid molecule has three acid sites having different pKa values; accordingly mono-, di- and tribasic salts, or any mixture thereof, or salts of any intermediate level of neutralization, can be used.
  • Glyphosate salts are commercially significant in part because they are water-soluble. Many ammonium, alkylammonium, alkanolammonium, alkylsulfonium and alkali metal salts are ' highly water-soluble, allowing for formulation as highly concentrated aqueous solutions which can be diluted in water at the point of use. Such concentrated aqueous solutions can contain about 50 to about 500 grams per liter of glyphosate, expressed as acid equivalent (g a.e./l). Higher glyphosate concentrations, for example about 300 to about 500 g a.e./l, are preferred.
  • Glyphosate salts are alternatively formulated as water-soluble or water-dispersible compositions, in the form for example of powders, granules, pellets or tablets. Such compositions are often known as dry formulations, although the term "dry” should not be understood in this context to imply the complete absence of water. Typically, dry formulations contain less than about 5% by weight of water, for example about 0.5% to about 2% by weight of water. Such formulations are intended for dissolution or dispersion in water at the point of use.
  • Contemplated dry glyphosate formulations can contain about 5% to about 80% by weight of glyphosate, expressed as acid equivalent (% a.e.). Higher glyphosate concentrations within the above range, for example about 50% to about 80% a.e., are preferred.
  • Especially useful salts of glyphosate for making dry formulations are sodium and ammonium salts.
  • Plant treatment compositions and liquid and dry concentrate compositions of the invention can optionally contain one or more desired excipient ingredients.
  • Especially useful excipient ingredients for glyphosate compositions are surfactants, which assist in retention of aqueous spray solutions on the relatively hydrophobic surfaces of plant leaves, as well as helping the glyphosate to penetrate the waxy outer layer (cuticle) of the leaf and thereby contact living tissues within the leaf.
  • surfactants can perform other useful functions as well.
  • surfactant there is no restriction in the type or chemical class of surfactant that can be used in glyphosate compositions of the invention. Noriionic, anionic, cationic and amphoteric types, or combinations of more than one of these types, are all useful in particular situations. However, it is generally preferred that at least one of the surfactants, if any, present should be other than anionic, i.e., at least one of the surfactants should be nonionic, cationic or amphoteric.
  • Standard reference sources from which one of skill in the art can select suitable surfactants include Handbook of Industrial Surfactants, Second Edition (1997) published by Gower, McCutcheon 's Emulsifiers and Detergents, North American and International Editions (1997) published by MC Publishing Company, and International Cosmetic Ingredient Dictionary, Sixth Edition (1995) Volumes 1 and 2, published by the Cosmetic, Toiletry and Fragrance Association.
  • Examples of commercial formulations of glyphosate include, without restriction, those sold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, all of which contain glyphosate as its isopropylammonium salt; those sold by Monsanto Company as ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as its ammonium salt; that sold by Monsanto Company as ROUNDUP® GEOFORCE, which contains glyphosate as its sodium salt; and that sold by Zeneca Limited as TOUCHDOWN® herbicide, which contains glyphosate as its trimethylsulfonium salt.
  • a glyphosate-containing herbicide is applied to the plant comprising the DNA constructs of the present invention, and the plants are evaluated for vegetative and reproductive tolerance to the glyphosate herbicide.
  • Any formulation of glyphosate can be used for testing plants comprising the DNA constructs of the present invention.
  • a glyphosate composition such as Roundup® Ultra can be used. The testing parameters for an evaluation of the glyphosate tolerance of the plant will vary depending on a number of factors.
  • Factors would include, but are not limited to the type of glyphosate formulation, the concentration and amount of glyphosate used in the formulation, the type of plant, the plant developmental stage during the time of the application, environmental conditions, the application method, and the number of times a particular formulation is applied.
  • plants can be tested in a greenhouse environment using a spray application method.
  • the testing range using Roundup® Ultra can include, but is not limited to 8 oz/acre to 256 oz/acre.
  • the preferred commercially effective range can be from 16 oz/acre to 64 oz/acre of Roundup® Ultra, depending on the crop and stage of plant development.
  • a crop can be sprayed with at least one application of a glyphosate formulation.
  • test parameters can be optimized for each crop in order to find the particular plant comprising the constructs of the present invention that confers the desired commercially effective glyphosate tolerance level.
