BR102013013974A2 - Plant growth improvement methods for 2,4-d resistant crops - Google Patents

Abstract

Methods of improving the growth of 2,4-d resistant crop plants Abstract of the disclosure 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. in particular, provided is a method using 2,4-d application to increase yield of crop plants which express aad-12 gene for 2,4-d resistance. The method provided is of particular interest for the treatment of crops including maize, soybean, spring and winter oil seed rape (canola), sugar beet, wheat, sunflower, barley, and rice.

Description

Report of the Invention Patent for "METHODS FOR IMPROVING GROWTH OF 2,4-D RESISTANT CROP PLANTS".

CROSS REFERENCE TO RELATED PATENT APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 61/656, 546, filed June 7, 2012, the disclosure of which is expressly incorporated into the present invention. by reference in its entirety. INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIAL

V A computer sequence list is simultaneously incorporated by reference in its entirety and annexed and identified as follows: an 11,342-byte AS-CII file (text) with the name "72747_ST25.txt", created on May 13, 2013.

BACKGROUND OF THE INVENTION

Weeds can quickly deplete the soil of valuable nutrients needed for crops and other desirable plants. There are many different types of herbicides currently used for weed control. An extremely popular herbicide is glyphosate.

Crops such as corn, soybean, canola, cotton, sugar beet, wheat, grass, rice have been developed which are resistant to glyphosate. In this way, glyphosate-resistant corn active-growing fields, for example, can be sprayed to control weeds without significantly harming maize plants.

With the introduction of genetically modified organisms, glyphosate tolerant crops (GCTs) in the mid-1990s, growers were empowered with a simple, convenient, flexible and cost effective tool to control a broad spectrum of larval leaves. gas and grass weeds unprecedented in agriculture. As a result, growers have been quick to adopt TCG and in many cases abandon many of the best accepted agronomic practices such as crop rotation, mode of action herbicide rotation, mixing tank, incorporation of mechanically controlled mechanics. cultural and chemical weeds. Glyphosate tolerant soybean, cotton, corn and canola are currently commercially available in the United States and elsewhere in the Western Hemisphere. More TCGs (eg wheat, rice, sugar beet, grass, etc.) are prepared for the pending introduction of global market acceptance. Many other species are resistant to glyphosate in experimental stages of development (eg, alfalfa, sugar cane, sunflower, beet, peas, cenourd, cucumber, lettuce, onion, strawberry, tomato and tobacco; forest species such as poplar and sweetgum, and V vegetable species such as marigold, petunia, and begonias, see the website "isb.vt.edu/cfdocs/fieldtests1.cfm 2005"). In addition, the cost of glyphosate has fallen dramatically in recent years, to the extent that some conventional weed control programs may compete effectively on price and performance with glyphosate GTC systems. Glyphosate has been successfully used in non-agricultural management areas and others for total vegetation control for over 15 years. In many cases, as with GCT, glyphosate has been used 1 to 3 times per year for 3, 5, 10, up to 15 years in a row. These circumstances have led to overconfidence in glyphosate and GTC technology and have placed heavy selection pressure on native weed species that are naturally more tolerant to glyphosate or have developed a mechanism to resist herbicide activity. of glyphosate. Extensive use of glyphosate-only weed control programs is, resulting in the selection of glyphosate resistant weeds, and is the selection for the propagation of weed species that are inherently more tolerant to glyphosate than most target species (eg weed changes). (Ng et al., 2003; Simamata et al., 2003; Lorraine Colwill et al., 2003; Sfiligoj, 2004; Miller et al., 2003; Heap, 2005, Murphy et al., 2002; Martin et al ., 2002) Although glyphosate has been widely used globally for over 15 years, only a small number of weeds have been reported to have developed glyphosate resistance (Heap, 2005); however, most of them have been identified in the last 3-5 years. Hardy weeds include both grass and leafy species, Lolium rigidum, Lolium multiflour, Eleusine indica, Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis and Plantago lanceolata. In addition, weeds, which previously had not been an agronomic problem before the widespread use of GCT, are now becoming more prevalent and difficult to control in the context of GCT, which comprises> 80% of cotton. US and hectares of soybeans and> 20% of US corn acres (Gianessi, 2005). These changes are occurring predominantly from weeds with (but not exclusively) difficult to control broadleaf weeds. Some examples include the species Ipomoea, Amaranthus, Chenopodium, Taraxacum, and Commelina.

In areas where growers are confronted with glyphosate resistant weeds or a shift to more difficult weed control species, growers can compensate for glyphosate weaknesses by mixing tank or alternating with other herbicides that may will control lost weeds. One of the popular and effective tank mixing partners to control broadleaf leakage has in many cases been 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D has been used agronomically and in broad spectrum non-harvesting situations, broadleaf weed control for over 60 years. Individual cases of more tolerant species have been reported, but 2,4-D remains one of the most widely used herbicides in the world. The limitation of using more than 2,4-D is that its dicotyledonous selectivity in crops such as soybean or cotton is very poor and therefore 2,4-D is not normally used in (and usually not near) sensitive dicotyledonous crops. . In addition, the use of 2,4-D in grasses is somewhat limited by the nature of crop damage that may occur. 2,4-D, in combination with glyphosate has been used to provide a more robust desiccation treatment before planting on soybean and cotton, however, due to the sensitivity of these dicotyledon species to 2,4-D , these management treatments must occur at least 14 to 30 days before planting (Agriliance, 2003). 2,4-D is the class of phenoxy acid herbicides as is MCPA. 2,4-D has been used in many monocot crops (such as maize, 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 normally deregulate cellular hormone homeostasis and prevent balanced and controlled growth; however, the exact mode of action is not yet known. Triclopyr and jfluroxipir are pyridyloxyacetic acid herbicides whose mode of action is as a synthetic auxin, as well.

These herbicides have different levels of selectivity in certain plants (for example, dicotyledons are more sensitive than grasses). Differential metabolism across different plants is one explanation for varying selectivity levels. In general, plants slowly metabolize 2,4-D, so varying plant response to 2,4-D can most likely be explained by different activity at the target site (s) (WSSA, 2002). ). Plant metabolism of 2,4-D normally occurs through a two-step mechanism, usually hydroxylation followed by conjugation with amino acids or glucose (WSSA, 2002).

Over time, microbial populations have developed an alternative and efficient pathway for the degradation of this particular xenobiotic, resulting in complete 2,4-D mineralization. Successive applications of the herbicide select for microbes that can use herbicide as a carbon source for growth, giving them a competitive advantage in the soil. For this reason, the currently formulated 2,4-D has a relatively short soil half-life, and no significant residual effects on subsequent crops are found. This contributes to the usefulness of 2,4-D herbicide.

One organism that has been extensively studied for its ability to degrade 2,4-D is Ralstonia eutropha (Streber et al, 1987). The gene that codes for the first enzymatic step in the mineralisation pathway is ADF. See U.S. Patent No. 6,153,401 and GENBANK Acc. No. M16730. TFDA catalyzes the conversion of 2,4-D acid to dichlorophenol (DCP) by an α-ketoglutarate-dependent dioxigenase reaction (Smejkal et al, 2001). PDD has little herbicidal activity compared to 2,4-D. ADHD has been used in transgenic plants to confer 2,4-D resistance in dicotyledonous plants (eg 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). A large number of TFDA-like genes encoding proteins capable of degrading 2,4-D have been identified from the environment and deposited in the Genbank database. Many homologs are similar to ADHD (> 85% amino acid identity) and have similar enzymatic properties to ADHD. However, there are a number of homologues that have a significantly lower ADHD identity (25-50%), yet have the characteristics associated with the acetoglutarate dioxigenase Fe 2 dioxigenases residues. Therefore, it is not obvious that the substrate specificities of these dioxigenases are divergent.

A unique example with low ADHD homology (31% amino acid identity) is SDPA from Delftia acidovorans (Kohler et al. From 1999, Westendorf et al. From 2002, Westendorf et al., 2003). This enzyme has been shown to catalyze the first step of (S) -dichlorprop (and others (S) -phenoxypropionic acids) as well as the mineralization of 2,4-D (a phenoxyacetic acid) (Westendorf et al. (2003) The transformation of this gene into plants has not been reported so far The development of new herbicide tolerant (HTC) crop technologies has been limited in success largely due to the efficacy, low cost and convenience of TCG. As a result, a high adoption rate for GTCs occurred among producers, which created little incentive for the development of new HTC technologies.

Aryloxyalkanoate chemical substructures are a common feature of many marketed herbicides, including phenoxyacetate auxins (such as 2,4-D and dichlorprop), auxins (such as fluroxipyr or triclopyr pyridyloxyacetate), aryloxyphenoxypropionates (AOPP) inhibitors coenzyme A carboxylase (ACCase) (eg haloxifope, quizalofope, and diclofop) and protoforphigen IX 5- substituted phenoxyacetate oxidase inhibitors (such as piraflufen and lumiclorac), However, these classes of herbicides are quite distinct, and do not exist evidence in the current literature on common degradation pathways between these chemical classes. The multifunctional enzyme for the degradation of herbicides aborted by various modes of action has recently been described (PCT US / 2005/014737, filed May 2, 2005.

SUMMARY OF THE PRESENT INVENTION The present invention relates to methods for improving plant height and / or crop yields that are resistant to the 2,4-D herbicide by treating plants with 2, 2, 3 4-D at application rates that are not harmful to plants. In particular, there is provided a method that utilizes the application of 2,4-D to increase the yield of AAD-12-expressing crop plants for the 2,4-D resistance gene. The present invention further relates to the use of 2,4-D for improving the productivity of 2,4-D resistant crop plants. The method provided is of particular interest for the treatment of plant crops including corn, soybean, winter and spring rapeseed (canola), sugar beet, wheat, sunflower, barley and rice.

In some embodiments, 2,4-D resistant crop plants are transgenic crop plants transformed with an aryloxyalkanoate dioxigenase (DAA). In another embodiment, the aryloxyalkanoate dioxigenase (DAA) is either AAD-1 or AAD-12. AAD-1 was previously described in US 2009/0093366 and AAD-12 was previously described in WO 2007/053482, the contents of which are incorporated by reference in their entirety. The productivity-enhancing effect of the 2,4-D treatment can be observed at application rates of 25 g ea / ha at 5000 g / ha, or 100 g ea / ha at 2500 g ea / ha, or particular 1,000 g ae / ha to 2,000 g ae / ha.

In one embodiment, 1000 g ea / ha to 1500 g ea / ha of 2,4-D is used.

In another mode, 2000 g ea / ha to 2500 g ea / ha is used. In addition, the effect of improving yield, the treatment of 2,4-D and D is particularly pronounced when 2,4-D is applied at the 2- and 8-leaf stage of crop plants. before flowering. However, the rate of application and / or leaf phase of the harvesting plant required will vary depending on a function of the plants, their height and climatic conditions. The increase in term yield refers to the plant yielding up to 50% or more. In one embodiment, the yield increase is at least 10%. In another embodiment, the income increase is at least 20%. In another embodiment, the income increase is from 10% to 60%. In another embodiment, the increase in income is from 20% to 50%. In another embodiment, the increase in income is statistically significant. The growth enhancing activity of 2,4-D to 2,4-D, resistant crop plants can be measured in pot tests or field trials. Herbicide Taking a different course of action, they are generally known to either have an adverse effect on yield or have no effect on yield.

In one aspect, there is provided a method of improving the yield of 2,4-D resistant crop plants, which comprises treating the plants with a stimulating amount of a herbicide comprising a portion of aryoxyoxanoate.

In one embodiment, 2,4-D-resistant crop plants are transgenic plants transformed with an aryloxyanoate dioxigease (DAA). In another embodiment, the aryoxyoxanoate dioxigenase (DA-A) is either AAD-1 or AAD-12. In another embodiment, the herbicide comprising a portion of aroxyoxanoate is a phenoxy herbicide or phenoxyacetic herbicide. In another embodiment, the herbicide comprising a moiety of aryoxyoxanoate is 2,4-D. In another embodiment, 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA).

In one embodiment, transgenic plants transformed with an aryloxyalkanoate dioxigenase (DAA) are selected from cotton, soybean, and canola oil. In another embodiment, purification is performed at least once at an application rate of 2,4-D as well as employed for weed control. In another embodiment, purification is performed twice at an application rate of 2,4-D as also employed for weed control. In another embodiment, 2,4-D is applied to soybean stages V3 and R2 with tolerance to 2,4-D. In another embodiment, the purification of at least three times at an application rate of '2,4-D is performed as also employed for weed control. In another embodiment, the herbicide comprising an aryloxyalkanoate moiety targets 2,4-D resistant crop plants by root absorption.

In another embodiment, 2.4-D resistant crop plants are also treated with a herbicide other than 2,4-D for weed control. In another embodiment, the herbicide other than 2.4-D is a phosphorus herbicide or aryloxyphenoxypropionic herbicide. In another embodiment, the phosphorus herbicide comprises glyphosate, glufosinate, their derivatives, or combinations thereof. In another embodiment, the phosphorus herbicide is in the form of ammonium salt, isopropylammonium salt, isopropylamine salt or potassium salt. In another embodiment, phosphorus herbicide targets 2.4-D-resistant crop plants through root absorption. In another embodiment, the aryloxyphenoxypropionic herbicide comprises chlorazifop, fenoxaprop, fluazifop, haloxyfope, quizalofope, or derivatives thereof, or combinations thereof. In another embodiment, the aryloxyphenoxypropionic herbicide targets 2,4-D-resistant crop plants through root absorption.

In one embodiment, 2,4-D resistant crop plants are treated at least once with 25 g ae / ha at 5000 g ae / ha of 2,4-D.

In another embodiment, 2,4-D resistant crop plants are treated at least once with 100 g ea / ha to 2000 g ea / ha of 2,4-D.

In another embodiment, 2,4-D resistant crop plants are treated at least once with 100 g ea / ha at 2500 g ea / ha of 2,4-D.

In another embodiment, 2,4-D resistant crop plants are treated at least once with 1000 g ea / ha to 2000 g ea / ha of 2,4-D.

In another embodiment, 2,4-D, 2,4-D comprises choline or 2,4-D dimethylamine (DMA).

In one embodiment, a method of improving yield of crop-resistant 2,4-D is provided. The method comprises: (A) transforming plant cells with a nucleic acid molecule comprising a nucleotide sequence encoding an aryloxyalkanoate dioxigenase (DAA); (j) selection of transformed cells; (C) plant regeneration from transformed cells; and (D) treating the plants with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety.

In one embodiment, the aryloxyalkanoate dioxigenase (DAA) is either AAD-1 or AAD-12. In another embodiment, the nucleic acid molecule comprises a selectable marker that is not an aryloxyalkanoate dioxygenase (DAA). In another embodiment or alternative embodiment, the selection marker is a phosphinothricin acetyltransferase (PAT) gene or bialaphos resistance (bar) gene. In another embodiment, the nucleic acid molecule is an optimized plant.

In another aspect, there is provided the use of a herbicide comprising a portion of aryloxyalkanoate in the production of higher yielding 2,4-D resistance transgenic plants compared to their non-transgenic parent plants. In one embodiment, a herbicide comprising an aryloxyalkanoate moiety is 2,4-D. In another embodiment, 2,4-D is applied at least once with 25 g ae / ha to 5000 g / ha of 2,4-D. In another embodiment, 2,4-D is applied at least once, with 100 g ea / ha to 2000 g ea / ha of 2,4-D. In another embodiment, 2,4-D is applied at least once, with 100 g ea / ha to 2500 g ea / ha of 2,4-D. In another embodiment, 2,4-D is applied at least once, with 1000 g ea / ha to 2000 g ea / ha of 2,4-D. In another embodiment, 2,4-D, 2,4-D comprises choline or 2,4-D dimethylamine (DMA). In another embodiment, plants of 2,4-D resistant crops are treated with 2,4-D at least twice before flowering. In another embodiment, 2,4-D resistant crop plants are transgenic plants transformed with an aryloxyalkanoate dioxigenase (DAA). In another embodiment, the aryloxyalkanoate dioxigenase (DAA) is AAD-1 or AAD-12.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCES Figure 1 illustrates the general chemical reaction that is catalyzed by AAD-12 enzymes of the present invention. Figure 2 shows a representative map for plasmid pDAB4468. Figure 3 shows a representative map for plasmid pDAS1740. SEQ ID NO: 1 is AAD-12 nucleotide sequence from Delftia acidovorans. SEQ ID NO: 2 is the translated protein sequence encoded by SEQ ID NO: 1. SEQ ID NO: 3 is the optimized AAD-12 (v1) plant nucleotide sequence. SEQ ID NO: 4 is the translated protein sequence encoded by SEQ ID NO: 3. SEQ ID NO: 5 is the AAD-12 (v2) optimized E. coli nucleotide sequence. SEQ ID NO: 6 is the M13 forward primer sequence. SEQ ID NO: 7 is the M13 reverse primer sequence. SEQ ID NO: 8 is AAD-12 (v1) forward PTU primer sequence. SEQ ID NO: 9 is the AAD-12 (v1) PTU reverse PTU primer sequence. SEQ ID NO: 10 is the PCR forward primer sequence encoding AAD-12 (v1). SEQ ID NO: 11 is the PCR reverse primer sequence coding for PCR primer coding AAD-12 (v1). SEQ ID NO: 12 shows the sequence of primer "sdpa-codF" AAD-12 (v1). SEQ ID NO: 13 shows the sequence of primer "sdpaco-dR" AAD-12 (v1) e. SEQ ID NO: 14 shows the sequence of primer "Ncol de Brady". SEQ ID NO: 15 shows the sequence of the "Brady SACI" primer.

DETAILED DESCRIPTION OF THIS INVENTION

As used in the present invention, the phrase "transformed" or "transformation" refers to the introduction of DNA into a cell. The term "transformant" or "transgenic" refers to plant cells, plants and the like, which have been transformed or have undergone a transformation procedure. The introduced DNA is generally in the form of a vector containing an inserted DNA fragment.

As used herein, the term "selectable marker" or "selectable marker gene" refers to a gene that is optionally used in plant transformation, for example, by protecting plant cells from a selective agent, or providing resistance / tolerance to a selective agent. Only cells or plants that receive a functional selection marker are able to divide or grow under conditions that have a selective agent. Examples of selective agents may include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paramomycin, gentamycin, and hygromycin.

These selection markers include genes for neomycin phosphotransferase (NPT II), which expresses an enzyme that confers kanamycin antibiotic resistance and the genes for neomycin, paromomycin, gentamicin, and G418 related antibiotics, or the hygromycin gene. - kin phosphotransferase (HPT), which expresses an enzyme that confers resistance to hygromycin. Other selection marker genes may include genes encoding herbicide resistance including Bar (resistance against Basta ® (ammonium glufosinate), or phosphinothricin (PPT)), acetolactate synthase (ALS, resistance to inhibitors such as sulfonylureas (SU), imidazolinones (Imis), triazolopyrimidines (TPS), pyrimidinyl oxybenzoates (Pobs), and sulfonylamino triazolinones carbonyl that prevent the first step in the synthesis of branched chain amino acids), glyphosate, 2,4-D, and metal strength or sensitivity. The term "positive marker" refers to plants that have been transformed to include the selectable marker gene. Several selectable or detectable markers may be incorporated into the expression vector chosen to allow identification and selection of transformed plants, or transformants. Several methods are available to confirm expression of transformed plant selection markers, including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blot, RNA blot, immunological methods for detection of a protein expressed from the vector, for example precipitated protein that mediates phosphinothricin resistance, or other proteins such as β-glucuronidase (GUS) reporter genes, luciferase, green fluorescent protein (GFP), DsRed, β-galactosidase , chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, New York, 2001, the contents of which are incorporated herein by in its entirety).

Selection marker genes are used for the selection of transformed cells or tissues. Selection 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 herbicide composites. Herbicide resistance genes code for a modified herbicide-insensitive target protein, or an enzyme that degrades or detoxifies the herbicide in the plant before it can act. See Deblock et al. (1987) EMBO J. 6: 2513 to 2518; Deblock et al. (1989) Plant Physiol, 91: 691 to 704. Fromm et al. (1990) 8: 833 to 839; Gordon-Kamm et al. (1990) 2: 603 to 618). For example, resistance to glyphosate or sulfonylurea herbicides has been obtained through genes encoding the mutant target enzymes, 5-enolpyruvyloxytimate-3-phosphate (EPSPS) and acetolactate synthase (ALS). Herbicide resistance to ammonia glufosinate, bromoxynil and 2,4-dichlorophenoxyacetate (2,4-D) were obtained using the bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase or a 2,4-dichlorophenoxyacetate monooxygenase that detoxifies the respective herbicides.

Enzymes / genes for 2,4-D resistance have been previously described in US 2009/0093366 and WO 2007/053482, the contents of which are incorporated in the present invention by reference in their entirety.

Other herbicides may inhibit the growth point or meristem, including imidazolinones and sulfonylureas. Examples of genes from this mutant ALS and AHAS enzyme category code as described, for example, Lee et al., EMBO J. 7: 1241 (1988). And Miki et al., Theon. Appl. Internet. 80: 449 (1990), respectively.

Glyphosate resistance genes include the 5-enolpyruvyloxytimate-3-phosphate (EPSP) mutant synthase genes (through the introduction of recombinant nucleic acids and / or various forms of in vivo mutagenesis of native EPSPS genes), the aroA genes and the glyphosate acetyl transferase (GAT) genes, respectively). Resistance genes from other phosphono composites include genes (phosphinothricin acetyl transfer (PAT) from Streptomyces species, including Streptomyces hygroscopy and Streptomyces viridichromogenes) glufosinate and phenoxy or pyridinoxy propionic acids and cyclohexones (Gene inhibitor). See, for example, US Patent No. 4,940,835 to Shah et al. and US Patent No. 6,248,876 to Barri et al. which disclose nucleotide sequences of EPSP forms which may confer glyphosate resistance to a plant. A DNA molecule encoding a mutant aroA gene can be obtained from the ATCC institution under accession number 39256, and the nucleotide sequence of the mutant gene is described in US Patent No. 4769061 to Cornai, European Patent Application No. 0 333 033 from Kumada et al, E

U.S. Patent No. 4,975,374 to Goodman et al., Describing the nucleotide sequences of the glutamine synthetase genes that confer herbicide resistance, such as L-phosphinothricin. The nucleotide sequence of a PAT gene is provided in European Patent Application No. 0 242 246 to Leemans et al. Also Degreef et al., Bio / Technology 7: 61 (1989), describes the production of transgenic plants that express chimeric bar genes encoding PAT activity. Exemplary genes that confer resistance to phenoxy and cyclohexone proprionic acids, including sethoxidim and haloxifop, are the ACC1-S1, ACC1-S2-S3 and ACC1 genes described by Marmor et al., Theon. Appl. Genet. 83: 435 (1992). GAT genes capable of conferring glyphosate resistance are described in WO 2005012515 in Castle et al. Genes conferring resistance to 2,4-D, fop and auxin pyridyloxy herbicides are described in WO 2005107437 and U.S. Patent Application Serial No. 11 / 587,893.

Other herbicides may inhibit photosynthesis, including triazine (psbA and 1s + genes) or benzonitrile (nitrilase gene). Przibila et al., Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are described in US Patent No. 4,810,648 to Stalker, and DNA molecules containing these genes are available from ATCC Nos. 53435, 67441 and 67442. Cloning and DNA expression encoding a glutathione S-transferase is described by Hayes et al. Biochem. J. 285: 173 (1992).

For purposes of the present invention, 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). to 25.); Cyanamide Hydratase (Maier-Greiner et al. (1991) Proc Natl Acad Sci USA, 88: 4250 to 4264); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) Bio / Technology, 11: 715 to 718.); tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Βίο, 22: 907 to 912); insensitive dihydrodipicolinate synthase kinase and aspartade (Perl et al. (1993) Bio / Technology, 11: 715 to 718); gene bar (Toki et al. (1992) Plant Physiol, 100: 1503 to 1507 and Meagher et al. (1996) and Crop Sci., 36: 1367), tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol., 22: 907 to 912), neomycin phosphotransferase (. Neo) (Southern et al. (1982) J. Mol. Appl Gen., 1: 327; hygromycin phosphotransferase (HPT or HYG) (Shimizu et (1986), Mol.

Cell Biol, 6: 1074), dihydrofolate reductase (DHFR) (Kwok et al (1986). PNAS USA 4552); phosphinothricin acetyltransferase (Deblock et al (1987.) EMBO J. 6: 2513), 2, 2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al (1989) J. Cell Biochem 13E: 330); acetohydroxy synthase acid (Anderson et al, U.S. Patent No. 4,761,373; Haughn et al (1988) Mol. Gen. Genet 221: 266); 5-enolpyruvyl-shikimate-phosphate (aroA) synthase (Comai et al (1985.) Nature 317: 741); haloaryl nitrylase (Stalker et al, PCT Application published WO87 / 04181), acetylcoenzyme A carboxylase (Parker et al (1990) Plant Phyiol 92: 1220); dihydropteroate synthetase (sul I) (Guerineau et al (1990) Plant Mol Biol 15: 127), and 32 kD photosystem II (psbA) polypeptide (Hirschberg et al (1983) Science, 222: 1346).

Also included are genes encoding resistance to: chloramphenicol; methotrexate (Herrera-Estrella et al. Nature, 303: 209 (1983) - (Herrera-Estrella et al. (1983) EMBO J. 2: 987 to 992.). 213. Meijer et al. (1991) Plant Mol. Bio , 16: 807 to 820 (1991), hygromycin (Waldron et al. (1985) Plant Mol. Biol, 5: 103-108, Zhijian et al. (1995.) Plant Science, 108: 219 to 227 and Meijer et al. (1991) Plant Mol. Bio 16: 807 to 820), streptomycin (Jones et al. (1987) Mol. Gen. Genet, 210: 86 to 91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res., 5: 131 to 137), bleomycin (Hille et al. (1986.) Plant Mol. Biol, 7: 171 to 176), sulfonamide (Guerineau et al. (1990) Plant Mol. Biol, 15: 127 to 136), bromoxy (Stalker et al. (1988.) Science, 242: 419 to 423), 2,4-D (Streber et al. (1989). Bio / Technology, 7: 811 to 816); glyphosate (Shaw et al. (1986) Science, 233: 478 to 481) and phosphinetricin (Deblock et al. (1987.) EMBO J. 6: 2513 to 2518) All references recited in the description are incorporated in the present invention. by reference in its entirety unless otherwise indicated. The above list of selectable marker and reporter genes are not intended to be a limiting factor. Any selectable reporter gene or marker is encompassed by the present invention. If necessary, these genes may be sequenced by methods known in the art.

