Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N39/00—Biocides, pest repellants or attractants, or plant growth regulators containing aryloxy- or arylthio-aliphatic or cycloaliphatic compounds, containing the group or, e.g. phenoxyethylamine, phenylthio-acetonitrile, phenoxyacetone
  • A01N39/02—Aryloxy-carboxylic acids; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G7/00—Botany in general
  • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N39/00—Biocides, pest repellants or attractants, or plant growth regulators containing aryloxy- or arylthio-aliphatic or cycloaliphatic compounds, containing the group or, e.g. phenoxyethylamine, phenylthio-acetonitrile, phenoxyacetone
  • A01N39/02—Aryloxy-carboxylic acids; Derivatives thereof
  • A01N39/04—Aryloxy-acetic acids; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
  • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
  • A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
  • C12N15/8275—Glyphosate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
• • 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)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

• Crops such as corn, soybeans, canola, cotton, sugar beets, wheat, turf, and rice, have been developed that are resistant to glyphosate.
• fields with actively growing glyphosate resistant corn for example, can be sprayed to control weeds without significantly damaging the corn plants.
• GTCs glyphosate tolerant crops
• GTCs glyphosate tolerant soybean, cotton, corn, and canola are commercially available in the United States and elsewhere in the Western Hemisphere. More GTCs (e.g., wheat, rice, sugar beets, turf, etc.) are poised for introduction pending global market acceptance.
• glyphosate resistant species are in experimental to development stages (e.g., alfalfa, sugar cane, sunflower, beets, peas, carrot, cucumber, lettuce, onion, strawberry, tomato, and tobacco; forestry species like poplar and sweetgum; and horticultural species like marigold, petunia, and begonias; see "isb.vt.edu/cfdocs/fieldtestsl .cfm, 2005" website). Additionally, the cost of glyphosate has dropped dramatically in recent years to the point that few conventional weed control programs can effectively compete on price and performance with glyphosate GTC systems.
• Glyphosate has been used successfully in burndown and other non-crop areas for total vegetation control for more than 15 years.
• glyphosate has been used 1-3 times per year for 3, 5, 10, up to 15 years in a row.
• Resistant weeds include both grass and broadleaf species— Lolium rigidum, Lolium multiflorum, Eleusine indica, Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis, and Plantago lanceolata.
• GTCs which comprise >80 of U.S. cotton and soybean acres and >20 of U.S. corn acres (Gianessi, 2005). These weed shifts are occurring predominantly with (but not exclusively) difficult-to-control broadleaf weeds.
• Some examples include Ipomoea, Amaranthus, Chenopodium, Taraxacum, and Commelina species.
• growers can compensate for glyphosate' s weaknesses by tank mixing or alternating with other herbicides that will control the missed weeds.
• One popular and efficacious tank mix partner for controlling broadleaf escapes in many instances has been 2,4- diclorophenoxyacetic acid (2,4-D).
• 2,4-D has been used agronomically and in non-crop situations for broad spectrum, broadleaf weed control for more than 60 years. Individual cases of more tolerant species have been reported, but 2,4-D remains one of the most widely used herbicides globally.
• 2,4-D selectivity in dicot crops like soybean or cotton is very poor, and hence 2,4-D is not typically used on (and generally not near) sensitive dicot crops. Additionally, 2,4-D' s use in grass crops is somewhat limited by the nature of crop injury that can occur. 2,4-D in combination with glyphosate has been used to provide a more robust burndown treatment prior to planting no-till soybeans and cotton; however, due to these dicot species' sensitivity to 2,4-D, these burndown treatments must occur at least 14-30 days prior to planting (Agriliance, 2003).
• 2,4-D is in the phenoxy acid class of herbicides, as is MCPA. 2,4-D has been used in many monocot crops (such as corn, wheat, and rice) for the selective control of broadleaf weeds without severely damaging the desired crop plants. 2,4-D is a synthetic auxin derivative that acts to deregulate normal cell-hormone homeostasis and impede balanced, controlled growth; however, the exact mode of action is still not known. Triclopyr and fluroxypyr are pyridyloxyacetic acid herbicides whose mode of action is as a synthetic auxin, also.
• 2,4-D mineralization of 2,4-D.
• Successive applications of the herbicide select for microbes that can utilize the herbicide as a carbon source for growth, giving them a competitive advantage in the soil.
• 2,4-D currently formulated has a relatively short soil half-life, and no significant carryover effects to subsequent crops are encountered. This adds to the herbicidal utility of 2,4-D.
• TfdA has been used in transgenic plants to impart 2,4-D resistance in dicot plants (e.g., cotton and tobacco) normally sensitive to 2,4-D (Streber et al. (1989), Lyon et al. (1989), Lyon (1993), and U.S. Patent No. 5,608,147).
• tfdA-type genes that encode proteins capable of degrading 2,4-D have been identified from the environment and deposited into the Genbank database. Many homologues are similar to tfdA (>85 amino acid identity) and have similar enzymatic properties to tfdA. However, there are a number of homologues that have a significantly lower identity to tfdA (25-50%), yet have the characteristic residues associated with cc-ketoglutarate dioxygenase Fe +2 dioxygenases. It is therefore not obvious what the substrate specificities of these divergent dioxygenases are.
• sdpA from Delftia acidovorans (Kohler et ah, 1999, Westendorf et ah, 2002, Westendorf et ah, 2003). This enzyme has been shown to catalyze the first step in (S)-dichlorprop (and other (S)- phenoxypropionic acids) as well as 2,4-D (a phenoxyacetic acid) mineralization (Westendorf et ah, 2003). Transformation of this gene into plants, has not heretofore been reported.
• HTC herbicide-tolerant crop
• Aryloxyalkanoate chemical substructures are a common entity of many
• aryloxyphenoxypropionates acetyl-coenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop), and 5-substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as pyraflufen and flumiclorac).
• ACCase carboxylase
• haloxyfop haloxyfop
• quizalofop quizalofop
• diclofop diclofop
• 5-substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors such as pyraflufen and flumiclorac
• This invention is related to methods for improving plant height and/or yield of crop plants which are resistant to herbicide 2,4-D by treating the plants with 2,4-D at application rates which are not harmful to the plants.
• a method using 2,4-D application to increase yield of crop plants which express AAD-12 gene for 2,4-D resistance.
• This invention further relates to the use of 2,4-D for improving the yield of crop plants which are 2,4-D resistant.
• the method provided is of particular interest for the treatment of crops plants including maize, soybean, spring and winter oil seed rape (canola), sugar beet, wheat, sunflower, barley, and rice.
• the 2,4-D resistant crop plants are transgenic crop plants transformed with an aryloxyalkanoate dioxygenase (AAD).
• AAD aryloxyalkanoate dioxygenase
• the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD-12.
• AAD-1 has been previously disclosed in US 2009/0093366 and AAD- 12 has been previously disclosed in WO 2007/053482, the contents of which are incorporated by reference in their entireties.
• the yield-improving effect of the treatment of 2,4-D can be observed at application rates from 25 g ae/ha to 5000 g/ha, or 100 g ae/ha to 2500 g ae/ha, or in particular, 1000 g ae/ha to 2000 g ae/ha.
• 1000 g ae/ha to 1500 g ae/ha of 2,4-D is used.
• 2000 g ae/ha to 2500 g ae/ha is used.
• the yield-improving effect of the treatment of 2,4-D is D is particularly pronounced when 2,4-D is applied in the 2- to 8- leaf stage of the crop plants before flowering.
• the application rate and/or leaf-stage of the crop plant required vary as a function of the plants, their height and the climate conditions.
• the term increase in yield refers to that the plant yield up to 50% or more. In one embodiment, the increase in yield is at least 10%. In another embodiment, the increase in yield is at least 20%. In another embodiment, the increase in yield is from 10% to 60%. In another embodiment, the increase in yield is from 20% to 50%. In another embodiment, the increase in yield is statistically significant.
• the growth-enhancing activity of 2,4-D to 2,4-D resistant crop plants can be measured in field trials or pot trials. Herbicide having different mode of action are generally known to either have an adverse effect on yield or have no effect on yield.
• a method of improving yield of 2,4-D resistant crop plants comprising treating the plants with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety.
• the 2,4-D resistant crop plants are transgenic plants transformed with an aryloxyalkanoate dioxygenase (AAD).
• AAD aryloxyalkanoate dioxygenase
• the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD- 12.
• the herbicide comprising an aryloxyalkanoate moiety is a phenoxy herbicide or phenoxyacetic herbicide.
• the herbicide comprising an aryloxyalkanoate moiety is 2,4-D.
• the 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA).
• the transgenic plants transformed with an aryloxyalkanoate dioxygenase are selected from cotton, soybean, and canola.
• the treating is performed at least once at an application rate of 2,4-D as employed also for weed control.
• the treating is performed twice at an application rate of 2,4-D as employed also for weed control.
• 2,4-D is applied at the V3 and R2 growth stages of soybean with 2,4-D tolerance.
• the treating is performed at least three times at an application rate of 2,4-D as employed also for weed control.
• the herbicide comprising an aryloxyalkanoate moiety reaches the 2,4-D resistant crop plants via root absorption.
• the 2,4-D resistant crop plants are also treated with a herbicide different than 2,4-D for weed control.
• the herbicide different than 2,4-D is a phosphor-herbicide or aryloxyphenoxypropionic herbicide.
• the phosphor-herbicide comprises glyphosate, glufosinate, their derivatives, or combinations thereof.
• the phosphor-herbicide is in form of ammonium salt, isopropylammonium salt, isopropylamine salt, or potassium salt.
• the phosphor-herbicide reaches the 2,4-D resistant crop plants via root absorption.
• the aryloxyphenoxypropionic herbicide comprises chlorazifop, fenoxaprop, fluazifop, haloxyfop, quizalofop, their derivatives, or combinations thereof.
• the aryloxyphenoxypropionic herbicide reaches the 2,4-D resistant crop plants via root absorption.
• the 2,4-D resistant crop plants are treated at least once with 25 g ae/ha to 5000 g ae/ha 2,4-D. In another embodiment, the 2,4-D resistant crop plants are treated at least once with 100 g ae/ha to 2000 g ae/ha 2,4-D. In another embodiment, the 2,4-D resistant crop plants are treated at least once with 100 g ae/ha to 2500 g ae/ha 2,4-D. In another embodiment, the 2,4-D resistant crop plants are treated at least once with 1000 g ae/ha to 2000 g ae/ha 2,4-D. In a further embodiment, the 2,4-D comprises 2,4-D choline or 2,4-D
• a method of improving yield of 2,4-D resistant crop plants comprises
• AAD aryloxyalkanoate dioxygenase
• the aryloxyalkanoate dioxygenase is AAD- 1 or AAD- 12.
• the nucleic acid molecule comprises a selectable marker which is not an aryloxyalkanoate dioxygenase (AAD).
• the selectable marker is phosphinothricin acetyltransferase gene (pat) or bialaphos resistance gene (bar).
• the nucleic acid molecule is plant-optimized.
• a herbicide comprising an aryloxyalkanoate moiety in the manufacture of transgenic plants with 2,4-D resistance with increased yield as compared to its non-transgenic parent plants.
• the a herbicide comprising an aryloxyalkanoate moiety is 2,4-D.
• the 2,4-D is applied at least once with 25 g ae/ha to 5000 g/ha 2,4-D.
• the 2,4-D is applied at least once with 100 g ae/ha to 2000 g ae/ha 2,4-D.
• the 2,4-D is applied at least once with 100 g ae/ha to 2500 g ae/ha 2,4-D. In another embodiment, the 2,4-D is applied at least once with 1000 g ae/ha to 2000 g ae/ha 2,4-D. In a further embodiment, the 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA). In a further embodiment, the 2,4-D resistant crop plants are treated with 2,4-D at least two times before flowering. In another embodiment, the 2,4-D resistant crop plants are transgenic plants transformed with an aryloxyalkanoate dioxygenase (AAD). In a further embodiment, the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD- 12.
• AAD aryloxyalkanoate dioxygenase
• FIG. 1 illustrates the general chemical reaction that is catalyzed by AAD- 12 enzymes of the subject invention.
• FIG. 2 shows a representative map for plasmid pDAB4468.
• FIG. 3 shows a representative map for plasmid pD AS 1740.
• SEQ ID NO: 1 is the nucleotide sequence of AAD- 12 from Delftia acidovorans.
• SEQ ID NO: 2 is the translated protein sequence encoded by SEQ ID NO: 1.
• SEQ ID NO: 3 is the plant optimized nucleotide sequence of AAD- 12 (vl).
• SEQ ID NO: 4 is the translated protein sequence encoded by SEQ ID NO: 3.
• SEQ ID NO: 5 is the E. coli optimized nucleotide sequence of AAD- 12 (v2).
• SEQ ID NO: 6 is the sequence of the M13 forward primer.
• SEQ ID NO: 7 is the sequence of the M13 reverse primer.
• SEQ ID NO: 8 is the sequence of the forward AAD- 12 (vl) PTU primer.
• SEQ ID NO: 9 is the sequence of the reverse AAD- 12 (vl) PTU primer.
• SEQ ID NO: 10 is the sequence of the forward AAD-12 (vl) coding PCR primer.
• SEQ ID NO: 11 is the sequence of the reverse AAD-12 (vl) coding PCR primer.
• SEQ ID NO: 12 shows the sequence of the "sdpacodF” AAD-12 (vl) primer.
• SEQ ID NO: 13 shows the sequence of the "sdpacodR” AAD-12 (vl) primer.
• SEQ ID NO: 14 shows the sequence of the "Ncol of Brady” primer.
• SEQ ID NO: 15 shows the sequence of the "Sacl of Brady” primer.
• transformed refers to the introduction of DNA into a cell.
• transformant or “transgenic” refers to plant cells, plants, and the like that have been transformed or have undergone a transformation procedure.
• the introduced DNA is usually in the form of a vector containing an inserted piece of DNA.
• selectable marker or “selectable marker gene” refers to a gene that is optionally used in plant transformation to, for example, protect the plant cells from a selective agent or provide resistance/tolerance to a selective agent. Only those cells or plants that receive a functional selectable marker are capable of dividing or growing under conditions having a selective agent.
• selective agents can include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and hygromycin.
• selectable markers include gene for neomycin phosphotransferase (npt II), which expresses an enzyme conferring resistance to the antibiotic kanamycin, and genes for the related antibiotics neomycin, paromomycin, gentamicin, and G418, or the gene for hygromycin phosphotransferase (hpt), which expresses an enzyme conferring resistance to hygromycin.
• npt II gene for neomycin phosphotransferase
• hpt hygromycin phosphotransferase
• selectable marker genes can include genes encoding herbicide resistance including Bar (resistance against BASTA ® (glufosinate ammonium), or phosphinothricin (PPT)), acetolactate synthase (ALS, resistance against inhibitors such as sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyl triazolinones that prevent the first step in the synthesis of the branched-chain amino acids), glyphosate, 2,4-D, and metal resistance or sensitivity.
• BASTA ® glufosinate ammonium
• PPT phosphinothricin
• ALS acetolactate synthase
• inhibitors such as sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrim
• Various selectable or detectable markers can be incorporated into the chosen expression vector to allow identification and selection of transformed plants, or transformants.
• Many methods are available to confirm the expression of selection markers in transformed plants, including for example DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, immunological methods for detection of a protein expressed from the vector, e g., precipitated protein that mediates phosphinothricin resistance, or other proteins such as reporter genes ⁇ -glucuronidase (GUS), luciferase, green fluorescent protein (GFP), DsRed, ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001, the content of which is incorporated herein by reference in its entirety).
• GUS ⁇ -glucuronidase
• Selectable marker genes are utilized for the selection of transformed cells or tissues.
• Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) as well as genes conferring resistance to herbicidal compounds.
• Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. See DeBlock et al. (1987) EMBO J., 6:2513-2518; DeBlock et al. (1989) Plant Physiol., 91:691-704; Fromm et al.
• EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
• ALS acetolactate synthase
• herbicides can inhibit the growing point or meristem, including imidazolinone or sulfonylurea.
• Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988); and Miki et al., Theon. Appl. Genet. 80:449 (1990), respectively.
• Glyphosate resistance genes include mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively).
• EPSPs 5-enolpyruvylshikimate-3-phosphate synthase
• GAT glyphosate acetyl transferase
• Resistance genes for other phosphono compounds include glufosinate (phosphinothricin acetyl transferase (PAT) genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes), See, for example, U.S. Pat. No. 4,940,835 to Shah, et al. and U.S. Pat. No. 6,248,876 to Barry et al., which disclose nucleotide sequences of forms of EPSPs which can confer glyphosate resistance to a plant.
• PAT phosphinothricin acetyl transferase
• a DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai, European patent application No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclosing nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin.
• the nucleotide sequence of a PAT gene is provided in European application No. 0 242 246 to Leemans et al.
• herbicides can inhibit photosynthesis, including triazine (psbA and ls+ genes) or benzonitrile (nitrilase gene).
• psbA and ls+ genes triazine
• benzonitrile nitrilase gene
• selectable marker genes include, but are not limited to genes encoding: neomycin phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in Plant Science, 4: 1-25); cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA, 88:4250-4264); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) Bio/Technology, 11:715-718); tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol.
• HPT hygromycin phosphotransferase
• HYG hygromycin phosphotransferase
• DHFR dihydrofolate reductase
• phosphinothricin acetyltransferase DeBlock et al. (1987) EMBO J., 6:2513
• 2,2- dichloropropionic acid dehalogenase Buchanan- Wollatron et al. (1989) J. Cell. Biochem.
• selectable marker and reporter genes are not meant to be limiting. Any reporter or selectable marker gene are encompassed by the present invention. If necessary, such genes can be sequenced by methods known in the art.
• the reporter and selectable marker genes are synthesized for optimal expression in the plant. That is, the coding sequence of the gene has been modified to enhance expression in plants.
• the synthetic marker gene is designed to be expressed in plants at a higher level resulting in higher transformation efficiency. Methods for synthetic optimization of genes are available in the art. In fact, several genes have been optimized to increase expression of the gene product in plants.
• the marker gene sequence can be optimized for expression in a particular plant species or alternatively can be modified for optimal expression in plant families.
• the plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research, 17: 477-498; U.S. Pat. No. 5,380,831; and U.S. Pat. No. 5,436,391, herein incorporated by reference.
• the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.
• the binary vector strategy is based on a two-plasmid system where T-DNA is in a different plasmid from the rest of the Ti plasmid.
• a co-integration strategy a small portion of the T-DNA is placed in the same vector as the foreign gene, which vector subsequently recombines with the Ti plasmid.
• the phrase "plant” includes dicotyledons plants and monocotyledons plants.
• Examples of dicotyledons plants include tobacco, Arabidopsis, soybean, tomato, papaya, canola, sunflower, cotton, alfalfa, potato, grapevine, pigeon pea, pea, Brassica, chickpea, sugar beet, rapeseed, watermelon, melon, pepper, peanut, pumpkin, radish, spinach, squash, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, and lettuce.