  • the present method for selecting DNA constructs in a model plant can be applied to selecting DNA constructs that are useful for producing male or female sterile transgenic crop plants and their use in the production of hybrid seed, including hybrid seed with restored fertility.
  • a method of producing hybrid seed of a crop plant that comprises first regenerating a crop plant from a transformed crop plant cell that contains a DNA construct selected by the method of the present invention. The method being applied to select a DNA construct wherein the transgenic model plant is vegetatively tolerant to a herbicide and reproductively sterile, preferably male sterile.
  • the method provides a DNA construct which when incorporated in a transgenic crop plant will result in plants that are, when exposed to glyphosate, produce male sterile, female fertile plants.
  • this transgenic crop plant serves as the seed parent plant and is sprayed with glyphosate and rendered male sterile, but remains female fertile and is pollinated by the pollen from a male fertile parent plant.
  • the DNA constructs used are either pUC cloning constructs or double border plant transformation constructs that contain DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an E. coli origin of replication such as or ⁇ ill, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spc/Str that encodes for Tn7 aminoglycoside adenyltransferase (aadA) confers resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker.
  • the host bacterial strain is Agrobacterium tumefaciens ABI or LBA4404.
  • DNA constructs used in the method of the current invention comprise any promoter known to function to cause the transcription in plant cells and any herbicide tolerance encoding polynucleotide sequence known to confer herbicide tolerance to plant cells.
  • the herbicide tolerance polynucleotide sequences include, but are not limited to polynucleotide sequences encoding for proteins involved in herbicide tolerance encoding for 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S.
  • Patent Number 4,810,648 phytoene desaturase (crtl (Misawa et al, (1993) Plant Journal 4:833-840, and (1994) Plant Jour 6:481-489) for tolerance to norflurazon, acetohydroxyacid synthase (AHAS, Sathasiivan et al. (1990) Nuc Acids Res. 18:2188-2193) and the bar gene for tolerance to glufosinate (DeBlock, et al. (1987) EMBO J. 6:2513-2519.
  • Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides.
  • transgene DNA constructs used for plant transformation and expression of transgenes in plants include, but are not limited to: the P-CaMV.35S promoter (U.S. Patent No. 5,858,742, herein incorporated by reference in its entirety), for example, the enhanced P-CaMV.35S from Cauliflower mosaic virus containing a duplication of the -90-300 region as described in U. S. Patent No. 5,424,200, herein incorporated by reference in its entirety; the Figwort mosaic virus promoter, P-FMV, as described in U. S. Patent No.
  • Patent No. 5,633,435, herein incorporated by reference in its entirety The method of the present invention enables one of skill in the art of plant molecular biology to design and assemble plant expression cassettes that contain promoters of known and unknown function.
  • the genetic elements of the DNA construct further comprise 5' leader polynucleotides for example, the Hsp70 non-translated leader sequence from Petunia hybrida as described in U. S. Patent No. 5,362,865, herein incorporated by reference in its entirety.
  • the genetic elements further comprise herbicide tolerance genes that include, but are not limited to, for example, the aroA:CP4 coding region for EPSPS glyphosate resistant enzyme isolated from Agrobacterium tumefaciens (AGRTU) strain CP4 as described in U. S. Patent No. 5,633,435, herein incorporated by reference in its entirety.
  • the genetic elements of the DNA construct further comprise termination regions that include, but are not limited to, the E9 3 ' termination region the pea RbcS gene that functions as a polyadenylation signal; the nos is the 3' end of the nopaline synthase gene that functions as a polyadenylation signal.
  • the genetic elements of the DNA construct further comprise the right border (RB) and left borders (LB) of the Ti plasmid of Agrobacterium tumefaciens octopine and nopaline strains.
  • Arabidopsis embryos have been transformed by an Agrobacterium mediated method described by Bechtold N, et al, CR Acad Sci Paris Sciences di la vie/life sciences 316: 1194- 1199, (1993). This method has been modified for use with the constructs of the present invention to provide a rapid and efficient method to transform Arabidopsis and select for a herbicide tolerant phenotype
  • An Agrobacterium strain ABI containing a DNA construct is prepared as inoculum by growing in a culture tube containing 10 mis Luria Broth and antibiotics, for example, 1 ml/L each of spectinomycin (100 mg/ml), chloramphenicol (25 mg/ml), kanamycin (50 mg/ml) or the appropriate antibiotics as determined by those skilled in the art. The culture is shaken in the dark at 28° C for approximately 16 - 20 hours.