The selection marker and reporter genes are synthesized for optimal expression in the plant. That is, the coding sequence of the gene has been modified in order to increase expression in plants. The synthetic marker gene is designed to be expressed in plants at a higher level, resulting in greater transformation efficiency. Methods for synthetic gene optimization are available in the art. In fact, several genes have been optimized to increase gene product expression in plants. The sequence of the marker gene may be optimized for expression in a particular plant species or alternatively may be modified for optimal expression in plant families. Preferred plant codons can be determined from the most frequent codons in proteins expressed in greater 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. Know. USA, 88: 3324 to 3328 and Murray et al (1989) Nucleic Acids Research, 17: 477 to 498;

U.S. Patent No. 5,380,831, and U.S. Patent No. 5,436,391, incorporated herein by reference. In this way, nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the genetic sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences can also be used.

In addition, various transformation strategies have been developed using the Agrobacterium-mediated transformation system. For example, the binary vector strategy is based on a two-plasmid system where T-DNA is on a different plasmid from the rest of the Ti plasmid. In a cointegration strategy, a small portion of the T-DNA is placed on the same plasmid. vector than the foreign gene, which vector subsequently recombines with the plasmid Ti.

As used herein, the term "plant" includes dicotyledonous plants and monocotyledonous plants. Examples of dicot plants include Tobacco, Arabidopsis, Soybean, Tomato, Papaya, Canola, Sunflower, Cotton, Alfalfa, Potato, Vine, Guandu, Pea, Brassica, Chickpeas, Beetroot, Rapeseed, Watermelon, Melon, Pepper, Peanut, Pumpkin , radish, spinach, squash, broccoli, cabbage, carrot, cauliflower, celery, cabbage, cucumber, eggplant and lettuce. Examples of monocotyledonous plants include maize, rice, wheat, sugar cane, barley, rye, sorghum, orchids, bamboo, banana, cattail, lilies, oats, onions, corn and triticale. Developing the objective of a 2,4-D resistance gene and subsequent resistant crops provides excellent options for controlling the broadleaf, herbicide resistant glyphosate (or highly tolerant and displaced) weeds for weed species applications. cultures. 2,4-D is a broad spectrum, relatively economical and robust broadleaf herbicide that provides excellent utility for growers if greater crop tolerance could be provided in equal dicotyledonous and monocotyledonous crops. Transgenic 2,4-D tolerant dicotyledonous cultures would also have greater flexibility in timing and application rate. An additional utility of the herbicide tolerance characteristic for 2,4-D is its usefulness in preventing damage to sensitive crops, usually from 2,4-D, drift, volatilization, inversion (or other movement phenomenon in deviation, vandalism and the like. An additional benefit of the AAD-12 gene is that unlike all TFDA homologs characterized to date, AAD-12 is capable of degrading the pyridyloxyacetates auxins (eg, tricyclopyr, fluoroxypyr), as well as aquiral phenoxy auxins (e.g. 2,4-D, MC-PA, 4-chlorophenoxyacetic acid). See Table 1. A general illustration of the chemical reactions catalyzed by the enzyme AAD-12 is shown in Figure 1. (Addition of O.sub.2 is stereospecific; Breakdown of phenol and glyoxylate intermediate is spontaneous) It should be understood that The chemical structures in Figure 1 illustrate the molecular structures and that the various R and similar groups (such as those shown in Table 1) are included, but are not necessarily specifically illustrated in Figure 1. Various combinations of different combinations of phenoxy auxin have have been used worldwide to treat specific weed spectra and environmental conditions in various regions. Use of the AAD-12 gene in plants protects a much broader spectrum of auxin herbicides, thereby increasing the flexibility and spectrum of weeds that can be controlled.

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 for allow the use of these herbicides. In addition, AAD-12 may provide plant protection to pyridyloxyacetate herbicides where natural tolerance was also not sufficient to permit selectivity, expansion of potential utility of these herbicides. Plants containing AAD-12 alone can now be treated sequentially or in a tank mix with one, two, or a combination of various phenoxy auxin herbicides. The rate for each of the phenoxy auxin herbicides can be between 25 to 4,000 g ae / ha, and more typically 100 to 2,000 g ae / ha for the control of a broad spectrum of dicotyledonous weeds. Similarly, one, two, or a mixture of various auxin pyridyloxyacetate composites may be applied to plants expressing AAD-12 with reduced risk of injury from said herbicide. The rate of each herbicide may range from 25 to 2000 g ae / ha pyridyloxyacetate, and more typically 35 to 840 g ae / ha for the control of additional dicotyledonous weeds. Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grassy weed species. However, repeated use of glyphosate in TCG and non-agricultural applications has, and will continue to, select for weed shifts for naturally more tolerant species or glyphosate resistant biotypes. Mixing tank herbicide partners used at effective rates that offer control of the same species but with different modes of action are prescribed by most herbicide resistance management strategies as a method for delaying the emergence of resistant weeds. AAD-12 stacking with a glyphosate tolerance trait (and / or other herbicide tolerance traits) can provide a mechanism for controlling glyphosate resistant dicotyledonous weed species in TCG, enabling the use of glyphosate, phenoxy auxin (s) (eg 2,4-D) and pyridyloxyacetates from auxin herbicides (eg triclopyr) - selectively in the same crop. Applications of such herbicides may be simultaneously in a mixing tank comprising two or more herbicides of different modes of action, individual applications of the single herbicidal composition in sequential applications such as pre-planting, pre-emergence or post-emergence and timing. applications ranging from about 2 hours to about 3 months, or alternatively in any combination of any number of representatives of each class of chemical herbicides may be applied at any time within approximately 7 months of planting the crop, until the crop is harvested (or the pre-harvest interval for the individual herbicide whichever is shorter). It is important to have flexibility in controlling a broad spectrum of broadleaf grasses and weeds in terms of application time, individual herbicide rate, and the ability to control difficult or resistant weeds. The herbicide application of a crop having a glyphosate / AAD-12 gene resistance may range from about 250 to 2500 g ae / ha; auxin herbicide phenoxy (s) (one or more) may be applied from about 25 to 4000 g ea / ha; auxin and herbicide pyridyloxyacetates (one or more) may be applied from 25 to 2000 g ae / ha. The optimal combination (s) and timing of their application (s) will depend on the particular situation, species, and environment, and will be better determined by a person who is versed in the weed control technique and having the benefit of the highlighted description.

Seedlings are typically hardy throughout the complete growth cycle. Transformed plants will typically be resistant to re-application of the herbicide anytime the gene is expressed. Tolerance is shown in the present invention for 2,4-D throughout the life cycle using the constitutive promoters tested so far (and primarily CsVMV AtUbi 10). One would normally expect this, but it is an improvement in other non-metabolic activities where tolerance can be significantly impacted by reducing expression of a resistance action mechanism site, for example. An example is Roundup Pronto cotton, where plants have been tolerant if sprayed early, but glyphosate concentrated in the meristems is sprayed too late (because it is not metabolized and translocated); Monsanto viral promoters used are not well expressed in flowers. The present invention provides an improvement on these subjects. Herbicidal formulations (e.g. ester, acid, or salt formulation, or soluble concentrate, emulsion concentrate, or soluble liquid) and mixing tank additives (e.g. adjuvants, surfactants, drift retardants, or agents can significantly affect weed control of a given herbicide or the combination of one or more herbicides. Any combination of these with any of the chemical herbicides mentioned above is within the scope of the present invention.

One skilled in the art also finds the advantage of combining two or more modes of action for the purpose of increasing the spectrum of controlled weeds and / or controlling naturally more tolerant or resistant weed species. This could also extend chemistry so that herbicide tolerance has been enabled in crops through human involvement (either transgenically or non-transgenically) in addition to GCT. In fact, the coding characteristics of glyphosate resistance (eg resistant plants or bacterial EPSPS, glyphosate oxidoreductase (GOX), GAT), glufosinate resistance (eg Pat, bar), acetolactate synthase (ALS) herbicide resistance inhibition agents (e.g. imidazolinones, sulfonylureas, triazolopyrimidine sulfonanilide, pyrmidinylbenzoates, and other chemicals = AHAS, Csr1, Sura et al), resistance to bromoxynil (eg BXN), resistance to HPPD inhibitors (4- hydroxyphenyl pyruvate dioxigenase) enzyme, resistance to phytoene desaturase inhibitors (PDS), resistance to herbicides that inhibit photosystem li (eg psbA), resistance to herbicides that inhibit photosystem I, resistance to protoporphyrigen oxidase IX (PPO) herbicide inhibitors (eg PPO-1), herbicide resistance (eg phenylurea, CYP76B1), dicamba degrading enzymes (see eg US 200301 35879), and others could be stacked alone or in various combinations to provide the ability to effectively control or prevent weed changes and / or herbicide resistance of any of the above classes. In vivo modified EPSPS may be used in some preferred embodiments, as well as glyphosate resistance class I, class II, and class III genes.

With regard to additional herbicides, some additional preferred ALS inhibitors include, but are not limited to sulfonylureas (such as chlororsulfurone, halosulfurone, nicosulfurone, sulfometurone, sulfosulfurone, trifloxysulfuron), imidazoloninones (such as imazamox, imazethair, imazaquin). ), triazolopyrimidine sulfonanilides (such as chloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam and penoxsulam), pyrimidinylthiobenzoates (such as bispiribac and piritiobaque), and flucarbazone. Some preferred HPPD inhibitors include, but are not limited to mesotrione, isoxaflutol, and sulcotrione. Some preferred PPO inhibitors include, but are not limited to, flumiclorac, flumioxazin, flufenpyr, pyraflufen, flutiacet, butafenacil, carfentrazone, sulfentrazone, and diphenyl ethers (such as acifluorfen, fomesafen, lactofen and oxyfluorfen).

In addition, AAD-12 alone or on top of one or more additional HTC features may be stacked with one or more additional entries (eg, insect resistance, fungus resistance, or stress tolerance, et al. ) or output traces (eg, increased yield, better oil profile, improved fiber quality, et al.). In this way, the present invention can be used to provide a complete crop quality improved agronomic package with the ability to flexibly and cost-effectively control any number of agronomic pests. The present invention relates in part to the identification of an enzyme that is not only capable of degrading 2,4-D, but also possesses the surprisingly novel properties which distinguish the enzyme of the present invention from TFDA proteins. previously known, for example. Even though this enzyme has very little homology to ADHD, the genes of the present invention can still generally be classified into the same general family of α-ketoglutarate-dependent dioxigenases. This family of proteins is characterized by three histidine residues conserved in a "HX (D / E), X23-26 (T / S) -X114 183HX10-13R" pattern, which comprises the active site. The coordinate Fe + 2 ion histidines in place (active, which is essential for catalytic activity (Hogan et al, 2000). The threshold% in in vitro expression experiments discussed in the present invention was adapted to help choose These experiments also indicate that the AAD-12 enzyme is unique to another disparate enzyme of the same class, described in an already filed patent application (PCT US / 2005/014737, filed May 2 The AAD-1 enzyme in this application shares only about 25% sequence identity with the highlighted AAD-12 protein.

More specifically, the present 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 α-ketog fighting rat-dependent dioxigenase enzyme has been previously reported to be able to degrade herbicides of different chemical classes and modes of action.

Preferred enzymes and genes for use in accordance with the present invention are referred to herein as 12-AAD (AryloxyAloanoate Dioxigenase) genes and proteins.

Subject proteins tested positive for the conversion of 2,4-D to 2,4-dichlorophenol ("DCP"; herbicidly inactive) in analytical assays.

Partially purified proteins of the present invention can rapidly convert 2,4-D to DCP in vitro. An additional advantage of AAD-12 transformed plants is that this parent herbicide (s) is metabolized to inactive forms, thus reducing the potential for harvesting herbicide residues in grain or Stover. The present invention also includes weed control methods wherein said methods comprise applying a pyridyloxyacetate and / or a plant herbicidal phenoxy auxin comprising an AAD-12 gene. In light of these findings, new plants that make up the polynucleotide encoding this type of enzyme are now provided. Until now, there has been no motivation for the production of such plants, and there has been no expectation that these plants can effectively produce this enzyme to make plants resistant to not only jfenoxy herbicides (such as 2.4 -D), but also pyridyloxyacetate herbicides. Accordingly, the present invention provides many advantages that were not previously thought possible in the art.

Publicly available strains (deposited in culture collections such as ATCC or DSMZ) can be purchased and selected using the techniques described in the present invention for the novel genes. The sequences described in the present invention may be used to amplify and clone homologous genes in a recombinant expression system for further screening and assays according to the present invention.

As discussed above in the Background section, an organism that has been extensively studied for its ability to degrade 2,4-D is Ralstonia eutropha (Streber et al, 1987). The gene that codes for the first enzyme in the degradation pathway is TFDA. See U.S. Patent No. 6,153,401 and GENBANK Acc. No. M16730. TFDA catalyzes the conversion of 2,4-D acid to herbicidly inactive DCP through an α-ketoglutarate-dependent deoxygenase reaction (Smejkal et al., 2001). TFDA has been used in transgenic plants to confer 2,4-D resistance in dicotyledonous plants (eg 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 encoding proteins capable of degrading 2,4-D have been identified from the environment and deposited in the Genbank database. Many homologs are quite similar to ADHD (> 85% amino acid identity) and have enzymatic properties similar to ADHD. However, a small collection of rat-fighting α-ketogens-dependent dioxigenase homologs is presently identified as having a low level of homology with ADHD. Part of the present invention relates to the surprising discoveries of novel uses for and functions of a distantly related enzyme, SDPA, from Delftia acidivorans (Westendorf et al., 2002, 2003), with low homology. with TFDA (31% amino acid identity). This purified α-ketoglutarate-dependent dioxigenase enzyme in its native form had previously been de-cloned to degrade 2,4-D and S-dichlorprop (Westendorf et al., 2002 and 2003). However, no α-ketoglutarate-dependent dioxigenase enzyme has previously been reported to have the ability to degrade pyridyloxyacetate chemical class herbicides. SDPA has never been expressed in plants, nor has there been any motivation to do so, in part because the development of new HTC technologies has been limited largely due to the effectiveness, low cost and convenience of TCG (Devine, 2005).

By virtue of the novel activity, the proteins and genes of the present invention are referred to herein as AAD-12 proteins and genes. AAD-12 has been confirmed to degrade a variety of auxin phenoxyacetate herbicides in vitro. However, this enzyme, as first reported in the present invention, has surprisingly been found to also be capable of degrading additional substrates of the aryloxyalkanoate class of molecules. Substrates of significant agronomic importance include the pyridyloxyacetate auxin herbicides. This highly new finding underpins the significant herbicide-tolerant (HTC) cultures and opportunities of marker marker selection. This enzyme is unique in its ability to deliver herbicide degradation activity to a range of broad spectrum herbicides (phenoxyacetate and auxin pyridyloxyacetate).

Accordingly, the present invention relates in part to the degradation of 2,4-dichlorophenoxyacetic acid, other phoxyacetic auxin herbicides, and pyridyloxyacetate herbicides by a recombinantly expressed aryloxyalkanoate dioxigenase enzyme ( ADF-12). The present invention also relates in part to the identification and use of genes encoding an aryloxyalkanoate dioxigenase degrading enzyme (AAD-12) capable of degrading phenoxy and / or pyridyloxy auxin herbicides. The subject enzyme allows transgenic expression resulting in tolerance to herbicide combinations that control almost all broadleaf weeds. AAD-12 can serve as an excellent herbicide tolerant (HTC) culture characteristic for stacking with other HTC characteristics [eg glyphosate resistance, resistance glufosinate, ALS inhibitor resistance (eg imidazolinones, sulfonylureas). , triazolopyrimidine sulfonanilide), bromoxynil resistance, HPPD inhibitor resistance, PPO inhibitor resistance, et al] and insect resistance traits (Cri1F, CrilAb, Cri 34/45, other proteins, or Bt proteins insecticides of non-Bacillis, et al.) origin, for example. In addition, AAD-12 may serve as a selection marker to assist in the selection of primary transformants from genetically modified plants with a second gene or group of genes.

In addition, the microbial subject gene has been redesigned such that the protein is encoded by codons that have a tendency to use both monocotyledonous and dicotyledonous plants (hemicot).

Arabidopsis, maize, tobacco, cotton, soybean, canola, and rice were transformed with 12 constructs containing DAA and demonstrated high levels of resistance to both phenoxy and pyridyloxy auxin herbicides. Thus, the present invention also relates to the optimized "plant" of the genes encoding the proteins of the present invention.

Oxyalkanoate groups are useful for introducing stable acid functionality into herbicides. The acid group can confer phloem mobility through "acid trapping", a desirable attribute for herbicide action and can therefore be incorporated into new herbicides for mobility purposes. Aspects of the present invention also provide a mechanism for creating HTCs. There are many potential commercial and experimental herbicides that can serve as substrates for AAD-12. Thus, the use of the genes under consideration may also result in tolerance to herbicides as well as other herbicides.

The HTC features of the present invention may be used in new combinations and with other HTC features (including but not limited to glyphosate tolerance). These combinations of traits give rise to new methods of controlling weed species (and similar ones) due to newly acquired inherent resistance or tolerance to herbicides (eg glyphosate). Thus, in addition to the HTC features, new methods for controlling weeds with herbicides, for which herbicide tolerance was created by said enzyme in transgenic crops, are within the scope of the present invention. The present invention may be applied in the context of marketing a resistance characteristic of 2,4-D stacked with current glyphosate resistance characteristics in soybeans, for example. Thus, this invention provides a tool for combating broadleaf weeds in species changes and / or selection of herbicide-resistant broadleaf weeds, which culminates in the very high dependence of glyphosate producers on weed control with multiple crops. The transgenic expression of the subject genes for AAD-12 is exemplified in, for example, Arabidopsis, tobacco, soy, cotton, rice, corn, and canola oil. Soy is a preferred crop for processing according to the present invention. However, the present invention may be used in various other monocotyledons (e.g., grass or lawn) and dicotyledonous crops such as alfalfa, clover, tree species, et al. Similarly, 2,4-D (or other AAD-12 substrates) can be used more positively on grasses where tolerance is moderated, with greater tolerance across this trait would provide producers with the opportunity to use these herbicides at more effective rates and a longer application time without the risk of crop damage.

Furthermore, the present invention provides a simple gene that can provide herbicide resistance that controls broadleaf weeds. This gene can be used in various cultures to allow the use of a broad spectrum herbicidal combination. The present invention can also control weeds resistant to current chemical products, and assists in weed control by changing the spectra resulting from current agronomic practices. AAD-12 can also be used in efforts to effectively detoxify herbicide substrates in addition to non-herbicide forms. Accordingly, the present invention provides for the development of additional IHTC features and / or selectable marker technology. % Separated from, or in addition to, using subject genes to produce HTCs, subject genes can also be used as selection markers for successful transformant selection in cell cultures, greenhouses, and in the field. There is inherent high value for the genes in question simply as a selectable marker for biotechnology projects. The promiscuity of AAD-12 for other auxin aryloxyalkanoate herbicides offers many opportunities to use this gene for HTC and / or selection marker purposes. One cannot easily argue the term "resistance" and not use the verb "tolerate" or the adjective "tolerant." The industry has spent countless hours defeating herbicide-tolerant (HTC) crops against herbicide-resistant crops ( HRC). HTC is a preferred term in the industry. However, the official definition of the American Society of Weed Science defines resistance as "the inherited ability of a plant to survive and reproduce following exposure to a normally lethal dose of wild-type herbicide. resistance may be occurring or induced by techniques such as genetic engineering or selection of variants produced by tissue culture or “naturally mutagenesis.” As used in the present invention, unless otherwise indicated, Herbicide "resistance" is hereditary and allows a plant to grow and reproduce in the presence of a typical herbicidally effective treatment of a particular plant herbicide, as suggested by editing the Herbicide Manual from the description depot. As recognized by those skilled in the art, a plant can still be considered "hardy", although Some degree of plant injury from herbicide exposure is apparent. As used in the present invention, the term "tolerance" is broader than the term "resistance", and includes "resistance" as defined in the present invention as well as a better ability of a plant to grow. particularly to withstand the varying degrees of herbicidal induced lesions that normally result in wild-type plants of the same genotype at the same herbicidal dose. Transfer of functional activity from plant or bacterial systems may involve a nucleic acid sequence, which encodes the amino acid sequence of a protein of the present invention, integrated into a protein expression vector suitable for the host in which the vector goes. reside. One way to obtain a nucleic acid sequence that encodes a protein with functional activity is to isolate native genetic material from the species of bacteria that produce the protein of interest, using the information derived from the amino acid sequence. protein acids as described in the present invention. Native sequences may 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. There are a number of methods for obtaining proteins for use in accordance with the present invention. For example, antibodies to the proteins described in the present invention may be used to identify and isolate other proteins from a protein mixture. Specifically, antibodies may be raised to portions of proteins that are more conserved or more distinct compared to other related proteins. These antibodies can then be used to specifically identify equivalent proteins with characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or immunoblotting. Antibodies to the proteins described in the present invention, or equivalent proteins, or fragments of these proteins can be readily prepared using standard procedures. Such antibodies constitute an aspect of the present invention. Antibodies of the present invention include preferably monoclonal and polyclonal antibodies produced in response to an exemplified or suggested protein.

One of ordinary skill in the art will readily recognize that the proteins (and genes) of the present invention may be obtained from a variety of sources. Since complete herbicide degradation operones are known to be encoded in transposable elements such as plasmids as well as genomically integrated, the proteins of the present invention may be obtained from a wide variety of microorganisms, for example, including wild type and / or recombinant bacteria.

Bacterial isolate mutants can be made by procedures that are well known in the art. For example, mutants may be obtained Asporogenic via ethyl methane sulfonate (EMS), mutagenesis of an isolate. Mutant strains can also be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

A "from" or "protein obtainable from" any of the isolated subjects referred to or suggested herein means that the protein (or a similar protein) may be obtained from the isolate or some other source such as another bacterial strain or a plant. The term "derived from" also has this connotation, and includes proteins that can be obtained from a particular type of bacteria that is modified for expression in a plant, for example. A person skilled in the art will readily recognize that, in view of the disclosure of a bacterial gene and protein, a plant may be modified for the purpose of producing the protein. Antibody preparations, nucleic acid probes (DNA, RNA or PNA, for example) and the like may be prepared using the polynucleotide and / or amino acid sequences described in this invention and used to screen and retrieve other related genes from other genes. sources (natural).

Conventional molecular biology techniques can be used to clone and sequence the genes and proteins described in the present invention. Additional information can be found in Sambrook et al., 1989, which is incorporated herein by reference.

Polynucleotides and Probes: The present invention further provides the protein-encoding nucleic acid sequences for use in accordance with the present invention. The present invention further provides methods of identifying and characterizing genes that encode proteins having the desired herbicidal activity. In one embodiment, the present invention provides unique nucleotide sequences, which are useful as hybridization probes and / or primers for PCR techniques. Primers produce the characteristic gene fragments that can be used to identify, characterize and / or isolate specific genes of interest. The nucleotide sequences of the present invention encode proteins that are distinct from the proteins described above.

The polynucleotides of the present invention may be used to form a complete system of "genes" encoding proteins or peptides in a desired host cell. For example, as one skilled in the art will readily recognize that the subject polynucleotides may be suitably 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 tissue / temporal specific expression can significantly impact the utility of the present invention. In general, higher levels of expression of a degradation protein 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 high expression has a consequent negative impact on plant health. Normally, one would like to have the AAD-12 gene constitutively expressed in all complete plant protection tissues at all stages of growth. Alternatively, however, a vegetatively expressed resistance gene could be used, which would allow the use of the target crop herbicide for weed control and would subsequently control the sexual reproduction of the target crop by application during the flowering phase. In addition, the desired levels and expression times may also depend on the type of plant and the degree of tolerance desired. Some preferred embodiments utilize strong constitutive promoters combined with transcription enhancers and the like for the purpose of increasing expression levels and increasing tolerance to desired levels. Some of these applications are discussed in more detail below, before the Examples section.

As one skilled in the art knows, DNA typically exists in a double stranded form. In this arrangement, one filament is complementary to the other filament 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 filament that binds with the antisense filament. The mRNA is transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (it is a three nucleotide codon that can be read as a three-residue unit to specify a particular amino acid), which can be read as an open reading frame ( ORF) to form a protein or peptide of interest. In order to produce a protein in vivo, a DNA strand is normally transcribed into a complementary mRNA strand, which is used as a template for the protein. Accordingly, the present invention includes the use of the exemplified polynucleotides shown in the attached sequence listing and / or equivalents, including complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA molecules are included in the present invention.

Proteins and genes for use in accordance with the present invention may be identified and obtained by using oligonucleotide probes, for example. These probes are detectable nucleotide sequences that may be detectable by virtue of a proper label or may be inherently fluorescent produced as described in International Application No. WO 93/16094. Probes (and the polynucleotides of the present invention) may be DNA, RNA or PNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U; for RNA molecules), and synthetic probes (polynucleotides) of the present invention may also have inosine (a neutral base). capable of pairing with all four bases, sometimes used instead of a mixture of all four synthetic probe bases) and / or other (non-natural) synthetic bases.

Thus, when a degenerate synthetic oligonucleotide is referred to in the present invention, and "N" or "n" is used generically, "N" or "n" may be G, A, T, C or inosine. Ambiguity codes as used in the present invention conform to IUPAC standard naming conventions from the filing of the subject application (eg R means A or G, Y means C or T, etc.). It is well known in the art that a hybrid probe molecule with a nucleic acid sample can reasonably be assumed that the probe and sample have substantial homology / similarity / identity. Preferably, hybridization of the first polynucleotide is then conducted under low, moderate, or high stringency conditions by techniques well known in the literature, as described, for example, by Keller, GH manak MM (1987). DNA, Stockton Press, New York, New York, pp 169 to 170. For example, as indicated therein, low severity conditions can be achieved by first washing with 2 x SSC (standard saline citrate). / 0.1% SDS (sodium dodecyl sulfate) for 15 minutes at room temperature. Two washes are typically performed. High accuracy can then be achieved by reducing the salt concentration and / or by increasing the temperature.