• Examples of monocotyledons plants include corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids, bamboo, banana, cattails, lilies, oat, onion, millet, and triticale.
• 2,4-D resistance gene and subsequent resistant crops provides excellent options for controlling broadleaf, glyphosate-resistant (or highly tolerant and shifted) weed species for in-crop applications.
• 2,4-D is a broad-spectrum, relatively
• 2,4-D-tolerant transgenic dicot crops would also have greater flexibility in the timing and rate of application.
• An additional utility of the subject herbicide tolerance trait for 2,4-D is its utility to prevent damage to normally sensitive crops from 2,4-D drift, volatilization, inversion (or other off-site movement phenomenon), misapplication, vandalism, and the like.
• AAD-12 is able to degrade the pyridyloxyacetates auxins (e.g., triclopyr, fluoroxypyr) in addition to achiral phenoxy auxins (e.g., 2,4-D, MCPA, 4-chlorophenoxyacetic acid). See Table 1.
• auxins e.g., triclopyr, fluoroxypyr
• achiral phenoxy auxins e.g., 2,4-D, MCPA, 4-chlorophenoxyacetic acid.
• FIG. 1 A general illustration of the chemical reactions catalyzed by the subject AAD-12 enzyme is shown in FIG. 1. (Addition of O.sub.2 is stereo specific; breakdown of intermediate to phenol and glyoxylate is spontaneous.) It should be understood that the chemical structures in FIG.
• FIG. 1 illustrate the molecular backbones and that various R groups and the like (such as those shown in Table 1) are included but are not necessarily specifically illustrated in FIG. 1.
• Multiple mixes of different phenoxy auxin combinations have been used globally to address specific weed spectra and environmental conditions in various regions. Use of the AAD-12 gene in plants affords protection to a much wider spectrum of auxin herbicides, thereby increasing the flexibility and spectra of weeds that can be controlled.
• AAD-12 A single gene (AAD-12) has now been identified which, when genetically engineered for expression in plants, has the properties to allow the use of phenoxy auxin herbicides in plants where inherent tolerance never existed or was not sufficiently high to allow use of these herbicides. Additionally, AAD-12 can provide protection in planta to
• pyridyloxyacetate herbicides where natural tolerance also was not sufficient to allow selectivity, expanding the potential utility of these herbicides.
• Plants containing AAD-12 alone now may be treated sequentially or tank mixed with one, two, or a combination of several phenoxy auxin herbicides.
• the rate for each phenoxy auxin herbicide may range from 25 to 4000 g ae/ha, and more typically from 100 to 2000 g ae/ha for the control of a broad spectrum of dicot weeds.
• one, two, or a mixture of several pyridyloxyacetate auxin compounds may be applied to plants expressing AAD-12 with reduced risk of injury from said herbicides.
• the rate for each pyridyloxyacetate herbicide may range from 25 to 2000 g ae/ha, and more typically from 35- 840 g ae/ha for the control of additional dicot weeds.
• Glyphosate is used extensively because it controls a very wide spectrum of broadleaf and grass weed species.
• repeated use of glyphosate in GTCs and in non-crop applications has, and will continue to, select for weed shifts to naturally more tolerant species or glyphosate-resistant biotypes.
• Tankmix herbicide partners used at efficacious rates that offer control of the same species but having different modes of action is prescribed by most herbicide resistance management strategies as a method to delay the appearance of resistant weeds.
• Stacking AAD-12 with a glyphosate tolerance trait could provide a mechanism to allow for the control of glyphosate resistant dicot weed species in GTCs by enabling the use of glyphosate, phenoxy auxin(s) (e.g., 2,4-D) and
• auxin herbicides e.g., triclopyr
• Applications of these herbicides could be simultaneously in a tank mixture comprising two or more herbicides of different modes of action; individual applications of single herbicide composition in sequential applications as pre-plant, preemergence, or postemergence and split timing of applications ranging from approximately 2 hours to approximately 3 months; or, alternatively, any combination of any number of herbicides representing each chemical class can be applied at any timing within about 7 months of planting the crop up to harvest of the crop (or the preharvest interval for the individual herbicide, whichever is shortest).
• Glyphosate applications in a crop with a glyphosate resistance gene/ AAD-12 stack could range from about 250-2500 g ae/ha; phenoxy auxin herbicide(s) (one or more) could be applied from about 25-4000 g ae/ha; and
• auxin herbicide(s) could be applied from 25-2000 g ae/ha.
• the optimal combination(s) and timing of these application(s) will depend on the particular situation, species, and environment, and will be best determined by a person skilled in the art of weed control and having the benefit of the subject disclosure.
• Plantlets are typically resistant throughout the entire growing cycle. Transformed plants will typically be resistant to new herbicide application at any time the gene is expressed. Tolerance is shown herein to 2,4-D across the life cycle using the constitutive promoters tested thus far (primarily CsVMV and AtUbi 10). One would typically expect this, but it is an improvement upon other non-metabolic activities where tolerance can be significantly impacted by the reduced expression of a site of action mechanism of resistance, for example.
• One example is Roundup Ready cotton, where the plants were tolerant if sprayed early, but if sprayed too late the glyphosate concentrated in the meristems (because it is not metabolized and is translocated); viral promoters Monsanto used are not well expressed in the flowers. The subject invention provides an improvement in these regards.
• Herbicide formulations e.g., ester, acid, or salt formulation; or soluble concentrate, emulsifiable concentrate, or soluble liquid
• tankmix additives e.g., adjuvants, surfactants, drift retardants, or compatibility agents
• Any combination of these with any of the aforementioned herbicide chemistries is within the scope of this invention.
• chemistries AHAS, Csrl, SurA, et al.), bromoxynil resistance (e.g., Bxn), resistance to inhibitors of HPPD (4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitors of phytoene desaturase (PDS), resistance to photosystem II inhibiting herbicides (e.g., psbA), resistance to photosystem I inhibiting herbicides, resistance to protoporphyrinogen oxidase IX (PPO) -inhibiting herbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g., CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), and others could be stacked alone or in multiple combinations to provide the ability to effectively control or prevent weed shifts and/or resistance to any herbicide of the aforementioned classes.
• In vivo modified EPSPS
• some additional preferred ALS inhibitors include but are not limited to the sulfonylureas (such as chlorsulfuron, halosulfuron, nicosulfuron, sulfometuron, sulfosulfuron, trifloxysulfuron), imidazoloninones (such as imazamox, imazethapyr, imazaquin), triazolopyrimidine sulfonanilides (such as cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, and penoxsulam), pyrimidinylthiobenzoates (such as bispyribac and pyrithiobac), and flucarbazone.
• sulfonylureas such as chlorsulfuron, halosulfuron, nicosulfuron, sulfometuron, sulfosulfuron, trifloxysulfuron
• Some preferred HPPD inhibitors include but are not limited to mesotrione, isoxaflutole, and sulcotrione.
• Some preferred PPO inhibitors include but are not limited to flumiclorac, flumioxazin, flufenpyr, pyraflufen, fluthiacet, butafenacil, carfentrazone, sulfentrazone, and the diphenylethers (such as acifluorfen, fomesafen, lactofen, and oxyfluorfen).
• AAD-12 alone or stacked with one or more additional HTC traits can be stacked with one or more additional input (e.g., insect resistance, fungal resistance, or stress tolerance, et al.) or output (e.g., increased yield, improved oil profile, improved fiber quality, et al.) traits.
• additional input e.g., insect resistance, fungal resistance, or stress tolerance, et al.
• output e.g., increased yield, improved oil profile, improved fiber quality, et al.
• the subject invention can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.
• the subject invention relates in part to the identification of an enzyme that is not only able to degrade 2,4-D, but also surprisingly possesses novel properties, which distinguish the enzyme of the subject invention from previously known tfdA proteins, for example. Even though this enzyme has very low homology to tfdA, the genes of the subject invention can still be generally classified in the same overall family of a-ketoglutarate-dependent dioxygenases. This family of proteins is characterized by three conserved histidine residues in a
• HX(D/E)X23-26(T/S)X114-183HX10-13R motif which comprises the active site.
• the histidines coordinate Fe+2 ion in the active site that is essential for catalytic activity (Hogan et al., 2000).
• the preliminary in vitro expression experiments discussed herein were tailored to help select for novel attributes. These experiments also indicate the AAD-12 enzyme is unique from another disparate enzyme of the same class, disclosed in a previously filed patent application (PCT US/2005/014737; filed May 2, 2005).
• the AAD-1 enzyme of that application shares only about 25% sequence identity with the subject AAD-12 protein.
• the subject invention relates in part to the use of an enzyme that is not only capable of degrading 2,4-D, but also pyridyloxyacetate herbicides.
• No a- ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of different chemical classes and modes of action.
• Preferred enzymes and genes for use according to the subject invention are referred to herein as AAD-12
• the subject proteins tested positive for 2,4-D conversion to 2,4-dichlorophenol ("DCP"; herbicidally inactive) in analytical assays.
• DCP 2,4-dichlorophenol
• Partially purified proteins of the subject invention can rapidly convert 2,4-D to DCP in vitro.
• An additional advantage that AAD-12 transformed plants provide is that parent herbicide(s) are metabolized to inactive forms, thereby reducing the potential for harvesting herbicidal residues in grain or stover.
• the subject invention also includes methods of controlling weeds wherein said methods comprise applying a pyridyloxyacetate and/or a phenoxy auxin herbicide to plants comprising an AAD-12 gene.
• TfdA has been used in transgenic plants to impart 2,4-D resistance in dicot plants (e.g., cotton and tobacco) normally sensitive to 2,4-D (Streber et al., 1989; Lyon et al., 1989; Lyon et al., 1993).
• a large number of tfdA-type genes that encode proteins capable of degrading 2,4-D have been identified from the environment and deposited into the Genbank database.
• Many homologues are quite similar to tfdA (>85 amino acid identity) and have similar enzymatic properties to tfdA.
• a small collection of a-ketoglutarate-dependent dioxygenase homologues are presently identified that have a low level of homology to tfdA.
• the subject invention relates in part to surprising discoveries of new uses for and functions of a distantly related enzyme, sdpA, from Delftia acidivorans (Westendorf et al., 2002, 2003) with low homology to tfdA (31% amino acid identity).
• This a-ketoglutarate-dependent dioxygenase enzyme purified in its native form had previously been shown to degrade 2,4-D and S-dichlorprop (Westendorf et al., 2002 and 2003).
• no a- ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of pyridyloxyacetate chemical class.
• SdpA has never been expressed in plants, nor was there any motivation to do so in part because development of new HTC technologies has been limited due largely to the efficacy, low cost, and convenience of GTCs (Devine, 2005).
• AAD-12 proteins and genes of the subject invention are referred to herein as AAD-12 proteins and genes.
• AAD-12 was presently confirmed to degrade a variety of phenoxyacetate auxin herbicides in vitro.
• this enzyme as reported for the first time herein, was surprisingly found to also be capable of degrading additional substrates of the class of aryloxyalkanoate molecules.
• Substrates of significant agronomic importance include the pyridyloxyacetate auxin herbicides.
• This highly novel discovery is the basis of significant Herbicide Tolerant Crop (HTC) and selectable marker trait opportunities.
• HTC Herbicide Tolerant Crop
• This enzyme is unique in its ability to deliver herbicide degradative activity to a range of broad spectrum broadleaf herbicides (phenoxyacetate and pyridyloxyacetate auxins).
• the subject invention relates in part to the degradation of 2,4- dichlorophenoxyacetic acid, other phenoxyacetic auxin herbicides, and pyridyloxyacetate herbicides by a recombinantly expressed aryloxyalkanoate dioxygenase enzyme (AAD-12).
• AAD-12 aryloxyalkanoate dioxygenase enzyme
• aryloxyalkanoate dioxygenase degrading enzyme capable of degrading phenoxy and/or pyridyloxy auxin herbicides.
• the subject enzyme enables transgenic expression resulting in tolerance to combinations of herbicides that would control nearly all broadleaf weeds.
• AAD-12 can serve as an excellent herbicide tolerant crop (HTC) trait to stack with other HTC traits [e.g., glyphosate resistance, glufosinate resistance, ALS-inhibitor (e.g., imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide) resistance, bromoxynil resistance, HPPD-inhibitor resistance, PPO-inhibitor resistance, et al.], and insect resistance traits (Cry IF, CrylAb, Cry 34/45, other Bt.
• HTC herbicide tolerant crop
• AAD-12 Proteins, or insecticidal proteins of a non-Bacillis origin, et al.
• AAD-12 can serve as a selectable marker to aid in selection of primary transformants of plants genetically engineered with a second gene or group of genes.
• the subject microbial gene has been redesigned such that the protein is encoded by codons having a bias toward both monocot and dicot plant usage (hemicot).
• the subject invention also relates to "plant optimized" genes that encode proteins of the subject invention.
• Oxyalkanoate groups are useful for introducing a stable acid functionality into herbicides.
• the acidic group can impart phloem mobility by "acid trapping," a desirable attribute for herbicide action and therefore could be incorporated into new herbicides for mobility purposes.
• Aspects of the subject invention also provide a mechanism of creating HTCs.
• the use of the subject genes can also result in herbicide tolerance to those other herbicides as well.
• HTC traits of the subject invention can be used in novel combinations with other HTC traits (including but not limited to glyphosate tolerance). These combinations of traits give rise to novel methods of controlling weed (and like) species, due to the newly acquired resistance or inherent tolerance to herbicides (e.g., glyphosate). Thus, in addition to the HTC traits, novel methods for controlling weeds using herbicides, for which herbicide tolerance was created by said enzyme in transgenic crops, are within the scope of the invention.
• This invention can be applied in the context of commercializing a 2,4-D resistance trait stacked with current glyphosate resistance traits in soybeans, for example.
• this invention provides a tool to combat broadleaf weed species shifts and/or selection of herbicide resistant broadleaf weeds, which culminates from extremely high reliance by growers on glyphosate for weed control with various crops.
• the transgenic expression of the subject AAD-12 genes is exemplified in, for example, Arabidopsis, tobacco, soybean, cotton, rice, corn and canola. Soybeans are a preferred crop for transformation according to the subject invention. However, this invention can be utilized in multiple other monocot (such as pasture grasses or turf grass) and dicot crops like alfalfa, clover, tree species, et al. Likewise, 2,4-D (or other AAD-12-substrates) can be more positively utilized in grass crops where tolerance is moderate, and increased tolerance via this trait would provide growers the opportunity to use these herbicides at more efficacious rates and over a wider application timing without the risk of crop injury.
• the subject invention provides a single gene that can provide resistance to herbicides that control broadleaf weed. This gene may be utilized in multiple crops to enable the use of a broad spectrum herbicide combination.
• the subject invention can also control weeds resistant to current chemicals, and aids in the control of shifting weed spectra resulting from current agronomic practices.
• the subject AAD-12 can also be used in efforts to effectively detoxify additional herbicide substrates to non-herbicidal forms.
• the subject invention provides for the development of additional HTC traits and/or selectable marker technology.
• the subject genes can also be used as selectable markers for successfully selecting transformants in cell cultures, greenhouses, and in the field. There is high inherent value for the subject genes simply as a selectable marker for biotechnology projects. The promiscuity of AAD-12 for other aryloxyalkanoate auxinic herbicides provides many opportunities to utilize this gene for HTC and/or selectable marker purposes.
• resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis.”
• herbicide “resistance” is heritable and allows a plant to grow and reproduce in the presence of a typical herbicidally effective treatment by a herbicide for a given plant, as suggested by the current edition of The Herbicide Handbook as of the filing of the subject disclosure. As is recognized by those skilled in the art, a plant may still be considered “resistant” even though some degree of plant injury from herbicidal exposure is apparent.
• the term “tolerance” is broader than the term “resistance,” and includes “resistance” as defined herein, as well an improved capacity of a particular plant to withstand the various degrees of herbicidally induced injury that typically result in wild-type plants of the same genotype at the same herbicidal dose.
• Transfer of the functional activity to plant or bacterial systems can involve a nucleic acid sequence, encoding the amino acid sequence for a protein of the subject invention, integrated into a protein expression vector appropriate to the host in which the vector will reside.
• One way to obtain a nucleic acid sequence encoding a protein with functional activity is to isolate the native genetic material from the bacterial species which produce the protein of interest, using information deduced from the protein's amino acid sequence, as disclosed herein.
• the native sequences can be optimized for expression in plants, for example, as discussed in more detail below.
• An optimized polynucleotide can also be designed based on the protein sequence.
• proteins for use according to the subject invention there are a number of methods for obtaining proteins for use according to the subject invention.
• antibodies to the proteins disclosed herein can be used to identify and isolate other proteins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the proteins that are most conserved or most distinct, as compared to other related proteins. These antibodies can then be used to specifically identify equivalent proteins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or immuno-blotting.
• ELISA enzyme linked immunosorbent assay
• Antibodies to the proteins disclosed herein, or to equivalent proteins, or to fragments of these proteins can be readily prepared using standard procedures. Such antibodies are an aspect of the subject invention.
• Antibodies of the subject invention include monoclonal and polyclonal antibodies, preferably produced in response to an exemplified or suggested protein.
• proteins (and genes) of the subject invention can be obtained from a variety of sources. Since entire herbicide degradation operons are known to be encoded on transposable elements such as plasmids, as well as genomically integrated, proteins of the subject invention can be obtained from a wide variety of microorganisms, for example, including recombinant and/or wild- type bacteria.
• Mutants of bacterial isolates can be made by procedures that are well known in the art.
• asporogenous mutants can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate.
• EMS ethylmethane sulfonate
• the mutant strains can also be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
• a protein "from” or “obtainable from” any of the subject isolates referred to or suggested herein means that the protein (or a similar protein) can be obtained from the isolate or some other source, such as another bacterial strain or a plant. "Derived from” also has this connotation, and includes proteins obtainable from a given type of bacterium that are modified for expression in a plant, for example.
• a plant can be engineered to produce the protein.
• Antibody preparations can be prepared using the polynucleotide and/or amino acid sequences disclosed herein and used to screen and recover other related genes from other (natural) sources.
• the subject invention further provides nucleic acid sequences that encode proteins for use according to the subject invention.
• the subject invention further provides methods of identifying and characterizing genes that encode proteins having the desired herbicidal activity.
• the subject invention provides unique nucleotide sequences that are useful as hybridization probes and/or primers for PCR techniques. The primers produce characteristic gene fragments that can be used in the identification, characterization, and/or isolation of specific genes of interest.
• the nucleotide sequences of the subject invention encode proteins that are distinct from previously described proteins.
• the polynucleotides of the subject invention can be used to form complete "genes" to encode proteins or peptides in a desired host cell.
• the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.
• the level of gene expression and temporal/tissue specific expression can greatly impact the utility of the invention. Generally, greater levels of protein expression of a degradative gene will result in faster and more complete degradation of a substrate (in this case a target herbicide). Promoters will be desired to express the target gene at high levels unless the high expression has a consequential negative impact on the health of the plant.
• AAD-12 gene constitutively expressed in all tissues for complete protection of the plant at all growth-stages.