  • the Agrobacterium inoculum is pelleted by centrifugation and resuspended in 25 ml Infiltration Medium (MS Basal Salts 0.5%, Gamborg's B-5 Vitamins 1%, Sucrose 5%, MES 0.5 g/L, pH 5.7) with 0.44 nM benzylaminopurine (10 ul of a 1.0 mg/L stock in DMSO per liter) and 0.02% Silwet L-77 to an OD 600 of 0.6.
  • the transgenic Arabidopsis plants produced by the infiltrated seed embryos are selected from the nontransgenic plants by a germination selection method.
  • the harvested seed is surface sterilized then spread onto the surface of selection media plates containing MS Basal Salts 4.3 g/L, Gamborg'a B-5 (500 X) 2.0 g/L, Sucrose 10 g/L, MES 0.5 g/L, and 8 g/L Phytagar with Carbenicillin 250mg/L, Cefotaxime 100 mg/L, and PPM 2 ml/L and appropriate selection agent added as a filter sterilized liquid solution, after autoclaving.
  • the selection agent can be an antibiotic or herbicide, for example kanamycin 60 mg/L, glyphosate 40-60 ⁇ M, or bialaphos 10 mg/L are appropriate concentrations to incorporate into the media depending on the DNA construct and the plant expression cassettes contained therein that are used to transform the embryos.
  • glyphosate selection the sucrose is deleted from the basal medium. Put plates into a box in a 4° C to allow the seeds to vernalize for ⁇ 2-4 days. After seeds are vernalized, transfer to a growth chamber with cool white light bulbs at a 16/8 light/dark cycle and a temperature of 23 C. After 5-10 days at ⁇ 23°C and a 16/8 light cycle, the transformed plants will be visible as green plants. After another 1-2 weeks, plants will have at least one set of true leaves. Transfer plants to soil, cover with a germination dome, and move to a growth chamber, keep covered until new growth is apparent, usually 5-7 days.
  • Micro-Tom seeds (Ball Seed Co., West Chicago, IL) are surface-sterilized by soaking in 25% Chlorox with 1-2 drops of Tween 20 for 10 minutes while stirring frequently. The seeds are then rinsed 3 times with sterile deionized water. The seeds are germinated in phytatrays maintained at 28°C under the dark for 5 days. The phytatray cultures are transferred and grown at 24°C under a 16h photoperiod until the seedlings are open in a very narrow "V shape to produce cotyledons which are at the appropriate stage for explanting. Typically, the seedlings will be explanted about six days after germination.
  • a loopful of the Agrobacterium bacterium cells transformed with the a DNA construct containing a glyphosate tolerance plant expression cassette is transferred from a plate culture and inoculated into 2 ml of Luria Broth + antibiotics in a 17 X 100 Falcon tube, and grown at about 28 C° on a rotator at the speed between medium to high for about 16 hours.
  • Approximately 0.2 ml of the liquid bacterium culture is inoculated into 2 ml of Luria Broth + 0.2 mM acetosyringone in a 17 X 100 mm Falcon tube, and grown at about 28 C° on a rotator at the speed between medium to high for 4 hours.
  • An O.D. 660 0.1 is the desired density for the Agrobacterium inoculum for use in the inoculation of the explants.
  • Micro-Tom cotyledon are excised from the narrow "V" shaped seedlings to make the explants for inoculation.
  • About 50 cotyledon explants are prepared by bathing them in 6 ml of TXD liquid medium and trimming them at both ends using a #15 feather i scapel blade. Immediately after the trimming, the 50 cotyledon explants are gently placed on "feeder plates".
  • the "feeder plates” are made by overlaying 2 ml of tobacco suspension cells and a sterile filter paper on the plates containing UC2PC medium.
  • the cotyledon explants are gently poked with a sharp forcep (ASIM5311, Stainless French), making six to ten pokes per explant cotyledon.
  • the explants are inoculated with 3 ml of Agrobacterium inoculum by pipetting the inoculum on to explants. The plates are incubated for 10 minutes at room temperature and the Agrobacterium inoculum is then aspirated off with a sterile pipette. The explants are then co-cultured for 2 days at 23 - 24 °C, 16 h photoperiod in plastic bags.