For example, the wash solution described above may be followed by two washes 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 may be used with other hybridization and wash protocols set forth in the present invention and, as would be known by persons skilled in the art (PES may be used in salt form instead of SSC for example). 2 x SSC / 0.1% SDS can be prepared by adding 50 ml of 20 x SSC and 5 ml 10% SDS and 445 ml 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 the pH to 7.0 with 10 N NaOH in then adjusting the volume to 1 liter. 10% SDS Ipode may be prepared by dissolving 10 g of SDS in 50 ml of sterile water followed by dilution to 100 ml. Probe detection provides a means for determining in a known manner whether hybridization has been maintained. Such probe analysis provides a rapid method for identifying genes of the present invention. Nucleotide segments used as probes according to the present invention may be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences may also be used as PCR primers to amplify the genes of the present invention.

Hybridization characteristics of a molecule may be used to define the polynucleotides of the present invention. Thus, the present invention includes polynucleotides (and / or their complements, preferably their full complement) which hybridize to a polynucleotide exemplified in the present invention. That is, one way of defining a gene (and the protein it encodes), for example, is by its ability to hybridize (under any of the conditions described in this invention specifically) to a known or specifically exemplified gene. .

As used herein, "stringent conditions" for hybridization refers to conditions that allow to achieve the same or nearly the same degree of hybridization specificity as the conditions employed by the present applicants. Specifically, DNA hybridization immobilized on Southern blots with gene specific 32 P-labeled probes can be performed by standard methods (see, for example, Maniatis et al 1982). In general, hybridization and subsequent washes may be performed under conditions that allow detection of target sequences. For double stranded genetic DNA probes, hybridization can be performed overnight at 20 to 25 ° C. below the fusion temperature (Tm) of the DNA hybrid in 6 x SSPE, 5 x Denhardt's solution, 0.1% SDS, 0.1 mg / ml denatured DNA.

The washes can usually be performed as follows: 1 (1) twice at room temperature for 15 minutes in 1 x SS-PE, 0.1% SDS (low severity wash), and (2) since the Tm-20 ° C for 15 minutes in 0.2 x SSPE, 0.1% SDS (moderate stringency wash).

With oligonucleotide probes, hybridization can be performed overnight at 10 to 20 ° C below the melting temperature (Tm) of the hybrid in 6-fold SSPE, 5 x Denhardt's solution, 0.1% SDS, 0.1 mg / ml denatured DNA.

The washes may typically be as follows: (1) twice at room temperature for 15 minutes 1 x SSPE, 0.1% SDS (low severity wash), and (2) once the temperature of hybridization for 15 minutes. minutes in 1 x SSPE, 0.1% SDS (moderate seriousness wash).

In general, salt and / or temperature may be changed to change accuracy. With a DNA fragment labeled> 70 or bases long, the following conditions may be used: (1) Low: 1 or 2 x SSPE, room temperature, (2) low:. 1 or 2 x SSPE, 42 oC, (3) moderate: 0.2 x or 1 x PEES, 65 ° C or (4) High: 0.1 x SSPE, 65 ° C formation and duplex stability depend the substantial complementarity between the two filaments of a hybrid and, as noted above, a certain degree of incompatibility can be tolerated. Therefore, the probe sequences of the present invention include both mutations (single and multiple), deletions, insertions of the described sequences and combinations thereof, wherein said mutations, insertions and deletions allow the formation of hybrids. stable with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a particular polynucleotide sequence in many ways, and these methods are known to one of ordinary skill in the art. Other methods may become known in the future. PCR: Polymerase Chain Reaction (PCR) technology is repetitive, enzymatic, prepared synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in the art (see Mullis, U.S. Patent No. 4,683,195, 4,683,202, and 4,800,159, Saiki et al., 1985). PCR is based on enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to the opaque filaments of the target sequence. The primers are preferably oriented with the 3 'ends pointing towards each other. Repeated cycles of template denaturation, primer hybridization to their complementary sequences, and extension of primers matched with a DNA polymerase sequence in amplification of the defined segment via the 5 'ends of the PCR primers. The extension product from each primer can serve as a template for the other initiator, so that each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in exponential accumulation of the specific target fragment up to several million times within hours. By using a thermostable DNA polymerase such as Tag polymerase, isolated from Thermus aquaticus thermophilic bacteria, the amplification process can be fully automated. Other enzymes that can be used are known to those of skill in the art.

Exemplified DNA sequences or segments thereof can be used as primers for PCR amplification. By performing PCR amplification, a certain degree of incompatibility may be tolerated between the primer and the template. Therefore, mutations, deletions and insertions (nucleotide additions, especially to the 5 'end) of the exemplified primers fall within the scope of the present invention.

Mutations, insertions and deletions can be produced in a particular primer by methods known to those skilled in the art. Gene and protein modification: The genes and proteins in question may be fused to other genes and proteins that produce chimeric or fusion proteins. Genes and proteins useful in accordance with the present invention include not only the specifically exemplified full length sequences, but also portions, segments and / or fragments thereof (including contiguous fragments and internal deletions and / or terminal in comparison to the full length molecules) of these sequences, variants, mutants, chimerics, and fusions thereof. The proteins of the present invention may have substituted amino acids as long as they maintain the desired functional activity.

"Variant" genes have nucleotide sequences encoding the same or equivalent proteins having equivalent or similar activity to an exemplified protein.

The first two results of BLAST searches with native aad-12 nucleotide sequence show a reasonable level of homology (about 85%), over 120 base pairs of sequence. Hybridization under certain conditions can be expected to include these two sequences. See GENBANK Acc. Nos. DQ406818.1 (89,329,742; Rhodoferax) and AJ6288601.1 (44,903,451; Sphingomonas). Rhodoferax is very similar to Dephtia, but Sphingomonas is a completely different class philaghetically.

The terms "variant proteins" and "equivalent proteins" refer to proteins having the same or essentially the same biological / functional activity against target substrates and sequences as the exemplified equivalent proteins. As used in the present invention, reference to a sequence of "equivalents" refers to sequences that have amino acid substitutions, deletions, additions, insertions or that negatively improve or negatively affect activity. significant. Fixation activity fragments are also included in this definition. Fragments and other equivalents that retain the same or similar function or activity as a fragment of a corresponding exemplified protein are within the scope of the present invention. Changes, such as amino acid substitutions or additions, may be made for a variety of purposes, such as increasing (or decreasing) the stability of the protease protein (without substantially decreasing atix / age). protein removal), the removal or addition of a local restriction, and the like. Gene variations can easily be constructed using conventional techniques for making point mutations, for example.

In addition, U.S. Patent No. 5,605,793, for example, describes methods for generating additional molecular diversity using reassembly DNA after random or focused fragmentation. This may be referred to as a "blending" gene, which typically involves mixing (fragments of a desired size) of two or more different DNA molecules, followed by repeated cycles of renaturation. This can improve the activity of a protein encoded by a start gene. The result is a chimeric protein that has improved activity, altered substrate specificity, increased enzyme stability, altered stereospecificity, or other characteristics. The term "scrambling" can be conceived and oriented after obtaining and analyzing the structure of a protein of interest atomic and crystal 3D (three-dimensional) coordinates. In this way, "focused shuffling" can be directed to certain segments of a protein that are ideal for modification, such as exposed surface segments, and internal segments that are preferably not involved with 3D fold structural integrity. and essential protein.

Changes specific to the "active site" of the enzyme may be made to affect inherent functionality with respect to activity or stereospecificity. Muller et al. (2006). The known tauD crystal structure was used as a model for determining active local dioxigease residues while bound to its inherent taurine substrate. Elkins et al. (2002) "the X-ray crystal structure of Escerichia coli dioxigenase taurine / alpha-ketoglutarate complexed to ferrous iron and substrates", Bio-chemistry 41 (16): 5185 to 5192. Concerning sequence optimization and designation of active enzyme sites, see Chakrabarti et al., PNAS (August 23, 2005), 102 (34): 12035 to 12040.

Fragments of the full length genes may be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or targeted mutagenesis may be used to systematically cut nucleotides off the ends of these genes. In addition, genes encoding the active fragments can be obtained using a variety of restriction enzymes. Proteases can be used to directly obtain active fragments of these proteins.

It is within the scope of the invention as described in the present invention that proteins may be truncated and still retain functional activity. By "truncated protein" is meant that a portion of a protein may be cleaved, while the remainder of the truncated protein retains and exhibits the desired activity after dividing. Divagem can be achieved by various proteases. In addition, effectively cleaved proteins can be produced using molecular biology techniques, wherein the DNA encoding said base protein is removed by restriction endonuclease digestion or other techniques available for a person skilled in the technique. After truncation, said proteins may be expressed in heterologous systems such as E. coli, baculovirus and plant-based, yeast, and similar viral systems and then placed in insect assays as described above. - feels invention to determine activity. It is well known in the art that truncated proteins can be successfully produced so that they maintain functional activity having less than the full length sequence. For example, B.t. proteins may be used in a truncated form (protein nucleus) (see, for example, Hofte et al (1989), and Adang et al. (1985)). As used herein, the term "protein" may include functionally active truncations. Unless otherwise specified as used in the present invention, percent sequence identity and / or similarity of two nucleic acids is determined using the modified Karlin and Altschul 1990 algorithm as modified by Karlin and Altschul. 1993

Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al 1990. BLAST nucleotide searches are performed with the NBLAST program, score = 100, word length = 12. BLAST columns can be used as described. in Altschul et al From 1997. Using BLAST and BLAST Gap programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See the NCBI / NIH website. To obtain gap alignments for comparison purposes, the AlignX Vector NTI Suite 8 function (InforMax, Inc., North Bethesda, Mariland, USA) was used employing the default parameters. These were: a gap gap penalty of 15, a gap gap penalty of 5.66, and a gap gap penalty ratio of 8. ___________________________ ~ ■ _______ Table 1 Amino Acid Class Amino Acid Examples Non-polar Wing, Go, Leu, Ile, Pro, Met, Fe, Trp Unloaded Polar Gly, Ser, Thr, Cys, Tyr, Asn, Asp Acidic Gin, Basic Glu Lys, Arg, His Various three-dimensional protein properties and characteristics they can also be altered without negatively affecting protein activity / functionality. Conservative amino acid substitutions can be tolerated / made so as not to adversely affect the activity and / or three-dimensional configuration of the molecule. Amino acids can be placed into the following classes: nonpolar, uncharged polar, basic and acidic. Conservative substitutions in which one amino acid of one class is replaced by another amino acid of the same type fall within the scope of the present invention, provided that the substitution is not detrimental to the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class. In some cases, non-conservative substitutions may also be made. However, preferential substitutions do not significantly decrease the functional / biological activity of the protein.

As used herein, reference to "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. , reference to "isolated" and / or "purified" means involvement of the "man's hand" as described in the present invention, for example, a bacterial "gene" of the present invention placed on a plant for the expression is "an isolated polynucleotide." Likewise, a protein derived from a bacterial protein produced by a plant is an "isolated protein".

Due to the degeneracy / redundancy of the genetic code, a variety of different DNA sequences may encode the amino acid sequences described in the present invention. It is well within the skill of a person skilled in the art to create alternative DNA sequences encoding the same or essentially the same proteins. These variant DNA sequences are within the scope of the present invention.

This is also discussed in more detail below in the section titled "Sequence Optimization for Plant Expression." Sequence optimization for plant expression: In order to achieve high expression of heterologous genes in plants it is generally preferred to reengineer the genes so that they are more efficiently expressed (in the cytoplasm) in plant cells. Corn is an example where the plant may be preferred to redesign the hemologue gene (s) prior to transformation in order to increase its level of expression in said plant. Therefore, an additional step in gene design that encodes a bacterial protein is a reengineering of the heterologous gene for optimal expression, using a codon bias more aligned with the target plant sequence, whether dichotidoneous or monocotyledonous species. . Sequences can also be optimized for expression in any of the more specific plant types discussed elsewhere in this document.

Transgenic Hosts: The genes encoding the protein of the present invention can be introduced into a wide variety of microbial or plant hosts. The present invention includes transgenic plant cells and transgenic plants. Preferred plants (and plant cells) are maize, Arabidopsife, tobacco, soybeans, cotton, canola, rice, wheat, grass, fodder legumes (e.g., alfalfa and clover), grasses, and the like. Other types of transgenic plants may also be made in accordance with the present invention, such as fruits, plants, ornamentals and trees. More generally, dicotyledons and / or single-cotyledons can be used in various aspects of the present invention.

In preferred embodiments, gene expression results, directly or indirectly, in the intracellular production (and maintenance) of the protein (s) of interest. Plants can be processed herbicide resistant in this manner. Such hosts may be referred to as transgenic, transformed, recombinant and / or transfected cells and / or hosts.

In some aspects of the present invention (when cloning and preparing the gene of interest, for example), preferably (bacterial) microbial cells may be produced and used according to conventional techniques, with the advantage of disclosure. in subject.

Plant cells transfected with a polynucleotide of the present invention may be regenerated into whole plants. The present invention includes cell culture, including tissue culture cells, liquid cultures, and metallized cultures. Seeds produced by and / or used to generate plants of the present invention are also included within the scope of the present invention. Other plant tissues and parts are also included in the present invention. The present invention also includes methods of producing plants or cells comprising a polynucleotide of the present invention. A preferred method of producing such plants is by planting a seed of the present invention.

Although plants may be preferred, the present invention also includes the production of highly active recombinant AAD-12 in a host Pseudomonas fluorescens (Pf) strain, for example. The present invention includes at preferred growth temperatures to keep soluble AAD-12 active in this host, a fermentation condition where A-AD-12 is produced as more than 40% total cellular protein, or at least 10%. g / l, a purification process results in high recovery of the recombinant AAD-12 pf host from a Pf host, a purification scheme yielding at least 10 g active AAD-12 per kg of cells, a purification scheme that can yield 20 g active AAD-12 per kg of cells, a formulation process that can store and retrieve AAD-12 activity in solution and a lyophilization process that can retain AAD-12 activity for long term storage and storage. Ptaxaleira's life. Gene insertion to form transgenic hosts: One aspect of the present invention is the transformation / transfection of plants, plant cells, and other polynucleotide host cells of the present invention expressing the proteins of the present invention. dog. Plants transformed in this way can be made resistant to a variety of herbicides with different modes of action.

A wide variety of methods are available for introducing a gene encoding a desired protein into the target host under conditions that permit the maintenance and stable expression of the gene.

These methods are well known to those of skill in the art and are described, for example, in U.S. Patent No. 5,135,867.

Vectors comprising an AAD-12 polynucleotide are included within the scope of the present invention. For example, a large number of cloning vectors comprising an E. coli replication system and a marker that allows selection of transformed cells are available for preparation for insertion of foreign genes into higher plants. Vectors comprise, for example, pBR322, pUC series, M13 series mp, pACYC184, etc. Thus, the protein-encoding sequence can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. E. coli cells are grown in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered by purifying distance from genomic DNA. Sequence analysis, restriction analysis, electrophoresis and other molecular biochemical-biological methods are generally performed as analysis methods. After each manipulation, the DNA sequence used can be digested by restriction and spliced to the next DNA sequence. Each plasmid sequence may be cloned into the same or other plasmids. Depending on the desired gene introduction method for the plant, other DNA sequences may be required. If, for example, plasmid Ti or Ri is used for transformation of the plant cell, then at least the right boundary, but often to the right and left margin of T-DNA plasmid Ti or Ri, must be joined as the flank region of the genes to be inserted. The use of T-DNA for plant cell transformation has been intensively studied and described in EP 120 516; Hoekema (1985), Fraleyefa. (1986), and An et al. (1985).

A large number of techniques are available for inserting DNA into a plant host cell. These techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, fusion, injection, biobased (microparticle bombardment), Swiss silicon carbide, radiant aerosols, PEG or electroporation, as well as other possible methods. If Agrobacterium are used for transformation, the DNA to be inserted must be cloned into special plasmids, namely either an intermediate vector or a binary vector. Intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination due to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the region that will be required for T-DNA transfer. Intermediate vectors cannot reproduce in Agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate in both E. coli and Agrobacterium. They comprise a selection marker gene and a ligand or polylinker that are framed via the right and left boundary regions of T-DNA. They can be transformed directly into Agrobacteria (Holsters, 1978). The Agrobacterium used as a host cell is comprised of a plasmid carrying a vir region. The vir region is required for T-DNA transfer in the plant cell. T-DNA supplements may be contained. The bacterium thus transformed is used for transformation of plant cells. Plant explants may be advantageously cultured with Agrobacterium tumefacides or Agrobacterium rhizogenes for DNA transfer in the plant cell. Whole plants can be regenerated from infected plant material (eg, leaf pieces, stem segments, roots, but also suspension of protoplasts or cultured cells) in a suitable medium which may contain antibiotics. or biocides for selection. Plants thus obtained can then be tested for the presence of the inserted DNA. No special requirements are placed on plasmids in the case of injection and electroporation. Common plasmids such as, for example, pUC derivatives may be used.

Transformed cells grow within plants in the usual way. They can form germ cells and transmit the transformed trait (s) to the seedlings. Such plants may be grown in the normal way and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. In some preferred embodiments of the present invention, genes encoding the bacterial protein are expressed from transcriptional units inserted into the plant genome. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the plant genome and allow selection of transformed plant lines expressing protein-encoding mRNA.

Once the introduced DNA has been integrated into the genome, it is relatively stable to arrive (not to return). It usually contains a selection marker that gives the transformed plant cells resistance to a biocide or antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol or, inter alia. Plant selection markers may also typically provide resistance to various herbicides such as glufosinate (e.g. PAT / bar), glyphosate (EPSPS), ALS inhibitors (e.g. imidazolinones, sulfonylureas, jtriazolopyrimidine sulfonanilide, et al). , Bromoxynil, HPPD resistance inhibitor, PPO-, ACC-ase inhibitors, and many others. The label used individually, should allow the selection of transformed cells and not cells that do not contain the inserted DNA. The gene (s) of interest are preferably both constitutive and inducible promoters in the plant cell. Once expressed, mRNA is translated into proteins, thereby incorporating amino acids into the protein of interest. Genes encoding a protein expressed in plant cells may be under the control of a constitutive promoter, a tissue specific promoter or an inducible promoter. Several existing techniques for introducing recombinant vectors into plant cells abroad, and for obtaining plants that stably express and maintain the introduced gene. Such techniques include the introduction of genetic material to coat the microparticles directly on cells (U.S. Patent No. 4,945,050 and 5,141,131 to Cornell to DowElanco, now Dow AgroSciences LLC). In addition, plants can be transformed using Agrobacterium technology, see U.S. Patent No. 5,177,010 to the University of Toledo; 5,104,310 from Texas A&M; European patent application 0131624B1; European patent applications 120,516, 159418B1 and 176,112 for Schilperoot; U.S. Patent Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500, all Max Planck;

European Patent Applications 604,662 and 627,752, and U.S. Patent No. 5591616, Japan Tobacco, European Patent Applications 0267159 and 0292435, and U.S. Patent No. 5231019, all Ciba Geigy, now Syngenta;

U.S. Patent Nos. 5,463,174 and 4,762,785, both for Calgene, and U.S. Patent Nos. 5,004,863 and 5,159,135, both for Agracetus. Other processing technology includes whisker technology. See U.S. Patent Nos. 5302523 and 5464765, both to Zeneca, now Syngenta. Another direct delivery DNA transformation technology includes aerosol beam technology. See U.S. Patent No. 6,802,932. Electroporation technology has also been used to transform plants. See WO 87/06614 for Boyce Thompson Institute, U.S. Patent Nos. 5,472,869 and 5,384,253, both V for Dekalb and WO 92/09696 and WO 93/21335 for both Plant Genetic Systems. In addition, viral vectors may also be used to produce transgenic plants expressing the protein of interest. For example, monocotyledonous plants can be transformed with a viral vector using the methods described in Mycogen Plant Science and Ciba-Geigy (now Syngenta) US Patent No. 5,569,597, as well as US Patent Nos. 5,589,367 and 5,316,931, both Biofonte, now Large Scale Biology.

As mentioned above, the manner in which the DNA construct is introduced into the host plant is not critical to the present invention. Any method that provides for efficient transformation may be employed. For example, various methods for transforming plant cells are described in the present invention and include the use of Ti or R1 plasmids and the like to effect Agrobacterium-mediated transformation. In many cases, it will be desirable to have the construct used for limited transformation on one or both sides via T-DNA boundaries, more specifically the right margin. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode of transformation, although T-DNA boundaries may find use with other modes of transformation. Where A-grobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host by homologous recombination with T-DNA or Ti or the plasmid R1 present in the host. Vector introduction can be accomplished by electroporation, tri-parental coupling, and other techniques for transforming gram-negative bacteria that are known to those skilled in the art. The form of the transformation vector in the Agrobacterium host is not critical to the present invention. The Ti or R1 plasmid containing the T-DNA for recombination may be capable or unable to cause gallbladder formation, and is not critical to said invention as long as the vir genes are present in said host.

In some cases, where Agrobotterium is used for transformation, the expression construct within the T-DNA boundaries will be inserted into a broad spectrum vector, such as pRK2 or derivatives, as described in Ditta et al. al. (1980) and EPO 0 120 515. Included within the expression construct and the T-DNA will be one or more markers as described in the present invention that allow selection of transformed Agrobacterium and transformed plant cells. The particular marker employed is not essential to the present invention, with the preferred marker depending on the host and the construct used.

For transformation of plant cells using Agrobacterium, the explants may be combined and incubated with the transformed Agrobacterium for a time sufficient to permit transformation thereof. After transformation, Agrobacterium is killed by selection with the appropriate antibiotic and the plant cells are cultured with the appropriate selective medium. Since calli are formed, bud formation can be encouraged by the use of appropriate plant hormones according to methods well known in the art of plant tissue culture and plant regeneration. However, an intermediate callus phase is not always necessary. After bud formation, such plant cells can be transferred to the medium that stimulates root formation thereby completing plant regeneration. Plants can then be grown to seed and said seed can be used to establish future generations. Regardless of the transformation technique, the gene encoding a bacterial protein is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by inclusion in the vehicle of a plant promoter regulatory element. as well as 3 'untranslated transcription termination regions such as numbers and the like.

In addition to a large number of plant transformation technologies, the type of tissue that is in contact with foreign genes can also vary. Such tissue would include, but are not limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, root tissue, for expression in phloem tissues, and the like. Almost all plant tissues can be transformed during de-differentiation V using the appropriate techniques described in the present invention.

As mentioned above, a variety of selection markers may be used if desired. Preference for a particular marker is at the discretion of the artisan, but any of the following selection markers may be used in conjunction with any other gene not enumerated in the present invention that could function as a selectable marker. Such selection markers include, but are not limited to, the amnoglycoside Tn5 transposon phosphotransferase gene (Aph II), which encodes kanamycin, neomycin or G41 antibiotic resistance, hygromycin resistance, methotrexate resistance, as well as genes. coding for glyphosate resistance or tolerance; phosphinothricin (glufosinate or bialaphos), ALS inhibitors (imidazolinones, sulphonylureas and triazolopyrimidine herbicides), ACC-ase inhibitors (e.g., airylaxipropionates or cyclohexanediones), and others such as bromoxynil, and for example PPHD ( , mesotrione) and the like.

In addition to a selectable marker, it may be desirable to use a reporter gene. In some cases, a reporter gene may be used with or without a selectable marker. Reporter genes are genes that are not normally present in the recipient organism or tissue and usually code for proteins that result from some phenotypic alteration or enzymatic property. Examples of such genes are provided in Weising et al. From 1988. Preferred reporter genes include the E. coli uidA beta-glucuronidase (GÜS) site, the E. coli TN9 chloramphenicol acetyltransferase gene, the bioluminescent jellyfish green protein Aequorea victoria, and the firefly luciferase Photinus piralis. An assay for detecting reporter gene expression may then be performed at the appropriate time after said gene has been introduced into receptor cells. One such preferred assay involves the use of the gene encoding the beta-glucuronidase (GUS) of the E. coli uidA site as described by Jefferson et al (1987) to identify the transformed cells. ’| In addition to plant promoter regulatory elements, V promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopin synthase promoter, the nopaline synthase promoter, the manopine synthase promoter; promoters of viral origin, such as cauliflower mosaic virus (35S and 19S), (35T, which is a 35S engineered remoter, see US Patent No. 6,166,302, especially Example 7E) and the like. can be used. Plant promoter regulatory elements include and are not limited to ribulose-1,6-bisphosphate (RuBP) small carboxylase subunit (SSU), beta-conglycinin promoter, phaseolin beta promoter, ADH promoter, heat shock promoters, and tissue specific promoters. Other elements, such as binding matrix regions, backbone binding regions, introns, promoters, polyadenylation sequences and the like may be present and therefore may improve transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they may provide better expression or DNA function, affecting transcription, mRNA stability, and the like. Such elements may be included in DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include, but are not limited to, Adh 1 intron, Adh 6 intron, alfalfa protein leader sequence mosaic virus coat, osmotin UTR sequences, maize protein leader sequence coat virus, as well as others available to a person skilled in the art. Constitutive promoter regulatory elements may also be used to thereby drive continuous gene expression in all cell types and at all times (e.g., atin, ubiquitin, CaMV 35S, and the like). Tissue-specific regulatory elements of promoters are responsible for gene expression in specific tissue types or cells, such as leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin, and the like), and these may (also used.

V

Promoter regulatory elements may also be active (or inactive) during a given stage of plant development, as well as active in plant tissues and organs. Examples of such include, but are not limited to, endosperm-specific, pollen-specific, embryo-specific, maize silk-specific, cotton-fiber-specific, root-specific, or vegetative promoter-specific regulatory elements. phase and the like. Under certain circumstances, it may be desirable to use an inducible promoter regulatory element which is responsible for gene expression in response to a specific signal, such as: physical stimuli (heat shock genes), light (RuBP carboxylase) ), hormone (Em), metabolites, chemicals (sensitive tetracycline) and stress. Other desirable transcriptional and translational elements that work in plants may be used. Numerous gene transfer plant specific vectors are known in the art.

Plant RNA virus-based systems can also be used to express bacterial protein. In doing so, the gene encoding a protein may be inserted into the region of a suitable plant virus that will infect the host plant of interest with the coat promoter. The protein can then be expressed thus providing protection of the plant against herbicide damage. Plant viral RNA-based systems are described in U.S. Patent No. 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Patent No. 5,316,931 and 5,589,367 to Biofonte, now Large Scale Biology.