• a vegetatively expressed resistance gene this would allow use of the target herbicide in-crop for weed control and would subsequently control sexual reproduction of the target crop by application during the flowering stage.
• desired levels and times of expression can also depend on the type of plant and the level of tolerance desired.
• Some preferred embodiments use strong constitutive promoters combined with transcription enhancers and the like to increase expression levels and to enhance tolerance to desired levels.
• DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. As DNA is replicated in a plant (for example), additional complementary strands of DNA are produced.
• the "coding strand” is often used in the art to refer to the strand that binds with the anti-sense strand.
• the mRNA is transcribed from the "anti-sense” strand of DNA.
• the "sense” or “coding” strand has a series of codons (a codon is three nucleotides that can be read as a three- residue unit to specify a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest.
• ORF open reading frame
• a strand of DNA is typically transcribed into a complementary strand of mRNA which is used as the template for the protein.
• the subject invention includes the use of the exemplified polynucleotides shown in the attached sequence listing and/or equivalents including the complementary strands.
• RNA and PNA peptide nucleic acids
• Proteins and genes for use according to the subject invention can be identified and obtained by using oligonucleotide probes, for example. These probes are detectable nucleotide sequences that can be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO 93/16094.
• the probes (and the polynucleotides of the subject invention) may be DNA, RNA, or PNA.
• synthetic probes (and polynucleotides) of the subject invention can also have inosine (a neutral base capable of pairing with all four bases; sometimes used in place of a mixture of all four bases in synthetic probes) and/or other synthetic (non-natural) bases.
• inosine a neutral base capable of pairing with all four bases; sometimes used in place of a mixture of all four bases in synthetic probes
• synthetic, degenerate oligonucleotide is referred to herein, and "N” or “n” is used generically, "N” or “n” can be G, A, T, C, or inosine.
• Ambiguity codes as used herein are in accordance with standard IUPAC naming conventions as of the filing of the subject application (for example, R means A or G, Y means C or T, etc.).
• hybridization of the polynucleotide is first conducted followed by washes under conditions of low, moderate, or high stringency by techniques well- known in the art, as described in, for example, Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
• low stringency conditions can be achieved by first washing with 2 x SSC (Standard Saline
• Citrate/0.1% SDS Sodium Dodecyl Sulfate
• SSC/0.1% SDS Sodium Dodecyl Sulfate
• Two washes are typically performed. Higher stringency can then be achieved by lowering the salt concentration and/or by raising the temperature.
• the wash described above can be followed by two washings with 0.1 x SSC/0.1% SDS for 15 minutes each at room temperature followed by subsequent washes with 0.1 x SSC/0.1% SDS for 30 minutes each at 55 °C.
• These temperatures can be used with other hybridization and wash protocols set forth herein and as would be known to one skilled in the art (SSPE can be used as the salt instead of SSC, for example).
• the 2 x SSC/0.1% SDS can be prepared by adding 50 ml of 20 x SSC and 5 ml of 10% SDS to 445 ml of water.
• 20 x SSC can be prepared by combining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), and water, adjusting pH to 7.0 with 10 N NaOH, then adjusting the volume to 1 liter.
• 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml of autoclaved water, then diluting to 100 ml. [0096] Detection of the probe provides a means for determining in a known manner whether hybridization has been maintained.
• nucleotide segments used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention.
• Hybridization characteristics of a molecule can be used to define polynucleotides of the subject invention.
• the subject invention includes polynucleotides (and/or their complements, preferably their full complements) that hybridize with a polynucleotide exemplified herein. That is, one way to define a gene (and the protein it encodes), for example, is by its ability to hybridize (under any of the conditions specifically disclosed herein) with a known or specifically exemplified gene.
• stringent conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants.
• hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods (see, e.g., Maniatis et al. 1982). In general, hybridization and subsequent washes can be carried out under conditions that allow for detection of target sequences.
• hybridization can be carried out overnight at 20-25 °C. below the melting temperature (Tm) of the DNA hybrid in 6 x SSPE, 5 x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
• Washes can typically be carried out as follows: (1) twice at room temperature for 15 minutes in 1 x SSPE, 0.1% SDS (low stringency wash); and (2) once at Tm-20 °C. for 15 minutes in 0.2 x SSPE, 0.1% SDS (moderate stringency wash).
• hybridization can be carried out overnight at 10-20 °C. below the melting temperature (Tm) of the hybrid in 6. times.
• SSPE 5 x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
• Washes can typically be out as follows: (1) twice at room temperature for 15 minutes 1 x SSPE, 0.1% SDS (low stringency wash); and (2) once at the hybridization temperature for 15 minutes in 1 x SSPE, 0.1% SDS (moderate stringency wash).
• salt and/or temperature can be altered to change stringency.
• a labeled DNA fragment>70 or so bases in length the following conditions can be used: (1) Low: 1 or 2 x SSPE, room temperature; (2) Low: 1 or 2 x SSPE, 42 °C; (3) Moderate: 0.2 x or 1 x SSPE, 65 °C. or (4) High: 0.1 x SSPE, 65 °C.
• Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated.
• the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
• PCR technology Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., 1985). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are preferably oriented with the 3' ends pointing towards each other.
• thermostable DNA polymerase such as Tag polymerase, isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated.
• Other enzymes which can be used are known to those skilled in the art.
• Exemplified DNA sequences, or segments thereof can be used as primers for PCR amplification.
• a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5' end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions, and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.
• genes and proteins can be fused to other genes and proteins to produce chimeric or fusion proteins.
• the genes and proteins useful according to the subject invention include not only the specifically exemplified full-length sequences, but also portions, segments and/or fragments (including contiguous fragments and internal and/or terminal deletions compared to the full-length molecules) of these sequences, variants, mutants, chimerics, and fusions thereof.
• Proteins of the subject invention can have substituted amino acids so long as they retain desired functional activity.
• "Variant" genes have nucleotide sequences that encode the same proteins or equivalent proteins having activity equivalent or similar to an exemplified protein.
• Hybridization under certain conditions could be expected to include these two sequences. See GENBANK Acc. Nos. DQ406818.1 (89329742; Rhodoferax) and AJ6288601.1 (44903451; Sphingomonas). Rhodoferax is very similar to Delftia but Sphingomonas is an entirely different Class phylogenetically.
• variant proteins and “equivalent proteins” refer to proteins having the same or essentially the same biological/functional activity against the target substrates and equivalent sequences as the exemplified proteins.
• reference to an "equivalent” sequence refers to sequences having amino acid substitutions, deletions, additions, or insertions that improve or do not adversely affect activity to a significant extent. Fragments retaining activity are also included in this definition. Fragments and other equivalents that retain the same or similar function or activity as a corresponding fragment of an exemplified protein are within the scope of the subject invention.
• Changes such as amino acid substitutions or additions, can be made for a variety of purposes, such as increasing (or decreasing) protease stability of the protein (without materially/substantially decreasing the functional activity of the protein), removing or adding a restriction site, and the like. Variations of genes may be readily constructed using standard techniques for making point mutations, for example.
• fragmentation This can be referred to as gene "shuffling,” which typically involves mixing fragments (of a desired size) of two or more different DNA molecules, followed by repeated rounds of renaturation. This can improve the activity of a protein encoded by a starting gene.
• the result is a chimeric protein having improved activity, altered substrate specificity, increased enzyme stability, altered stereospecificity, or other characteristics.
• “Shuffling” can be designed and targeted after obtaining and examining the atomic 3D (three dimensional) coordinates and crystal structure of a protein of interest.
• focused shuffling can be directed to certain segments of a protein that are ideal for modification, such as surface-exposed segments, and preferably not internal segments that are involved with protein folding and essential 3D structural integrity.
• Specific changes to the "active site” of the enzyme can be made to affect the inherent functionallity with respect to activity or stereospecificity. Muller et. al. (2006). The known tauD crystal structure was used as a model dioxygenase to determine active site residues while bound to its inherent substrate taurine. Elkins et al. (2002) "X-ray crystal structure of Escerichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates," Biochemistry 41(16):5185-5192. Regarding sequence optimization and
• Fragments of full-length genes can be made using commercially available exonucleases or endonuc leases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes that encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these proteins.
• proteins can be truncated and still retain functional activity.
• truncated protein it is meant that a portion of a protein may be cleaved off while the remaining truncated protein retains and exhibits the desired activity after cleavage. Cleavage can be achieved by various proteases.
• effectively cleaved proteins can be produced using molecular biology techniques wherein the DNA bases encoding said protein are removed either through digestion with restriction endonucleases or other techniques available to the skilled artisan. After truncation, said proteins can be expressed in heterologous systems such as E.
• truncated proteins can be successfully produced so that they retain functional activity while having less than the entire, full-length sequence.
• B.t. proteins can be used in a truncated (core protein) form (see, e.g., Hofte et al. (1989), and Adang et al. (1985)).
• core protein truncated protein
• protein can include functionally active truncations.
• amino acids can be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution is not adverse to the biological activity of the compound.
• Table 1 provides a listing of examples of amino acids belonging to each class. In some instances, non-conservative substitutions can also be made. However, preferred substitutions do not significantly detract from the functional/biological activity of the protein.
• isolated polynucleotides and/or purified proteins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to “isolated” and/or “purified” signifies the involvement of the "hand of man” as described herein.
• a bacterial "gene” of the subject invention put into a plant for expression is an “isolated polynucleotide.”
• a protein derived from a bacterial protein and produced by a plant is an “isolated protein.”
• DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create alternative DNA sequences that encode the same, or essentially the same, proteins. These variant DNA sequences are within the scope of the subject invention. This is also discussed in more detail below in the section entitled
• Transgenic hosts The protein-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts.
• the subject invention includes transgenic plant cells and transgenic plants.
• Preferred plants (and plant cells) are corn, Arabidopsis, tobacco, soybeans, cotton, canola, rice, wheat, turf, legume forages (e.g., alfalfa and clover), pasture grasses, and the like.
• Other types of transgenic plants can also be made according to the subject invention, such as fruits, vegetables, ornamental plants, and trees. More generally, dicots and/or monocots can be used in various aspects of the subject invention.
• expression of the gene results, directly or indirectly, in the intracellular production (and maintenance) of the protein(s) of interest.
• Plants can be rendered herbicide-resistant in this manner.
• Such hosts can be referred to as transgenic, recombinant, transformed, and/or transfected hosts and/or cells.
• microbial (preferably bacterial) cells can be produced and used according to standard techniques, with the benefit of the subject disclosure.
• Plant cells transfected with a polynucleotide of the subject invention can be regenerated into whole plants.
• the subject invention includes cell cultures including tissue cell cultures, liquid cultures, and plated cultures. Seeds produced by and/or used to generate plants of the subject invention are also included within the scope of the subject invention. Other plant tissues and parts are also included in the subject invention.
• the subject invention likewise includes methods of producing plants or cells comprising a polynucleotide of the subject invention. One preferred method of producing such plants is by planting a seed of the subject invention.
• the subject invention also includes production of highly active recombinant AAD-12 in a Pseudomonas fluorescens (Pf) host strain, for example.
• the subject invention includes preferred growth temperatures for maintaining soluble active AAD-12 in this host; a fermentation condition where AAD-12 is produced as more than 40% total cell protein, or at least 10 g/L; a purification process results high recovery of active recombinant AAD-12 from a Pf host; a purification scheme which yields at least 10 g active AAD-12 per kg of cells; a purification scheme which can yield 20 g active AAD-12 per kg of cells; a formulation process that can store and restore AAD-12 activity in solution; and a lyophilization process that can retain AAD-12 activity for long-term storage and shelf life.
• Insertion of genes to form transgenic hosts is the transformation/transfection of plants, plant cells, and other host cells with polynucleotides of the subject invention that express proteins of the subject invention. Plants transformed in this manner can be rendered resistant to a variety of herbicides with different modes of action.
• Vectors comprising an AAD-12 polynucleotide are included in the scope of the subject invention.
• a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants.
• the vectors comprise, for example, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly, the sequence encoding the protein can be inserted into the vector at a suitable restriction site.
• the resulting plasmid is used for transformation into E. coli.
• the E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed.
• the plasmid is recovered by purification away from genomic DNA. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be restriction digested and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and described in EP 120 516; Hoekema (1985); Fraley et al. (1986); and An et al. (1985).
• a large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agwbacterium tumefaciens or Agwbacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), silicon carbide whiskers, aerosol beaming, PEG, or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector.
• the intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T- DNA.
• the Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T- DNA.
• Intermediate vectors cannot replicate themselves in Agrobacteria.
• the intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
• Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters, 1978).
• the Agrobacterium used as host cell is to comprise a plasmid carrying a vir region.
• the vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
• the bacterium so transformed is used for the transformation of plant cells. Plant explants can be cultivated advantageously with Agrobacterium tumefaciens or
• Agrobacterium rhizogenes for the transfer of the DNA into the plant cell.
• Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
• the plants so obtained can then be tested for the presence of the inserted DNA.
• No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
• genes encoding the bacterial protein are expressed from transcriptional units inserted into the plant genome.
• said transcriptional units are recombinant vectors capable of stable integration into the plant genome and enable selection of transformed plant lines expressing mRNA encoding the proteins.
• Plant selectable markers also typically can provide resistance to various herbicides such as glufosinate (e.g., PAT/bar), glyphosate (EPSPS), ALS -inhibitors (e.g., imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide, et al.), bromoxynil, HPPD-inhibitor resistance, PPO-inhibitors, ACC-ase inhibitors, and many others.
• glufosinate e.g., PAT/bar
• EPSPS glyphosate
• ALS -inhibitors e.g., imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide, et al.
• bromoxynil e.g., imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide,
• the mRNA is translated into proteins, thereby incorporating amino acids of interest into protein.
• the genes encoding a protein expressed in the plant cells can be under the control of a constitutive promoter, a tissue-specific promoter, or an inducible promoter.
• Syngenta Other direct DNA delivery transformation technology includes aerosol beam technology. See U.S. Pat. No. 6,809,232. Electroporation technology has also been used to transform plants. See WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both to Plant Genetic
• viral vectors can also be used to produce transgenic plants expressing the protein of interest.
• monocotyledonous plants can be transformed with a viral vector using the methods described in U.S. Pat. No. 5,569,597 to Mycogen Plant Science and Ciba-Geigy (now Syngenta), as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to
• Agrobacterium mediated transformation In many instances, it will be desirable to have the construct used for transformation bordered on one or both sides by T-DNA borders, more specifically the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although T-DNA borders may find use with other modes of transformation.
• Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host.
• the manner of vector transformation into the Agrobacterium host is not critical to this invention.
• the Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation, and is not critical to said invention so long as the vir genes are present in said host.
• the expression construct being within the T-DNA borders will be inserted into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al. (1980) and EPO 0 120 515. Included within the expression construct and the T-DNA will be one or more markers as described herein which allow for selection of transformed Agrobacterium and transformed plant cells. The particular marker employed is not essential to this invention, with the preferred marker depending on the host and construction used.
• explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof. After transformation, the Agrobacteria are killed by selection with the appropriate antibiotic and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encouraged by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary. After shoot formation, said plant cells can be transferred to medium which encourages root formation thereby completing plant regeneration. The plants may then be grown to seed and said seed can be used to establish future generations.
• the gene encoding a bacterial protein is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by including in the vector a plant promoter regulatory element, as well as 3' non- translated transcriptional termination regions such as Nos and the like.
• tissue that is contacted with the foreign genes may vary as well.
• tissue would include but would not be limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, root tissue, tissues for expression in phloem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
• selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G41; hygromycin resistance; methotrexate resistance, as well as those genes which encode for resistance or tolerance to glyphosate;
• phosphinothricin biashos or glufosinate
• ALS -inhibiting herbicides imidazolinones, sulfonylureas and triazolopyrimidine herbicides
• ACC-ase inhibitors e.g., ayryloxypropionates or cyclohexanediones
• HPPD-inhibitors e.g., mesotrione
• reporter gene In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes that are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al., 1988. Preferred reporter genes include the beta- glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E.
• GUS beta- glucuronidase
• an assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells.
• a preferred such assay entails the use of the gene encoding beta- glucuronidase (GUS) of the uidA locus of E. coli as described by Jefferson et al., (1987) to identify transformed cells.
• GUS beta- glucuronidase
• promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes.
• promoter regulatory elements of bacterial origin such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter
• promoters of viral origin such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No. 6,166,302, especially Example 7E) and the like may be used.
• Plant promoter regulatory elements include but are not limited to ribulose-l,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissue specific promoters.
• Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration.
• Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like.
• Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant.
• Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, osmotin UTR sequences, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
• Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like).
• Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these may also be used.
• Promoter regulatory elements may also be active (or inactive) during a certain stage of the plant's development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo -specific, corn-silk-specific, cotton-fiber- specific, root-specific, seed-endosperm-specific, or vegetative phase-specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemical (tetracycline responsive), and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant- specific gene transfer vectors are known in the art.
• Plant RNA viral based systems can also be used to express bacterial protein.
• the gene encoding a protein can be inserted into the coat promoter region of a suitable plant virus which will infect the host plant of interest. The protein can then be expressed thus providing protection of the plant from herbicide damage.
• Plant RNA viral based systems are described in U.S. Pat. No. 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Pat. Nos.
• plants of the subject invention can be imparted with novel herbicide resistance traits without observable adverse effects on phenotype including yield. Such plants are within the scope of the subject invention. Plants exemplified and suggested herein can withstand 2 x, 3 x, 4 x, and 5 x typical application levels, for example, of at least one subject herbicide. Improvements in these tolerance levels are within the scope of this invention. For example, various techniques are know in the art, and can forseeably be optimized and further developed, for increasing expression of a given gene.
• One such method includes increasing the copy number of the subject AAD-12 genes (in expression cassettes and the like). Transformation events can also be selected for those having multiple copies of the genes.
• promoters examples include the preferred 35T promoter which uses 35S enhancers. 35S, maize ubiquitin, Arabidopsis ubiquitin, A.t. actin, and CSMV promoters are included for such uses. Other strong viral promoters are also preferred. Enhancers include 4 OCS and the 35S double enhancer. Matrix attachment regions (MARs) can also be used to increase transformation efficiencies and transgene expression.
• Shuffling directed evolution
• transcription factors can also be used for embodiments according to the subject invention.
• Variant proteins can also be designed that differ at the sequence level but that retain the same or similar overall essential three-dimensional structure, surface charge distribution, and the like. See e.g. U.S. Pat. No. 7,058,515; Larson et al., Protein Sci. 2002 11: 2804-2813, "Thoroughly sampling sequence space: Large-scale protein design of structural ensembles”; Crameri et al., Nature Biotechnology 15, 436-438 (1997), “Molecular evolution of an arsenate detoxification pathway by DNA shuffling"; Stemmer, W. P. C. 1994. "DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution” Proc. Natl. Acad.
• the activity of recombinant polynucleotides inserted into plant cells can be dependent upon the influence of endogenous plant DNA adjacent the insert.