  • delay medium which is an MS based medium that is supplemented after autoclaving with 500 mg/L carbenicillin, 100 mg/L cefotaxime, 0.1 mg/L IAA, and 2 mg/L zeatin riboside (Biosynth), at a density of 12-16 explants per plate for 7 days at 23 - 24° C, 16 h photoperiod.
  • Explants are transferred to shoot induction medium supplemented after autoclaving with 500 mg/L carbenicillin, 100 mg/L cefotaxime, 0.1 mg/L IAA, and 2 mg/L zeatin riboside (Biosynth, ), and 0.03 mM glyphosate, at a density of 9 explants per plate. All culture plates are placed inside a plastic bag and cultured at 23 - 24 °C, 16 h photoperiod
  • Explants with callus and/or small buds are separated and transferred to shoot elongation medium (CPEl) that is supplemented after autoclaving with 500 mg/L carbenicillin, 100 mg/L cefotaxime, 0.1 mg/L IAA, and 0.5 mg/L zeatin riboside (Biosynth, ), and 0.03 mM glyphosate, at a density of 5 explants per plate. They are cultured for three to four weeks, for shoot elongation. All plates are cultured in plastic bags, at 23 - 24° C, 16 h photoperiod.
  • CPEl shoot elongation medium
  • shoots elongate (about 2 to 3 cm in length) they are excised from explants, and transferred to rooting medium, that has been supplemented after autoclaving with
  • This medium is used for delay, selection and elongation steps
  • Micro Tom Rooting Medium Basal Recipe This medium is used for the rooting medium. For 1 liter
  • Cotton transformation is performed essentially as described in WO/0036911, herein incorporated by reference in its entirety, or as described in U.S. Patent No. 5,846,797, herein incorporated by reference in its entirety.
  • a modification of these methods can include but is not limited to the following example.
  • Coker 130 seed is surface sterilized and germinated in the dark. Hypocotyl explants are cut from the germinated seedlings to lengths of about 1-1.5 cm.
  • Agrobacterium tumefaciens strain ABI transformed to contain one of the DNA constructs pMON15737, pMON26140, pMON45331, pMON51915, and pMON52060 is grown in Luria broth without antibiotics forl ⁇ hours at 28 °C, then diluted to approximately 2 X 10 8 bacteria/ml.
  • the hypotocyl explant is submersed in the Agro inoculum for 2-5 minutes, then co- cultivated for about 45 hours on MS+1.9mg/l KN03+3%glucose (TRM), 30 explants per plate, 24C, in the dark.
  • the explants are transferred to TRM containing 150 mg/1 cefotaxime and 300 ⁇ M glyphosate for four culture periods, each period for approximately six weeks.
  • Embryogenic calli is segregated from the primary explant at the end of 3 rd or 4th culture periods and placed onto same medium.
  • the embryogenic calli are subcultured once by briefly suspending in liquid TRM +3% glucose, followed by pouring suspension onto 'TRM' + 150mg/l cefotaxime + 300 ⁇ M glyphosate plates.
  • the somatic embryos are harvested 3-8 weeks after the liquid subculture, then grown on Stewart and Hsu media with 0.5% glucose. Plantlets derived from the somatic embryos are matured to about 4-7cm (3-6 leaves) in Magenta boxes with Stewart & Hsu modified with 40mM NO3/10mM NH4 + 2% sucrose. These plants are then transplanted to potting soil, 4" pots, 100% humidity, 16hours of light per day, for 4-6 days, followed by 50% humidity 5-10 days. Tobacco transformation
  • Tobacco transformation is performed as follows: stock tobacco plants maintained by in- vitro propagation. Stems are cut into sections and placed into phytatrays. Leaf tissue is cut and placed onto solid pre-culture plates, MS 104 to which 2 mis of liquid TXD meduim and a sterile Whatman filter disc have been added. Pre-culture the explants in warm room (23° Celsius, continuous light) for 1-2 days. The day before Agro inoculation, a 10 ⁇ l loop of a transformed Agrobacterium containing one of the DNA constructs is placed into a tube containing 10 mis of YEP media with appropriate antibiotics to maintain selection of the DNA construct. The tube is put into a shaker to grow overnight at 28 °C. The OD 600 of the Agrobacterium is adjusted to
  • Soybean transformation is performed essentially as described in WO 00/42207, herein incorporated by reference in its entirety.