Additional means of increased tolerance or resistance levels. It is demonstrated in the present invention that the plants of the present invention can be transmitted with novel herbicide resistance characteristics, with no observable adverse effects on the phenotype including yield. Such plants are within the scope of the present invention. The plants and exemplified in the present invention may support x 2, x 3, x 4, and 5 x typical levels of application of, for example, at least one subject killer. Improvements in tolerance at these levels are within the scope of the present invention. For example, various techniques are known in the literature, and predictably can be optimized and further developed to increase expression of a particular gene.

Such a method includes increasing the copy number of the subject AAD-12 genes (in expression cassettes and the like). Transformation cases can also be selected for those with multiple gene copies.

Strong stimulatory promoters may be used to "turbine" expression. Examples of such promoters include the preferred 35T promoter using 35S enhancers. 35S, corn ubiquitin, Arabidopsis ubiquitin, A.T. and CSMV promoters are included for such uses. Other strong viral promoters are also preferred. Enhancers include 4 OCS and dual 35S enhancer. Matrix attachment regions (MARs) can also be used to increase transgene transformation and expression efficiency. Shuffling (directed evolution) and transcription factors may also be used for embodiments in accordance with the present invention.

Variant proteins may also be designed which differ in sequence but which maintain the same or similar three-dimensional essential general structure, surface charge distribution, and the like. See, for example, U.S. Patent No. 7,058,515; Larson et al., Protein Sci. 2002 11: 2804 to 2813, "Absolute Space Sequence Sampling: Large-scale Protein Design of Structural Assemblies";

Crameri et al., Nature Biotechnology 15, 436 to 438 (1997), "The molecular evolution of a scrambled DNA arsenate detoxification pathway.

Stemmer, WPC 1994. "DNA shuffled through random fragmentation and reassembly: in vitro recombination for molecular evolution" Proc.

Natl. Acad. Know. USA 91: 10747 to 10751; Stemmer, W. P.C. 1994. "Rapid evolution of a protein in vitro through scrambled DNA" Nature 370: 389 to 391; Stemmer, WPC 1995. Search for sequence space. Bio / Technology 13: 549 to 553, Crameri, A. et al. 1996. "The Construct and Evolution of Scrambled DNA Phage Antibody Libraries" Nature Medici 2: 100-103, and Crameri, A., et al. 1996. "Molecular evolution-enhanced fluorescent green protein using scrambled DNA" Nature Biotechnology 14: 315 to 319. The activity of recombinant polynucleotides inserted into plant cells may be dependent on the influence of endogenous plant DNA production adjacent to the tablet. Thus, another option is to take advantage of cases that are known to be excellent locations in a plant genome for insertions. See, for example, WO 2005/103266 A1, relating to cri1F cases and cri 1 Ac cotton; AAD-12 gene can be substituted at these genomic sites in place of cri1F and / or cri 1 Ac insertions. Thus, the homologous recombination target, for example, may be used in accordance with the present invention. This type of technology is the subject of, for example, WO 03/080809 A2 and the corresponding publication US 20030232410, concerning the use of zinc fingers for specific recombination. The use of recombineses (cre-10 x and FLP-FRT, for example) is also known. AAD-12 detoxification is believed to occur in the cytoplasm.

Accordingly, means for further stabilizing this protein and mRNA (including blocking mRNA degradation) are included in aspects of the present invention, and techniques known in the art may be applied accordingly. The subject proteins may be designed to resist degradation by proteases and the like (proteinase splitting sites can be effectively removed by reengineering the amino acid sequence of the protein). Such variants include the use of 5 'and 3' as loop structures of osmotin and per5 UTRs (UA-rich 5 'untranslated sequences). 7-methyl type buffers or 2'-O-methyl groups, for example 7-methylguanylic acid residue, may also be used. See, for example: Proc. Natl. Acad. Know. USA vol. 74, No. 7, pp 2734-2738 (July 1977) Importance of the 5 'terminal blocking structure for stabilizing mRNA in eukaryotic protein synthesis. Protein complexes or ligand blocking groups may also be used. The most suitable 5 'or 3' RTU computational design for AAD-12 (synthetic clamps) may also be carried out within the scope of the present invention. Computer modeling in general, as well as scrambled gene and directed evolution, are discussed in the present invention elsewhere. More specifically with respect to RTUs and computer modeling, computer modeling techniques for use in predicting / evaluating 5 'and 3' RTU derivatives of the present invention include, but are not limited to: MFold version 3.1 available from from Genetics Corporation Group, Madison, Wis (see Zucker et al., Algorithms and Thermodynamics for Secondary RNA Structure Prediction: A Practical Guide in Biochemical RNA and Biotechnology, 11-43, J. Barciszewski & BFC

Clark, eds, NATO ASI Series, Kluwer Academic Publishers, Dordrecht, NL., (1999); Zucker et al, Extended Sequence Dependence on Thermodynamic Parameters Improves prediction of RNA secondary structure J. Mol Biol 288, 911 to 940 (1999); Zucker et al. Secondary RNA prediction of the current structure. Protocols of Chemical Nucleic Acid S. Beaucage, DE

Bergstrom, GD Glick and RA Jones eds., John Wiley & Sons, New York, 11.2.1 to 11.2.10, (2000)), ANGRA (RNA structure analysis using covariance models (context of free grammar stochastic methods) ) v 2.4.2 (Eddi & Durbin, Nucl Acids 1994, 22: 2079 to 2088), which is distributed free of charge as source code and can be downloaded from the Genetics.wustl .edu / EddI / software website. / e FOLDALIGN, also freely distributed and available for download from the bioinf.au.dk website. FOLDALIGN / (see Finding the Most Significant Common Sequence and Structure Motifs of an RNA Sequence Set J. Gorodkin, LJ Heyer, and GD Stormo Acids Research, Vol. 25, No. 18 pp 3724 to 3732, 1997; Finding a Common Sequence and Structure Motifs in a Set of RNA Sequences J. Gorodkin, LJ Heyer, and GD Stormo ISMB 5; 120 to 123, 1997).

The embodiments of the present invention may be used in conjunction with naturally evolving or chemically enhanced mutants (mutants may be selected by V-screening techniques, then transformed with AAD-12 genes and possibly others). The plants of the present invention may be combined with ALS resistance and / or have developed glyphosate resistance. Aminopiralide resistance, for example, may also be combined or "stacked" with an AAD-12 gene.

Traditional breeding techniques can also be combined with the present invention to powerfully combine, introduce, and enhance desired characteristics.

Improvements also include the use of suitable protective agents to further protect plants and / or to add cross-resistance to additional herbicides. Protective agents usually act to increase plant immune systems by activating / expressing cP450. Protective agents are chemical agents that reduce the phytotoxicity of herbicides to plants grown through a physiological or molecular mechanism without compromising the effectiveness of weed control.

Phytoprotective herbicides include benoxacor, cloquintocet, cyometrinyl, dichlormid, dicyclonon, dietolate, fenclorazole, fenclorime, flurzol, fluxophenim, furilazole, isoxadifen, mefenpir, mefenate, naphthalic anhydride, and oxabetriniia. Plant activators (a new class of compounds that protect plants by activating their defense mechanisms) may also be used in embodiments of the present invention. These include acibenzolar and probenazole.

The commercialized protectors can be used for the protection of large grass seeds such as maize, sorghum, moist sown rice, against constituted pre-plants or pre-emergence applied herbicides of thiocarbamate and chloroacetanilide families. Protectors have also been developed to protect cereal crops such as winter wheat from post-emergence applications of aryloxyphenoxypropionate and sulfonylurea herbicides. The use of protective agents for the protection of maize and rice against sulfonylurea, imidazolinone, cyclohexadione, isoxazole, and tricetone herbicides is also well established. % The valorization of the induced herbicide detoxification protector in safety plants is widely accepted as the main mechanism involved in the protective action. Protectors induce cofactors such as glutathione and herbicides such as glutathione S-transferase detoxification enzymes, cytochrome P450 monooxygenases, and glycosyl transferases.

Hatzios KK, Burgos N (2004) "Metabolism-based herbicide resistance: regulation through protective agents." Weed Herb Science: vol. 52, No. 3 pp 454 to 467. The use of a cytochrome Ρ450 monooxygenase gene stacked with AAD-12 is a preferred embodiment. There are P450 involved in herbicide metabolism; cP450 may be of plant or mammalian origin, for example. In higher plants, cytochrome P450 monooxygenase (P450) is known to drive secondary metabolism. It also plays an important role in the oxidative metabolism of xenobiotics in cooperation with NADPH cytochrome P450 (reductase) oxidoreductase. Resistance to some herbicides was classified as a result of P450 metabolism as well as glutathione S-transferase. A number of P450 microsomal species involved in xenobiotic metabolism in mammals have been characterized by molecular cloning. Some of them have been reported to efficiently metabolize various herbicides.

In this way, transgenic plants with P450 plants or mammals may show resistance to various herbicides.

A preferred embodiment of the foregoing is the use of cP450 for acetochlor chlorine resistance (acetochlor chlorine products include Surpass ®, Keystone ®, Keystone LA, ® FulTime ® and first line herbicides) and / or trifluralin (such as Treflan ®). Such resistance of soy and / or corn is included in some preferred embodiments. For further guidance on such embodiments, see, for example, Inui et al, "Using a Selectable Cytochrome P450 Monooxygenase Marker for Arabidopsis Transformation," Plant Biotechnology 22, 281 to 286 (2005) (in Regarding a selection system for the transformation of Arabidopsis thaliana, through Ide Agrobacterium tumefaciens using cytochrome P450 monooxygenases that metabolize herbicides, herbicide tolerant propagules were transformed and selected with acetochlor chlorine, amiprophos methyl, chlorprofam, chlororsulfurone , norflurazon, and pendimethalin); Siminszky et al, "Expression of soybean cytochrome P450 mono-oxygenase of light cDNA and tobacco increases metabolism of phenylurea herbicides", PNAS vol. 96, Issue 4, 1750 to 1755, February 16, 1999; Sheldon et al, Weed Science: vol. 48, No. 3, pp 291 to 295, "A cytochrome monooxygenase P450 cDNA (CYP71A10) confers resistance to nicotiana tabacum transgenic linurone" and "Phytoremediation of atrazine and metolachlor herbicides by transgenic rice plants expressing CYP1A1 hu- mana, CYP2B6 and CYP2C19, "J Agric Food Chem. April 19, 2006; 54 (8): 2985 to 91 (relating to testing a human cytochrome P450 monooxygenase in rice, where rice plants reportedly showed a high tolerance to chloroacetomides (acetochlor, alachlor, methoachlor, pretylachlor and tenylchlor), oxyacetamides (mefenacet), pyridazinones (norflurazon), 2,6-dinitroanalines (trifluralin and pendimethalin), phosphamidates (amiprofos-methyl, thiocarbamates (piributicarb), and urea (chlortolurone)) There is also the possibility of altering or using different chemicals of 2 , 4-D to make AAD-12 genes into subject more efficient.

Such possible changes include creating better substrates and better performing groups (higher electronegativity). Amino acid transport inhibitors (eg diflufenzopyr) may also be used for the purpose of increasing the herbicidal activity of 2,4-D. Unless specifically stated or implied, the terms "one", "one" and "a" means "at least one" used in the present invention.

All patents, patent applications, provisional applications, and publications referred to above or cited in the present invention are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification. Following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. All percentages are by weight and all proportions of V solvent mixtures are by volume unless otherwise specified.

EXAMPLES

Example 1 Method for Identifying 2.4-D In-Plant Resistance Genes As a way of identifying genes that have plant-degrading herbicide activities, it is possible to extract current public databases such as NCBI (National Center for Biotechnology Information).

To start the process, an already identified functional gene sequence that encodes a protein with the desired characteristics (ie α-ketoglutarate dioxigenase activity) is required. This protein sequence is then used as input to the BLAST (Basic Locald Alignment and Search Tool) (Altschul et al, 1997), the algorithm for comparison with deposited available NCBI protein sequences. Using the default settings, this search returns over 100 homologous protein sequences at different levels. These range from highly identical (85 to 98%) to very low identity (23 to 32%) at the amino acid level. Traditionally, unique sequences with a high homology would be expected to retain properties similar to the input sequence. In this case, they were chosen with sequences only.

Gorek. 50% homology. As exemplified in the present invention, cloning and recombinant expressing homologues with as little as 31% amino acid conservation (relative to Ralstonia eutropha TFDA) can be used to transmit commercial levels of resistance, not only to the herbicide to which but also on substrates not previously tested with these enzymes.

A single gene (SDPA) has been identified in the NCBI database (see ncbi.nlm.nih.gov; adhesion # AF516752) as a 31% amino acid identity homologue for TFDA. The percent identity was determined by both the first DNA sequences translating SDPA and TFDA deposited into the protein database, then using ClustalW in the VetorNTI software package to perform multiple sequence alignment.

Example 2 Sequence Optimization for Plant and Bacterial Expression With the aim of obtaining high levels of heterologous gene expression in plants, it may be preferable to restructure the protein coding sequence of the genes so that they are more efficiently expressed in human cells. plant. Corn is an example where the plant may be preferred to redraw the coding region of the heterologous protein prior to transformation in order to increase the level of gene expression and the level of the coded protein in the plant. Therefore, an additional step in gene design that encodes a bacterial protein is a reengineering of the heterologous gene for optimal expression. the number of genes of the class given in parentheses. b standard deviations in parentheses. and middle of combined groups ignored in averaging.

One reason for the reengineering of a bacterial protein for maize expression is due to the non-optimal G + C content of the native gene.

For example, the very low content of many native bacterial gene (s) (and consequently high T + distortion) results in the generation of sequences that mimic the sequences of control and duplication of plant genes known to be highly A + G + C T rich. The presence of some A + T rich sequences within the DNA of gene (s) introduced into plants (e.g. TATA box regions normally found in gene promoters) may result in aberrant V transcription of the gene (s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (eg, polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to instability. RNA. Therefore, a goal in gene design that encodes a bacterial protein for maize expression, more preferably referred to as an optimized plant gene (s), is to generate a DNA sequence having a higher G + C content and preferably a close content of maize genes encoding metabolic enzymes. Another objective in designing the optimized gene of the plant (s) encoding a bacterial protein is to generate a DNA sequence in which sequence modifications do not prevent translation. Table 2 illustrates how high the G + C content is for maize.

To obtain the data from Table 2, gene coding regions were extracted from GenBank (version 71), inputs and base compositions were calculated using the MacVetor ™ program (Acceleris, San Diego, Calif.). intron sequences were ignored in the calculations.

Due to the plasticity provided by the redundancy / degeneracy of the genetic code (ie some amino acids are specified by more than one codon), the evolution of the genomes of different organisms or classes of organisms has resulted in the differential use of re - abundant codons. This "redundant codon" is reflected in the composition of the average base of protein coding regions. For example, organisms with relatively low G + C content use codes having one or T in the third position of the redundant codons, while those with higher G + C content use codes having G or C in the third position. The presence of "smaller" codons within an mRNA is thought to be able to reduce the mRNA translation rate absolutely, especially when the relative abundance of the loaded codon corresponding to the minor codon is accessible. An extension of this situation is that decreasing the translation rate from individual smaller codons would be at least additive to multiple smaller codons. Therefore, mRNAs with high relative levels of lower codons have correspondingly low conversion rates.

This rate would be reflected by the subsequent low levels of the encoded protein.

In engineering genes that encode a bacterial expression protein from maize (or from another plant, such as cotton or soy), the plant's polarization codon was determined. Corn codon bias is the codon of statistical distribution that the plant uses for coding its proteins and the use of the preferred codon is shown in Table 3. After determining the bias, the percent frequency of codons in ( The gene (s) of interest is determined. The codons preferred by the plant primaries should be determined, as well as the second, third, and fourth preferred codon choices when there are several options. A new DNA sequence can then be designed that encodes the amino acid sequence of the bacterial protein, but the new DNA sequence that differs from the native bacterial (encoding the protein) DNA sequence by replacing the plant (from preferably first, the second preferred, third preferred or fourth preferred codons) to specify the amino acid at each position within the amino acid sequence of the protein. The new sequence is then parsed through the restriction enzyme sites that may have been created by this modification. Identified sites are further modified by replacing the codons with the first, second, third, or fourth preferred codons of choice. Other sites in the sequence that may affect transcription or translation of the gene of interest are exone: armchair (5 'or 3') junctions, poly A addition signals, or RNA polymerase termination signals. THE

The sequence is further analyzed and modified to reduce the frequency of paired AT or GC. In addition to doublets, G or C sequence blocks that have more than about four residues are the same that may affect sequence transcription. Therefore, these blocks are also modified by replacing the first or second choice codons, etc., with the next preferred codon of choice. It is preferable that the optimized gene of the plant (s) coding for a bacterial protein contain from about 63% of the first codons of choice, from about 22% to about 37% of the second codons of choice, and between about 15% to about 0% or third choice fourth codons, where the total percentage is 100%. Most preferred is the optimized plant gene (s) containing about 63% of the first codons to choose from, at least about 22% from the second choice codons, about 7.5% from the third choice codons, and about 7.5% choose fourth codons, where the total percentage is 100%. The above method allows one skilled in the art to modify the gene (s) that are foreign to a particular plant so that the genes are perfectly expressed in plants. The method is further illustrated in PCT application WO 97/13402.

Thus, in order to create the optimized plant genes encoding a bacterial protein, a DNA sequence is designed to encode the amino acid sequence of said protein using a redundant genetic code established from a bias table. codons compiled from gene sequences for the particular plant or plants. The resulting DNA sequence has a greater degree of codon diversity, a desirable base composition, may radically contain placed restriction enzyme recognition sites, and lacks sequences that may interfere with gene transcription, or the mRNA translation of the product. Thus, synthetic genes that are functionally equivalent to the proteins / genes of the present invention can be used to transform hosts, including plants. Further guidance with respect to synthetic gene production can be found in, for example, U.S. Patent No. 5,380,831.

Analysis of AAD-12 Plant Reconstruction: An extensive analysis of the 876 base pairs (bp) of the native coding AAD-12 region DNA sequence (SEQ ID NO: 1) revealed the presence of several sequence motifs that thought to be detrimental to optimal plant expression as well as non-optimal codon composition. The protein encoded by SEQ ID NO: 1 (AAD-12) is shown as SEQ ID NO: 2.

To improve recombinant protein production in monocotyledons such as dicotyledons, in this way, a DNA sequence of the AAD-12 (v1) and "optimized" plant (SEQ ID NO: 3) was developed that encodes a protein (SEQ ID NO: 4) which is the same as native SEQ ID NO: 2, except for the addition of an alanine residue in the second position (underlined in SEQ ID NO: 4). The additional alanine codon (TCG; underlined in SEQ ID NO: 3) encodes part of an Ncol restriction enzyme recognition site (CCATGG), which measures the translation initiation ATG codon. Thus, it serves the dual purpose of facilitating subsequent cloning operations by improving the sequence context around the ATG initiation codon V to optimize translation initiation. The proteins encoded by (v1) and native and optimized plant coding regions are 99.3% identical, differing only in amino acid number 2. In contrast, the sequences of the native and optimized plant DNA coding regions (v1 ) are only 79.7% identical. Table 4 shows differences in native codon compositions (columns A and D), and optimized plant sequences (columns B and E), and allows comparison with a theoretical optimized plant sequence (columns C and F) . It is evident from the examination of Table 4 that the native and plant-optimized coding regions, while encoding almost identical proteins, are substantially different from each other. The Optimized Plant (v1) version mimics the codon composition of a theoretical optimized plant coding region encoding the AAD-12 protein.

E. coli Expression Reconstruction: Specially designed Escherichia coli strains and associated vector systems are often used to produce relatively large amounts of protein 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, although the source organism for the gene may be another genus of bacteria. In such cases, it is possible and desirable to restructure the protein coding region of the gene to make it more suitable for expression in E. coli '. Class 1 E. coli genes are defined as those which are highly expressed and continuously during the exponential growth phase of E. coli cells. (Henaut, A. and Danchin, A. (1996) in Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, Vol. 2, pp. 2047-2066. Neidhardt, F., Curtiss III, R., Ingrham, J. Lin, E., Bass, B., Magasanik, B., Reznikoff, W., Riley, M., Schacherter, M. and Umbarger, H. (eds.), American Society of Microbiology, Washington, DC ). By examining the codon compositions of the Class II E. coli gene coding regions, an average codon composition can be designed for these gene II coding regions.

E. coli class.

A protein coding region having the following average codon composition that mimics that of the Class II genes is thought to be favored for expression during the exponential growth phase of E. coli. Using these guidelines, a novel DNA sequence encoding the AAD-12 protein (SEQ ID NO: 4), including additional second position alanine, as mentioned above), was designed according to the codon average composition. coli genes from the Class II gene coding region. The initial sequence, whose design is based solely on codon composition, was further designed to include certain restriction enzyme recognition sequences suitable for cloning into E. coli expression vectors. Harmful sequence characteristics, such as highly stable loop tail structures, were avoided, as well as intragenic sequences homologous to the 3 'end of 16S RNA (Le. Hine Dalgarno sequences) Ribosomal. The optimized E. coli sequence (V2) is described as SEQ ID NO: 5, and encodes the protein described in SEQ ID NO: 4.

The native and optimized E. coli (v2) DNA sequences are 84.0% identical, while the optimized plant (v1) and optimized E. coli (v2) DNA sequences are 76.0% identical. Table 5 shows AAD-12 codon compositions, the native coding region (columns A and D), an AAD-12 coding region optimized for expression in E. coli (v2, columns B and E ) and the composition of the AAD-12 protein-coding region coding codon with an optimal codon composition of V Class II E. coli genes (columns C and F). It is evident from the examination of Table 6 that the native coding regions and optimized E. coli, while encoding almost identical proteins, are substantially different from each other. The optimized version E. coli (v2) mimics the composition of an E. coli codon, the optimized theoretical coding region that encodes the AAD-12 protein. The design of a biased codon soy DNA sequence encodes a soy EPSPS with mutations that confer glyphosate tolerance. This example describes the design of a novel DNA sequence encoding a soybean 5-enolpyruvoylxiquimate 3-phosphate (EPSPS) synthase, which has been optimized for expression in soybean cells. The amino acid sequence of a triple mutated soybean EPSPS is described as SEQ ID NO: 5 of WO 2004/009761. Amino acids mutated in the sequence are thus described at residue 183 (isoleucine-substituted threonine native protein), from residue 186 (lysine-substituted inactive protein arginine), and residue 187 (serine-substituted native protein proline). In this way, the amino acid sequence of the native EPSPS soy protein can be deduced by substituting the substituted amino acids of SEQ ID NO: 5 of WO 2004/009761 with the native amino acids at the appropriate positions. Such native protein sequence is described as PCT / US2005 / 014737 SEQ ID NO: 20 (filed May 2, 2005). The double protein mutated EPSPS soy sequence containing a mutation at residue 183 (native protein substituted by threonine substituted) and at residue 187 (proline native protein substituted by serine) is described as PCT / US2005 SEQ ID NO: 21 / 014737. The codon usage table for the soy protein (Glycine max) coding sequences, calculated from 362,096 codons (approximately 870 coding sequences), was obtained from the "kazasa.or.jp/códon" World Wide Web site. These data were reformatted as shown in Table 6. Columns D and H of Table 6 show the distributions (in% utilization of all codons for that amino acid) of synonymous codons for each of the amino acids as found in the region protein. coding of soy genes. It is evident that some codons synonymous with some amino acids (one amino acid may be specified by 1,2,3, 4, or 6 codons) are present relatively rare in soy protein coding regions (for example, the use of comparisons). GCT and GCG codons to specify alanine).

A table of biased soybean codon utilization was calculated from the data in Table 6. Codons found in soybean genes less than about 10% of total occurrences of particular amino acids were ignored. To balance the distribution of the remaining codon choices for an amino acid, a weighted average representation of each codon was calculated using the formula: weight% C1 = 1 / (% C1 +% C2 +% C3 + etc) x % C1 x 100 where C1 is the codon in question, C2, C3, etc. represent the remaining synonymous codons, and the% values for the relevant codons are taken from columns D and H of table 6 (ignoring the rare codon values V in bold). The weight% value for each codon is given in columns C and G of Table 6. TGA was arbitrarily chosen as the translation terminator. The frequencies of partial codon use were then entered into a specialized genetic code table for use of the OptGene ™ gene design program (Ocimum Biosolutions LLC, Indianapolis, Indiana).

To obtain an optimized soybean DNA sequence encoding the doubly mutated EPSPS protein, the protein sequence of SEQ ID NO: 21 from PCT / US2005 / 014737 was reverse translated by the OptGene ™ program using the genetic code of biased soy derived above. The initial DNA sequence thus obtained was then modified by compensating codon changes (maintaining the weighted average global representation for codons) to reduce the number of CG and TA junctions between adjacent codons, increase the number of CTs and TG pairs between adjacent codons, remove highly stable intrachain secondary structures, remove or add restriction enzyme recognition sites, and to remove other sequences that may be detrimental to expression or cloning manipulations of genetic engineering. Other sequence enhancements were made to eliminate potential intron sites of splice plants, long ATT or C / G residues, and other motifs that could interfere with RNA stability, transcription or translation of the coding region. in plant cells. Other changes have been made to eliminate long internal open reading frames (except one frames). These changes were all made within the retention limits of the biased soybean codon composition as described above, while preserving the amino acid sequence described as PCT / US2005 / 014737 SEQ ID NO: 21. The biased soybean DNA sequence encoding the EPSPS protein of SEQ ID NO: 21 is described as bases 1 to 1575 of PCT / US2005 / 014737 SEQ ID NO: 22. The synthesis of a DNA fragment comprising PCT / US2005 / 014737 SEQ ID NO: 22 was performed by a commercial supplier (PicoScript, Houston Texas). EXAMPLE 3 Vector Expression Cloning and Transformation The E. coli construct, the PET expression vector: Using restriction enzymes corresponding to the sites added with the additional cloning ligands (Xba 1, Xho 1) AAD-12 ( v2) was cut from the picoscript vector, and ligated into a streptomycin / spectinomycin resistant to the pET280 vector. Bound products were then transformed into E. coli TOP10F 'and plated for Broth + 50 µg / ml streptomycin and spectkinomycin (LB S / S) from Luria agar plates.