• another option is taking advantage of events that are known to be excellent locations in a plant genome for insertions. See e.g. WO 2005/103266 Al, relating to crylF and crylAc cotton events; the subject AAD-12 gene can be substituted in those genomic loci in place of the crylF and/or crylAc inserts.
• targeted homologous recombination for example, can be used according to the subject invention.
• This type of technology is the subject of, for example, WO 03/080809 A2 and the corresponding published U.S. application 20030232410, relating to the use of zinc fingers for targeted recombination.
• the use of recombinases (ere- 10 x and flp-frt for example) is also known.
• AAD-12 detoxification is believed to occur in the cytoplasm.
• means for further stabilizing this protein and mRNAs are included in aspects of the subject invention, and art-known techniques can be applied accordingly.
• the subject proteins can be designed to resist degradation by proteases and the like (protease cleavage sites can be effectively removed by re-engineering the amino acid sequence of the protein).
• Such embodiments include the use of 5' and 3' stem loop structures like UTRs from osmotin, and per5 (AU-rich untranslated 5' sequences).
• 5' caps like 7-methyl or 2'-0-methyl groups, e.g., 7-methylguanylic acid residue, can also be used. See, e.g.: Proc.
• Embodiments of the subject invention can be used in conjunction with naturally evolved or chemically induced mutants (mutants can be selected by screening techniques, then transformed with AAD-12 and possibly other genes). Plants of the subject invention can be combined with ALS resistance and/or evolved glyphosate resistance. Aminopyralid resistance, for example, can also be combined or "stacked" with an AAD-12 gene.
• Safeners typically act to increase plants immune system by activating/expressing cP450. Safeners are chemical agents that reduce the phytotoxicity of herbicides to crop plants by a physiological or molecular mechanism, without compromising weed control efficacy.
• Herbicide safeners include benoxacor, cloquintocet, cyometrinil, dichlormid, dicyclonon, dietholate, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, and oxabetrinil.
• Plant activators a new class of compounds that protect plants by activating their defense mechanisms
• Plant activators include acibenzolar and probenazole.
• Safeners also have been developed to protect winter cereal crops such as wheat against postemergence applications of aryloxyphenoxypropionate and sulfonylurea herbicides.
• the use of safeners for the protection of corn and rice against sulfonylurea, imidazolinone, cyclohexanedione, isoxazole, and triketone herbicides is also well-established.
• a safener-induced enhancement of herbicide detoxification in safened plants is widely accepted as the major mechanism involved in safener action.
• Safeners induce cofactors such as glutathione and herbicide-detoxifying enzymes such as glutathione S-transferases, cytochrome P450 monooxygenases, and glucosyl transferases.
• herbicide-detoxifying enzymes such as glutathione S-transferases, cytochrome P450 monooxygenases, and glucosyl transferases.
• cytochrome p450 monooxygenase gene stacked with AAD-12 is one preferred embodiment.
• P450s involved in herbicide metabolism cP450 can be of mammalian or plant origin, for example.
• cytochrome P450 monooxygenase P450
• P450 cytochrome P450 monooxygenase
• NADPH-cytochrome P450 oxidoreductase reductase
• Resistance to some herbicides has been reported as a result of the metabolism by P450 as well as glutathione S-transferase.
• a number of microsomal P450 species involved in xenobiotic metabolism in mammals have been characterized by molecular cloning. Some of them were reported to metabolize several herbicides efficiently. Thus, transgenic plants with plant or mammalian P450 can show resistance to several herbicides.
• acetochlor-based products include Surpass®, Keystone®, Keystone LA, FulTime® and TopNotch® herbicides) and/or trifluralin (such as Treflan®).
• Such resistance in soybeans and/or corn is included in some preferred embodiments.
• NCBI National Center for Biotechnology Information
• RNA instability may lead to RNA instability.
• genes encoding a bacterial protein for maize expression more preferably referred to as plant optimized gene(s)
• plant optimized gene(s) is to generate a DNA sequence having a higher G+C content, and preferably one close to that of maize genes coding for metabolic enzymes.
• Another goal in the design of the plant optimized gene(s) encoding a bacterial protein is to generate a DNA sequence in which the sequence modifications do not hinder translation.
• Table 2 illustrates how high the G+C content is in maize.
• coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the Mac VectorTM program (Accelerys, San Diego, Calif.). Intron sequences were ignored in the calculations.
• codon bias of the plant is the statistical codon distribution that the plant uses for coding its proteins and the preferred codon usage is shown in Table 3. After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined, as well as the second, third, and fourth choices of preferred codons when multiple choices exist.
• a new DNA sequence can then be designed which encodes the amino sequence of the bacterial protein, but the new DNA sequence differs from the native bacterial DNA sequence (encoding the protein) by the substitution of the plant (first preferred, second preferred, third preferred, or fourth preferred) codons to specify the amino acid at each position within the protein amino acid sequence.
• the new sequence is then analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with first, second, third, or fourth choice preferred codons. Other sites in the sequence which could affect transcription or translation of the gene of interest are the exondntron junctions (5' or 3'), poly A addition signals, or RNA polymerase termination signals.
• the sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred cod
• the plant optimized gene(s) encoding a bacterial protein contain about 63% of first choice codons, between about 22% to about 37% second choice codons, and between about 15% to about 0% third or fourth choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contains about 63% of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100%.
• the method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in PCT application WO
• a DNA sequence is designed to encode the amino acid sequence of said protein utilizing a redundant genetic code established from a codon bias table compiled from the gene sequences for the particular plant or plants.
• the resulting DNA sequence has a higher degree of codon diversity, a desirable base composition, can contain strategically placed restriction enzyme recognition sites, and lacks sequences that might interfere with transcription of the gene, or translation of the product mRNA.
• synthetic genes that are functionally equivalent to the proteins/genes of the subject invention can be used to transform hosts, including plants. Additional guidance regarding the production of synthetic genes can be found in, for example, U.S. Pat. No.
• AAD-12 Plant Rebuild Analysis Extensive analysis of the 876 base pairs (bp) of the DNA sequence of the native AAD-12 coding region (SEQ ID NO: 1) revealed the presence of several sequence motifs that are thought to be detrimental to optimal plant expression, as well as a non-optimal codon composition.
• the protein encoded by SEQ ID NO: 1 (AAD-12) is presented as SEQ ID NO: 2.
• a "plant- optimized" DNA sequence AAD-12 (vl) (SEQ ID NO: 3) was developed that encodes a protein (SEQ ID NO: 4) which is the same as the native SEQ ID NO: 2 except for the addition of an alanine residue at the second position (underlined in SEQ ID NO: 4).
• the additional alanine codon (GCT; underlined in SEQ ID NO: 3) encodes part of an Ncol restriction enzyme recognition site (CCATGG) spanning the ATG translational start codon.
• CCATGG Ncol restriction enzyme recognition site
• Table 4 shows the differences in codon compositions of the native (Columns A and D) and plant- optimized sequences (Columns B and E), and allows comparison to a theoretical plant-optimized sequence (Columns C and F).
• E. coli Expression Specially engineered strains of Escherichia coli and associated vector systems are often used to produce relatively large amounts of proteins for biochemical and analytical studies. It is sometimes found that a native gene encoding the desired protein is not well suited for high level expression in E. coli, even though the source organism for the gene may be another bacterial genus. In such cases it is possible and desirable to reengineer the protein coding region of the gene to render it more suitable for expression in E. coli.
• E. coli Class II genes are defined as those that are highly and continuously expressed during the exponential growth phase of E. coli cells. (Henaut, A. and Danchin, A.
• composition was further engineered to include certain restriction enzyme recognition sequences suitable for cloning into E. coli expression vectors.
• Detrimental sequence features such as highly stable stemloop structures were avoided, as were intragenic sequences homologous to the 3' end of the 16S ribosomal RNA (L e. Shine Dalgarno sequences).
• the E. co/i-optimized sequence (v2) is disclosed as SEQ ID NO: 5 and encodes the protein disclosed in SEQ ID NO: 4.
• the native and E. co/i-optimized (v2) DNA sequences are 84.0% identical, while the plant-optimized (vl) and E. co/i-optimized (v2) DNA sequences are 76.0% identical.
• Table 5 presents the codon compositions of the native AAD-12 coding region (Columns A and D), an AAD-12 coding region optimized for expression in E. coli (v2; Columns B and E) and the codon composition of a theoretical coding region for the AAD-12 protein having an optimal codon composition of E. coli Class II genes (Columns C and F).
• This example teaches the design of a new DNA sequence that encodes a mutated soybean 5-enolpyruvoylshikimate 3-phosphate synthase (EPSPS), but is optimized for expression in soybean cells.
• the amino acid sequence of a triply- mutated soybean EPSPS is disclosed as SEQ ID NO: 5 of WO 2004/009761.
• the mutated amino acids in the so-disclosed sequence are at residue 183 (threonine of native protein replaced with isoleucine), residue 186 (arginine in native protein replaced with lysine), and residue 187 (proline in native protein replaced with serine).
• soybean EPSPS protein by replacing the substituted amino acids of SEQ ID NO:5 of WO 2004/009761 with the native amino acids at the appropriate positions.
• native protein sequence is disclosed as SEQ ID NO: 20 of PCT/US2005/014737 (filed May 2, 2005).
• a doubly mutated soybean EPSPS protein sequence, containing a mutation at residue 183 (threonine of native protein replaced with isoleucine), and at residue 187 (proline in native protein replaced with serine) is disclosed as SEQ ID NO: 21 of PCT/US2005/014737.
• a codon usage table for soybean (Glycine max) protein coding sequences calculated from 362,096 codons (approximately 870 coding sequences), was obtained from the "kazusa.or.jp/codon" World Wide Web site. Those data were reformatted as displayed in Table 6. Columns D and H of Table 6 present the distributions (in % of usage for all codons for that amino acid) of synonymous codons for each amino acid, as found in the protein coding regions of soybean genes. It is evident that some synonymous codons for some amino acids (an amino acid may be specified by 1, 2, 3, 4, or 6 codons) are present relatively rarely in soybean protein coding regions (for example, compare usage of GCG and GCT codons to specify alanine).
• Codons found in soybean genes less than about 10% of total occurrences for the particular amino acid were ignored.
• a weighted average representation for each codon was calculated, using the formula:
• Weighted % of CI 1/(%C1 + %C2 + %C3 + etc.) x %C1 x 100
• CI is the codon in question
• C2, C3, etc. represent the remaining synonymous codons
• % values for the relevant codons are taken from columns D and H of Table 6 (ignoring the rare codon values in bold font).
• soybean-biased DNA sequence that encodes the EPSPS protein of SEQ ID NO: 21 is disclosed as bases 1-1575 of SEQ ID NO: 22 of PCT/US2005/014737. Synthesis of a DNA fragment comprising SEQ ID NO: 22 of PCT/US2005/014737 was performed by a commercial supplier (PicoScript, Houston Tex.).
• AAD-12 v2
• pET280 and pCR2.1 pET280 ligations
• approximately 20 isolated colonies were picked into 6 ml of LB-S/S, and grown at 37 °C. for 4 hours with agitation.
• Each culture was then spotted onto LB + Kanamycin 50 ⁇ g/ml plates, which were incubated at 37 °C. overnight. Colonies that grew on the LB-K were assumed to have the pCR2.1 vector ligated in, and were discarded. Plasmids were isolated from the remaining cultures as before, and checked for correctness with digestion by Xbal/Xhol. The final expression construct was given the designation pDAB3222.
• [00181] Construction of Pseudomonas Expression Vector The AAD-12 (v2) open reading frame was initially cloned into the modified pET expression vector (Novagen), "pET280 S/S," as an Xbal-Xhol fragment. The resulting plasmid pDAB725 was confirmed with restriction enzyme digestion and sequencing reactions. The AAD-12 (v2) open reading frame from pDAB725 was transferred into the Pseudomonas expression vector, pMYC1803, as an Xbal- Xhol fragment. Positive colonies were confirmed via restriction enzyme digestion. The completed construct pDAB739 was transformed into the MB217 and MB324 Pseudomonas expression strains.
• the resulting binary vector, pDAB724, containing the following cassette [AtUbilO promoter: Nt OSM5'UTR: AAD-12 (vl): Nt OSM 3'UTR: ORF1 polyA 3'UTR: CsVMV promoter: PAT: ORF25/26 3'UTR] was restriction digested (with Bam HI, Nco I, Not I, Sad, and Xmn I) for verification of the correct orientation.
• the verified completed construct (pDAB724) was used for transformation into Agrobacterium.
• Arabidopsis thaliana Growth Conditions Wild type Arabidopsis seed was suspended in a 0.1% Agarose (Sigma Chemical Co., St. Louis, Mo.) solution. The suspended seed was stored at 4 °C. for 2 days to complete dormancy requirements and ensure synchronous seed germination (stratification).
• Sunshine Mix LP5 Sun Gro Horticulture, Bellevue, Wash.
• the soil mix was allowed to drain for 24 hours.
• Stratified seed was sown onto the vermiculite and covered with humidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days.
• Seeds were germinated and plants were grown in a Conviron (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 hours light/8 hours dark) at a light intensity of 120-150 ⁇ / ⁇ sec under constant temperature (22 °C.) and humidity (40-50%). Plants were initially watered with Hoagland's solution and subsequently with deionized water to keep the soil moist but not wet.
• Agrobactenum Transformation An LB + agar plate with erythromycin (Sigma Chemical Co., St. Louis, Mo.) (200 mg/L) or spectinomycin (100 mg/L) containing a streaked DH5a colony was used to provide a colony to inoculate 4 ml mini prep cultures (liquid LB + erythromycin). The cultures were incubated overnight at 37 °C. with constant agitation.
• Qiagen (Valencia, Calif.) Spin Mini Preps, performed per manufacturer's instructions, were used to purify the plasmid DNA.
• Electro-competent Agrobacterium tumefaciens (strains Z707s, EHAlOls, and LBA4404s) cells were prepared using a protocol from Weigel and Glazebrook (2002).
• the competent Agrobacterium cells were transformed using an electroporation method adapted from Weigel and Glazebrook (2002). 50 ⁇ of competent agro cells were thawed on ice and 10- 25 ng of the desired plasmid was added to the cells.
• the DNA and cell mix was added to pre- chilled electroporation cuvettes (2 mm).
• An Eppendorf Electroporator 2510 was used for the transformation with the following conditions, Voltage: 2.4 kV, Pulse length: 5 msec.
• YEP broth per liter: 10 g yeast extract, 10 g Bacto- peptone, 5 g NaCl
• the cells were incubated at 28 °C. in a water bath with constant agitation for 4 hours. After incubation, the culture was plated on YEP + agar with erythromycin (200 mg/L) or spectinomycin (100 mg/L) and streptomycin (Sigma Chemical Co., St. Louis, Mo.) (250 mg/L). The plates were incubated for 2-4 days at 28 °C.
• Colonies were selected and streaked onto fresh YEP + agar with erythromycin (200 mg/L) or spectinomycin (100 mg/L) and streptomycin (250 mg/L) plates and incubated at 28 °C. for 1-3 days. Colonies were selected for PCR analysis to verify the presence of the gene insert by using vector specific primers.
• Qiagen Spin Mini Preps performed per manufacturer's instructions, were used to purify the plasmid DNA from selected Agrobacterium colonies with the following exception: 4 ml aliquots of a 15 ml overnight mini prep culture (liquid YEP + erythromycin (200 mg/L) or spectinomycin (100 mg/L)) and streptomycin (250 mg/L)) were used for the DNA purification.
• An alternative to using Qiagen Spin Mini Prep DNA was lysing the transformed Agrobacterium cells, suspended in 10 ⁇ of water, at 100 °C. for 5 minutes. Plasmid DNA from the binary vector used in the Agwbacterium transformation was included as a control.
• PCR reaction was completed using Taq DNA polymerase from Takara Minis Bio Inc. (Madison, Wis.) per manufacturer's instructions at 0.5 x concentrations. PCR reactions were carried out in a MJ Research Peltier Thermal Cycler programmed with the following conditions; 1) 94 °C. for 3 minutes, 2) 94 °C. for 45 seconds, 3) 55 °C. for 30 seconds, 4) 72 °C. for 1 minute, for 29 cycles then 1 cycle of 72 °C. for 10 minutes. The reaction was maintained at 4 °C. after cycling. The amplification was analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. A colony was selected whose PCR product was identical to the plasmid control.
• Arabidopsis Transformation Arabidopsis was transformed using the floral dip method. The selected colony was used to inoculate one or more 15-30 ml pre-cultures of YEP broth containing erythromycin (200 mg/L) or spectinomycin (100 mg/L) and streptomycin (250 mg/L). The culture(s) was incubated overnight at 28 °C. with constant agitation at 220 rpm. Each pre-culture was used to inoculate two 500 ml cultures of YEP broth containing
• Tl seed Freshly harvested Tl seed [AAD-12 (vl) gene] was allowed to dry for 7 days at room temperature. Tl seed was sown in 26.5 x 51-cm germination trays (T.O. Plastics Inc., Clearwater, Minn.), each receiving a 200 mg aliquots of stratified Tl seed (.about.10,000 seed) that had previously been suspended in 40 ml of 0.1% agarose solution and stored at 4 °C. for 2 days to complete dormancy requirements and ensure synchronous seed germination.
• AAD-12 (vl) gene Freshly harvested Tl seed [AAD-12 (vl) gene] was allowed to dry for 7 days at room temperature. Tl seed was sown in 26.5 x 51-cm germination trays (T.O. Plastics Inc., Clearwater, Minn.), each receiving a 200 mg aliquots of stratified Tl seed (.about.10,000 seed) that had previously been suspended in 40 ml of 0.1% agarose solution and stored at 4
• Tl plants were then randomly assigned to various rates of 2,4-D.
• 50 g ae/ha 2,4-D is an effective dose to distinguish sensitive plants from ones with meaningful levels of resistance. Elevated rates were also applied to determine relative levels of resistance (50, 200, 800, or 3200 g ae/ha).
• Tl individuals were subjected to alternative commercial herbicides instead of a phenoxy auxin.
• One point of interest was determining whether the pyridyloxyacetate auxin herbicides, triclopyr and fluoroxypyr, could be effectively degraded in planta.
• Herbicides were applied to Tl plants with use of a track sparyer in a 187 L/ha spray volume. Tl plants that exhibited tolerance to 2,4-D DMA were further accessed in the T2 generation.
• Table 7 compares the response of AAD-12 (vl) and control genes to impart 2,4-D resistance to Arabidopsis Tl transformants. Response is presented in terms of % visual injury 2 WAT. Data are presented as a histogram of individuals exhibiting little or no injury ( ⁇ 20%), moderate injury (20-40%), or severe injury (>40%). Since each Tl is an independent transformation event, one can expect significant variation of individual Tl responses within a given rate. An arithmetic mean and standard deviation is presented for each treatment. The range in individual response is also indicated in the last column for each rate and
• PAT /Cry IF -transformed Arabidopsis served as an auxin-sensitive transformed control.