  • DNA constructs are selected from a plurality of DNA constructs that will perform in a manner predictive of the herbicide tolerance performance observed in Arabidopsis for a glyphosate herbicide tolerant phenotype.
  • DNA constructs for example, pMON15737 ( Figure 1), pMON26140 ( Figure 2), pMON40500 (Figure 3), pMON40501 ( Figure 4), pMON51915 (Figure 5), pMON52059 ( Figure 6), pMON52060 ( Figure 7), pMON52065 ( Figure 8), pMON54042 ( Figure 9), pMON54045 (Figure 10), pMON54047 ( Figure 11), and pMON54049 (Figure 12) are transformed into Arabidopsis plants by vacuum infiltration and into crop plants by Agrobacterium mediated methods as described herein for each crop.
  • callus tolerance, vegetative tolerance and reproductive tolerance are scored or rated as good, poor, or percent reproductive tolerance (generally measured as number of seeds produced relative to the unsprayed check plants, except for cotton where a boll plant map rating system is used to measure glyphosate tolerance.
  • the efficacy of the DNA constructs, pMON52059 plant expression cassette P-FMV- At.EFla /At.CTP2- r ⁇ :CP4/E9 3' and pMON15737 plant expression cassette P- FMV7At.CTP2- ⁇ r ⁇ /4:CP4/E93' is compared in transgenic Arabidopsis thaliana.
  • the transgenic Arabidopsis thaliana plants are produced by the vacuum infiltration (Bechtold et al, C R Acad Paris Life Sci 316: 1194-1199) seeds are potted in soil in trays in a growth chamber adjusted for 24°C, 16 hour light (120 ⁇ E m "2 s "1 ) cycle to permit normal growth and development of the plants.
  • the pMON52059 VI event glyphosate tolerant transgenic Arabidopsis plants are selected by spray application of glyphosate herbicide at a rate of 24 ounces/acre, the surviving plants are transplanted into individual pots. Eight pMON52059 VI plants and eight pMON15737 homozygous plants are sprayed a second time corresponding to the observation of bolting, approximately 16 days after the at a rate of 24 ounces/acre. The second spray will determine the efficacy of the two constructs for conferring reproductive tolerance. The plants are observed for vegetative effects of glyphosate application. All plants had complete vegetative tolerance and no abnormal flowers are observed. However, abortion of siliques occurred indicated that seed had not been set in the aborted siliques.
  • DNA constructs are transformed into Arabidopsis and transgenic lines assayed for vegetative and reproductive tolerance. Selected constructs are transformed into cotton plants, then are tested in a greenhouse spray test using Roundup UltraTM a glyphosate formulation with a Track Sprayer device (Roundup Ultra is a registered trademark of Monsanto Company). Plants are treated at the "two" true leaf or greater stage of growth and the leaves are dry before applying the Roundup® spray.
  • the formulation used is Roundup UltraTM as a 3 lb/gallon a.e. (acid equivalent) formulation.
  • the calibration used is as follows:
  • the spray concentrations will vary, depending on the desired testing ranges. For example, for a desired rate of 8 oz/acre a working solution of 3.1 ml/L is used, and for a desired rate of 64 oz/A a working range of 24.8 ml/L is used.
  • the treated plants are evaluated for vegetative tolerance to glyphosate injury and for reproductive tolerance. Reproductive tolerance is determine by counting the number of first position bolls remaining after treatment for the first five branches.
  • pMON26140 and pMON52061 show good vegetative tolerance, but no reproductive tolerance in Arabidopsis. Constructs demonstrating this phenotype are useful in a hybrid seed production system so pMON26140 was tested in transgenic cotton. The results confirm that vegetative tolerance is good, but reproductive tolerance is markedly reduced. The boll rating of " ⁇ 1" indicates that less than one cotton boll is set at any first boll position of the first five branches.
  • pMON40500 and pMON40501 show low vegetative tolerance and no reproductive tolerance in Arabidopsis and are not transformed into cotton.
  • pMON51915 and pMON52060 show good vegetative tolerance in Arabidopsis and 70% and 78% reproductive tolerance, respectively.
  • DNA constructs are transformed into Arabidopsis and transgenic lines assayed for vegetative and reproductive tolerance.
  • the same constructs are transformed into tomato to confirm the glyphosate tolerant phenotype observed in Arabidopsis relative to tomato (Table 3).