To differentiate between AAD-12 (v2): pET280 and p-CR2.1: pET280 bonds, about 20 isolated colonies were picked in 6 ml LB-S / S, and grown at 37 ° C for 4 hours with shaking . Each culture was then stained in 50 µg / ml LB + kanamycin, the plates incubated at 37 ° C overnight. Colonies that grew on LB-K were assumed to have the pCR2.1 vector turned on, and were discarded. Plasmids (were isolated from the remaining cultures as before and checked for correctness by XbaI / XhoI digestion. The final expression construct was given the designation pDAB3222. The Pseudomonas Expression Vector construct: A structure - open reading frame AAD-12 (v2) was initially cloned into the modified pET expression vector (Novagen), "pET280 S / S", as an XbaI-XhoI fragment.The resulting PDAB725 plasmid was confirmed with enzymatic digestion. The AAD-12 (v2) open reading frame of pDAB725 was transferred to the Pseudomonas expression vector, pMYC1803, as an Xbal-Xhol fragment .The positive colonies were confirmed by restriction enzyme digestion The completed construct pDAB739 was transformed into MB217 and MB324-expressing Pseudomonas strains.Binary vector completion: The optimized plant gene AAD-12 (v1) was received from Picoscrip t (breeding gene reconstruction has been completed (see above) and out of source for Picoscript for the construct) and the sequence verified (SEQ ID NO: 3) internally to confirm that none of the expected sequence changes were present . Sequencing reactions were performed with forward M13 (SEQ ID NO: 6) and reverse M13 (SEQ ID NO: 7) using the Beckman "Color Terminator Cycle Sequencing Quick Start Kit" reagent primers. Coulter as before. Sequence data were analyzed and results indicated that no anomalies were present in the optimized AAD-12 (v1) plant and DNA sequence. A (v1) AAD-12 gene pDAB726 was cloned as a Sac I-NcoI fragment. The resulting construct was designated pDAB723, containing: [AtUbilO promoter: Nt OSM 5'UTR: AAD-12 (v1): NT OSM3'UTR: ORF1 polyA 3'ÜTR] (verified with a Pvull restriction digestion and Not I). A Not I-Not I fragment containing the described cassette was then cloned into the Not I site of the pDAB3038 binary vector. The resulting binary vector, pDAB724, containing the following cassettes [Actuator promoter: NT OSM5'UTR: AAD-12 (v1): NT OSM 3'UTR: ORF1 polyA 3'UTR: CsVMV promoter: PAT: ORF25 / 26 3 'UTR] has been digested constraint (with Bam Jhi, not me, not me, Saci and Xmn I) to verify correct orientation. The verified completed construct (pDAB724) was used for transformation into Agrobacterium. Cloning Additional Transformation Constructs: All other constructs created by transformation into appropriate plant species were constructed using procedures similar to those previously described in the present invention, and other standard molecular cloning methods (Maniatis et al., 1982). .).

Example 4 Arabidopsis Transformation and Selection Growth Conditions of Arabidopsis thaliana: Wild-type Arabidopsis seed was suspended in a 0.1% agarose solution (Sigma Chemical Co., St. Louis, Mo). Seed suspension was stored at 4 ° C for 2 days to complete dormancy requirements and ensure synchronous seed germination (stratification). The Sunshine LP5 mixture (Sun Gro Florticulture, Bellevue, Washington) was covered with fine vermiculite and sub-irrigated with Hagland's solution until wet. The soil mixture was allowed to drain for 24 hours. The stratified seed was sown on vermiculite and covered with moisture domes (Kord Products, Bramalea, Ontario, Canada) for 7 days.

Seeds were germinated and plants were grown in a Conviron (models CMP4030 and CMP3244, Limited Controlled Environments, Winnipeg, Manitoba, Canada) under long day conditions (16 hours light / 8 hours dark) with a light intensity of 120-150 pmol / m2 sec under constant temperature (22 ° C) and humidity (40-50%). The plants were initially watered with Hoagland's solution and, subsequently, with deionized water to keep the soil moist but not wet. Agrobacterium transformation: An LB + agar plate with erythromycin (Sigma Chemical Co., St. Louis, MO) (200 mg / L) or specinomycin (100 mg / L) containing a streaked DH5a colony was used. to provide a colony to inoculate 4 ml mini-culture preparation i (liquid LB + erythromycin). Cultures were incubated overnight at 37 ° C with constant shaking. Qiagen (Valencia, California) Mini Preps Spin, performed by manufacturer's instructions, were used to purify plasmid DNA.

Electro-competent agrobacterium tumefaciens (strains Z707s, EHA101s and LBA4404s), cells were prepared using a protocol by Weigel and Glazebrook (2002). The competent Agrobacterium cells were transformed using an adapted electroporation method and Glazebrook Weigel (2002). 50 µl of agro competent cells were frozen in ice and 10 to 25 ng of the desired plasmid was added to the cells. DNA and cell mixtures were added to pre-cooled (2 mm) electrotransplant cuvettes. An Eppendorf Eletroporator 2510 was used for transformation under the following conditions, voltage: 2.4 kV, pulse length: 5 ms.

After electroporation, 1 ml YEP broth (per liter: 10 g yeast extract, 10 g Bacto-peptone, 5 g NaCl) was added to the beaker and the YEP cell suspension was transferred to a 15 ml culture. Cells were incubated at 28 ° C in a constantly shaking water bath for 4 hours. After incubation, the culture was seeded on YEP + agar with erythromycin (200 mg / L) or spectinomycin (100 mg / L) and streptomycin (Sigma Chemical Co., St. Louis, MO) (250 mg / L). Plates were incubated for 2 to 4 days at 28 ° C

Colonies were selected and seeded on fresh YEP + agar with erythromycin (200 mg / L) or spectinomycin (100 mg / L) and streptomycin (250 mg / L) and plates incubated at 28 ° C for 1 to 3 days. . Colonies were selected for PCR analysis to verify the presence of gene insertion using the specific primers of the vector. Mini Preps rotation, performed according to the manufacturer's instructions, were used to purify plasmid DNA from selected Agrobacterium colonies, with the following exception: 4 ml aliquots of 1 ml prep 15 mini culture during overnight (YEP liquid + erythromycin (200 mg / L) or spectinomycin (100 mg / L)), and streptomidine (250 mg / L)) were used for DNA purification. An alternative to using DNA rotation

Qiagen Mini-Prep was lysing the transformed Agrobacterium cells suspended in 10 µl of water at 100 ° C for 5 minutes. Plasmid DNA from the binary vector used in Agrobacterium transformation was included as a control. The PCR reaction was completed using Takara Bio Inc.'s Taq DNA polymerase Mirus (Madison, Wis) per manufacturer's instructions at 0.5 x concentrations. PCR reactions were performed in an MJ Research Peltier Thermal Cycler thermal cycler programmed under 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 a 72 ° C cycle for 10 minutes. The reaction was kept at 4 ° C after cyclization. Amplification was analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. One colony was selected whose PCR product was identical to that of the control plasmid. The Arabidopsis: Arabidopsis transformation was transformed using the floral dip method. The selected colony was used to inoculate one or more 15-30 ml of YEP broth precultures 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 shaking at 220 rpm. Each preculture was used to inoculate two 500 ml cultures of YEP broth containing erythromycin (200 mg / l) or spectinomycin (100 mg / l) and streptomycin (250 mg / l)) and the cultures were incubated for overnight at 28 ° C with constant agitation. The cells were then dried at approx. 8700 xg for 10 minutes at room temperature, and the resulting supernatant is discarded. The cell pellet was resuspended gently in 500 ml infiltration media containing: 1/4 x Murashige and Skoog / Gamborg's salts B5 vitamins, 10% (w / v) sucrose, 0.044 µM benzylamino purine (10 µl / ml). 1 mg / ml in DMSO) and 300 µl / liter of Silwet L-77. Plants about 1 month old were dipped for a mean of 15 seconds, making sure to submerge the newest inflorescence. The plants were then laid on their sides and covered (transparent or opaque) for 24 hours, then washed with water, and placed upright. The plants were grown at 22 ° C, at a 16-hour light / 8-hour dark period. Approximately 4 weeks after immersion, the seeds were harvested.

Selection of Transformed Plants: The newly harvested T1 seed [gene AAD-12 (v1)] was allowed to dry for 7 days at room temperature. The T1 seed was sown in 26.5 x 51 cm germination trays (TO Plastics Inc., Clearwater, Minn), each receiving 200 mg of stratified T1 seed aliquots (about 10,000 seeds), which it had 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. The Sunshine LP5 mixture (Sun Gro Horticulture Inc., Bellevue, Washington) was covered with fine vermiculite, irrigated with Hoagland solution until wet, then left the gravity drain. Each 40 ml aliquot of stratified seeds was evenly seeded onto the vermiculite with a pipette and covered with moisture domes (Kord, Bramalea, Ontario, Canada) for 4 to 5 days. The domes were removed one day prior to initial transformant selection using post-emergence glufosinate spray (choosing for the cotransformed PAT gene).

Seven days after planting (DAP) and 11 DAP again, T1 plants (cotyledons and phase 2-4-1-F, respectively) were sprayed with a 0.2% Free herbicide solution ( 200 g ai / l glufosate, Bayer Crop Sciences, Kansas City, Missouri) in a 10 ml syrup volume (703 l / ha) using a DeVilbiss tip compressed air spray to deliver an effective rate 280 g ai / ha glufosinate per application. Survivors (actively growing plants) were identified 4 to 7 days after the end of spraying and transplanted to 3-well pots prepared with potting medium (Metro Mix 360). Transplanted plants were covered with moisture domes for 3 to 4 days and placed in a 22 ° C growth chamber as before or moved directly to the greenhouse. Domes were later removed and greenhouse grown (22 ± 5 ° C, 50 ± 30% RH and 14 h of light: 10 h Dark, minimum 500 pE / m2 natural light + supplementary s1) for at least one day prior to testing for the ability of AAD-12 (v1) and (plant-optimized gene) to provide resistance to phenoxy auxin herbicides. T1 plants were then randomly divided into various rates of 2,4-D. For Arabidopsis, 50 g ea / ha of 2,4-D is an effective dose to distinguish sensitive plants from those with significant levels of resistance. High rates were also applied to determine relative resistance levels (50, 200, 800, or 3200 g ae / ha).

All applications of auxin herbicides were made using the DeVilbiss spray as described above to apply 703 L / ha of spray solution (0.4 ml of pot / 3 inch solution) or applied by the sprayer to a 187 L. spray / volume of spray solution. 2,4-D, either technical grade (Sigma, St. Louis, MO) dissolved in DMSO and diluted with water (<1% DMSO, final concentration) or commercial dimethylamine salt formulation (456 g ea / ml) was used. L, Nufarm, St. Joseph, Missouri). Dichlorprope used was commercial grade formulated as a potassium salt of R-dichlorprop (600 g ai / l, AH Marks). As herbicide rates increased beyond 800 g ea / ha, the pH of the spray solution became extremely acidic, burning of younger leaves, softening of Arabidopsis plants, and complication of evaluating the primary effects of herbicides. It has become common practice to apply these high herbicide rates in 200 mM HEPES buffer pH 7.5.

Some T1 individuals were subjected to alternative commercial herbicides instead of a phenoxy auxin. A point of interest was to determine whether auxin pyridyloxyacetate, Triclopir and fluoroxypir herbicides could be effectively degraded in the plant. Herbicides were applied to T1 plants using a range of a sprayer 187 L / ha spray volume. T1 plants that exhibited tolerance to 2,4-D DMA were later accessed in T2 generation.

Results of selection of transformed plants: The first transformations of Arabidopsis were performed using AAD-12 (v1) | (optimized plant gene). T1 transformants were selected from the first background of unprocessed seeds using a glufosinate selection scheme. More than 300,000 T1 seeds were selected and 316 glufosinate resistant plants were identified (PAT gene), which corresponds to a frequency of 0.10%, which is in the normal construct selection frequency range, where PAT + Freedom are used for transformation selection / selection. T1 plants selected above were later transplanted into individual pots and sprayed with various rates of commercial aryloxyalkanoate herbicides. Table 7 compares the AAD-12 (v1) response and control genes to give the 2,4-D resistance for T1 Arabidopsis transformants. The answer is presented in terms of visual% of lesion 2 WAT.

Data are presented as a histogram of individuals who exhibit little or no injury (<20%), moderate injury (20 to 40%), or severe injury (> 40%). Since each T1 is a case of independent transformation, a significant variation of individual T1 responses can be expected within a given rate. An arithmetic mean and standard deviation are presented for each treatment. The range in individual response is also indicated in the last column for each rate and transformation. Transformed PAT / Cri1F arabidopsis served as an auxin-sensitive transformed control. A (v1), herbicide resistance gene AAD-12 conferred to individual Arabidopsis T1 plants. Within a given treatment, the level of plant response varied widely and can be attributed to the fact that each unit represents a case of independent transformation.

Of important note, at each rate of 2,4-D tested, there were individuals who were affected, while some were severely affected. The overall mean lesion population by rate is shown in Table 7 only to demonstrate the significant difference between plants transformed with AAD-12 (v1) versus wild type or transformed PAT / Cri1F controls. Injury levels tend to be higher and the frequency of unharmed plants was below high rates up to 3,200 g ea / ha (or ~ 6x field rate). Also at these high rates, the syrup becomes highly acidic less buffered. Preliminarily grown Arabidopsis in the growth chamber has a very thin cuticle and severe burn effects can complicate testing at such high rates. However, many individuals have survived 3200 g ea / ha of 2,4-D, with little or no injury. Table 8 shows a response to the similarly performed dose of Arabidopsis T1 to phenoxypropionic acid, dichlorprop. The data show that the active herbicide (R-) dichlorprope isomer does not serve as a suitable substrate for AAD-12 (v1). The fact that AAD-1 metabolizes R-dichlorprop well enough to confer commercially acceptable tolerance is a discriminatory feature that separates the two genes. (Table 8). AAD-1 and AAD-12 are considered to be specific α-ketoglutarate R and S dioxigenases, respectively. AAD-12 (v1) as selection marker: The ability to use AAD-12 (v1) as a selectable marker using 2,4-D as the selection agent was initially analyzed with transformed Arabidopsis, as described above. About 50 seeds of generation T4 Arabidopsis (homozoto for A AD-12 (v1)) were increased by about 5,000 wild-type (sensitive) seeds. Several treatments were compared in each tray of plants that received one or two seasons of 2,4-D application in one of the following treatment regimens: 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 from 2,4-D can be directly compared with glufosinate-selected PAT.

Treatments were applied with a DeVilbiss spray tip as described above. Plants were identified as resistant or sensitive DAP 17. The ideal treatment was 75 g ae / ha 2,4-D applied 7 and 11 days after planting (DBH), was equally effective at the frequency of selection, and resulted in less herbicidal injuries to those who transformed the method. checkbox Freedom. These results indicate AAD-12 (V1) can be effectively used as an alternative selectable marker for a transformed Arabidopsis population. Heritability: A variety of T1 cases were self-pollinated to produce T2 seed. These serrients were by the offspring tested by applying 2,4-D (200 g ea / ha) to 100 random T2 brothers. Each individual T2 plant was transplanted to 7.5 cm of square pots prior to spray application (spray rate range 187 L / ha). Seventy-five percent of the secreted plantas1 (T2 plants) families in the long-awaited resistant 3: a sensitive model for a single dominant inheritance site with Mendelian inheritance as determined by Chi square analysis (P> 0.05).

The seeds were collected between 12 and 20 T2 individuals (only T3). Twenty-five T3 siblings from each of the eight randomly selected T2 families were progeny tested as previously described. About one third of the T2 families predicted to be homozygous (non-segregating populations) were identified in each row. These data show that AAD-12 (V1) is stably integrated and Mendelian inherited for at least three generations.

Additional applications of leaf herbicide resistance to Arabidopsis AAD-12: The ability of AAD-12 (V1) to provide resistance to other auxin aryloxyalkanoate herbicides in transgenic Arabidopsis was determined by foliar application of several substrates. Arabidopsis T2 seed generation was stratified and sown in selection trays very similar to Arabidopsis. The transformed PAT control line containing the Cri1F insect resistance gene was planted in a similar manner. The seedlings were transferred to individual 3-inch pots in the greenhouse. All plants were sprayed using a trajectory spray set at 187 L / ha. Plants were sprayed with a range of pyridyloxyacetate herbicides: 280-2240 g a- and ha triclopyr (Garlon 3A, Dow AgroSciences) and 280-2240 g ae / ha fluoroxipir (Starane, Dow AgroSciences), and 2,4- D metabolite resulting from the activity of AAD-12, 2,4-dichlorophenol (DCP, Sigma) (at a technical grade molar equivalent of 280 to 2240 g ea / ha of 2,4-D, DCP was used). All requests were formulated in water. Each treatment was repeated 3 to 4 times. Plants were evaluated at 3 and 14 days after treatment. There is no effect of the 2,4-D metabolite, 2,4-dichlorophenol (DCP), on control of transgenic non-AAD-12 Arabidopsis (Pat / Cri1F). Transformed AAD-12 plants were also clearly protected from triclopyr and fluoroxipir from herbicide injury that was viscous in ittransformed non-resistant controls (see Table 9). These results confirm that AAD-12 V (v1) in Arabidopsis provides resistance to tested pyridyloxyacetic auxins. This is the first report of an enzyme with significant activity in pyridyloxyacetic acid herbicides. No other 2.4-D degrading enzyme has been reported with similar activity.

Molecular Analysis of Arabidopsis AAD-12 (v1): The PAD Invader Assay (Third Wave Methods Procedures Kit) Assay for gene copy number analysis was performed with total DNA obtained from the Qiagen DNeasy kit in several AAD-12 lines (v1) homozygotes to determine the stable integration of the plant transformation unit containing PAT and AAD-12 (v1). The analysis assumed the direct physical binding of these genes that were contained in the same plasmid.

The results showed that all 2.4-D resistant plants tested contained PAT (and therefore, by inference, AAD-12 (v1)). Copy number analysis showed a total of inserts ranging from 1 to 5 copies. This also correlates with AAD-12 (v1) and protein expression data, indicating that the presence of the enzyme produces significantly high levels of resistance to all commercially available pyridyloxyacetic phenoxyacetic acids.

AAD-12 (V1) Molecular Cell transformed Arabidopsis and a glyphosate resistance gene: Arabidopsis T1 seed was produced, as described above, containing the plasmid pDAB3759 (AAD-12 (v1) + EPSPS), which encodes a trace of putative glyphosate resistance. T1 transformants were selected using AAD-12 (v1) as the selectable marker as described. T1 plants (individually transformed cases) were recovered from the first selection attempt and transferred to three-inch pots in the greenhouse as previously described. Three different control Arabidopsis lines were also tested: Columbia-O wild type, AAD-12 (v1) + PAT T4 (transformed pDAB724) homozygous lines and PAT + Cri1F (transformed control) homozygous lines. Transformed plants pDAB3759 and pDAB724 were pre-selected in the seedling phase for 2,4-D tolerance. Four days after transplantation, the plants were divided equally for% foliar spray treatment as described above with 0, 26.25, 105, 420, or 1,680 g ea / ha of glyphosate (Glyphomax Plus, Dow AgroSciences) in Water.

All treatments were repeated 5 to 20 times. The plants were evaluated seven and 14 days after treatment. Initial evaluation resistance indicated 2,4-D tolerant plants that were subsequently tolerant to glyphosate when compared to the response of the three control lines. These results indicate that resistance can be given to plants to two herbicides with different modes of action, including 2,4-D and glyphosate tolerance, allowing the application of both post-emergence herbicides. In addition, AAD-12 + 2,4-D has been used effectively as a selectable marker for true resistance selection. AAD-12 Arabidopsis genetically stacked with AAD-1 to give broader spectrum of herbicide tolerance: Plants AAD-12 (v1) (pDAB724) and AAD-1 (v3) (pDAB721) were reciprocally crossed and F1 seed was collected. . Eight F1 seeds were planted and grown to produce seeds. Tissue samples were taken from the eight F1 plants and subjected to Western analysis to confirm the presence of both genes. It was concluded that the total of eight plants tested expressed both AAD-1 and AAD-12 proteins. The seed was large and allowed to dry for one week before planting.

One hundred F2 seeds were sown and 280 g ai / ha of glufosinate was applied. Ninety-six F2 plants survived glufosinate selection by fitting an expected segregation ratio to two sites chosen regardless of glufosinate resistance (15 R: 1 S). Glufosinate resistant plants were then treated with 560 µg ea / ha R-dichlorprop + 560 g ea / ha triclopyr, applied to the plants under the same spray regime used for the other test. The plants were classified in 3 and 14 DAT. Sixty-three of the 96 plants that survived glufosinate selection also survived herbicide application. These data are consistent with an expected dominant segregation pattern (9R: 6S) of two independently chosen traits where each gene confers resistance to a single auxin herbicide (either R-dichlroprop or triclopyr). The results indicate that AAD-12 (pDAB724) can be successfully stacked with AAD-1 (pDAB721), thereby increasing the herbicide spectra that can be applied to plants of interest [(2,4- D + dichlorprop (P) and (2,4-D + + fluoroxypyr triclopyr), respectively]. This may be useful in bringing 2,4-D tolerance to a very sensitive species by conventionally stacking two separate 2,4-D resistance genes. In addition, if either gene has been used as a selectable marker for a third and fourth gene of interest through independent transformation activities, then each pair of genes can be assembled by conventional breeding activities and, subsequently selected in generation F1 through herbicide-paired sprayers that are unique between AAD-1 and AAD-12 enzymes (as shown with R-dichlorprope and triclopyr for AAD-1 and AAD-12, respectively) .

Other AAD batteries are also within the scope of the present invention. The TFDA protein has been discussed in the present invention elsewhere (Streber et al). For example, it can be used in conjunction with the subject AAD-12 genes to give herbicide resistance spectra in transgenic plants of the present invention.

Example 5 WHISKERS-Mediated Transformation Using Imazetapir Selection Cloning AAD-12 (v1): A (v1), AAD-12 gene was cut from intermediate vector pDAB3283 as a Nco1 / Sac1 fragment. This was directionally ligated in the same form by cutting off vector pDAB3403 containing the monocotyledonous ZmUbil promoter. The two fragments were ligated together using DNA ligase and transformed T4 into DH5a cells. Minimal preparations were performed on the resulting colonies using the Qiagen preparation QIA rotation mini-kit, and the colonies were digested for guidance. This first construct intermediate (pDAB4100) contains the ZmUbil cassette: AAD-12 (v1). This construct was digested with Pvu1 not1 to release the gene cassette and digest the unwanted mainframe. This was attached to the not1 cut pDAB2212, which contains the selectable AHAS marker driven by the OsActl rice atin promoter. The final construct was designated pDAB4101 or pDAS1863, and contains ZmUb1 / AAD-12 (v1) / ZmPer5: OsAct1 / AHAS / LZmLip.

Callus Initiation / Suspension: To obtain the immature embryos for callus culture initiation, F1 crosses between the grown greenhouse of Hi-11 relatives A and B (Armstrong et al, 1991) were performed. When the embryos were 1.0 to 1.2 mm in size (about 9 to 10 days after pollination), the ears were harvested and surface sterilized by washing with Liqui-Nox ® soap, immersed in 70% ethanol. % for 2 to 3 minutes, then immersed in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes.

The ears were washed in sterile distilled water and immature zygotic embryos were aseptically excised and cultured in 15Ag10 medium (N6 medium (Chu et al., 1975), 1.0 mg / L 2,4-D, 20 g / L saccharin, 100 mg / L casein hydrolyzate (enzymatic digest), 25 mM L-proline, 10 mg / L AgNO3, 2.5 g / L Gelrite, pH 5.8) for 2 to 3 weeks, with the scutellum facing outward from the middle. Tissue showing the appropriate morphology (Welter et al, 1995) was selectively transferred biweekly intervals to fresh 15Ag10 medium for about 6 weeks, then transferred to medium 4 (N6, 1.0 mg / L of 2,4-D, 20 g / L sucrose, 100 mg / L casein hydrolyzate (enzymatic digestion), 6 mM L-proline, 2.5 g / L Gelrite, pH 5.8) at biweekly intervals approximately 2 months.

To initiate embryogenic suspension cultures, approximately 3 æl of callus tissue compacted cell (PCV) volume from a single embryo was added to approximately 30 ml H9CP + liquid medium (MS basal salt mixture (Murashige and Skoog, 1962), the modified vitamins MS containing 10 times and 5 times higher nicotinic acid thiamine-HCI, 2.0 mg / L 2,4-D, 2.0 mg / L α-naphthalenoacetic acid (NAA ), 30 g / l sucrose, 200 mg / l caesine hydrolysate (digestive acid) with 100 mg / l myo-inositol, 6 mM L-proline, 5% v / v coconut water (added immediately before subculture), pH 6.0). Suspension cultures were maintained under dark conditions at 125 ml in Erlenmeyer flasks of a controlled temperature stirrer 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 the addition of 3 ml of cell PCV and 7 ml of conditioned medium to 20 ml of fresh H9CP + liquid medium using a large-sized pipette. Once initiated by folding the growing tissue, the suspensions were intensified and kept in 500 ml flasks in which 12 ml of cell PCV and 28 ml of conditioned medium was transferred to 80 ml of H9CP + medium. Once the suspensions were fully established, they were cryopreserved for future use. Cryoconservation and thawing of the suspensions: Two days after subculturing, 4 ml suspended cell PCV and 4 ml conditioned medium were added to 8 ml cryoprotectant (dissolved in H9CP + medium without coconut water, 1 M glycerol, 1 M DMSO, 2 M sucrose, sterile filtered) and allowed to stir at 125 rpm at 4 ° C. for 1 hour in a 125 ml flask. After 1 hour, 4.5 ml of a 5.0 ml cooled solution of Corning was added to the vial. Once the individual vials were filled, they were held for 15 minutes at 4 ° C in a rate controlled freezer, and then allowed to freeze at a speed of - 0,5 ° C / minute to a final temperature of - 40 ° C After reaching the final temperature, the vials were transferred into 4-holder boxes inside a storage unit (Crioplus Form a Scientific) filled with liquid nitrogen vapors.