• the AAD-12 (vl) gene imparted herbicide resistance to individual Tl Arabidopsis plants.
• the level of plant response varied greatly and can be attributed to the fact each plant represents an independent transformation event.
• Table 8 shows a similarly conducted dose response of Tl Arabidopsis to the phenoxypropionic acid, dichlorprop.
• the data shows that the herbicidally active (R-) isomer of dichlorprop does not serve as a suitable substrate for AAD-12 (vl).
• AAD-1 will metabolize R-dichlorprop well enough to impart commercially acceptable tolerance is one distinguishing characteristic that separates the two genes.
• AAD-1 and AAD-12 are considered R- and S-specific a-ketoglutarate dioxygenases, respectively.
• AAD-12 (vl) as a Selectable Marker The ability to use AAD-12 (vl) as a selectable marker using 2,4-D as the selection agent was analyzed initially with Arabidopsis transformed as described above. Approximately 50 T4 generation Arabidopsis seed (homozygous for A AD-12 (vl)) were spiked into approximately 5,000 wild type (sensitive) seed. Several treatments were compared, each tray of plants receiving either one or two application timings of 2,4-D in one of the following treatment schemes: 7 DAP, 11 DAP, or 7 followed by 11 DAP. Since all individuals also contained the PAT gene in the same transformation vector, AAD-12 selected with 2,4-D could be directly compared to PAT selected with glufosinate.
• Treatments were applied with a DeVilbiss spray tip as previously described. Plants were identified as Resistant or Sensitive 17 DAP. The optimum treatment was 75 g ae/ha 2,4-D applied 7 and 11 days after planting (DAP), was equally effective in selection frequency, and resulted in less herbicidal injury to the transformed individuals than the Liberty selection scheme. These results indicate AAD-12 (vl) can be effectively used as an alternative selectable marker for a population of transformed Arabidopsis.
• Tl events were self -pollinated to produce T2 seed. These seed were progeny tested by applying 2,4-D (200 g ae/ha) to 100 random T2 siblings. Each individual T2 plant was transplanted to 7.5-cm square pots prior to spray application (track sprayer at 187 I/ha applications rate). Seventy-five percent of the Tl families (T2 plants) segregated in the anticipated 3 Resistant: 1 Sensitive model for a dominantly inherited single locus with Mendelian inheritance as determined by Chi square analysis (P>0.05).
• T3 seed were collected from 12 to 20 T2 individuals (T3 seed). Twenty- five T3 siblings from each of eight randomly- selected T2 families were progeny tested as previously described. Approximately one-third of the T2 families anticipated to be homozygous (non- segregating populations) have been identified in each line. These data show AAD-12 (vl) is stably integrated and inherited in a Mendelian fashion to at least three generations.
• AAD-12 (vl) The ability of AAD-12 (vl) to provide resistance to other aryloxyalkanoate auxin herbicides in transgenic Arabidopsis was determined by foliar application of various substrates. T2 generation Arabidopsis seed was stratified, and sown into selection trays much like that of Arabidopsis. A transformed-control line containing PAT and the insect resistance gene CrylF was planted in a similar manner. Seedlings were transferred to individual 3-inch pots in the greenhouse. All plants were sprayed with the use of a track sprayer set at 187 L/ha.
• the plants were sprayed with a range of pyridyloxyacetate herbicides: 280-2240 g ae/ha triclopyr (Garlon 3A, Dow AgroSciences) and 280-2240 g ae/ha fluoroxypyr (Starane, Dow AgroSciences); and the 2,4-D metabolite resulting from AAD-12 activity, 2,4-dichlorophenol (DCP, Sigma) (at a molar equivalent to 280-2240 g ae/ha of 2,4-D, technical grade DCP was used). All applications were formulated in water. Each treatment was replicated 3-4 times. Plants were evaluated at 3 and 14 days after treatment.
• AAD-12 (vl) Arabidopsis Molecular Analysis of AAD-12 (vl) Arabidopsis: Invader Assay (methods of Third Wave Agbio Kit Procedures) for PAT gene copy number analysis was performed with total DNA obtained from Qiagen DNeasy kit on multiple AAD-12 (vl) homozygous lines to determine stable integration of the plant transformation unit containing PAT and AAD-12 (vl). Analysis assumed direct physical linkage of these genes as they were contained on the same plasmid.
• Results showed that all 2,4-D resistant plants assayed, contained PAT (and thus by inference, AAD-12 (vl)). Copy number analysis showed total inserts ranged from 1 to 5 copies. This correlates, too, with the AAD-12 (vl) protein expression data indicating that the presence of the enzyme yields significantly high levels of resistance to all commercially available phenoxyacetic and pyridyloxyacetic acids.
• Tl Arabidopsis seed was produced, as previously described, containing the pDAB3759 plasmid (AAD-12 (vl) + EPSPS) which encodes a putative glyphosate resistance trait. Tl transformants were selected using AAD-12 (vl) as the selectable marker as described. Tl plants (individually transformed events) were recovered from the first selection attempt and transferred to three-inch pots in the greenhouse as previously described.
• each gene pair could be brought together through conventional breeding activities and subsequently selected in the Fl generation through paired sprays with herbicides that are exclusive between the AAD- 1 and AAD-12 enzymes (as shown with R-dichlorprop and triclopyr for AAD-1 and AAD-12, respectively).
• AAD stacks are also within the scope of the subject invention.
• the TfdA protein discussed elsewhere herein (Streber et al.), for example, can be used together with the subject AAD-12 genes to impart spectrums of herbicide resistance in transgenic plants of the subject invention.
• AAD-12 (vl) Cloning of AAD-12 (vl): The AAD-12 (vl) gene was cut out of the intermediate vector pDAB3283 as an Ncol/Sacl fragment. This was ligated directionally into the similarly cut pDAB3403 vector containing the ZmUbil monocot promoter. The two fragments were ligated together using T4 DNA ligase and transformed into DH5a cells. Minipreps were performed on the resulting colonies using Qiagen's QIA Spin mini prep kit, and the colonies were digested to check for orientation. This first intermediate construct (pDAB4100) contains the ZmUbil:AAD-12 (vl) cassette.
• This construct was digested with Notl and Pvul to liberate the gene cassette and digest the unwanted backbone. This was ligated to Notl cut pDAB2212, which contains the AHAS selectable marker driven by the Rice Actin promoter OsActl. The final construct was designated pDAB4101 or pDAS1863, and contains ZmUbil/AAD-12 (vl)/ZmPer5::OsActl/AHAS/LZmLip.
• Ears were rinsed in sterile, distilled water, and immature zygotic embryos were aseptically excised and cultured on 15AglO medium (N6 Medium (Chu et al., 1975), 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/L casein hydrolysate (enzymatic digest), 25 mM L-proline, 10 mg/L AgNC"3, 2.5 g/L Gelrite, pH 5.8) for 2-3 weeks with the scutellum facing away from the medium.
• 15AglO medium N6 Medium (Chu et al., 1975), 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/L casein hydrolysate (enzymatic digest), 25 mM L-proline, 10 mg/L AgNC"3, 2.5 g/L Gelrite, pH 5.8 for 2-3 weeks with the scutellum facing away from the medium.
• Tissue showing the proper morphology was selectively transferred at biweekly intervals onto fresh 15AglO medium for about 6 weeks, then transferred to 4 medium (N6 Medium, 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/L casein hydrolysate (enzymatic digest), 6 mM L-proline, 2.5 g/L Gelrite, pH 5.8) at bi-weekly intervals for approximately 2 months.
• PCV packed cell volume
• H9CP + liquid medium MS basal salt mixture (Murashige and Skoog, 1962), modified MS Vitamins containing 10-fold less nicotinic acid and 5-fold higher thiamine-HCl, 2.0 mg/L 2,4-D, 2.0 mg/L a-naphthaleneacetic acid (NAA), 30 g/L sucrose, 200 mg/L casein hydrolysate (acid digest), 100 mg/L myo-inositol, 6 mM L-proline, 5% v/v coconut water (added just before subculture), pH 6.0).
• Suspension cultures were maintained under dark conditions in 125 ml Erlenmeyer flasks in a temperature-controlled shaker set at 125 rpm at 28 °C. Cell lines typically became established within 2 to 3 months after initiation. During establishment, suspensions were subcultured every 3.5 days by adding 3 ml PCV of cells and 7 ml of conditioned medium to 20 ml of fresh H9CP+ liquid medium using a wide-bore pipette. Once the tissue started doubling in growth, suspensions were scaled-up and maintained in 500 ml flasks whereby 12 ml PCV of cells and 28 ml conditioned medium was transferred into 80 ml H9CP+ medium. Once the suspensions were fully established, they were cryopreserved for future use.
• Cryopreservation and Thawing Of Suspensions Two days post-subculture, 4 ml PCV of suspension cells and 4 ml of conditioned medium were added to 8 ml of cryoprotectant (dissolved in H9CP+ medium without coconut water, 1 M glycerol, 1 M DMSO, 2 M sucrose, filter sterilized) and allowed to shake at 125 rpm at 4 °C. for 1 hour in a 125 ml flask. After 1 hour 4.5 ml was added to a chilled 5.0 ml Corning cryo vial. Once filled individual vials were held for 15 minutes at 4 °C.
• cryoprotectant dissolved in H9CP+ medium without coconut water, 1 M glycerol, 1 M DMSO, 2 M sucrose, filter sterilized
• tissue with promising morphology was transferred off the filter paper directly onto fresh GN6 medium.
• This tissue was subcultured every 7-14 days until 1 to 3 grams was available for suspension initiation into approximately 30 ml H9CP+ medium in 125 ml Erlenmeyer flasks.
• Three milliliters PCV was subcultured into fresh H9CP+ medium every 3.5 days until a total of 12 ml PCV was obtained, at which point subculture took place as described previously.
• GN6 liquid media was removed and replaced with 72 ml GN6 S/M osmotic medium (N6 Medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 45.5 g/L sorbitol, 45.5 g/L mannitol, 100 mg/L myo-inositol, pH 6.0) per flask in order to plasmolyze the cells.
• N6 Medium 2.0 mg/L 2,4-D, 30 g/L sucrose, 45.5 g/L sorbitol, 45.5 g/L mannitol, 100 mg/L myo-inositol, pH 6.0
• the flasks were placed on a shaker shaken at 125 RPM in the dark for 30-35 minutes at 28 °C, and during this time a 50 mg/ml suspension of silicon carbide whiskers was prepared by adding the appropriate volume 8.1 ml of GN6 S/M liquid medium to -405 mg of pre-autoclaved, sterile silicon carbide whiskers (Advanced Composite Materials, Inc.).
• the cocktail of cells, media, whiskers and DNA was added to the contents of the 1-L flask along with 125 ml fresh GN6 liquid medium to reduce the osmoticant.
• the cells were allowed to recover on a shaker at 125 RPM for 2 hours at 28 °C. before being filtered onto Whatman #4 filter paper (5.5 cm) using a glass cell collector unit that was connected to a house vacuum line.
• GN6 (3P) medium N6 Medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 100 mg/L myo-inositol, 3 ⁇ imazethapyr from Pursuit® DG, 2.5 g/L Gelrite, pH 5.8. Plates were placed in boxes and cultured for an additional week.
• the tissue was embedded by scraping all cells on the plate into 3.0 ml of melted GN6 agarose medium (N6 medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 100 mg/L myo-inositol, 7 g/L Sea Plaque agarose, pH 5.8, autoclaved for only 10 minutes at 121 °C.) containing 3 ⁇ imazethapyr from Pursuit® DG.
• the tissue was broken up and the 3 ml of agarose and tissue were evenly poured onto the surface of a 100 x 15 mm plate of GN6 (3P). This was repeated for all remaining plates.
• plates were individually sealed with Nescofilm® or Parafilm M®, and then cultured until putative isolates appeared.
• Protocol for Isolate Recovery and Regeneration Putatively transformed events were isolated off the Pursuit®-containing embedded plates approximately 9 weeks post- transformation by transferring to fresh selection medium of the same concentration in 60 x 20 mm plates. If sustained growth was evident after approximately 2-3 weeks, the event was deemed to be resistant and was submitted for molecular analysis. Table 11. Characterization of TO corn plants transformed with AAD-12
• Regeneration was initiated by transferring callus tissue to a cytokinin-based induction medium, 28 (3P), containing 3 ⁇ imazethapyr from Pursuit.RTM. DG, MS salts and vitamins, 30.0 g/L sucrose, 5 mg/L BAP, 0.25 mg/L 2,4-D, 2.5 g/L Gelrite; pH 5.7. Cells were allowed to grow in low light (13 ⁇ - ' 2 s- " 1 ) for one week, then higher light (40 ⁇ - ' 2 s- " 1 ) for another week, before being transferred to regeneration medium, 36 (3P), which was identical to 28 (3P) except that it lacked plant growth regulators.
• Invader assay analysis The DNA samples are diluted to 20 ng/ ⁇ then denatured by incubation in a thermocycler at 95 °C. for 10 minutes. Signal Probe mix is then prepared using the provided oligo mix and MgCl 2 (Third Wave Technologies). An aliquot of 7.5 ⁇ is placed in each well of the Invader assay plate followed by an aliquot of 7.5 ⁇ of controls, standards, and 20 ng/ ⁇ diluted unknown samples. Each well is overlaid with 15 ⁇ of mineral oil (Sigma). The plates are then incubated at 63 °C. for 1 hour and read on the fluorometer (Biotek).
• Calculation of % signal over background for the target probe divided by the % signal over background internal control probe will calculate the ratio.
• the ratio of known copy standards developed and validated with Southern blot analysis is used to identify the estimated copy of the unknown events.
• ACATGGTCTA AAGG (SEQ ID NO: 8) and Reverse-GCTGCAACAC TGATAAATGC CAACTGG (SEQ ID NO: 9).
• the PCR reaction is carried out in the 9700 Geneamp
• thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 63 °C. for 30 seconds, and 72 °C. for 1 minute and 45 seconds followed by 72 °C. for 10 minutes.
• Primers for AAD-12 (vl) Coding Region PCR are Forward- ATGGCTCAGA CCACTCTCCA AA (SEQ ID NO: 10) and Reverse- AGCTGCATCC ATGCCAGGGA (SEQ ID NO: 11).
• the PCR reaction is carried out in the 9700 Geneamp thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 65 °C. for 30 seconds, and 72 °C. for 1 minute and 45 seconds followed by 72 °C. for 10 minutes.
• PCR products are analyzed by electrophoresis on a 1% agarose gel stained with EtBr.
• Southern Blot Analysis Southern blot analysis is performed with genomic DNA obtained from Qiagen DNeasy kit. A total of 2 ⁇ g of genomic leaf DNA or 10 ⁇ g of genomic callus DNA is subjected to an overnight digestion using BSM I and SWA I restriction enzymes to obtain PTU data.
• the membrane is then washed in 0.1% SDS, 0.1 SSC for 45 minutes. After the 45 minute wash, the membrane is baked for 3 hours at 80 °C. and then stored at 4 °C. until hybridization.
• the hybridization template fragment is prepared using the above coding region PCR using plasmid DNA.
• the product is run on a 1% agarose gel and excised and then gel extracted using the Qiagen (28706) gel extraction procedure.
• the membrane is then subjected to a pre-hybridization at 60 °C. step for 1 hour in Perfect Hyb buffer (Sigma H7033).
• the Prime it RmT dCTP-labeling rxn (Stratagene 300392) procedure is used to develop the p32 based probe (Perkin Elmer).
• the probe is cleaned up using the Probe Quant. G50 columns (Amersham 27-5335-01). Two million counts CPM are used to hybridize the southern blots overnight. After the overnight hybridization the blots are then subjected to two 20 minute washes at 65 °C. in 0.1% SDS, 0.1 SSC. The blots are then exposed to film overnight, incubating at -80 °C.
• Postemergence Herbicide Tolerance in AAD-12 Transformed TO Corn Four TO events were allowed to acclimate in the greenhouse and were grown until 2-4 new, normal looking leaves had emerged from the whorl (i.e., plants had transitioned from tissue culture to greenhouse growing conditions). Plants were grown at 27 °C. under 16 hour light: 8 hour dark conditions in the greenhouse. Plants were then treated with commercial formulations of either Pursuit® (imazethapyr) or 2,4-D Amine 4. Pursuit® was sprayed to demonstrate the function of the selectable marker gene present within the events tested. Herbicide applications were made with a track sprayer at a spray volume of 187 L/ha, 50-cm spray height.
• Hi-II is the genetic background of the transformants of the present invention.
• Tl AAD-12 (vl) seed were planted into 3-inch pots containing Metro Mix media and at 2 leaf stage were sprayed with 70 g ae/ha imazethapyr to eliminate nulls.
• S urviving plants were transplanted to 1 -gallon pots containing Metro Mix media and placed in the same growth conditions as before.
• the plants were sprayed in the track sprayer set to 187 L/ha at either 560 or 2240 g ae/ha 2,4-D DMA. Plants were graded at 3 and 14 DAT and compared to 5XH751 x Hi II control plants.
• Brace Root grades were taken on 14DAT to show 2,4-D tolerance. 2,4-D causes brace root malformation, and is a consistent indicator of auxinic herbicide injury in corn. Brace root data (as seen in the table below) demonstrates that 2 of the 3 events tested were robustly tolerant to 2240 g ae/ha 2,4-D DMA. Event
• AAD-12 (vl) Heritability in Corn A progeny test was also conducted on seven AAD-12 (vl) Tl families that had been crossed with 5XH751. The seeds were planted in three-inch pots as described above. At the 3 leaf stage all plants were sprayed with 70 g ae/ha imazethapyr in the track sprayer as previously described. After 14 DAT, resistant and sensitive plants were counted. Four out of the six lines tested segregated as a single locus, dominant Mendelian trait (1R: 1S) as determined by Chi square analysis. Surviving plants were subsequently sprayed with 2,4-D and all plants were deemed tolerant to 2,4-D (rates > 560 g ae/ha). AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species when reciprocally crossed to a commercial hybrid.
• AAD-12 (vl) can be conventionally stacked with a glyphosate tolerance gene (such as the Roundup CP4-EPSPS gene) or other herbicide tolerance genes to provide an increased spectrum of herbicides that may be applied safely to corn.
• a glyphosate tolerance gene such as the Roundup CP4-EPSPS gene
• other herbicide tolerance genes to provide an increased spectrum of herbicides that may be applied safely to corn.
• imidazolinone + 2,4-D + glyphosate tolerance was observed in Fl plants and showed no negative phenotype by the molecular or breeding stack combinations of these multiple transgenes.
• Herbicide treatments were 2,4-D (dimethylamine salt) at 1120, 2240 and 4480 g ae/ha, triclopyr at 840 g ae/ha, fluoroxypyr at 280 g ae/ha and an untreated control.
• the AAD-12 (vl) events contained the AHAS gene as a selectable marker.
• the F2 corn events were segregating so the AAD-12 (vl) plants were treated with imazethapyr at 70 g ae/ha to remove the null plants.