  • Transgenic lines produced from Arabidopsis and tomato are treated with a track sprayer at 48 oz/ac or an effective dose of glyphosate at the 5-6 leaf stage or prior to flowering.
  • pMON26140 shows good vegetative tolerance in Arabidopsis, low vegetative tolerance in tomato, and no reproductive tolerance in either plant.
  • pMON52065, pMON51915, andpMON52060 show good vegetative and reproductive tolerance in Arabidopsis and similarly good tolerance in tomato (percent seed set relative to unsprayed check).
  • DNA constructs, pMON26140, pMON40500, pMON-40501, pMON51915, pMON52050, and pMON52060 are transformed into Arabidopsis and soybean to confirm the method of the present invention for use in soybean (Table 4).
  • DNA constructs, pMON40500 and pMON40501 show poor vegetative tolerance in Arabidopsis and no reproductive tolerance.
  • pMON26140 shows good vegetative tolerance in Arabidopsis, but no reproductive tolerance.
  • This construct shows a similar activity in soybean.
  • the constructs, pMON51915, pMON52059 and pMON52060 show good vegetative and reproductive tolerance in Arabidopsis and the first two of these constructs have similar efficacy in soybean. Soybean plants transformed with pMON51915 also showed good vegetative tolerance to glyphosate, but were not tested for reproductive tolerance.

Abstract

L'invention concerne un procédé amélioré qui permet de choisir parmi des constructions d'ADN conférant une tolérance aux herbicides celles qui confèrent à des plantes cultivées une tolérance végétative et reproductive supérieure. Elle concerne en outre un procédé amélioré qui permet de choisir parmi des constructions d'ADN conférant une tolérance aux herbicides celles qui confèrent une tolérance supérieure à des tissus végétatifs de plantes cultivées mais une tolérance minime ou nulle à des tissus reproductifs mâles de plantes cultivées dans le but de produire des plantes mâles stériles utiles dans un système de production de cultures hybrides.
PCT/US2002/014793 2001-05-15 2002-05-10 Procede de selection de constructions d'adn pouvant conferer une tolerance aux herbicides chez des plantes WO2002092856A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006023869A2 (fr) 2004-08-24 2006-03-02 Monsanto Technology Llc Elements regulateurs non codants de gene de proteine de translocateur d'adenylate utilises dans des vegetaux
EP1794310A2 (fr) * 2004-08-25 2007-06-13 Monsanto Technology, LLC Elements regulateurs non codants de genes proteiques de l'aspartique proteinase destines a etre utilises dans des plantes
US7622641B2 (en) 2005-08-24 2009-11-24 Pioneer Hi-Bred Int'l., Inc. Methods and compositions for providing tolerance to multiple herbicides
US9944945B2 (en) 2005-05-27 2018-04-17 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof

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US5633435A (en) * 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

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US5633435A (en) * 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006023869A2 (fr) 2004-08-24 2006-03-02 Monsanto Technology Llc Elements regulateurs non codants de gene de proteine de translocateur d'adenylate utilises dans des vegetaux
EP1794310A2 (fr) * 2004-08-25 2007-06-13 Monsanto Technology, LLC Elements regulateurs non codants de genes proteiques de l'aspartique proteinase destines a etre utilises dans des plantes
EP1794310A4 (fr) * 2004-08-25 2010-07-21 Monsanto Technology Llc Elements regulateurs non codants de genes proteiques de l'aspartique proteinase destines a etre utilises dans des plantes
US9944945B2 (en) 2005-05-27 2018-04-17 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US10738320B2 (en) 2005-05-27 2020-08-11 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US11390881B2 (en) 2005-05-27 2022-07-19 Monsanto Technology, Llc Soybean event MON89788 and methods for detection thereof
US7622641B2 (en) 2005-08-24 2009-11-24 Pioneer Hi-Bred Int'l., Inc. Methods and compositions for providing tolerance to multiple herbicides
US7803992B2 (en) 2005-08-24 2010-09-28 Pioneer Hi-Bred International, Inc. Methods and compositions for expressing an herbicide-tolerant polynucleotide
US7973218B2 (en) 2005-08-24 2011-07-05 Pioneer Hi-Bred International, Inc. Methods and compositions for controlling weeds
US8203033B2 (en) 2005-08-24 2012-06-19 Pioneer Hi-Bred International, Inc. Methods and compositions for providing tolerance to multiple herbicides

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