To thaw, the vials were removed from the storage unit and placed on dry ice in a sealed container, then immersed in a water bath maintained at 40 to 45 oC until the "boil" subsided. When thawed, the contents were poured onto a stack of 8 ~ sterile 70mm Whatman filter papers (No. 4) on the object 100 x 25mm Petri dishes. Liquid was allowed to absorb the filters for several minutes, and then the cell top filter was transferred to GN6 medium (N6, 2.0 mg / L 2,4-D, 30 g / L sucrose, 2, 5 g / l Gel, pH 5.8) for 1 week. After 1 week, only tissue with promising morphology was transferred out of directly fresh filter paper from GN6 medium. This tissue was subcultured every 7 to 14 days, up to 1 to 3 grams was available for initiation suspension in approximately 30 ml medium + H9CP in 125 ml Erlenmeyer flasks. Three milliliters of PCV were subcultured in fresh medium to H9CP every 3.5 days until a total of 12 ml PCV was obtained, which at the subculture point took place as described above.

Stable Transformation: Approximately 24 hours prior to transformation, 12 ml PCV of previously cryopreserved suspended embryogenic maize cells plus 28 ml conditioned medium was subcultured in 80 ml GN6 liquid medium (GN6 medium without Gelrite) in a flask. Erlenmeyer 500 ml, and placed on a shaker at 125 rpm at 28 ° C. This was repeated twice using the same cell line such that a total of 36 ml PCV was distributed between three vials. After 24 hours, the GN6 liquid media was removed and replaced with 72 ml GN6 S / M osmotic medium (N6, 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 vial to plasmolyze the cells. The vials were placed on a shaker shaken at 125 rpm in the dark for 30-35 minutes at 28 ° C, and during this time 50 mg / ml Swiss silicon carbide suspension was prepared by adding 8.1 ml volume. S-GN6 / M liquid medium for ~ 405 mg of pre-autoclaved, silicon carbide sterile whiskers (Advanced Composite Materials, Inc.).

After GN6 P / M incubation, the contents of each flask were pooled in a 250 ml centrifuge bottle. Once all cells were bottomed, but all ~ 44 ml of liquid GN6 S / M was removed and collected in a sterile 1 L flask for future use. The pre-moistened whisker suspension was vortexed for 60 seconds at full speed and 8.1 ml was then added to the flask, to which 170 µg of DNA was added as a last step. The vial was immediately placed in a commercially modified Red Devil 5400 ink mixer and shaken for 10 seconds. After shaking, cocktail of cells, media, tangled filaments and DNA was added to the contents of the 1 L flask with 125 ml of fresh GN6 liquid medium to reduce osmoticant. Cells were allowed to recover on a shaker at 125 rpm for 2 hours at 28 ° C before being filtered over Whatman # 4 filter paper (5.5 cm) using a glass cell collector unit that was connected to a domestic vacuum line.

Approximately 2 ml of dispersed suspension was pipetted to the filter surface, the vacuum was drawn. The filters were placed in 60 x 20 mm of GN6 media plates. The plates were grown for one week at 28 ° C in a dark box.

After one week, the filter papers were transferred to 60 x 20 mm plates of GN6 (3P) medium (N6 medium, 2.0 mg / l 2,4-D, 30 g / l sucrose, 100 mg / L myo-inositol, 3 µM imazetapir ® DG Activity, 2.5 g / L Gelrite, pH 5.8). The plates were placed in boxes and grown for one week.

Two weeks after transformation, tissue was incorporated by scraping all cells from the plate into 3.0 ml of fused GN6 agarose (N6, 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 only for 10 minutes at 121 ° C) containing 3 µM imazetapyr from Pursuit ® GD. The tissue was cleaved and 3 ml of agarose and the tissue was evenly poured onto the surface of a plate x 15 mm of GN6 (3P) 100. This was repeated for all rack plates. Once incorporated, the plates were individually sealed with Nescofilm ® or Parafilm M ® and cultured until putative isolates appeared.

Protocol to Isolate Recovery and Regeneration: Positively transformed cases were isolated outside the embedded Pursuit ® plaques about nine weeks post-transformation, transferring the new selection medium of the same concentration to 60-µl plates. x 20mm. If sustained growth was evident after approximately 2 to 3 weeks, the case was considered to be resistant, and subjected to molecular analysis. Regeneration was initiated by transfer of callus to a cytokine induction medium, 28 (3P) containing 3 µM Pursuit.RTM. DG, MS 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 pEm-2 s-1) for one week, then the upper light (40 pEm-2 s-1) for another week before being transferred to regeneration medium. , 36 (3P), which was identical to 28 (3P), except that it had no plant growth regulators. Small (3 to 5 cm) seedlings were removed and placed in 150 x 25 mm culture tubes containing SHGA free selection medium (Schenk and Hildebrandt basal salts and vitamins, 1972; 1 g / L myo-inositol, 10 g / L sucrose, 2.0 g / L Gelrite, pH 5.8). Once the seedlings developed a sufficient root and shoot system, they were transplanted to the soil in the greenhouse. From 4 experiments, the complete seedlings, composed by an aerial part and roots, were formed in vitro on the selection plates embedded in dark conditions, without going through a traditional callus phase. The leaf tissues of nine of these "first" regenerators were subjected to the PCR coding region and plant transcription unit (PTU) PCR for the AAD-12 gene and cassette gene, respectively. All had an intact AAD-12 coding region, while 3 did not have a full-length PTU (Table 11). These "early regenerators" were identified as 4101 cases to distinguish them from traditionally derived cases, which were identified as "1283" cases. Plants from 19 additional cases, obtained through standard selection and regeneration, were sent to the greenhouse effect, grown to maturity and cross-pollinated with a property strain to produce T1 seeds. Some of the cases that appear to be clones of each other due to similar band patterns in the Southern blot sequence, so only 14 unique cases were represented. Case T0 plants were tolerant 70 g / ha imazetapyr.

Invader analysis (AHAS gene) indicated insertion complexity ranging from 1 to> 10 copies. Thirteen cases contained the coding region to compete AAD-12, however, a post-inferior analysis indicated the complete plant transformation unit had not been incorporated for nine cases.

None of the compromised 1,863 cases were advanced beyond the T1 stage and post-characterization used the 4,101 cases.

Molecular Analysis - Materials and Methods of Corn: Harvesting tissue for DNA isolation and quantification. Fresh tissue is placed into tubes and lyophilized at 4 ° C for 2 days. After the fabric is completely dried, a tungsten bead (Valenite) is placed in the tube and the samples are subjected to 1 minute dry milling using a Kelco ball mill. The standard DNeasy DNA isolation process is followed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA is then stained with Green Pico (Molecular Probes P7589) and read at the fluorometer (BioTek) with known standards to obtain the concentration in ng / ml. Invader Assay Analysis: DNA samples are diluted to 20 ng / ul, then denatured by incubation in a thermocycler at 95 ° C for 10 minutes. Mixture of signal probes is then prepared using the provided oligo mixture and MgCl2 (third Wave Technologies). A 7.5 µl aliquot is placed into each well of the Invader Assay Plate followed by a 7.5 µl aliquot of standards, controls, and 20 ng / mL diluted unknown samples. Each well is coated with 15 µl of mineral oil (Sigma). The plates are then incubated at 63 ° C. For 1 hour and read in the fluorometer (Biotek). Calculating the% signal on the background for the target probe divided by the% signal relative to the internal control background probe will calculate the ratio. The proportion of known copy patterns developed and validated with Southern blot analysis is used to identify the estimated copy of unknown cases.

Polymerase Chain Reaction: A total of 100 ng of total DNA is used as a template. 20 mM of each primer is used with the Takara PCR Ex Taq Polymerase Kit (Mirus TAKRR001 A). Primers for AAD-12 (v1) PTU are Forward-GAACAGTTAG ACATGGTCTA AAGG

(SEQ ID NO: 8) and reverse GCTGCAACAC TGATAAATGC CAACTGG (SEQ ID NO: 9). The PCR reaction is performed on the GeneAmp 9700 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 PCR AAD-12 (v1) Encoding Region are Forward-ATGGCTCAGA CCACTCTCCA AA (SEQ ID NO: 10) and reverse AGCTGCATCC ATGCCAGGGA (SEQ ID NO: 11). The PCR reaction is performed on the GeneAmp 9700 thermocycler (Applied Biosystems) by subjecting the samples to 94 ° C for 3 min. 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 EtBr stained 1% agarose gel electrophoresis. Southern Blot analysis: Southern blot analysis is performed with genomic DNA obtained from Qiagen's DNeasy kit. A total of 2 µg leaf genomic DNA or 10 µg callus genomic DNA is subjected to overnight digestion using restriction enzymes BSM I and I SWA to obtain PTU data.

After overnight digestion an aliquot of ~ 100 ng is run on a 1% gel to ensure complete digestion. After this warranty the samples are run on a large 0.85% agate 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-HCl, 1.5 M NaCl pH 7.5 for 30 minutes. A gel apparatus containing 20 x SSC is then configured to obtain a gravity gel for nylon membrane (Millipore INYC00010) overnight transfer. After overnight transfer, the membrane is then subjected to UV light by a crosslinking agent (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 template hybridization fragment is prepared using the above PCR coding region using the plasmid DNA. The product is run on a 1% agarose gel and removed and then gel extracted using the (28,706) Qiagen gel extraction procedure. The membrane is then prehybridized at 60 ° C for 1 hour in perfect Hyb buffer (Sigma H7033). The it RMT primer of the rxn labeling dCTP procedure (Stratagene 300.392) is used to develop the p32 based probe (Perkin Emer). The probe is cleaned using G50 Column Probe Quantity (Amersham 27-5335-01). Two million CPM counts are used to hybridize Southern blots at night. After overnight hybridization the spots are then subjected to two s20 minute washes at 65 ° C in 0.1% SDS, 0.1 SSC. The spots are then exposed to a film overnight by incubating at -80 ° C. Herbicide tolerance Post-emergence in AAD-12 Corn T0 Transforms: Four T0 cases were allowed to acclimate in the greenhouse. and up to 2 to 4 new, normal leaves emerged from the spiral (ie, plants had the transition from tissue culture to greenhouse growth conditions). Plants were grown at 27 ° C under 16 hours of light: 8 hours dark ambient in the greenhouse. The plants were then treated with commercial formulations of an Activity ® (imazetapyr), or 2,4-D amine 4. Pursuit ® was sprayed to demonstrate the function of the selectable marker gene present within the ca - We are tested. Herbicide applications were made with a track sprayer in a spray volume of 187 L / ha, spray height 50 cm.

Plants were sprayed with a lethal dose of imazethapyr (70 g a- and / ha) or a rate of 2,4-D DMA salt capable of significant injury to untransformed maize lines (2240 g ea / ha). A lethal dose is defined as the rate that causes injury> 95% of pure Hi-11. Hi-11 is the genetic background of the transformants of the present invention. Several individuals were safely placed on the herbicides to which their genes went to provide resistance. The individual clone '001' from case '001' (aka, 4101 (0) -001-001), however, did not incur minor injuries but recovered at 14 DAT. Three of the four cases were moved forward and subjects were crossed with 5XH751 and taken to the next generation. Each herbicide tolerant plant was positive for the presence of the AAD-12 coding region (PCR), or the presence of the 2,4-D AHAS gene (invader assay) and imazetapyr tolerant plants. , in a respective way. AAD-12 protein was detected in all TO-tolerant plants of 2,4-D-containing cases in an intact coding region. The copy number of the transgene (s) (AHAS, and, by default, AAD-12) varied significantly from 1 to 15 copies. Individual TO plants were grown to maturity and cross-pollinated with a pure row of proprietary plants to produce T1 seeds. j High tolerance verification of 2,4-D in T1: T1 AAD-12 (v1) maize, the seeds were planted in 3 inch pots containing Metro Mix medium and on the 2nd stage leaf were sprayed with 70 g ae / ha of imazethapyr eliminating the nulls. The urviving S plants were transplanted into 1 gallon pots containing Metro Mix media and placed under the same growth conditions as before. In phase V3 V4 the plants were sprayed on the trajectory sprayer set at 187 L / ha, or 560 or 2240 g ea / ha of 2,4-D DMA. The plants were classified in 3 and 14 DAT and compared with 5XH751 x Hi control II plants. The 0-10 rating scale (without extreme auxin injury) was developed to distinguish support root injury. Supporting root notes were taken in 14DAT to show 2.4-D tolerance. 2,4-D causes root support malformation, and is a consistent indicator of auxin herbicide injury in corn crop.

Supporting root data (as shown in the table below) demonstrates that 2 of the 3 tested cases were robustly tolerant to 2240 g ea / ha of 2,4-D DMA. The case "pDAB4101 (0) 001.001" was apparently unstable, however the other two cases were robustly tolerant to 2,4-D and 2,4-D + imazetapyr or 2,4-D + glyphosate (see Table 12).

AAD-12 (v1) heritability in maize: A progeny test was also performed on seven AAD-12 (v1) T1 families that were crossed with 5XH751. The seeds were planted in three inch pots as described above. In the three leaf phase the plants were sprayed with 70 g ea / ha of imazetapyr the trajectory sprayer as previously described. After 14 DAT, resistant and sensitive plants were counted. Four of the six lines tested segregated as a single local, dominant Mendelian trait (1R: 1S), as determined by Chi square analysis. Surviving plants were subsequently sprayed with 2,4-D and plants were found to be tolerant to 2,4-D (rates> 560 g ea / ha). AAD-12 is hereditary as a robust auxin-resistant aryloxyalkanoate resistance gene in multiple species mutually when crossed with a commercial hybrid.

Stacking AAD-12 (v1) to increase herbicide spectrum: AAD-12 (v1) (pDAB4101) and Inborn Ready Roundup elite (BE1146RR) were reciprocally crossed and the F1 seed was collected. Seed from two F1 rows were planted and treated with 70 g ea / ha of imazetapyr in phase V2 to eliminate nodules. For surviving plants, the representatives were separated and or treated with 1,120 g ea / ha 2,4-D DMA + 70 g ae / ha imazetapyr (to confirm the presence of the AHAS gene) or 1,120 g ea / ha 2,4- D. D DMA 1680 g ea / ha glyphosate (to confirm the presence of the Round Up Ready gene) in a trajectory calibrated to 187 L / ha. The plants were classified 3 and 16 DAT.

Spray data showed that AAD-12 (V1) can be conveniently stacked with a glyphosate tolerance gene (such as the Roundup-CP4 EPSPS gene) or other herbicide tolerance genes to provide a broader spectrum of herbicides. that can be safely used for maize. Similarly imidazolinones + 2,4-D + glyphosate tolerance was observed in F1 plants and showed no negative phenotype by the stacked or breeding moledular combinations of these transgenes .______________________________________________________ 2,4-D, Triclopyr and Fluoroxipir Herbicides: Level tolerance field trials were performed in two cases AAD-12 (v1) pDAB4101 (4101 (0) 003.R.003.AF and 4101 (0 ) 005.R001.AF) and a Ready Hybrid Roundup (RR) control (2P782) in Fowler, Ind. And Wayside, Mississippi, seeds were planted with a 40 inch Wayside spacer and 40 inch spacing. Fowler spacing. The experimental design was in randomized blocks with three replications. Herbicidal treatments were 2,4-D (dimethylamine salt) at 1120, 2240 and 4480 g ae / ha, triclopyr at 840 g ea / ha, fluoroxipir at 280 g ea / ha and a control. The AAD-12 (v1) cases contained the AH AS gene as a selectable marker.

Maize events were F2 to secrete AAD-12 (v1), plants were treated with imazetapyr at 70 g ae / ha to remove null plants. Herbicide treatments were applied when maize reached the V6 stage using costal spray compressed air delivering 187 L / transport volume at 130-200 kPa. Visual classifications of the lesion were made at 7, 14 and 21 days after treatment. The L-V ratings of the support root were taken 28DAT on a scale from 0 to 10, with 0 to 1 being a slight fusion of the support root, 1 to 3 being a moderate / errant swelling support root and proliferation of the root. root, 3 to 5 being moderate support root fusion, 5 to 9 severe support root fusion and malformation, and 10 being total inhibition of support roots. Case response AAD-12 (v1) and for 2,4-D, triclopyr, fluoroxyr and at 14 days after treatment are shown in Table 14. Harvest injury was more severe at 14 DAT. RR maize control (2P782) was severely injured (44% at 14 DAT) by 2,4-D at 4480 g ea / ha, which is 8 times (8 x) normal field use rate. The AAD-12 cases (v1) all demonstrated excellent tolerance to 2,4-D at 14 DAT with 0% lesion at 1, 2 and 4 x doses, respectively. Corn control (2P782) was severely injured (31% at 14 DAT) at the 2 x triclopyr rate (840 g ea / ha). The AAD-12 (v1) cases showed tolerance at 2 x doses of triclopyr, with an average lesion of 3% at 14 DAT between the two cases. Fluoroxipir at 280 g ea / ha caused visual impairment of 11% for wild type maize at 14 DAT. AAD-12 cases (v1) demonstrated greater tolerance with an average lesion of 8% at 5 DAT.

Applications of auxin herbicides to V6 growing maize may cause support root malformation. Table 15 shows the severity of the supporting root lesion caused by 2,4-D, triclopyr and fluoroxipir. Triclopyr at 840 g ea / ha caused the most severe support root fusion and malformation resulting in a supportive mean root lesion score of 7 in the 2P782 corn type control.

Both AAD-12 (v1) maize cases showed no root damage from the triclopyr treatment root. Root support lesion in 2P782 corn increased with increasing doses of 2,4-D. At 4480 g ae / ha of 2,4-D, AAD-12 cases showed no support root lesion and severe support root fusion and malformation were seen in the 2P782 hybrid.

Fluoroxipir caused only moderate swelling support root and wandering in wild-type corn with AAD-12 (v1) cases showed no support root lesion. These data clearly show that AAD-12 (v1) transmits high level tolerance. in maize for 2,4-D, triclopyr and fluoroxipir at rates much higher than those commercially used and that cause non-AAD-12 (v1) severe visual maize and root support lesion.

Example 6 Tobacco Transformation Tobacco transformation with Agrobacterium tumefaciens was performed by a similar but not identical method to published methods (Horsch et al., 1988). To provide the source tissue for transformation of tobacco seeds (Nicotiana tabacum cv. KY160) was sterilized surface and planted on the TOB-support surface, which is free of Murashige and Skoog hormones (Murashige and Skoog , 1962), solidified with agar. The plants were grown for 6-8 weeks in a light incubator room at 28-30 ° C and allowed to be sterile collected for use in the transformation protocol. Pieces of approximately one square centimeter were sterilized from these cut leaves, excluding the central rib. Cultures of Agrobacterium strains (EHA101S containing pDAB3278, or pDAS1580, AAD-12 (v1) + PAT), grown overnight in a shaker flask at 250 rpm at 28 ° C. in a centrifuge and resuspended in sterile Murashige medium and Skoog salts, and adjusted to a final optical density of 0.5 to 600 nm. Leaf pieces were dipped into this bacterial suspension for approximately 30 seconds, then dried with absorbent paper on sterile paper towels and placed right side on top TOB + medium (Murashige and Skoog, containing 1 mg / l indole acetic acid and 2, 5 mg / l benzylanine) and incubated in the dark at 28 ° C Two days later, the leaf pieces were transferred to TOB + medium containing 250 mg / l cefotaxime (Agri-Bio, North Miami, Florida) and 5 mg / l ammonium glufosinate (active ingredient in Basta, Bayer Crop Sciences) and incubated at 28 - 30 ° C in light.

Leaf pieces were transferred to new TOB + medium with cefotaxime and Basta twice a week for the first two weeks and once a week thereafter. Four to six weeks after the leaf pieces were treated with bacteria, the small plants resulting from transformed foci were removed from this tissue preparation and planted in TOB medium containing 250 mg / l cefotaxime and 10 mg / l Basta in pots. Phytatray ™ II (Sigma). These seedlings were grown in a lighted incubator. After 3 weeks, the cuttings were removed and relocated in the same media. The plants were left ready to send to the greenhouse after an additional 2-3 weeks.

The plants were transferred to the greenhouse, washing the agar from the roots, transplanting into the soil in 13.75 square cm pots, placing the pot in a Ziploc ® bag (SC Johnson & Son, Inc.), placing the water from the tap to the bottom of the bag, and placing in indirect light in a 30 ° C greenhouse for a 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 acclimatized, at which time the bag was removed. The plants were grown in common warm greenhouse (30 ° C, Day 16 hours, 8 pm, natural minimums + supplementary light pE / m2 s1 = 500).

Prior to propagation, T0 plants were sampled for DNA analysis to determine insert copy number. The PAT gene that was molecularly linked to AAD-12 (v1) was tested for convenience. Fresh tissue was tubeed and lyophilized at 4 ° C for 2 days. After the fabric was completely dried, a tungsten bead (Valenite) was placed in the tube and the samples were subjected to 1 minute dry milling using a Kelco ball mill. The standard DNeasy DNA isolation process was followed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA was then stained with Pico Green (Molecular Probes P7589) and read at the fluorometer (BioTek) with known standards to obtain the concentration in ng / mL.

DNA samples were diluted to 9 ng / mL and then denatured by incubating in a thermocycler at 95 ° C for 10 minutes. Mixture of signal probes was then prepared using the provided oligo mixture and MgCl2 (third Wave Technologies). A 7.5 µl aliquot was placed in each well of the Invader Assay Plate followed by a 7.5 µl aliquot of standards, controls, and 20 ng / mL of unknown diluted samples. Each well was coated with 15 µl mineral oil (Sigma). The plates were then incubated at 63 ° C. for 1.5 hours and read on fluorometer (Biotek). Calculating the% Signal on the Background for the Target Probe Divided by the% signal relative to the internal control background probe will calculate the ratio. The proportion of known copy standards developed and validated with Southern blot analysis was used to identify the estimated copy of unknown cases.

Cases were also assayed for the presence of the (v1) AAD-12 gene by PCR using the same extracted DNA samples.

A total of 100 ng of total DNA was used as a template. 20 mM of each primer was used with the Takara Ex Taq PCR polymerase kit. Primers for the AAD-12 PCR Plant Transcription Unit (PTU) were (SdpacodF: ATGGCTCATG CTGCCCTCAG CC) (SEQ ID NO: 12) and (Sdpa-codR: CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 13). The PCR reaction was performed on the GeneAmp 9700 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 an EtBr stained 1% agarose gel.

Four to 12 clonal strains from each of 18 PCR-positive cases with 1-3 copies of the PAT gene (and presumably AAD-12 (v1) because these genes are physically linked) were regenerated and moved to the greenhouse. Post-emergence herbicide tolerance of AAD-12 (v1) TO Transformed Tobacco: The TO plants in each of the 19 cases were challenged with a wide range of sprayed 2,4-D, triclopyr, or fluoroxipyr on the plants that were 3-4 inches tall. Spray applications were made as previously described using a trajectory sprayer at a spray volume of 187 L / ha. 2,4-D Dimethylamine salt (Riverside Corp) was applied at 0, 140, 560, or 2240 g ea / ha of representative dogs of each case mixed in deionized water. Fluoroxypir was also applied at 35, 140, or 560 g ae / ha. Triclopyr was applied at 70, 280, or 1120 g ea / ha. Each treatment was repeated 1-3 times. Injury ratings were recorded 3 and 14 DAT. Each case tested was more tolerant to 2,4-D than the unprocessed KY160 line control. In several cases, some of the initial related herbicidal epinepsy auxin occurred at doses of 560 g ea / ha of 2,4-D or less. Some cases were unharmed 2.4-D applied at 2,240 g ea / ha (equivalent to 4 x field rate). Overall, AAD-12 (v1) cases were more sensitive to fluoroxipir, followed by jtriclopir, and less affected by 2,4-D. The quality of the cases with respect to the magnitude of the resistance was discerned using T0 responses from plants at 560 g ea / ha fluoroxipir. The cases were categorized as "low" (> 40% injury 14 DAT), "medium" (20 to 40% injury), "high" (<20% injury). Some cases were inconsistent in response between repetitions and were considered "variable". High Tolerance Check for 2,4-D in T1 Tobacco: Two to four T0 individuals who survive high rates of 2,4-D and fluoroxipir were saved from each case and allowed to autofertilize to give rise to T1 seeds. The T1 seed was stratified and sown in selection trays very similar to that of Arabidopsis, followed by selective removal of unprocessed nulls in these 560 g ai / ha glufosinate (PAT gene selection) segregating populations. Survivors were transferred to individual 3 inch pots in the greenhouse. These high levels of lines provided resistance to 2,4-D in generation T0. Better response consistency is predicted in T1 not coming directly from tissue culture. These plants were compared against wild type tobacco KY160. All plants were sprayed with a defined trajectory sprayer at 187 L / ha. 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 requests were formulated in water.

Each treatment was repeated 2 to 4 times. Plants were evaluated at 3 and 14 days after treatment. The plants were assigned the lesion classification in relation to dwarfism, chlorosis and necrosis. The T1 generation is secreted, in no way is a response variable expected due to the difference in zygosity. __________________________________________________________________ No lesions were observed at 4 field rate x (2,240 g ea / ha) for 2,4-D or below. Some lesion with Triclopir treatments was observed in one case line, but the largest lesion with fluoroxipir was observed. The fluoroxipyr lesion was short-lived and new growth in one case was almost indistinguishable from untreated 14 DAT control (Table 17). It is important to note that unprocessed tobacco is extremely sensitive to fluoroxipir. These results indicated a commercial level of 2.4-D tolerance can be provided by AAD-12 (v1), even in a culture very sensitive to dicotyledonous auxin such as tobacco. These results also show that resistance can be transmitted to the herbicides pyridyloxyacetic acid, triclopyr and fluoroxypyr. Having the ability to prescribe treatments in an AAD-12 protected herbicide tolerant crop with several active ingredients having varying weed control spectrum is extremely useful for growers. The heritability of AAD-12 (v1) in Tobacco: A 100 plant progeny test was also performed on seven T1 lines of AAD-> 12 (v1) lines. The seeds were stratified, sown, and transplanted with respect to the process described above, except that non-zero plants were removed by Freedom selection. All plants were then sprayed with 560 g ea / ha of 2,4-D DMA as described above. After 14 DAT, resistant and sensitive plants were contacted. Five of the seven lines tested segregated as a single dominant Mendelian trait (3R: 1S) as determined by Chi square analysis. AAD-12 is hereditary as a robust auxin aryloxyalkanoate resistance gene in multiple species. Tobacco Plant Tolerance Field pDAS1580 for 2,4-D, Herbicides Dicloprop, Triclopir and Fluoroxipir: Level tolerance field trials were performed in three AAD-12 (v1) lines (pDAS1580- [1] -018,001, pDAS1580- [1] -004,001 and pDAS1580- [1] -020,016) and a wild-type line (KY160) at field stations in Indiana and Miss tobacco transplants were grown in greenhouse with T1 seed planting in 72 flattened transplant wells (Hummert International) containing Metro 360 medium according to the above growth conditions. Null plants were selectively removed by freedom selection as described above. Transplant plants were transported to field stations and planted 14 or 24 centimeters apart using industrial plant planters.