• Herbicide treatments were applied when corn reached the V6 stage using compressed air backpack sprayer delivering 187 L/ha carrier volume at 130-200 kpa pressure. Visual injury ratings were taken at 7, 14 and 21 days after treatment.
• Brace root injury ratings were taken at 28DAT on a scale of 0-10 with 0-1 being slight brace root fusing, 1-3 being moderate brace root swelling/wandering and root proliferation, 3-5 being moderate brace root fusing, 5-9 severe brace root fusing and malformation and 10 being total inhibition of brace roots.
• AAD-12 (vl) event response to 2,4-D, triclopyr, and fluoroxypyr at 14 days after treatment are shown in Table 14. Crop injury was most severe at 14 DAT. The RR control corn (2P782) was severely injured (44% at 14 DAT) by 2,4-D at 4480 g ae/ha, which is 8 times (8 x) the normal field use rate. The AAD-12 (vl) events all demonstrated excellent tolerance to 2,4-D at 14 DAT with 0% injury at the 1, 2 and 4 x rates, respectively. The control corn (2P782) was severely injured (31% at 14 DAT) by the 2 x rate of triclopyr (840 g ae/ha).
• AAD- 12 (vl) events demonstrated tolerance at 2 x rates of triclopyr with an average of 3% injury at 14 DAT across the two events. Fluoroxypyr at 280 g ae/ha caused 11% visual injury to the wild- type corn at 14 DAT. AAD-12 (vl) events demonstrated increased tolerance with an average of 8% injury at 5 DAT.
• Both AAD- 12 (vl) corn events showed no brace root injury from the triclopyr treatment. Brace root injury in 2P782 corn increased with increasing rates of 2,4-D. At 4480 g ae/ha of 2,4-D, the AAD- 12 events showed no brace root injury; whereas, severe brace root fusing and malformation was seen in the 2P782 hybrid. Fluoroxypyr caused only moderate brace root swelling and wandering in the wild- type corn with the AAD- 12 (vl) events showing no brace root injury.
• Tobacco transformation with Agrobacterium tumefaciens was carried out by a method similar, but not identical, to published methods (Horsch et al., 1988).
• tobacco seed Nicotiana tabacum cv. KY160
• TOB -medium which is a hormone-free Murashige and Skoog medium (Murashige and Skoog, 1962) solidified with agar.
• Plants were grown for 6-8 weeks in a lighted incubator room at 28-30 °C. and leaves collected sterilely for use in the transformation protocol. Pieces of approximately one square centimeter were sterilely cut from these leaves, excluding the midrib.
• Murashige & Skoog salts and adjusted to a final optical density of 0.5 at 600 nm.
• Leaf pieces were dipped in this bacterial suspension for approximately 30 seconds, then blotted dry on sterile paper towels and placed right side up on TOB+ medium (Murashige and Skoog medium containing 1 mg/L indole acetic acid and 2.5 mg/L benzyladenine) and incubated in the dark at 28 °C.
• TOB+ medium Middlerashige and Skoog medium containing 1 mg/L indole acetic acid and 2.5 mg/L benzyladenine
• Two days later the leaf pieces were moved to TOB+ medium containing 250 mg/L cefotaxime (Agri-Bio, North Miami, Fla.) and 5 mg/L glufosinate ammonium (active ingredient in Basta, Bayer Crop Sciences) and incubated at 28-30 °C.
• Leaf pieces were moved to fresh TOB+ medium with cefotaxime and Basta twice per week for the first two weeks and once per week thereafter.
• Plants were moved into the greenhouse by washing the agar from the roots, transplanting into soil in 13.75 cm square pots, placing the pot into a Ziploc® bag (SC Johnson & Son, Inc.), placing tap water into the bottom of the bag, and placing in indirect light in a 30 °C. greenhouse for one week. After 3-7 days, the bag was opened; the plants were fertilized and allowed to grow in the open bag until the plants were greenhouse-acclimated, at which time the bag was removed. Plants were grown under ordinary warm greenhouse conditions (30 °C,
• the DNA samples were diluted to 9 ng/ ⁇ and then denatured by incubation in a thermocycler at 95 °C. for 10 minutes. Signal Probe mix was then prepared using the provided oligo mix and MgCl 2 (Third Wave Technologies). An aliquot of 7.5 ⁇ was placed in each well of the Invader assay plate followed by an aliquot of 7.5 ⁇ of controls, standards, and 20 ng/ ⁇ diluted unknown samples. Each well was overlaid with 15 ⁇ of mineral oil (Sigma). The plates were then incubated at 63 °C. for 1.5 hours and read on the fluorometer (Biotek).
• CTGCCCTCAG CC (SEQ ID NO: 12) and (SdpacodR: CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 13).
• the PCR reaction was carried out in the 9700 Geneamp thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 64 °C. for 30 seconds, and 72 °C. for 1 minute and 45 seconds followed by 72 °C. for 10 minutes.
• PCR products were analyzed by electrophoresis on a 1% agarose gel stained with EtBr. Four to 12 clonal lineages from each of 18 PCR positive events with 1-3 copies of PAT gene (and presumably AAD-12 (vl) since these genes are physically linked) were regenerated and moved to the greenhouse.
• Survivors were transferred to individual 3-inch pots in the greenhouse. These lines provided high levels of resistance to 2,4-D in the TO generation. Improved consistency of response is anticipated in Tl plants not having come directly from tissue culture. These plants were compared against wild type KYI 60 tobacco. All plants were sprayed with a track sprayer set at 187 L/ha. The plants were sprayed from a range of 140-2240 g ae/ha 2,4-D dimethylamine salt (DMA), 70-1120 g ae/ha triclopyr or 35-560 g ae/ha fluoroxypyr. All applications were formulated in water. Each treatment was replicated 2-4 times. Plants were evaluated at 3 and 14 days after treatment. Plants were assigned injury rating with respect to stunting, chlorosis, and necrosis. The Tl generation is segregating, so some variable response is expected due to difference in zygosity.
• DMA dimethylamine salt
• Table 17 Segregating AAD-12 T ⁇ tobacco plants' response to phenoxy and pyridyloxy auxin herbicides.
• AAD-12 (vl) Heritability in Tobacco A 100 plant progeny test was also conducted on seven Tl lines of AAD-12 (vl) lines. The seeds were stratified, sown, and transplanted with respect to the procedure above with the exception that null plants were not removed by Liberty selection. All plants were then sprayed with 560 g ae/ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. Five out of the seven lines tested segregated as a single locus, dominant Mendelian trait (3R: 1S) as determined by Chi square analysis. AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species.
• the experimental design was a split plot design with 4 replications.
• the main plot was herbicide treatment and the sub-plot was tobacco line.
• the herbicide treatments were 2,4- D (dimethylamine salt) at 280, 560, 1120, 2240 and 4480 g ae/ha, triclopyr at 840 g ae/ha, fluoroxypyr at 280 g ae/ha and an untreated control.
• Plots were one row by 25-30 ft.
• Herbicide treatments were applied 3-4 weeks after transplanting using compressed air backpack sprayer delivering 187 I/ha carrier volume at 130-200 kpa pressure. Visual rating of injury, growth inhibition, and epinasty were taken at 7, 14 and 21 days after treatment.
• Table 18 AAD-12 tobacco plants response to 2,4-D, triclopyr, and fluroxypyr under field conditions.
• AAD-12 (vl) event response to 2,4-D, triclopyr, and fluoroxypyr are shown in Table 18.
• the non-transformed tobacco line was severely injured (63% at 14 DAT) by 2,4-D at 560 g ae/ha which is considered the l.times. field application rate.
• the AAD-12 (vl) lines all demonstrated excellent tolerance to 2,4-D at 14 DAT with average injury of 1, 4, and 4% injury observed at the 2, 4 and 8. times, rates, respectively.
• the non-transformed tobacco line was severely injured (53% at 14 DAT) by the 2 x rate of triclopyr (840 g ae/ha); whereas, AAD-12 (vl) lines demonstrated tolerance with an average of 5% injury at 14 DAT across the three lines.
• Fluoroxypyr at 280 g ae/ha caused severe injury (99%) to the non-transformed line at 14 DAT.
• AAD-12 (vl) lines demonstrated increased tolerance with an average of 11% injury at 14 DAT.
• AAD- 12 (v 1 ) Protection Against Elevated 2,4-D Rates results showing AAD- 12 (vl) protection against elevated rates of 2,4-D DMA in the greenhouse are shown in Table 19.
• Tl AAD-1 (v3) seed was also planted for transformed tobacco controls (see PCT/US2005/014737). Untransformed KY160 was served as the sensitive control. Plants were sprayed using a track sprayer set to 187 L/ha at 140, 560, 2240, 8960, and 35840 g ae/ha 2,4-D DMA and rated 3 and 14 DAT.
• AAD-12 (vl) clearly demonstrated a marked advantage over AAD-1 (v3) by protecting up to 64 x the standard field rates.
• AAD-12 to Increase Herbicide Spectrum: Homozygous AAD-12 (vl) (pDAS1580) and AAD-1 (v3) (pDAB721) plants (see PCT/US2005/014737 for the latter) were both reciprocally crossed and Fl seed was collected.
• the Fl seed from two reciprocal crosses of each gene were stratified and treated 4 reps of each cross were treated under the same spray mitine as used for the other testing with one of the following treatments: 70, 140, 280 g ae/ha fluoroxypyr (selective for the AAD-12 (vl) gene); 280, 560, 1120 g ae/ha R-dichloroprop (selective for the AAD-1 (v3) gene); or 560, 1120, 2240 g ae/ha 2,4-D DMA (to confirm 2,4-D tolerance). Homozygous T2 plants of each gene were also planted for use as controls. Plants were graded at 3 and 14 DAT. Spray results are shown in Table 20.
• AAD-12 (vl) can be successfully stacked with AAD-1 (v3), thus increasing the spectrum herbicides that may be applied to the crop of interest (phenoxyactetic acids + phenoxypropionic acids vs penoxyacetic acids + pyridyloxyacetic acids for AAD-1 and AAD-12, respectively).
• the complementary nature of herbicide cross resistance patterns allows convenient use of these two genes as complementary and stackable field- selectable markers. In crops where tolerance with a single gene may be marginal, one skilled in the art recognizes that one can increase tolerance by stacking a second tolerance gene for the same herbicide.
• Soybean improvement via gene transfer techniques has been accomplished for such traits as herbicide tolerance (Padgette et al., 1995), amino acid modification (Falco et al., 1995), and insect resistance (Parrott et al., 1994).
• Introduction of foreign traits into crop species requires methods that will allow for routine production of transgenic lines using selectable marker sequences, containing simple inserts.
• the transgenes should be inherited as a single functional locus in order to simplify breeding.
• This system needs a high level of 2,4-D, 40 mg/L concentration, to initiate the embryogenic callus and this poses a fundamental problem in using the AAD-12 (vl) gene since the transformed locus could not be developed further with 2,4-D in the medium. So, the meristem based transformation is ideal for the development of 2,4-D resistant plant using AAD-12 (vl).
• Gateway Cloning of Binary Constructs The AAD-12 (vl) coding sequence was cloned into five different Gateway Donor vectors containing different plant promoters. The resulting AAD-12 (vl) plant expression cassettes were subsequently cloned into a Gateway Destination Binary vector via the LR Clonase reaction (Invitrogen Corporation, Carlsbad Calif., Cat #11791-019).
• Ncol-Sacl fragment containing the AAD-12 (vl) coding sequence was digested from DASPIC012 and ligated into corresponding Ncol-Sacl restriction sites within the following Gateway Donor vectors: pDAB3912 (attLl//CsVMV promoter//AtuORF23
• pDAB4463 attLl//CsVMV promoter//AAD-12 (vl)//AtuORF23
• the plant expression cassettes were recombined into the Gateway Destination Binary vector pDAB4484 (RB7 MARv3//attRl-ccdB-chloramphenicol resistance- attR2//CsVMV promoter//PATv6//AtuORFl 3'UTR) via the Gateway LR Clonase reaction.
• Gateway Technology uses lambda phage-based site-specific recombination instead of restriction endonuclease and ligase to insert a gene of interest into an expression vector.
• the DNA recombination sequences (attL, and attR,) and the LR Clonase enzyme mixture allows any DNA fragment flanked by a recombination site to be transferred into any vector containing a corresponding site.
• the attLl site of the donor vector corresponds with attRl of the binary vector.
• the attL2 site of the donor vector corresponds with attR2 of the binary vector.
• the plant expression cassette (from the donor vector) which is flanked by the attL sites can be recombined into the attR sites of the binary vector.
• the resulting constructs containing the following plant expression cassettes were labeled as: pDAB4464 (RB7 MARv3//CsVMV promoter//AAD-12 (vl)//AtuORF23
• promo ter//PATv6// AtuORFl 3'UTR were confirmed via restriction enzyme digestion and sequencing.
• Transformation Method 1 Agrobacterium-mediated Transformation: The first reports of soybean transformation targeted meristematic cells in the cotyledonary node region (Hinchee et al., 1988) and shoot multiplication from apical meristems (McCabe et al., 1988). In the A. tumefciciens-based cotyledonary node method, explant preparation and culture media composition stimulate proliferation of auxiliary meristems in the node (Hinchee et al., 1988). It remains unclear whether a truly dedifferentiated, but totipotent, callus culture is initiated by these treatments.
• the soybean shoot multiplication method originally based on microprojectile bombardment (McCabe et al., 1988) and, more recently, adapted for Agrobacterium- mediated transformation (Martinell et al., 2002), apparently does not undergo the same level or type of dedifferentiation as the cotyledonary node method because the system is based on successful identification of germ line chimeras. Also, this is a non 2,4-D based protocol which would be ideal for 2,4-D selection system. Thus, the cotyledonary node method may be the method of choice to develop 2,4-D resistant soybean cultivars.
• AAD-12 (vl) tolerant phenotypes Plant transformation production of AAD-12 (vl) tolerant phenotypes. Seed derived explants of "Maverick” and the Agrobacterium mediated cot-node transformation protocol was used to produces AAD-12 (vl) transgenic plants.
• Agrobacterium Preparation and Inoculation Agrobacterium strain EHA101 (Hood et al. 1986), carrying each of five binary pDAB vectors (Table 8) was used to initiate transformation. Each binary vector contains the AAD-12 (vl) gene and a plant-selectable gene (PAT) cassette within the T-DNA region. Plasmids were mobilized into the EHA101 strain of Agrobacterium by electroporation. The selected colonies were then analyzed for the integration of genes before the Agrobacterium treatment of the soybean explants. Maverick seeds were used in all transformation experiments and the seeds were obtained from University of
• Agrobacterium-mediated transformation of soybean (Glycine max) using the PAT gene as a selectable marker coupled with the herbicide glufosinate as a selective agent was carried out.
• the seeds were germinated on B5 basal medium (Gamborg et al. 1968) solidified with 3 g/L Phytagel (Sigma-Aldrich, St. Louis, Mo.). Selected shoots were then transferred to the rooting medium.
• the optimal selection scheme was the use of glufosinate at 8 mg/L across the first and second shoot initiation stages in the medium and 3-4 mg/L during shoot elongation in the medium.
• glufosinate injury can be made 1-7 days after treatment. Plants can also be tested for 2,4-D tolerance in a non-destructive manner by selective application of a 2,4-D solution in water (0.25-1% v/v commercial 2,4-D
• This assay allows assessment of 2,4-D sensitive plants 6 hours to several days after application by assessment of leaf flipping or rotation >90 degrees from the plane of the adjacent leaflets. Plants tolerant to 2,4-D will not respond to 2,4-D. TO plants will be allowed to self fertilize in the greenhouse to give rise to Tl seed.
• Tl plants (and to the extent enough TO plant clones are produced) will be sprayed with a range of herbicide doses to determine the level of herbicide protection afforded by AAD-12 (vl) and PAT genes in transgenic soybean.
• Rates of 2,4-D used on TO plants will typically comprise one or two selective rates in the range of 100-1120 g ae/ha using a track sprayer as previously described.
• Tl plants will be treated with a wider herbicide dose ranging from 50-3200 g ae/ha 2,4-D.
• TO and Tl plants can be screened for glufosinate resistance by postemergence treatment with 200-800 and 50-3200 g ae/ha glufosinate, respectively.
• Glyphosate resistance in plants transformed with constructs that contain EPSPS
• another glyphosate tolerance gene can be assessed in the Tl generation by postemergence applications of glyphosate with a dose range from 280-2240 g ae/ha glyphosate.
• Individual TO plants were assessed for the presence of the coding region of the gene of interest (AAD-12 (vl) or PAT v6) and copy number.
• 2,4-D was applied to a subset of the plants that were of similar size to the wild type control plants with either 560 or 1120 g ae 2,4-D. All AAD-12 (vl)-containing plants were clearly resistant to the herbicide application versus the wild type Maverick soybeans. A slight level of injury (2 DAT) was observed for two AAD-12 (vl) plants, however, injury was temporary and no injury was observed 7 DAT. Wild type control plants were severely injured 7-14 DAT at 560 g ae/ha 2,4-D and killed at 1120 g ae/ha. These data are consistent with the fact that AAD-12 (vl) can impart high tolerance (>2. times, field rates) to a sensitive crop like soybeans. The screened plants were then sampled for molecular and biochemical analyses for the confirmation of the AAD12 (vl) genes integration, copy number, and gene expression levels.
• PCR reaction is carried out in the 9700 Geneamp thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 63 °C. for 30 seconds, and 72 °C. for 1 minute and 45 seconds followed by 72 °C. for 10 minutes.
• Primers for Coding Region PCR AAD-12 are (Forward- ATGGCTCATG CTGCCCTCAG CC) (SEQ ID NO: 10) and (Reverse-CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 11).
• the PCR reaction is carried out in the 9700 Geneamp thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 65 °C. for 30 seconds, and 72 °C. for 1 minute and 45 seconds followed by 72 °C. for 10 minutes.
• PCR products are analyzed by electrophoresis on a 1% agarose gel stained with EtBr.
• Southern blot analysis Southern blot analysis is performed with total DNA obtained from Qiagen DNeasy kit. A total of 10 ⁇ g of genomic DNA is subjected to an overnight digestion to obtain integration data. After the overnight digestion an aliquot of -100 ng is run on a 1% gel to ensure complete digestion. After this assurance the samples are run on a large 0.85% agarose gel overnight at 40 volts. The gel is then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gel is then neutralized in 0.5 M Tris HC1, 1.5 M NaCl pH of 7.5 for 30 minutes.
• a gel apparatus containing 20 x SSC is then set up to obtain a gravity gel to nylon membrane (Millipore INYCOOOIO) transfer overnight. After the overnight transfer the membrane is then subjected to UV light via a crosslinker (Stratagene UV stratalinker 1800) at 1200 x 100 microjoules. The membrane is then washed in 0.1% SDS, 0.1 SSC for 45 minutes. After the 45 minute wash, the membrane is baked for 3 hours at 80 °C. and then stored at 4 °C. until hybridization. The hybridization template fragment is prepared using the above coding region PCR using plasmid DNA. The product is run on a 1% agarose gel and excised and then gel extracted using the Qiagen (28706) gel extraction procedure.
• Qiagen 28706
• the membrane is then subjected to a pre-hybridization at 60 °C. step for 1 hour in Perfect Hyb buffer (Sigma H7033).
• the Prime it RmT dCTP-labeling rxn (Stratagene 300392) procedure is used to develop the p32 based probe (Perkin Elmer).
• the probe is cleaned up using the Probe Quant. G50 columns (Amersham 27-5335-01). Two million counts CPM are used to hybridize the southern blots overnight. After the overnight hybridization the blots are then subjected to two 20 minute washes at 65 °C. in 0.1% SDS, 0.1 SSC. The blots are then exposed to film overnight, incubating at -80 °C.
• Biochemical Analyses - Soybean Tissue Sampling and Extracting AAD-12 (vl) protein from soybean leaves. Approximately 50 to 100 mg of leaf tissue was sampled from the N-2 leaves that were 2,4-D leaf painted, but after 1 DAT. The terminal N-2 leaflet was removed and either cut into small pieces or 2- single-hole-punched leaf discs (-0.5 cm in diameter) and were frozen on dry ice instantly. Protein analysis (ELISA and Western analysis) was completed accordingly.
• Tl Progeny evaluation TO plants will be allowed to self fertilize to derive Tl families. Progeny testing (segregation analysis) will be assayed using glufosinate at 560 g ai/ha as the selection agent applied at the VI- V2 growth stage. Surviving plants will be further assayed for 2,4-D tolerance at one or more growth stages from V2-V6. Seed will be produced through self fertilization to allow broader herbicide testing on the transgenic soybean.
• AAD-12 (vl) transgenic Maverick soybean plants have been generated through Agrobacterium-mediated transformation system.
• the TO plants obtained tolerated up to 2 x levels of 2,4-D field applications and developed fertile seeds.
• the frequency of fertile transgenic soybean plants was up to 5.9%.
• the integration of the AAD1-12 (vl) gene into the soybean genome was confirmed by Southern blot analysis. This analysis indicated that most of the transgenic plants contained a low copy number.
• the plants screened with AAD-12 (vl) antibodies showed positive for ELISA and the appropriate band in Western analysis.
• Embryogenic Soybean Callus Tissue Culture of embryogenic soybean callus tissue and subsequent beaming can be accomplished as described in U.S. Pat. No. 6,809,232 (Held et al.) to create transformants using constructs provided herein.
• Transformation Method 3 Biolistic Bombardment of Soybean: This can be accomplished using mature seed derived embryonic axes meristem (McCabe et al. (1988)). Following established methods of biolistic bombardment, one can expect recovery of transformed soybean plants.
• Transformation Method 4 Whisker preparation and whisker transformation can be performed according to methods described previously by Terakawa et al. (2005)). Following established methods of biolistic
• Maverick seeds were surface- sterilized in 70% ethanol for 1 min followed by immersion in 1% sodium hypochlorite for 20 minutes and then rinsed three times in sterile distilled water. The seeds were soaked in distilled water for 18-20 hours. The embryonic axes were excised from seeds, and the apical meristems were exposed by removing the primary leaves. The embryonic axes were positioned in the bombardment medium [BM: MS
• Transformation Method 5 Particle bombardment-mediated transformation for embryogenic callus tissue can be optimized for according to previous methods (Khalafalla et al., 2005; El-Shemy et al., 2004, 2006).
• Cotton Transformation Protocol Cotton seeds (Co310 genotype) are surface- sterilized in 95% ethanol for 1 minute, rinsed, sterilized with 50% commercial bleach for twenty minutes, and then rinsed 3 times with sterile distilled water before being germinated on G-media (Table 21) in Magenta GA-7 vessels and maintained under high light intensity of 40- 60 ⁇ /m 2 , with the photoperiod set at 16 hours of light and 8 hours dark at 28 °C.
• Cotyledon segments ( ⁇ 5 mm) square are isolated from 7-10 day old seedlings into liquid M liquid media (Table 21) in Petri plates (Nunc, item #0875728). Cut segments are treated with an Agrobacterium solution (for 30 minutes) then transferred to semi-solid M-media (Table 21) and undergo co-cultivation for 2-3 days. Following co-cultivation, segments are transferred to MG media (Table 21). Carbenicillin is the antibiotic used to kill the
• Agrobacterium and glufosinate-ammonium is the selection agent that would allow growth of only those cells that contain the transferred gene.
• Agrobacterium preparation Inoculate 35 ml of Y media (Table 21) (containing streptomycin (100 mg/ml stock) and erythromycin (100 mg/ml stock)), with one loop of bacteria to grow overnight in the dark at 28 °C, while shaking at 150 rpm. The next day, pour the Agrobacterium solution into a sterile oakridge tube (Nalge-Nunc, 3139-0050), and centrifuge for in Beckman J2-21 at 8,000 rpm for 5 minutes. Pour off the supernatant and resuspend the pellet in 25 ml of M liquid (Table 21) and vortex.
• MG media can be supplemented with dichlorprop (added to the media at a concentration of 0.01 and 0.05 mg/L) to supplement for the degradation of the 2,4-D, since dichlorprop is not a substrate for to the AAD-12 enzyme, however dichlorprop is more active on cotton than 2,4-D.
• dichlorprop is not a substrate for to the AAD-12 enzyme, however dichlorprop is more active on cotton than 2,4-D.
• Callus is then transferred to CG-media (Table 21), and transferred again to fresh selection medium after three weeks. After another three weeks the callus tissue is transferred to D media (Table 21) lacking plant growth regulators for embryogenic callus induction. After 4-8 weeks on this media, embryogenic callus is formed, and can be
• AAD-12 (vl) cotton lines that have produced plants according to the above protocol will be sent to the greenhouse.
• AAD-12 (vl) gene provides resistance to 2,4-D in cotton
• both the AAD-12 (vl) cotton plant and wild- type cotton plants will be sprayed with a track sprayer delivering 560 g ae/ha 2,4-D at a spray volume of 187 L/ha.
• the plants will be evaluated at 3 and 14 days after treatment. Plants surviving a selective rate of 2,4-D will be self pollinated to create Tl seed or outcrossed with an elite cotton line to produce Fl seed.
• the subsequent seed produced will be planted and evaluated for herbicide resistance as previously described.
• AAD-12 (vl) events can be combined with other desired HT or IR trants.
• additional crops can be transformed according to the subject invention using techniques that are known in the art.
• Agrobacterium-mediated trans-formation of rye see, e.g., Popelka and Altpeter (2003).
• Agrobacterium-mediated transformation of soybean see, e.g., Hinchee et al., 1988.
• Agrobacterium-mediated transformation of sorghum see, e.g., Zhao et al., 2000.
• Agrobacterium-mediated transformation of barley see, e.g., Tingay et al., 1997.
• Agrobacterium-mediated trans-formation of rye see, e.g., Popelka and Altpeter (2003).
• Agrobacterium-mediated transformation of soybean see, e.g., Hinchee et al., 1988.
• Agrobacterium-mediated transformation of sorghum see, e.g., Zhao et al., 2000.
• Agrobacterium-mediated transformation of barley see,
• Oats (Avena sativa and strigosa), Peas (Pisum, Vigna, and Tetragonolobus spp.), Sunflower (Helianthus annuus), Squash (Cucurbita spp.), Cucumber (Cucumis sativa), Tobacco (Nicotiana spp.), Arabidopsis (Arabidopsis thaliana), Turfgrass (Lolium, Agrostis, Poa, Cynadon, and other genera), Clover (Tifolium), Vetch (Vicia).
• Such plants, with AAD-12 (vl) genes, for example, are included in the subject invention.
• AAD-12 (vl) has the potential to increase the applicability of key auxinic herbicides for in-season use in many deciduous and evergreen timber cropping systems.
• Triclopyr, 2,4-D, and/or fluoroxypyr resistant timber species would increase the flexibility of over-the-top use of these herbicides without injury concerns.
• Alder Alder (Alnus spp.), ash (Fraxinus spp.), aspen and poplar species (Populus spp.), beech (Fagus spp.), birch (Betula spp.), cherry (Prunus spp.), eucalyptus (Eucalyptus spp.), hickory (Carya spp.), maple (Acer spp.), oak (Quercus spp), and pine (Pinus spp).
• Use of auxin resistance for the selective weed control in ornamental and fruit-bearing species is also within the scope of this invention.
• Examples could include, but not be limited to, rose (Rosa spp.), burning bush (Euonymus spp.), petunia (Petunia spp), begonia (Begonia spp.), rhododendron (Rhododendron spp), crabapple or apple (Malus spp.), pear (Pyrus spp.), peach (Prunus spp), and marigolds (Tagetes spp.).
• AAD-2 (vl) Initial Cloning: Another gene was identified from the NCBI database (see the ncbi.nlm.nih.gov website; accession #AP005940) as a homologue with only 44% amino acid identity to tfdA. This gene is referred to herein as AAD-2 (vl) for consistency. Percent identity was determined by first translating both the AAD-2 and tfdA DNA sequences (SEQ ID NO: 12 of PCT/US2005/014737 and GENBANK Accession No. M16730, respectively) to proteins (SEQ ID NO: 13 of PCT/US2005/014737 and GENBANK Accession No. M16730, respectively), then using ClustalW in the VectorNTI software package to perform the multiple sequence alignment.
• the strain of Bradyrhizobium japonicum containing the AAD-2 (vl) gene was obtained from Northern Regional Research Laboratory (NRRL, strain #B4450).
• the lyophilized strain was revived according to NRRL protocol and stored at -80 °C. in 20% glycerol for internal use as Dow Bacterial strain DB 663. From this freezer stock, a plate of Tryptic Soy Agar was then struck out with a loopful of cells for isolation, and incubated at 28 °C. for 3 days. A single colony was used to inoculate 100 ml of Tryptic Soy Broth in a 500 ml tri-baffled flask, which was incubated overnight at 28 °C.
• Fail Safe Buffer 25 ⁇ ea. primer 1 ⁇ (50 ng/ ⁇ ), gDNA 1 ⁇ (200 ng/ ⁇ ), H.sub.20 21 ⁇ , Taq polymerase 1 ⁇ (2.5 units/ ⁇ ).
• Three Fail Safe Buffers-A, B, and C- were used in three separate reactions.
• PCR was then carried out under the following conditions: 95 °C. 3.0 minutes heat denature cycle; 95 °C. 1.0 minute, 50 °C. 1.0 minute, 72 °C. 1.5 minutes, for 30 cycles; followed by a final cycle of 72 °C. 5 minutes, using the FailSafe PCR System (Epicenter cat. #F599100).
• the resulting ⁇ 1 kb PCR product was cloned into pCR 2.1 (Invitrogen cat. #K4550-40) following the included protocol, with chemically competent TOPI OF' E. coli as the host strain, for verification of nucleotide sequence.
• AAD-2 (vl) Binary Vector The AAD-2 (vl) gene was PCR amplified from pDAB3202. During the PCR reaction alterations were made within the primers to introduce the Afllll and Sacl restriction sites in the 5' primer and 3' primer, respectively. See PCT/US2005/014737.
• the primers "Ncol of Brady” [5' TAT ACC ACA TGT CGA TCG CCA TCC GGC AGC TT 3'] (SEQ ID NO: 14) and “Sad of Brady” [5' GAG CTC CTA TCA CTC CGC CGC CTG CTG CTG CAC 3'] (SEQ ID NO: 15) were used to amplify a DNA fragment using the Fail Safe PCR System (Epicentre).
• the PCR product was cloned into the pCR2.1 TOPO TA cloning vector (Invitrogen) and sequence verified with Ml 3 Forward and Ml 3 Reverse primers using the Beckman Coulter "Dye Terminator Cycle Sequencing with Quick Start Kit” sequencing reagents.
• Sequence data identified a clone with the correct sequence (pDAB716).
• the Afllll/SacI AAD-2 (vl) gene fragment was then cloned into the Ncol/Sacl pDAB726 vector.
• the resulting construct (pDAB717); AtUbilO promoter: Nt OSM 5'UTR: AAD-2 (vl): Nt OSM3'UTR: ORF1 polyA 3'UTR was verified with restriction digests (with Ncol/Sacl). This construct was cloned into the binary pDAB3038 as a Notl-Notl DNA fragment.
• AAD-12 (vl) and AAD-2 (vl) did provide detectable 2,4-D resistance versus the transformed and untransformed control lines; however, individual constructs were widely variable in their ability to impart 2,4-D resistance to individual Tl Arabidopsis plants.
• AAD-2 (vl) and AAD-2 (v2) transformants were far less resistant to 2,4-D than the AAD-12 (vl) gene, both from a frequency of highly tolerant plants as well as overall average injury.
• AAD-12 (vl) had a population injury average of about 6% when treated with 3,200 g ae/ha 2,4-D. Tolerance improved slightly for plant- optimized AAD- 2 (v2) versus the native gene; however, comparison of both AAD-12 and AAD-2 plant optimized genes indicates a significant advantage for AAD-12 (vl) in planta.
• AAD-12 can enable the use of phenoxy auxin herbicides (e.g., 2,4-D and MCPA) and pyridyloxy auxins (triclopyr and fluoroxypyr) for the control of a wide spectrum of broadleaf weeds directly in crops normally sensitive to 2,4-D.
• 2,4-D at 280 to 2240 g ae/ha would control most broadleaf weed species present in agronomic environments. More typically, 560-1120 g ae/ha is used.
• triclopyr application rates would typically range from 70-1120 g ae/ha, more typically 140-420 g ae/ha.
• fluoroxypyr application rates would typically range from 35-560 g ae/ha, more typically 70-280 ae/ha.
• An advantage to this additional tool is the extremely low cost of the broadleaf herbicide component and potential short-lived residual weed control provided by higher rates of 2,4-D, triclopyr, and fluoroxypyr when used at higher rates, whereas a non-residual herbicide like glyphosate would provide no control of later germinating weeds.
• This tool also provides a mechanism to combine herbicide modes of action with the convenience of HTC as an integrated herbicide resistance and weed shift management strategy.
• a further advantage this tool provides is the ability to tankmix broad spectrum broadleaf weed control herbicides (e.g., 2,4-D, triclopyr and fluoroxypyr) with commonly used residual weed control herbicides. These herbicides are typically applied prior to or at planting, but often are less effective on emerged, established weeds that may exist in the field prior to planting. By extending the utility of these aryloxy auxin herbicides to include at-plant, preemergence, or pre-plant applications, the flexibility of residual weed control programs increases. One skilled in the art would recognize the residual herbicide program will differ based on the crop of interest, but typical programs would include herbicides of the
• chloracetmide and dinitroaniline herbicide families but also including herbicides such as clomazone, sulfentrazone, and a variety of ALS -inhibiting PPO-inhibiting, and HPPD- inhibiting herbicides.
• grass species such as, but not limited to, corn, rice, wheat, barley, or turf and pasture grasses
• AAD-12 vl
• transformation of grass species would allow the use of highly efficacious phenoxy and pyridyloxy auxins in crops where normally selectivity is not certain.
• Most grass species have a natural tolerance to auxinic herbicides such as the phenoxy auxins (i.e., 2,4-D.).
• phenoxy auxins i.e., 2,4-D.
• a relatively low level of crop selectivity has resulted in diminished utility in these crops due to a shortened window of application timing or unacceptable injury risk.
• AAD-12 (vl)-transformed monocot crops would, therefore, enable the use of a similar combination of treatments described for dicot crops such as the application of 2,4-D at 280 to 2240 g ae/ha to control most broadleaf weed species. More typically, 560-1120 g ae/ha is used.
• application rates would typically range from 70-1120 g ae/ha, more typically 140-420 g ae/ha.
• fluoroxypyr application rates would typically range from 35-560 g ae/ha, more typically 70-280 ae/ha.
• An advantage to this additional tool is the extremely low cost of the broadleaf herbicide component and potential short-lived residual weed control provided by higher rates of 2,4-D, triclopyr, or fluoroxypyr.
• a non-residual herbicide like glyphosate would provide no control of later-germinating weeds.
• This tool would also provide a mechanism to rotate herbicide modes of action with the convenience of HTC as an integrated-herbicide- resistance and weed- shift- management strategy in a glyphosate tolerant crop/AAD-12 (vl) HTC combination strategy, whether one rotates crops species or not.
• a further advantage this tool provides is the ability to tankmix broad spectrum broadleaf weed control herbicides (e.g., 2,4-D, triclopyr and fluoroxypyr) with commonly used residual weed control herbicides. These herbicides are typically applied prior to or at planting, but often are less effective on emerged, established weeds that may exist in the field prior to planting. By extending the utility of these aryloxy auxin herbicides to include at-plant, preemergence, or pre-plant applications, the flexibility of residual weed control programs increases. One skilled in the art would recognize the residual herbicide program will differ based on the crop of interest, but typical programs would include herbicides of the
• chloracetmide and dinitroaniline herbicide families but also including herbicides such as clomazone, sulfentrazone, and a variety of ALS -inhibiting PPO-inhibiting, and HPPD- inhibiting herbicides.
• PRH50 medium
• BAP 2,4-dichlorophenoxyacetic acid
• NAA a-naphthaleneacetic acid
• ABA abscisic acid
• RNH50 regeneration medium
• RNH50 Regeneration of plantlets followed via culture on regeneration medium (RNH50) comprising NB medium without 2,4-D, and supplemented with 3 mg/L BAP, 0.5 mg/L NAA, and 50 mg/L hygromycin B until shoots regenerated.
• Shoots were transferred to rooting medium with half-strength Murashige and Skoog basal salts and Gamborg's B5 vitamins, supplemented with 1% sucrose and 50 mg/L hygromycin B (1/2MSH50).
• Tissue Culture Development Mature desiccated seeds of Oryza sativa L. japonica cv. Taipei 309 were sterilized as described in Zhang et al. 1996. Embryogenic tissues were induced by culturing sterile mature rice seeds on NB medium in the dark. The primary callus approximately 1 mm in diameter, was removed from the scutellum and used to initiate cell suspension in SZ liquid medium. Suspensions were then maintained as described in Zhang 1995. Suspension-derived embryogenic tissues were removed from liquid culture 3-5 days after the previous subculture and placed on NBO osmotic medium to form a circle about 2.5 cm across in a Petri dish and cultured for 4 hous prior to bombardment.
• tissues were transferred from NBO medium onto NBH50 hygromycin B selection medium, ensuring that the bombarded surface was facing upward, and incubated in the dark for 14-17 days.
• Newly formed callus was then separated from the original bombarded explants and placed nearby on the same medium.
• relatively compact, opaque callus was visually identified, and transferred to PRH50 pre-regeneration medium for 7 days in the dark.
• Growing callus, which became more compact and opaque was then subcultured onto RNH50 regeneration medium for a period of 14-21 days under a 16-hour photoperiod. Regenerating shoots were transferred to Magenta boxes containing 1/2 MSH50 medium.