Drip irrigation at the Mississippi site and sprinkler irrigation at the Indiana site were used to keep the plants growing vigorously. The experimental design was in split plots with four replications. The main plot was the herbicide treatment and the subplot was the tobacco line. Herbicidal treatments were 2,4-D (dimethylamine salt) at 280, 560, 1,120, 2,240 and 4,480 g ea / ha, triclopyr at 840 g ea / ha, fluo-roxipir at 280 g ea / ha and a control. The plots were a 25 to 30 foot line of herbicide treatments were applied 3 to 4 weeks after transplantation using costal spray compressed air delivering 187 L / ha of transport volume at 130 to 200'kPa. Visual classification of the lesion, growth inhibition, and epinastia were taken at 7, 14, and 21 days after treatment. AAD-12 (v1) and case response for 2,4-D, triclopyr and fluoroxy- pir are shown in Table 18. The unprocessed tobacco line was severely injured (63% at 14 DAT) by 2,4-D at 560 g ea / ha, which is considered once the field application rate. The AAD-12 (v1) lines all showed excellent tolerance to 2,4-D at 14 DAT with an average lesion of 1.4 and a 4% lesion observed at 2, 4 and 8 times the rates, in a resilient manner. - positive. The unprocessed tobacco line was severely injured (53% at 14 DAT) at the 2 x triclopyr rate (840 g ea / ha), while the AAD-12 (v1) lines showed tolerance with an average injury of 5%. % at 14 DAT on all three lines. Fluoroxipir at 280 g ea / ha caused serious injury (99%) to the 14 DAT unprocessed line. The AAD-12 (v1) lines demonstrated greater tolerance with an injury rate of 11% at 14 IDAT.

These results indicate that cases of transformed AAD-12 (v1) lines showed a high tolerance level for 2,4-D, triclopyr and fluoroxipir at multiples of commercial use rates or were lethal causing severe epinastic malformations for unprocessed tobacco under representative field conditions.

AAD-12 (v1) protection against high 2,4-D tariffs: Results show AAD-12 (v1) protection against high greenhouse 2,4-D DMA rates are presented in Table 19. T1 AAD -12 (V1) from a case of 3R: IS segregating plants when selected, with 560 g ai / h freedom using the same protocol as previously described. T1 AAD-1 seed (v3) was also planted to control transformed tobacco (see PCT / US2005 / 014737). Unprocessed KY160 was served as the sensitive control. Plants were sprayed using a lane-adjusted sprayer of 187 L / ha at 140, 560, 2240, 8960, and 35,840 g ae / ha 2,4-D DMA and nominal 3 and 14 DAT.

Both AAD-12 (v1) and AAD-1 (v3) tobaccos are effectively protected against 2,4-D lesions at doses up to 4 x commercial use rates. AAD-12 (v1), however, clearly demonstrated a marked advantage over AAD-1 (v3) by protecting at 64 x standard field rates.

Stacking AAD-12 for Increasing the Herbicide Spectrum: The AAD-12 (v1) (pDAS1580) and AAD-1 (v3) homozygotes (pDAB721) plants (see PCT / US2005 / 014737 for last) were reciprocally crossed and the F1 seed was collected. F1 progeny from two reciprocal crosses of each gene were stratified and treated. Four replicates of each crossover were treated with the same spraying regime as used for the other test with one of the following treatments: 70, 140, 280 g. ae / ha fluoroxipir (selective for the AAD-12 (v1) 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 tolerance of 2.4 -D). Homozygous T2 plants of each gene were also planted for use as controls. The plants were classified in 3 and 14 DAT. Spray results are shown in Table 20.

The results confirm that AAD-12 (V1) can be successfully stacked with AAD-1 (v3), thereby increasing herbicide spectra that can be applied to plants of interest (phenoxyacetic acids + phenoxypropionic acids. vs penoxyacetic acids + pyridyloxyacetic acids for AAD-1 and AAD-12, respectively). The complementary nature of herbicide cross-resistance patterns allows the practical use of these two genes as complementary and stackable selection field markers. In cultures where tolerance with a single gene may be marginal, one skilled in the art recognizes that tolerance can be increased by stacking a second inherited tolerance gene for the same. This can be done using the same gene with the same or different promoters, however, as in the present invention observed, the stacking and tracking of two complementary characteristics can be facilitated by cross-differential protection of phenoxypropionic acids [from AAD-1 ( v3) 1 or p-idylaxiacetic acids [AAD-12 (v1) 1.

Example 7 Soybean transformation Soybean improvement by gene transfer techniques has been performed for such characteristics as herbicide tolerance (Padgette et al, 1995), amino acid modification (Falco et al., 1995), and insect resistance (Parrott et al., 1994). The introduction of foreign traits into the crop species requires methods that will allow the routine production of transgenic lines using selection marker sequences containing simple insertions. Transgenes should be inherited as a single functional site in order to simplify breeding. Delivery of foreign genes in soybeans grown by microprojectile bombardment of zygotic axis embryos (McCabe et al., 1988) or somatic embryogenic cultures (Finer and McMullen, 1991), and Agrobacterium-mediated transformation of cotyledonary explants (Hinchee et al., 1988) or zygotic embryos (Chee et al., 1989) have been reported.

Transformants from Agrobacterium-mediated transformations tend to have simple low copy number insertions (Birch 1991). There are advantages and disadvantages associated with each of the three target tissues investigated for gene transfer in soybean, the zygotic embryonic axis (Chee et al., 1989; McCabe et al., 1988), cotyledon embryogenic cultures (Hinchee et al, 1988). .) And somatic (Finer and McMullen, 1991). The latter has been extensively investigated as a target tissue for direct gene transfer. Embryogenic cultures tend to be quite prolific and can be maintained for a prolonged period. However, the chromosomal aberrations and sterility aberrations of primary transformants have been associated with the age of embryogenic suspensions (Singh et al, 1998) and, consequently, continuous initiation of new cultures seems to be necessary for systems. soy processing using this fabric. This system needs a high level of 2,4-D, 40 mg / L concentration to initiate embryogenic callus and this poses a fundamental problem in using the AAD-12 (v1) and since the site of the transformed gene could not be developed with 2,4-D in the medium. Thus, the meristem base transformation is ideal for the development of a 2,4-D resistant plant using AAD-12 (v1).

Portal Binary Construct Cloning: The AAD-12 (v1) coding sequence was cloned into five different Gateway Donor vectors containing the different plant promoters. The resulting AAD-12 plant expression cassettes (v1) were post-ligated into a binary vector Destination portal by the Clonase LR reaction (Invitrogen Corporation, Carlsbad, California, Cat # 11791-019).

An Ncol-Sacl fragment containing the AAD-12 (v1) coding sequence was digested from DASPIC012 and ligated into corresponding Ncol-Saci restriction sites within the following Portal Donor vectors: pDAB3912 (attL1 // CsVMV promoter // AtuORF23 3 'UTR // attl_2); pDAB3916 (attL1 // Promoter Ability // AtuORF23 3'UTR // attL2); pDAB4458 (attL1 // A-tUbi3 promoter // AtuORF23 3'UTR // attL2); pDAB4459 (attL1 // ZmUbil promoter // AtuORF23 3'UTR // attL2) and pDAB4460 (attL1 // AtAct2 promoter // A-tuORF23 3'UTR // attL2). The resulting constructs containing the following plant expression cassettes were designated: pDAB4463 (attL1 // CsVMV promoter // AAD-12 (V1) // AtuORF23 3'UTR // attL2); pDAB4467 (atTL1 // Promoter Activation // AAD-12 (v1) // AtuORF23 3'UTR // attL2); pDAB4471 (attL1 // AtUbi3 promoter // AAD-12 (v1) // AtuORF23 3'UTR // attL2); pDAB4475 (attL1 // ZmUbil promoter // AAD -12 (v1) // AtuORF23 3'UTR // attL2) and pDAB4479 (attL1 // AtAct2 promoter // AAD-12 (v1) // AtuORF23 3'UTR / / attL2). These constructs were confirmed by restriction enzyme digestion and sequencing.

Plant expression cassettes were recombined into destinopDAB4484 Binary Portal Vector (RB7 MARv3 // attr1-ccdB-chloramphenicol resistance attR2 // CsVMV promoter // PATv6 // AtuORFI 3'UTR) by Gateway LR Clonase reaction. Gateway uses site-specific recombination lambda phage-based technology rather than restriction endonuclease and ligase to insert a gene of interest into an expression vector. Invitrogen Corporation, Gateway Technology: Universal Technology to Clone DNA Sequences for Functional Analysis and Expression in Various Systems, Technical Manual, Catalog ri ° 12535-019 and 12535-027, Gateway Version Technology, September 22, 2003, n 0 25 to 022. The DNA recombination sequences (attL and attR1) and the LR Clonase enzyme mixture allows any DNA fragment flanked by a recombination site to be transferred to any vector containing a corresponding site. The attL1 location of the donor vector corresponds to attrl of the binary vector. Similarly, the donor vector's attL2 location corresponds to attR2 of the binary vector. Using gateway technology the plant expression cassette (from the donor vector) that 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 identified as: pDAB4464 (RB7 MARv3 // CsVMV promoter // AAD-12 (v1) // Atu ORF23 3'UTR // CsVMV promoter // PATv6 AtuORFI 3'UTR ); pDAB4468 (RB7 MARv3 // Promoter Actuator // AAD-12 (v1) // AtuORF23 3'UTR // CsVMV Promoter // PATv6 // AtuORFI 3'UTR); pDAB4472 (RB7 MARv3 // AtUbi3 promoter // AAD-12 (v1) // AtuORF23 3'UTR // CsVMV promoter // PATv6 // Atu- ORF1 3'UTR); pDAB4476 (RB7 MARv3 // ZmUbil promoter // AAD-12 (v1) // AtuORF23 3'UTR // CsVMV promoter // PATv6 AtuORFI 3'UTR) and pDAB4480 (RB7 MARv3 // AtAct2 promoter // AAD-12 (v1 ) // AtuORF23 3'UTR // CsVMV promoter // ΡΑΤνδ // AtuORFI 3'UTR). These constructs were confirmed by restriction enzyme digestion and sequencing. Transformation Method 1 - Agro-bacterium-Mediated Transformation: Early reports of target soybean meristatic cell transformation in the cotyledon node region (Hinchee et al, 1988.) And multiplication of footage from apical meristems (McCabe et al ., 1988.). In the A. tumefaciens-based cotiledonar node method, the preparation of the explants and composition of the meristema culture medium to stimulate auxiliary proliferation in the node (Hinchee et al, 1988). It is still unclear whether a true undifferentiated but totipotent callus culture is initiated by these treatments. Recovery of several clones from a single explant transformation case and frequent recovery of chimeric plants (Clemente et al, 2000, Olhoft et al, 2003) indicate a single cell origin, followed by multiplication of transgenic cell, either to produce the proliferation of transgenic meristem culture or a uniformly transformed session that undergoes more multiplication shot. The soybean filming multiplication method, initially based on microprojectile bombardment (McCabe et al, 1988) and, more recently, adapted for Agrobacterium-mediated transformation (Martinell et al, 2002), apparently does not submit to the same level or de-differentiation type as cotiledonar node method because the system relies on the successful identification of germline chimeras. Also, this is a 2.4-D based protocol, which would not be ideal for the 2.4-D selection system. Thus, the cotiledonar node method may be the method of choice for developing 2,4-D resistant soybean cultivars. AAD-12 tolerant phenotype transformation plant (v1). Maverick-derived explant seeds and the Agrobacterium-mediated node transformation protocol were used to produce transgenic AAD-12 (v1) plants. Preparation and Inoculation of Agrobacterium: Agrobacterium strain EHA101 (Hood et al 1986), carrying each of the five binary AOR vectors (Table 8) was used to initiate the transformation. Each binary vector contains the AAD-12 (v1) and a selectable plant gene cassette (PAT) gene within the T-DNA region. Plasmids were mobilized into the Agrobacterium EHA101 strain by electroporation. The selected colonies were then analyzed for gene integration before treatment with Agrobacterium, soybean explants. Maverick seeds were used in all transformation experiments and seeds were obtained from the University of Missouri, Columbia, Mo. Soybean Agrobacterium (Glycine max) -mediated transformation using the PAT gene as a selectable marker along with the herbicide glufosinate as a selective agent was performed. Seeds were germinated in basic medium B5 (Gamborg et al. 1968) solidified with 3 g / l Phytagel (Sigma-Aldrich, St. Louis, Mo). The selected shoots were then transferred to the rooting medium. The optimal selection regimen was the use of 8 mg / L glufosinate between the first and second stages of initiation in the medium shot and 3-4 mg / L during the shoot lengthening in the medium.

Before transferring the elongated shoots (3 to 5 cm) to rooting medium, the excised end of the internodes were dipped in 1 mg / l indole-3-butyric acid for 1-3 min to promote rooting (Khan et al 1994 .). The shoots were rooted in 25 x 100 mm glass culture tubes containing rooting medium and then transferred to the soil mix for seedling acclimation in Metro-mix 200 (Hummert International, Earth City, Missouri). ) in open Magenta boxes in Convirons. Glufosinate, the active ingredient in the herbicide Freedom (Bayer Crop Science), was used for selection during branch initiation and stretching. Rooted seedlings were acclimated in Magenta boxes opened for several weeks before they were selected and transferred to the greenhouse for later acclimation and establishment.

Assay of supposedly transformed seedlings, and established analyzes of TO plants in the greenhouse: The selected leaflet terminal leaflets of these seedlings were leaf painted with glufosinate 50 mg / L twice with a one week interval to observe the results for the screen for putative transformants. The seedlings were then screened transferred to the greenhouse and after acclimatization the leaves were stained with glufosinate again to confirm the tolerance status of these seedlings in GH and considered putative transformants. Plants that are transferred to the greenhouse can be tested for the presence of an active non-destructive PAT gene by painting a leaf section of the primary transformant TO, or progeny thereof, with a glufosinate solution. 0.05% v / v herbicide freedom, preferably 0.25-1.0% (v / v) = 500-2000 ppm glufosinate, Bayer Crop Science], Depending on the concentration used, damage assessment Glufosinate can be taken 1-7 days after treatment. Plants can also be tested for 2,4-D tolerance in a non-destructive manner by selectively applying a 2,4-D solution in water (0.25-1% v / v of 2,4-D D commercial dimethylamine salt formulation, preferably 0.5% v / v = 2280 ppm 2,4-D AE) for the newly expanding trifoliolate leaflet terminal, one or two, preferably two, knots below of the newest emerging trifolioate. This assay allows the evaluation of 2,4-D sensitive plants 6 hours to several days after application by an evaluation of the inversion leaf or rotation> 90 degrees from the plane of adjacent cusps. Plants tolerant to 2,4-D will not respond to 2,4-D. T0 plants will be allowed to self-fertilize in the greenhouse to give rise to T1 seeds. T1 plants (and as far as T0 are clones of the plant produced) will be sprayed with a range of herbicide doses to determine the level of protection provided by the herbicide AAD-12 (v1) and the PAT genes in soybean. transgenic. 2,4-D rates used in T0 plants will typically comprise one or two selective rates in the range 100-1120 g ea / ha using a trajectory sprayer as described above. T1 plants will be treated with a higher dose of herbicide ranging from 50-3200 g ea / ha of 2,4-D. Similarly, the TO and T1 plants can be evaluated for glufosinate resistance by post-emergence treatment with e 200-800 50-3200 g ea / ha of glufosinate, respectively. Glyphosate resistance (in plants transformed with constructs containing EPSPS) or another glyphosate tolerance gene may be evaluated in T1 generation by post-emergence glyphosate applications with a glyphosate 280-2240 g / ha dose range. Individual T0 plants were evaluated for the presence of the gene of interest coding region (AAD-12 (v1) or PAT v6) and copy number. Determination of AAD-12 (v1) inheritance will be done using T1 and T2 progeny segregation with respect to herbicide tolerance as described in the previous examples.

A subset of the initial transformants were evaluated at generation T0 according to the above methods. Any plant confirmed to have the AAD-12 (V1) coding region, regardless of the conduction gene promoter, did not respond to leaf painting 2.4-D, whereas wild-type Maverick soybean did. Only transformed PAT plants responded the same to wild-type plants for 2,4-D leaf painting applications. 2,4-D was applied to a subset of plants that were similar in size to wild-type control plants, either 560 or 1120 g and 2,4-D. All plants containing AAD-12 (v1) were clearly resistant to herbicide application against independent jungle-type soybeans. A slight lesion level (2 DAT) was observed by two AAD-12 (v1) plants, however, the lesion was temporary and no lesion was observed 7 DAT. Wild control plants were severely injured 7-14 DAT at 560 g ae / ha of 2,4-D and killed at 1120 g ae / ha.

These data are consistent with the fact that AAD-12 (v1) and may confer high tolerance (> 2 x field rates.) To a sensitive crop such as soy. The plants were then screened for molecular and biochemical analysis to confirm AAD12 (ν1) gene integration, ο copy number, and gene expression levels.

Molecular analyzes - Soybeans: DNA isolation tissue harvesting and quantification. Fresh tissue is placed in tubes and lyophilized at 4 ° C for 2 days. After the fabric is completely dried, a tungsten bead (Valenite) is placed in the tube and the samples are subjected to 1 minute dry milling using a Kelco ball mill. The standard DNeasy DNA isolation process is followed (Qiagen, DNeasy 69109).

An aliquot of the extracted DNA is then stained with Green Pico (Molecular Probes P7589) and read at the fluorometer (BioTek) with known standards for over concentration in ng / mL.

The polymerase chain reaction: A total of 100 ng of total DNA is used as a template. 20 mM of each primer is used with the Takara PCR Ex Taq Polymerase Kit (Mirus TAKRR001A). Initiators for AAD-12 (v1) UTP are (Front-ATAATGCCAG CCTGTTAAAC CCG) (SEQ

ID NO: 8) and (Re-CT CAAGCATA TGAATGACCT CG A) (SEQ ID NO: 9). The PCR reaction is performed on the GeneAmp 9700 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 the AAD-12 (v1) PCR coding region are (Forward-ATGGCTCATG CTGCCCTCAG CC) (SEQ ID NO: 10) and (Reverse-CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 11). The PCR reaction is performed on the GeneAmp 9700 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 EtBr stained 1% agarose gel electrophoresis. Southern blot analysis: Southern blot analysis is performed with the total DNA obtained from Qiagen's DNeasy kit. A total of 10 æg of genomic DNA is digested overnight to obtain integration data. After overnight digestion a ~ 100 ng aliquot is run on a 1% gel to ensure complete digestion. Following this warranty the samples are run on a large 0.85% overnight agarose gel 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 to 0.5 M Tris-HCl, 1.5 M NaCl pH 7.5 for 30 minutes. A gel apparatus containing 20 x SSC is then configured to obtain a nylon membrane gravity gel (Millipore INYC00010) overnight transfer. After overnight transfer, the membrane is then subjected to UV light by a crosslinking agent (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 template hybridization fragment is prepared using the above PCR coding region using the plasmid DNA. The product is run on a 1% agarose gel and removed and then gel extracted using the (28,706) Qiagen gel extraction procedure. The membrane is then subjected to a prehybridization at 60 ° C step for 1 hour in perfect Hyb buffer (Sigma H7033). The First It RMT dCTP labeling rxn (Stratagene 300.392) procedure is used to develop the p32 based probe (Perkin Elmer). The probe is cleaned using the Probe Quant: G50 Columns (Amersham 27-5335-01). Two million CPM counts are used to hybridize Southern blots at night. After overnight hybridization of the spots are then subjected to two 20 minute washes at 65 ° C in 0.1% SDS, 0.1 SSC. The spots are then exposed to a film overnight by incubating at -80 ° C.

Biochemical Analysis - Soy: Tissue Sampling and extracting AAD-12 (v1) protein from soybean leaves. About 50 to 100 mg of leaf tissue was sampled from N-2 leaves that were 2.4-D spotted leaf, but after a DAT. The N-terminus of sheet 2 was removed and cut into small single-punctured sheet pieces or discs (~ 0.5 cm in diameter) and were instantly frozen on dry ice. Protein analysis (ELISA and Western analysis) were completed accordingly.

Evaluation of T1 progeny: TO plants will be allowed to self-fertilize T1 families. Testing (segregation analysis) will be tested using glufosinate at 560 g ai / ha as the selection agent applied in the V1-V2 growth phase. Surviving plants will be further analyzed for 2,4-D tolerance in one or more growth stages from V2-V6. Seed will be produced through self-fertilization to allow broader herbicide testing on transgenic soybeans. AAD-12 (v1) and Maverick transgenic soybean plants were generated through the Agrobacterium-mediated transformation system. The TO plants obtained tolerated up to 2 x 2,4-D levels and field applications developed fertile seeds. The frequency of fertile transgenic soybean plants was up to 5.9%. The integration of (v1), AAD1-12 gene in the soybean genome was confirmed by Southern blot analysis. This analysis indicated that most transgenic plants contained a low copy number. Plants screened with AAD-12 (v1) tested positive for ELISA antibodies and the appropriate band in Western analysis. Transformation Method 2 - Beam Aerosol Mediated Transformation of Soybean Embryogenic Callus Tissue: Culture of soybean embryogenic callus tissue and subsequent irradiation can be performed as described in US Patent No. 6,809,232 (Held et al). transformers using constructs in the present invention provided. Transformation Method 3 - Biolistic Soybean Bombardment: This can be accomplished using mature seed derived embryonic axes meristem (McCabe et al. (1988). Following established methods of biobalistic bombardment, recovery of transformed soybean plants can be expected. [Transformation Method 4 - whisker-mediated transformation: Mustache preparation and Swiss transformation can be performed according to methods previously described by Terakawa et al (2005)).

Following established methods of biobalistic bombardment, recovery of transformed soybean plants can be expected.

Maverick seeds were surface sterilized with 70% ethanol for 1 minute followed by soaking in 1% sodium hypochlorite solution for 20 minutes and then washed three times in sterile distilled water. The seeds were soaked in distilled water for 18-20 hours. Embryo axes were excised from seeds, and apical microsystems were exposed by removal of primary leaves. Embryonic axes were placed in the bombardment medium [BM: MS (Murashige and Skoog, 1962) basal salt medium, 3% sucrose and 0.8% phytagel Sigma, pH 5.7], with the apical region directed upwards in 5 cm high culture dishes containing 12 ml Culture medium. Transformation Method 5 - Particular callus-mediated bombardment transformation can be optimized according to previous methods (Khalafalla et al, 2005; El-Shemy et al., 2004, 2006.).

Example 8 AAD-12 (v1) on Cotton Cotton Transformation Protocol: Cotton seeds (Co310 genotype) are surface sterilized in 95% ethanol for 1 minute, rinsed, sterilized with 50% commercial bleach for 20 minutes. then washed 3 times with sterile distilled water before being germinated on G-media (Table 21) on Magenta GA-7 vessels and maintained under high light intensity of 40-60 pE / m2 with the photoperiod set with 16 hours of light and 8 hours of darkness at 28 ° C

Square cotyledon segments (~ 5 mm) are isolated 7-10 days from germination in liquid liquid medium M (Table 21) in Petri dishes (Nunc, item # 0875728). Cut segments are treated with an Agrobacterium solution (for 30 minutes), then transferred to medium-H semi-solid (Table 21) and cocultured for 2-3 days. After co-cultivation, the segments are transferred to MG media (Table 21). Carbenicillin is the antibiotic used to kill Agrobacterium and ammonium glufosinate is the screening agent that allows only those cells containing the transferred gene to grow.

Agrobacterium preparation: inoculate 35 ml of Y medium (Table 21) (containing streptomycin (100 mg / ml stock) and erythromycin (100 mg / ml stock)), with a bacterial loop growing overnight in the dark at 28 ° C, stirring at 150 rpm. The next day, pour the Agrobacterium solution into a sterile Oakridge tube (Nalge Nunc-, 3139-0050), and centrifuge into Beckman J2-21 at 8,000 rpm for 5 minutes.

Decant the supernatant and resuspend the pellet in 25 ml of liquid M (Table 21) and vortex. Place an aliquot into a glass culture tube (Fisher, 14-961-27) for Klett reading (Klett-Summerson, model 800-3). The resuspension is diluted using M liquid media to a Klett reading of 108 meters colony forming units per ml with a total volume of up to 40 ml.

V

After three weeks, corns from cotyledon segments are isolated and transferred to fresh MG media. The callus is transferred for an additional 3 weeks in Mg medium. In a side-by-side comparison, MG media can be supplemented with dichlorprop (added to the media at a concentration of 0.01 and 0.05 mg / L) to complete 2,4-D degradation once It is not a dichlorprope substrate for AAD-12 enzyme, however dichlorprope is more active than cotton in 2,4-D. In a separate comparison, the segments that were plated on media containing MG no growth regulator relative to standard MG media showed reduced callus but still callus growth.

Callus is then transferred to mean CG (Table 21), and again transferred to fresh selection medium after three weeks. After another three weeks callus tissue is transferred to D media (Table 21) without growth regulators for callus induction. After 4-8 weeks on this support, embryogenic callus is formed, and can be distinguished from non-embryogenic callus by its yellowish white color and granular cells.

Embryos begin to regenerate soon after and are distinct in green color.

Cotton may take some time to regenerate and form embryos, one of the ways to speed up this process is to stress the tissue. Desiccation is a common way to do this through changes in tissue and plate microenvironment, using fewer culture media and / or adopting various plate cabinet modes (paraphilm tapping).