• Microprojectile Bombardment All bombardments were conducted with the Biolistic PDS-1000/HeTM system (Bio-Rad, Laboratories, Inc.). Three milligrams of 1.0 micron diameter gold particles were washed one with 100% ethanol, twice with sterile distilled water and resuspended in 50 ⁇ water in a siliconized Eppendorf tube. Five micrograms plasmid DNA representing a 1:6 molar ratio of pDOW3303 (Hpt-containing vector) to pDAB4101 (AAD-12 (vl)+AHAS), 20 ⁇ spermidine (0.1 M) and 50 ⁇ calcium chloride (2.5 M) were added to the gold suspension.
• AAD-12 (vl) in Tl Rice A 100-plant progeny test was conducted on five Tl lines of AAD-12 (vl) lines that contained both the AAD-12 (vl) PTU and AHAS coding region. The seeds were planted with respect to the procedure above and sprayed with 140 g ae/ha imazethapyr using a track sprayer as previously described. After 14 DAT, resistant and sensitive plants were counted. Two out of the five lines tested segregated as a single locus, dominant Mendelian trait (3R: 1S) as determined by Chi square analysis. AAD-12 coseregated with the AHAS selectable marker as determined by 2,4-D tolerance testing below.
• pDAB4101(20)003 was more tolerant to high levels of 2,4-D than the line pDAB4101(27)002.
• the data also demonstrates that tolerance of 2,4-D is stable for at least two generations.
• Tissue Harvesting, DNA Isolation and Quantification Fresh tissue was placed into tubes and lyophilized at 4 °C. for 2 days. After the tissue was fully dried, a tungsten bead (Valenite) was placed in the tube and the samples were subjected to 1 minute of dry grinding using a Kelco bead mill. The standard DNeasy DNA isolation procedure was then followed (Qiagen, Dneasy 69109). An aliquot of the extracted DNA was then stained with Pico Green (Molecular Probes P7589) and scanned in the florometer (BioTek) with known standards to obtain the concentration in ng/ ⁇ .
• AAD-12 (vl) Expression All 33 TO transgenic rice lines and 1 non-transgenic control were analyzed for AAD-12 expression using ELISA blot. AAD-12 was detected in the clones of 20 lines, but not in line Taipai 309 control plant. Twelve of the 20 lines that had some of the clones tolerant to imazethapyr were expressing AAD-12 protein, were AAD-12 PCR PTU positive, and AHAS coding region positive. Expression levels ranged from 2.3 to 1092.4 ppm of total soluble protein.
• Tl generation pDAB4101[20] and two rows of Clearfield rice were planted using a small plot drill with 8-inch row spacing.
• the pDAB4101 [20] rice contained the AHAS gene as a selectable marker for the AAD-12(vl) gene.
• Imazethapyr was applied at the one leaf stage as selection agent to remove any AAD-12 (vl) null plants from the plots.
• Herbicide treatments were applied when the rice reached the 2 leaf stage using compressed air backpack sprayer delivering 187 I/ha carrier volume at 130-200 kpa pressure. Visual ratings of injury were taken at 7, 14 and 21 days after application.
• AAD-12 (vl) event response to 2,4-D and triclopyr are shown in Table 23.
• the non-transformed rice line (Clearfield) was severely injured (30% at 7DAT and 35% at 15DAT) by 2,4-D at 2240 g ae/ha which is considered the 4 x commercial use rate.
• the AAD-12 (vl) event demonstrated excellent tolerance to 2,4-D with no injury observed at 7 or 15DAT.
• the non-transformed rice was significantly injured (15% at 7DAT and 25% at 15DAT) by the 2 x rate of triclopyr (560 g ae/ha).
• the AAD-12 (vl) event demonstrated excellence tolerance to the 2 x rates of triclopyr with no injury observed at either 7 or 15DAT.
• Canola Transformation The AAD-12 (vl) gene conferring resistance to 2,4-D was used to transform Brassica napus var. Nexera*710 with Agwbacterium-mediated
• the construct contained AAD-12 (vl) gene driven by CsVMV promoter and Pat gene driven by AtUbilO promoter and the EPSPS glyphosate resistance trait driven by AtUbilO promoter.
• Seeds were surface-sterilized with 10% commercial bleach for 10 minutes and rinsed 3 times with sterile distilled water. The seeds were then placed on one half concentration of MS basal medium (Murashige and Skoog, 1962) and maintained under growth regime set at 25 °C, and a photoperiod of 16 hours light/8 hours dark.
• MS basal medium Merashige and Skoog, 1962
• the hypocotyl segments After 30 min treatment of the hypocotyl segments with Agwbacterium, these were placed back on the callus induction medium for 3 days. Following co-cultivation, the segments were placed on K1D1TC (callus induction medium containing 250 mg/L CarbeniciUin and 300 mg/L Timentin) for one week or two weeks of recovery. Alternately, the segments were placed directly on selection medium K1D1H1 (above medium with 1 mg/L Herbiace). CarbeniciUin and Timentin were the antibiotics used to kill the Agwbacterium. The selection agent Herbiace allowed the growth of the transformed cells.
• K1D1TC callus induction medium containing 250 mg/L CarbeniciUin and 300 mg/L Timentin
• B3Z1H3 medium MS medium, 3 mg/L benzylamino purine, 1 mg/L Zeatin, 0.5 gm/L MES [2-(N-morpholino) ethane sulfonic acid], 5 mg/L silver nitrate, 3 mg/L Herbiace, CarbeniciUin and Timentin) for another 2-3 weeks.
• Primers for Coding Region PCR AAD-12 (vl) were (SEQ ID NO: 10) (forward) and (SEQ ID NO: 11) (reverse).
• the PCR reaction was carried out in the 9700 Geneamp thermocycler (Applied Biosystems), by subjecting the samples to 94 °C. for 3 minutes and 35 cycles of 94 °C. for 30 seconds, 65 °C. for 30 seconds, and 72 °C. for 2 minutes followed by 72 °C. for 10 minutes.
• PCR products were analyzed by electrophoresis on a 1% agarose gel stained with EtBr. 35 samples from 35 plants with AAD-12 (vl) events tested positive. Three negative control samples tested negative.
• ELISA Using established ELISA described in previous section, AAD-12 protein was detected in 5 different canola transformation plant events. Expression levels ranged from 14 to over 700 ppm of total soluble protein (TSP). Three different untransformed plant samples were tested in parallel with no signal detected, indicating that the antibodies used in the assay have minimal cross reactivity to the canola cell matrix. These samples were also confirmed positive by Western analysis. A summary of the results is presented in Table 24.
• a lethal dose is defined as the rate that causes >95 injury to the untransformed controls. [00337] Twenty-four of the events were tolerant to the 2,4-D DMA herbicide application. Some events did incur minor injury but recovered by 14 DAT. Events were progressed to the Tl (and T2 generation) by selfpollination under controlled, bagged, conditions.
• AAD-12 (vl) Heritability in Canola A 100 plant progeny test was also conducted on 11 Tl lines of AAD-12 (vl). The seeds were sown and transplanted to 3-inch pots filled with Metro Mix media. All plants were then sprayed with 560 g ae/ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. Seven out of the 11 lines tested segregated as a single locus, dominant Mendelian trait (3R: 1S) as determined by Chi- square analysis. AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes.
• AAD-12 (vl) Heritability in Canola A 100 plant progeny test was also conducted on 11 Tl lines of AAD-12 (vl). The seeds were sown and transplanted to 3-inch pots filled with Metro Mix media. All plants were then sprayed with 560 g ae/ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. Seven out of the 11 lines tested segregated as a single locus, dominant Mendelian trait (3R: 1S) as determined by Chi- square analysis. AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes.
• Canola response to 2,4-D, triclopyr, and fluoroxypyr are shown in Table 26.
• the wild-type canola (Nex710) was severely injured (72% at 14DAT) by 2,4-D at 2240 g ae/ha which is considered the 4 x rate.
• the AAD-12 (vl) events all demonstrated excellent tolerance to 2,4-D at 14DAT with an average injury of 2, 3 and 2% observed at the 1, 2 and 4 x rates, respectively.
• the wild- type canola was severely injured (25% at 14DAT) by the 2 x rate of triclopyr (840 g ae/ha).
• AAD-12 (vl) events demonstrated tolerance at 2 x rates of triclopyr with an average of 6% injury at 14DAT across the two events. Fluoroxypyr at 280 g ae/ha caused severe injury (37%) to the non-transformed line at 14DAA. AAD-12 (vl) events demonstrated increased tolerance with an average of 8% injury at 5DAT.
• AAD-12 (vl) transformed events displayed a high level of resistance to 2,4-D, triclopyr and fluoroxypyr at rates that were lethal or caused severe epinastic malformations to non-transformed canola.
• AAD-12 has been shown to have relative efficacy of 2,4-D > triclopyr > fluoroxypyr.
• Transgenic soybean (Glycine max) Event DAS-68416-4 was generated through Agwbacterium-mediated transformation of soybean cotyledonary node explants.
• Agwbacterium-mediated transformation was carried out. Briefly, soybean seeds (cv Maverick) were germinated on basal media and cotyledonary nodes were isolated and infected with Agwbacterium. Plant initiation, shoot elongation, and rooting media were supplemented with cefotaxime, timentin and vancomycin for removal of Agwbacterium. Glufosinate selection was employed to inhibit the growth of non-transformed shoots. Selected shoots were transferred to rooting medium for root development and then transferred to soil mix for acclimatization of plantlets.
• Terminal leaflets of selected plantlets were leaf painted with glufosinate to screen for putative transformants.
• the screened plantlets were transferred to the greenhouse, allowed to acclimate and then leaf-painted with glufosinate to reconfirm tolerance and deemed to be putative transformants.
• the screened plants were sampled and molecular analyses for the confirmation of the selectable marker gene and/or the gene of interest were carried out. TO plants were allowed to self fertilize in the greenhouse to give rise to Tl seed.
• soybean Event DAS-68416-4 was generated from an independent transformed isolate. The event was selected based on its unique characteristics such as single insertion site, normal Mendelian segregation and stable expression, and a superior combination of efficacy, including herbicide tolerance and agronomic performance in broad genotype backgrounds and across multiple environmental locations. Additional description of soybean Event DAS-68416- 4 has been disclosed in WO 2011/066384, which is incorporated by reference in its entirety.
• Agronomic Experiment S 1 An agronomic study with Event DAS-68416-4 soybean and a non-transgenic control (var. Maverick) was conducted in 2008 at six sites located in Iowa, Illinois, Indiana, California and Ontario, Canada (2 sites). Agronomic determinants, including stand/population count, seedling/plant vigor, plant height, lodging, disease incidence, insect damage, and days to flowering were evaluated to investigate the equivalency of the soybean Event DAS-68416-4 (with and without herbicide treatments) as compared to the control line Maverick. This study is referred to as Agronomic Experiment S 1.
• Seedling vigor VC-V2 Visual estimate of 1-10 scaled based on average vigor of growth of the non- emerged plants per transformed soybeans plot 10 Growth equivalence to non-transformed
• the test and control soybean seed were planted at a seeding rate of approximately 112 seeds per 25 ft row with a row spacing of approximately 30 inches (75 cm).
• a seeding rate of approximately 112 seeds per 25 ft row with a row spacing of approximately 30 inches (75 cm).
• Plots were arranged in a randomized complete block (RCB) design, with a unique randomization at each site.
• RBC randomized complete block
• Each soybean plot was bordered by two rows of a non-transgenic soybean of similar maturity. The entire trial site was surrounded by a minimum of 10 ft of a non-transgenic soybean of similar relative maturity.
• Herbicide treatments were applied with a spray volume of approximately 20 gallons per acre (187 L/ha). These applications were designed to replicate maximum label rate commercial practices.
• 2,4-D was applied as three broadcast over-the-top applications for a seasonal total of 3 lb ae/A.
• Individual applications of 1.0 lb ae A (1,120 g/ha) were made at pre-emergence and approximately V4 and R2 growth stages.
• Glufosinate was applied as two broadcast over-the-top applications for a seasonal total of 0.74 lb ai/A (828 g ai/ha).
• Individual applications of 0.33 lb ai/A and 0.41 lb ai/A (374 and 454 g ai/ha) were made at approximately V6 and Rl growth stages.
• Paired contrasts were made between the control and unsprayed soybean Event DAS-68416-4 (unsprayed), soybean Event DAS-68416-4 sprayed with glufosinate (soybean Event DAS-68416-4 + glufosinate), soybean Event DAS-68416-4 sprayed with 2,4-D (soybean Event DAS-68416-4 + 2,4-D) and soybean Event DAS-68416-4 sprayed with both glufosinate and 2,4-D (soybean Event DAS-68416-4 + both) transgenic entries using t- tests. Adjusted P-values were also calculated using the False Discovery Rate (FDR) to control for multiplicity (Benjamini and Hochberg, 1995).
• FDR False Discovery Rate
• soybean Event DAS-68416-4 was agronomically equivalent to the near-isogenic non-transgenic control. Table 28. Analysis of agronomic characteristics from Agronomic Experiment SI.
• Seedling vigor VI - V3 General seedling vigor 1 (low) to 10 B
• a randomized-complete-block design was used for trials. Entries were soybean Event DAS-68416-4, a Maverick control line, and commercially available non-transgenic soybean lines. The test, control and reference soybean seed were planted at a seeding rate of approximately 112 seeds per row with row spacing of approximately 30 inches (75 cm). At each site, 4 replicate plots of each treatment were established, with each plot consisting of 2-25 ft rows. Each soybean plot was bordered by 2 rows of a non-transgenic soybean (Maverick). The entire trial site was surrounded by a minimum of 4 rows (or 10 ft) of non-transgenic soybean (Maverick). Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop.
• the herbicide was applied at a rate of 1.0 lb ae /A (1,120 g ae/ha) at the V4 and R2 growth stages.
• applications were made to plants at the V4 and V6-R2 growth stages.
• glufosinate was applied at a rate of 0.33 lb ai/A (374 g ai/ha) and 0.41 lb ai/A (454 g ai/ha) for the V4 and V6-R2 applications, respectively.
• Entries for both herbicide applications were soybean Event DAS-68416-4 and the controls including non-transgenic Maverick. Maverick plots were expected to die after herbicide application.
• Paired contrasts were made between unsprayed AAD-12 (unsprayed), AAD-12 sprayed with glufosinate (AAD- 12 + glufosinate), AAD- 12 sprayed with 2,4-D (AAD- 12 + 2,4-D) and AAD- 12 sprayed with both glufosinate and 2,4-D (AAD-12 + 2,4-D + glufosinate) transgenic entries and the control entry using T- tests.
• the AAD1 event pDAS 1740-278 was produced by WHISKER - mediated transformation of maize line Hi-II.
• the transformation method used is described in US Patent Application # 20090093366.
• An Fspl fragment of plasmid pD AS 1740 (FIG. 3), also referred to as pDAB3812, was transformed into the maize line.
• This plasmid construct contains the plant expression cassette containing the RB7 MARv3 : : Zea mays Ubiquitin 1 promoter v2 // AAD1 v3 // Zea mays PER5 3'UTR :: RB 7 MARv4 plant transcription unit (PTU).
• Tl plants were taken for molecular analysis to verify the presence of the AAD-I transgene by Southern Blot, DNA border confirmation, and genomic marker assisted confirmation. Positive TO plants were pollinated with inbred lines to obtain Tl seed. Tl plants of Event pDAS 1470-278-9 (DAS-40278-9) was selected, self -pollinated and characterized for five generations. Meanwhile, the Tl plants were backcrossed and introgressed into elite germplasm (XHH 13) through marker-assisted selection for several generations. This event was generated from an independent transformed isolate.
• DAS-40278-9 was selected, self -pollinated and characterized for five generations. Meanwhile, the Tl plants were backcrossed and introgressed into elite germplasm (XHH 13) through marker-assisted selection for several generations. This event was generated from an independent transformed isolate.
• Herbicide treatments were applied with a spray volume of approximately 20 gallons per acre (187 L/ha).
• Root Lodging Approximately R6 Visual estimate of percent of plants in the plot leaning approximately 30° or more in the first -1/2 meter above the soil surface
• Crop Injury (0.431) 0 0 0 0 0.28 - 2nd app. e (1.00, 1.00) (1.00, 1.00) (1.00, 1.00) (0.130, 0.819)
• Root (0.431) 0.44 0.17 0.72 0.17 0.11 Lodging (%) (0.457, 0.819) (0.457, 0.819) (0.457, 0.819) (0.373, 0.819)
• Agronomic characteristics of corn line 40278 compared to a near-isoline corn line were evaluated across diverse environments. Treatments included 4 genetically distinct hybrids and their appropriate near-isoline control hybrids tested across a total of 21 locations.
• the four test hybrids were medium to late maturity hybrids ranging from 99 to 113 day relative maturity.
• Experiment A tested event DAS-40278-9 in the genetic background Inbred C x BC3S1 conversion. This hybrid has a relative maturity of 109 days and was tested at 16 locations (Table 32).
• Experiment B tested the hybrid background Inbred E x BC3S1 conversion, a 113 day relative maturity hybrid. This hybrid was tested at 14 locations, using a slightly different set of locations than Experiment A.
• Experiments C and D tested hybrid backgrounds BC2S1 conversion x Inbred D and BC2S1 conversion x Inbred F, respectively. Both of these hybrids have a 99 day relative maturity and were tested at the same 10 locations.
• Results from these agronomic characterization trials can be found in Table 32. No statistically significant differences were found for any of the four 40278 hybrids compared to the isoline controls (at p ⁇ 0.05) for the parameters of ear height, stalk lodging, root lodging, grain moisture, test weight, and yield. Final population count and plant height were statistically different in Experiments A and B, respectively, but similar differences were not seen in comparisons with the other 40278 hybrids tested. Some of the variation seen may be due to low levels of genetic variability remaining from the backcrossing of the DAS-40278-9 event into the elite inbred lines. The overall range of values for the measured parameters are all within the range of values obtained for traditional corn hybrids and would not lead to a conclusion of increased weediness. In summary, agronomic characterization data indicate that 40278 corn is biologically equivalent to conventional corn.
• Test Weight AAD-1 56.96 50.90 59.50 0.2796 (lb/bushel) Control 56.67 52.00 60.10
• Test Weight AAD-1 54.62 42.10 58.80 0.1715 (lb/bushel) Control 55.14 52.80 58.40
• Test Weight AAD-1 56.59 54.80 58.30 0.0992 (lb/bushel) Control 55.50 52.70 57.40
• Agronomic characteristics for the hybrid corn lines containing event DAS-40278-9 and null plants sprayed with the herbicides quizalofop (280 g ae/ha) at the V3 stage of development and 2,4-D (2,240 g ae/ha) sprayed at the V6 stage of development are in Table 34.
• Transgenic soybean with AAD-12 transgene provides protection to the soybean plant while weeds are destroyed by application of 2,4-D. It has been unexpectedly observed that 2,4-D also increase growth in 2,4-D tolerant soybean. This increased growth has resulted in increases in plant height and/or yield of sprayed plots compared to non- sprayed plots.
• Seedling vigor Percent vigor with 0% representing VI - V3

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