Well-developed Larger embryos are isolated and transferred to NS medium (Table 21) for embryo development. After 3 weeks (or when the embryos have developed), the germinated embryos are transferred to fresh means for shoot and root development. After 4-8 weeks, well-developed plants are transferred to soil and grown to maturity. After two months, the plant has grown to a point that can be sprayed to determine if it has resistance to 2,4-D.

Cell Transformation: Several experiments were started in which cotyledonary segments were treated with pDAB724 containing Agrobacterium. Over 2000 of the resulting segments were treated with various auxin options for pDAB724 cotton callus proliferation, either: 0.1 or 0.5 mg / L R-dichlorprope, 2.4-D concentration standard and none. auxin treatment. The callus was selected with glufosinate-ammonium due to the inclusion of the PAT gene in the construct. The callus line analysis in the form of PCR and Invader will be used to determine if, and to ensure that the gene was present in the callus formation phase, then which are embryogenic callus lines will be sent for Western analysis. Embryogenic cotton callus was emphasized using drying techniques to improve the quality and quantity of the recovered tissue. Almost 200 callus cases were selected for intact PTU and expression using western analysis for the AAD-12 gene (v1). Plant regeneration: AAD-12 (v1) and cotton lines, which have produced plants in accordance with the above protocol, will be sent to the greenhouse. To demonstrate the (v1) and the AAD-12 gene provides resistance to 2,4-D in the case of cotton, both the AAD-12 (v1) of wild type cotton plants and cotton plants will be sprayed. - with a trajectory sprayer deliver 560 g ea / ha 2,4-D in a syrup volume of 187 l / ha. Plants will be evaluated on days 3 and 14 days after treatment. Surviving plants at the selective rate of 2,4-D will be self pollinated to create T1 seed or outcrossed with an elite cotton line for F1 seed production. Subsequent produced seed will be planted and evaluated for herbicide resistance as described above. AAD-12 cases (v1) and can be combined with other desired HT or IR Trants.

Example 9 Agrobacterium Transformation from Other Cultures Depending on the description of the subject, additional crops may be transformed in accordance with the present invention using techniques which are known in the art. For Agro-bacterium-mediated transformation of rye, see, for example, Popelka and Altpeter (2003). For Agrobacterium-mediated transformation of soybean, see, for example, Hinchee et al., 1988. For Agrobacterium-Initiated transformation, of Jfeorgo, see, for example, Zhao et al. From 2000. For Agrobacterium-mediated transformation of barley, see, for example, Tingay et al., 1997.

For Agrobacterium-mediated transformation of wheat, see, for example, Cheng et al. De 1997. For Agrobacterium-mediated transformation of rice, see, for example, Hiei et al.

The Latin names for these and other plants are given below.

It should be clear that these and other (non-Agrobacterium) transformation techniques can be used to transform AAD-12 (v1), for example, these and other plants, including but not limited to maize (Zea mays), wheat (Triticum spp.), rice (Oriza spp. and zizania spp.), barley (Hordeum spp.), cotton (Abroma augusta and Gossypium spp.), soybean (Giycine max), sugar and beet (Beta spp. ), sugar cane (Arenga pinnata), tomato (Lycopersicon esculentum and other spp., Physalis ixocarpa, Solanum incanum and other spp., and Cyphomandra betacea), potato (Solanum tubersoum), sweet potato (Ipomoea betatas), rye (Secale spp.), Peppers (Capsicum annuum, sinense and frutescens), Lettuce (Lactuca sativa, perennis, and pulchella), Cabbage (Brassica spp), Celery (Apium graveolens), Eggplant (Solanum melongena), Peanut (Arachis hypogea) , sorghum (Sorghum all species), Alfalfa (Medicago sativua), carrot (Daucus carota), beans (Phaseolus spp. and other genera), oats (Aven sativa and strigosa), peas (Pisum, Vigna and tetragonolobus spp.), sunflower (Helianthus annuus), squash (Cucurbita spp.), cucumber (Cucumis sativa), tobacco (Nicotiana spp.), Arabidopsis (Arabidopsis thaliana) , Lawn (Lolium, Agrostis, Poa, Cynadon and other genera), Clover (Tifolium), Vetch (Vicia). Such plants, with gene AAD-12 (v1), for example, are included in the present invention. AAD-12 (v1) has the potential to increase the applicability of major auxin herbicides for use in the season in many deciduous and green wood cultivation systems. Triclopyr, 2,4-D, and / or fluoroxipir resistant wood species would increase the flexibility of their use during the tops of these herbicides without injury problems. These species include, but are not limited to: alder (Alnus spp.), Ashes (Fraxinus spp.), Poplar and poplar species (Populus dp.), Beech (Fagus spp.), Birch | l3etula spp.), Cherry (Prunus spp.), Eucalyptus (Eucalyptus spp.), Walnut (Caria spp.), Maple (Acer spp.), Oak (Quercus spp) and Pine (Pinus spp). The use of auxin resistance for selective weed control in ornamental plants and fruit is also within the scope of the present invention. Examples may include, but are not limited to, Rose (Rosa spp.), Burning Bush (Euonymus spp.), Petunia (Petunia spp), Begonia (Begonia spp.), Rhododendron (Rhododendron spp), Sour Apple or Apple ( Malus spp.), Pear (Pirus spp.), Peach (Prunus spp) and marigolds (Tagetes spp.).

Example 10 Other evidence of surprising results: AAD-12 vs AAD-2 Initial AAD-2 (v1) cloning: Another gene was identified in the NCBI database (see ncbi.nlm.nih.gov; adhesion # AP005940) as an amino acid identity homologue only 44% for TFDA. This gene is in the present invention referred to as AAD-2 (v1) and for consistency. The percent identity was determined by the first translation of AAD-2 and TFDA DNA sequences (SEQ ID NO: 12 and PCT / US2005 / 014737 GenBank M16730 access, respectively) to proteins (SEQ ID NO : 13 PCT / US2005 / 014.737 and GenBank M16730 access, respectively), then using ClusterW in the VetorNTI software package to perform multiple sequence alignment. The Bradirhizobium japonicum strain containing the AAD-2 (v1) gene was obtained from Northern Regional Research Laboratory (NRRL, strain # B4450). The lyophilized strain was taken up NRRL according to the protocol and stored at -80 ° C in 20% glycerol for internal use, such as Dow bacterial strain DB 663. From this freezer stock, a tryptic soy agar plate was then struck. with a cell loop for isolation, and incubated at 28 ° C for 3 days. A single colony was used to inoculate 100 ml of Triptic Soy Broth into a ml of 500 ml tri-vortex flask, which was incubated overnight at 28 ° C on a shaker stage at 150 rpm. Thereafter, total DNA was isolated with the iQiagen DNeasy Gram-negative Kit protocol (Qiagen cat. # 69504). The following primers were used to amplify the target gene from genomic DNA, forward: 5 'ACT AGT AAC AAA GAA ATA CCA GAT GGA CGA TGA T 3' {(brjap 5 * (Spel) PCT SEQ ID NO: 14 /US2005/014.737 (Spe I restriction site and added ribosome binding site (RBS))] and Reverse: 5 AGC TTC TCG TAT CCG GCC CAC TCC TGC TGC TGC 3 '[(br jap 3 1 (Xhol) to SEQ] PCT ID NO: 15 (US2005 / 014737 (Xhol site added)).

Fifty microliter reactions were constituted as follows: 25 mL buffer safety failure, ea. 1 µl primer (50 ng / mL), 1 µg gDNA (200 ng / mL), H.sub.20 21 µl, 1 µl Taq polymerase (2.5 units / µl). Three faults of Buffer B, and C-were used in three distinct reactions. The PCR was then performed under the following conditions: 95 ° C. 3.0 minutes heat denaturing cycle; 95 ° C 1.0 minutes, 50 ° C 1.0 minutes, 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 ~ 1 kb of the resulting PCR product was cloned into pCR 2.1 (Invitrogen cat. # K4550-40), following the included protocol, with chemically competent E. coli TOP10F 'as the host strain for verification. nucleotide sequence.

Ten of the resulting white colonies were collected in 3 µl Luria Broth + 1000 µg / ml ampicillin (amp LB) and grown overnight at 37 ° C with shaking. Plasmids were purified from each culture using NucleoSpin Plus Plasmid Miniprep Kit (BD Biosciences cat. # K3063-2) and following the included protocol. Restriction digestion of the isolated DNA was completed to confirm the presence of the PCR product in the pCR2.1 vector. Plasmid DNA was digested with the restriction enzyme EcoRI (New England Biolabs, cat. # R0101S). Sequencing was performed with Beckman CEQ Quick Start Kit (Beckman Coulter cat # 608120). Using front M13 [5 'GTA AAA CG A CGG CCA G 3'] (SEQ ID NO: 6) and Reverse [5 'CAG GAA ACA GCT ATG CA 3'] (SEQ ID NO: 7), initiators as manufacturers instructions. This gene sequence and its corresponding protein were given a new general designation AAD-jh (v1) for internal consistency.

Conclusion of the AAD-2 Binary Vector (v1): The AAD-2 (v1) gene was PCR amplified from pDAB3202. During PCR reaction changes were made within the primers to introduce the Afll11 and Saci restriction sites into the primer 5 'and 3' primer, respectively.

See PCT / US2005 / 014737. The Primers "Bradi Ncol" [5 ΎΑΤ ACC ACA TGT CGA TCG CCA CCA TCC GGC AGC TT 3 '] (SEQ ID NO: 14) and "Bradi Saci" [5' GAG CTC CTA TC A CTC CGC CGC CTG CTG CTG CTG CAC 3 '] (SEQ ID NO: 15) were used to amplify a DNA fragment using the intrinsic safety PCR system (Epiceiitre). The PCR product was cloned into the pCR2.1 TOPO TA cloning vector (Invitrogen) and the sequence verified with M13 and M13 Advanced Reverse Primers using the Beckman Coulter "Die Terminator Cycle Sequencing Kit with Quick Start" sequencing reagents. Data string has identified a clone with the correct sequence (pDAB716). The AAD-2 (v1) and AfllII / Saci gene fragment was then cloned into Ncol / Saci pDAB726 vector. The resulting construct (pDAB717); Promoter Active: NT OSM 5'UTR: AAD-2 (v1): NT OSM3'UTR: ORF1 polyA 3'UTR was verified with digest restriction (with Ncol / Sac). This construct was cloned into binary pDAB3038 as a Notl-Notl DNA fragment. The resulting construct (pDAB767); ATUbilO promoter: NT OSM5'UTR: AAD-2 (v1): NT OSM 3'UTR: ORF1 polyA 3'UTR: CsVMV promoter: PAT: ORF25 / 26 3'UTR was digested restriction (with Nod, EcoRI, HindIII Ncol, Pvull, and Sall) to verify the correct orientation. The complete construct (pDAB767) was then used for transformation into Agrobacterium.

Evaluation of Transformed Arabidopsis: Recently harvested T1 seeds transformed with an optimized native gene plant AAD-12 (v1) and AAD-2 (v1) were planted and selected for glufosinate herbicide resistance as the plants described above were then , divided into various rates of 2,4-D (50-3200 g ea / ha). Herbicide applications were applied by a sprayer labeled at a volume of 187 L / ha in spray. The 2,4-D used was the commercial formulation of dimethylamine salt (456 g w / l, Nufarm, St Joseph, tVlissouri) mixed in 200 mM ide Tris buffer (pH 9.0) or 200 mM HEPES buffer (pH 7.5). AAD-12 (v1) and AAD-2 (v1) provided detectable 2,4-D resistance with both transformed and untransformed control lines, however individual constructs were highly variable in their ability to transmit resistance. of 2,4-D to individual Arabidopsis T1 plants. Surprisingly, the AAD-2 (v1) and AAD-2 (v2) transformants were much less resistant to 2,4-D than the AAD-12 (v1) gene, both from a highly tolerant plant frequency. , as well as the global average lesion. Without plants transformed with AAD-2 (v1), 200 g ea / ha of relatively uninjured 2,4-D survived (<20% visual toxicity), and the overall population loss was about 83% (see PCT / US2005 / 014737). Conversely, AAD-12 (v1) had an average population lesion of about 6% when treated with 3200 g ea / ha of 2,4-D. Tolerance slightly improved from AAD-2 (v2) optimized plants versus the native gene, however, comparison of the two optimized AAD-12-2 and AAD gene plants indicates a significant advantage for AAD-12 (v1). in the plant.

These results are unexpected as the in vitro comparison of AAD-2 (v1) and AAD-12 (v2) (see PCT / US2005 / 014737) indicated that both were highly effective in the degradation of 2,4-D and both. shared a S-type specificity with respect to chiral aryloxyalkanoate substrates. AAD-2 (v1) and is expressed in individual T1 plants at various levels, however, little protection from the 2,4-D lesion is offered by this expressed protein. No significant differences were evident in protein expression level (in plant) for native plant and AAD-2 optimized gene (see PCT / US2005 / 014737). These data corroborate previous findings that make the functional expression of AAD-12 (v1) in the plant and cause resistance to the unexpected 2,4-D herbicide and pyridyloxyacetate herbicides.

Example 11 Use of phenoxy auxin herbicides in soybean, cotton and other Dicot-transformed crops with AAD-12 (v1) i AAD-12 (v1) alone may allow the use of phenoxy auxin herbicides (eg 2 , 4-D and MCPA) and pyridyloxy auxins (triclopyr and fluoroxypyr) for the control of a broad spectrum of broadleaf weeds in crops normally directly sensitive to 2,4-D. Applying 2,4-D at 280-2240 g ae / ha would control most broadleaf weed species present in agronomic environments. Most typically, 560-1120 g ea / ha is used. For triclopyr application rates typically range from 70-1120 g ae / ha, more typically 140-420 g ae / ha.

For fluoroxypyr, application rates typically ranging 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 component and short-term herbicidal potential of residual weeds provided by higher rates of 2,4-D, triclopyr and fluoroxipir when used at higher rates, while that a non-residual herbicide such as glyphosate would not provide weed control later can germinate. This tool also provides a mechanism for combining herbicide modes of action with HTC's convenience such as integrated herbicide resistance and weed management strategy.

Another advantage of this tool provides is the ability of broad-spectrum broadleaf weed control herbicide mixing tanks (eg 2,4-D, triclopyr and fluoroxipir) to control weeds with herbicides. used waste. These herbicides are typically applied before or during planting, but are often less effective in emerging established weeds that may exist in the field prior to planting. By extending the usefulness of these aryloxil auxin herbicides to include pre-plant, pre-emergence or pre-plant applications, the flexibility of residual weed control programs increases. One of ordinary skill in the art would recognize the residual herbicide program will differ based on the crop of interest, but typical programs would include chloracetmide family herbicides and dinitroaniline herbicides, but also including herbicides such as clomazone, sulfentrazone, and a variety of inhibiting PPO inhibitor herbicidal ALS, and HPPD inhibitor.

Other benefits may include the tolerance to 2,4-D, triclopyr or fluoroxipyr required prior to planting following application of aryloxyacetic acid-assisted herbicides (see previous example), and less contamination damage to dicotyledonous crops from ponds. with incomplete cleaning expansion contained 2,4-D, triclopyr or fluoroxipir. Dicamba (and many other herbicides) can also be used for subsequent control of volunteer transformed AAD-12 (v1) in dicotyledonous crops.

Those skilled in the art will also recognize that the above example can be applied to any crop of 2,4-D-sensitive (or other aryloxy auxin herbicide) that must be protected by the AAD-12 (V1) gene if stably. transformed. One person skilled in the weed control technique now recognizes that the use of a variety of commercial phenine or pyridyloxy auxin herbicides alone or in combination with a herbicide is activated by the transformation AAD-12 (v1). Specific rates of other representative herbicides of these chemicals may be determined by the herbicide labels compiled in the (Crop Protection Reference) book CPR or similar compilation or any commercial or academic crop protection references, such as the Agriliance Crop Protection Guide ( 2005). Each alternative herbicide activated for use on HTCs by AAD-12 (v1), whether used alone, mixing tank, or sequentially, is considered within the scope of the present invention.

Example 12 Use In Herbicide Cultures Phenoxy auxin and pyridyloxy auxin in AAD-12 (v1) Only in maize, rice and other Transformed Monocot species Similarly, the transformation of grass species (such as, but not limited to, maize, rice, wheat, barley, grass or herbs and pasture) with AAD-12 (v1) would allow the use of highly effective phenoxy and pyridyloxy auxins in crops where normally the selectivity is not unclear. Most natural grassy species have a tolerance to herbicides such as auxin phenoxy auxins (eg 2,4-D.). However, a relatively low level of crop selectivity has resulted in decreased utility in these crops due to a reduced window of application time or risk of unacceptable injury. AAD-12 (v1) transformed monocotyledonous plants would therefore allow the use of a combination of similar treatments described for di-cotyledonous crops, such as the application of 2,4-D, 280-2240 g ea / ha for the control of broadleaf weeds most species. More typically, 560-1120 g ea / ha is used. For triclopyr, application rates typically range from 70-1120 g ae / ha, more typically 140-420 g ae / ha. For fluoroxypyr, application rates typically ranging from 35-560 g ae / ha, more typically 70-280 ae / ha.

An advantage of this additional tool is the extremely low cost of the leafy herbicide component and short-term potential for residual weed control provided by higher rates of 2,4-D, triclopyr, or fluoroxipir. In contrast, a non-residual herbicide such as glyphosate would not provide control of later germinating weeds. This tool also provides a mechanism for rotating herbicide modes of action with HTC's convenience as an integrated strategy, herbicides and weed resistance management change into a glyphosate tolerant HTC / AAD-12 combination strategy. (v1), whether one rotates the plant species or not.

Another advantage of this tool provides the broad-spectrum weed control herbicide mixing tank capability (eg 2,4-D, triclopyr and fluoroxipir) weed control with used residual herbicides. These herbicides are typically applied before or during planting, but are often less effective on emerged, established weeds that may exist in the field prior to planting. By extending the utility of these auxin aryloxy herbicides to include pre-plant, pre-emergence or pre-plant applications, the flexibility of residual weed control programs increases. One of ordinary skill in the art would recognize the residual herbicide program will differ based on the crop of interest, but typical programs would include chloracetmide family herbicides and dinitroaniline herbicides, but also including herbicides such as clomazone, sulfentrazone, and a variety of ALS inhibiting PPO inhibitor herbicides, and HPPD inhibitor. Increasing the tolerance of maize, rice and other monocotyledonous to phenoxy or pyridyloxy auxins should enable the use of these herbicides in unrestricted growth phase cultivation or the potential for sloping crop, unfurling phenomena such as "rag tail" sloping crop. - da, fragility-induced growth regulator on corn stalk or deformed support roots. Each alternative herbicide activated for use on HTCs by AAD-12 (v1), whether used alone, mixing tank, or sequentially, is considered to be within the scope of the present invention.

Example 13 AAD-12 (V1) in rice Description: The culture media used were adjusted to pH 5.8 with 1 M KOH and solidified with 2.5 g / l Phytagel (Sigma). Embryogenic calli were grown in 100 x 20 mm Petri dishes containing 40 ml semisolid medium. Rice seedlings were grown in 50 ml medium in magenta boxes. Cell suspensions were kept in 125 ml conical flasks containing 35 ml of spinning medium at 125 rpm. Induction and maintenance of embryogenic cultures occurred in the dark at 25-26 ° C, and plant regeneration and whole plant culture occurred within a 16 hour photoperiod (Zhang et al, 1996). The induction and maintenance of embryogenic callus occurred in basal NB medium as described previously (Li et al. 1,993), but adapted to contain 500 mg / L glutamine. Suspension cultures were started and maintained in SZ liquid medium (Zhang et al. 1998) with the inclusion of 30 g / l sucrose instead of maltose. Osmotic medium (NBO) consisted of an NB medium with the addition of 0.256 M of each fnanitol and sorbitol. Hygromycin-B-resistant callus was selected with NB medium supplemented with 50 mg / L hygromycin B for 3-4 weeks. Pre-regeneration was performed in medium (PRH50) consisting of NB medium without 2,4-dichlorophenoxyacetic acid (2,4-D), but with the addition of 2 mg / L 6-benzylaminopurine (BAP), 1 mg / L L

α naphthalenoacetic acid (NAA) at 5 mg / L abscisic acid (ABA) and 50 mg / L hygromycin B for 1 week. Seedling regeneration followed by culture in regeneration medium (RNH50) comprising NB medium without 2,4-D, supplemented with 3 mg / l BAP, 0,5 mg / l NAA, and 50 mg / l hygromycin B until regenerated shoots. The buds were transferred to rooting medium with half the concentration of basal Murashige and Skoog salts and Gamborg vitamins B5, supplemented with 1% saccharide and 50 mg / L hygromycin B (1 / 2MSH50).

Culture tissue development: the desiccated mature seeds of Oriza sativa L. cv japonica. Taipei 309 were sterilized as described in Zhang et al. 1996. Embryonic tissues were induced by sterile cultivation of mature rice seeds in NB medium in the dark. The primary callus, approximately 1 mm in diameter, was removed from the scutellum and used to initiate the suspension of SZ cells in liquid medium. The suspensions were then maintained as described in Zhang 1995. Suspension-derived embryonic tissues were removed from the liquid culture 3-5 days after the previous subculture and placed in osmotic NBO medium to form a circle about 2.5 cm in diameter. in a Petri dish and grown for 4 h before bombardment.

Sixteen to 20 hours after bombardment, tissues were transferred to NBO medium NBH50 hygromycin B selection medium, ensuring that the surface was bombarded upwards and incubated in the dark for 14-17 days. Freshly formed callus was then separated from the bombed original explants and placed in the vicinity of the same medium. After an additional period of 8-12 days, relatively compact, opaque callus was visually identified, and transferred to the pre-PRH50 regeneration medium for 7 days in the dark. Callus growth, which became more compact and opaque, was then subcultured in NH 4 O regeneration medium over a period of 14-21 days under a 16 hour photoperiod.

Regenerated shoots were transferred to magenta boxes containing 1/2 medium MSH50. Several plants regenerated from a single explant are considered siblings and were treated as an independent plant line. One plant was classified as positive for the hph gene that produced thick, white roots and grew vigorously in 1/2 M-SH50 medium. Once the seedlings reached the top of the Magenta boxes, they were transferred to the soil in a 6 cm pot at 100% humidity for one week, then moved to the growth chamber with a period of 10%. light for 14 hours at 30 ° C and in the dark at 21 ° C for 2-3 weeks before transplanting in 13 cm pots in the greenhouse. The seeds were collected and dried at 37 ° C for one week prior to storage.

Microprojectile bombardment: All bombardments were performed with the PDS-1000 / He Biolistic ™ system (Bio-Rad, Laboratories, Inc.). Three milligrams of 1.0 micron diameter gold particles were washed once with 100% ethanol, twice with sterile distilled water and resuspended in 50 µl water in a siliconized Eppendorf tube. Five micrograms of plasmid DNA representing a molar ratio of 1:06 pDOW3303 (Hpt-containing vector) to pDAB4101 (AAD-12 (v1) + AHAS), 20 µl spermidine (0.1 M) and 50 µl of Calcium (2.5 M) was added to the gold suspension. The mixture was incubated at room temperature for 10 min, pelleted at 10,000 rpm for 10 s, resuspended in cold 100% ethanol and 8-9 µl was distributed into each macro vehicle. Tissue samples were bombarded at 1100 psi and 27 Hg in vacuum as described by Zhang et al. (1996). Post-emergence herbicide tolerance of AAD-12 (v1) Transformed T0 to Rice; 3-5 leaf rice seedlings were sprayed with a lethal dose of 0.16% (v / v) Activity solution (to confirm the presence of the AHAS gene) containing 1% Sunit II (v / v ) and 1.25% UAN (v / v) using a trajectory sprayer calibrated to 187 L / ha. Sensitivity or resistance evaluation was performed at 36 days after treatment (DAT). Ten of the 33 cases sent for greenhouse effect were robustly tolerant to persecution, others suffered different levels of herbicidal injury. Plants were harvested and molecular characterization was performed which identified seven of these 10 cases as containing both AAD-12 (v1) and UTP AHAS throughout the coding region. The heritability of AAD-12 (v1) in T1 Rice: A 100 plant progeny test was performed on five T1 lines of the AAD-12 (v1) lines that contained both the AAD-12 (v1) OCT and the region. AHAS coder.

The seeds were planted as described above and sprayed with 140 g ea / ha of imázetapir using a trajectory sprayer as described above. After 14 DAT, resistant and sensitive plants were counted. Two of the five tested lines segregated as a single, Mendelian dominant trait (3R: 1S), as determined by Chi square analysis. AAD-12 confirmed with the selectable AHAS marker as determined by 2,4-D the following tolerance test.

High Tolerance 2.4-D Check on T1 Rice: The following T1 AAD-12 (v1) single site segregating lines were planted in 3 inch pots containing Metro Mix media: pDAB4101 (20) 003 and pDAB4101 (27) 002. In phase 2-3 sheets were sprayed with 140 g ae / ha imazethapyr. Nulls were eliminated and subjects were sprayed at the V3 V4 stage in the set spray range at 187 l / ha, 1120, 2240 or 4480 g ea / ha of 2,4-D DMA (2 x, x 4, and 8 x typical commercial use the rates in a respective manner). The plants were classified at 7 and 14 DAT and compared to unprocessed commercial rice cultivars, 'La mont' as negative control plants.

Injury data (Table 22) show that AAD-12 (v1) -transformed lines are more tolerant to the high rates of 2,4-D DMA than untransformed controls. The pDAB4101 (20) 003 line was more tolerant to high levels of 2,4-D than the pDAB4101 (27) 002 line. Data also show that the tolerance of 2,4-D is stable for at least two generations. Tissue collection, DNA isolation and quantitation: Fresh tissue was placed in tubes and lyophilized at 4 ° C for 2 days. After the fabric was completely dried, a tungsten bead (Valenite) was placed in the tube and the samples were subjected to 1 minute dry milling using a Kelco ball mill. The standard DNe- asy DNA isolation process was followed (Qiagen, Dneasy 69109). An aliquot of the extracted DNA was then stained with green peaks (Molecular Probes P7589) and digitized on the florometer (BioTek) to known standards to obtain the concentration in ng / mL.

AAD-12 Expression (v1): All 33 T0 transgenic and one non-transgenic control rice lines were analyzed for AAD-12 expression using ELISA blot. AAD-12 was detected in the 20 line clones, but not in the 309 Taipai control plant line. Twelve of the 20 lines that had some of the imazetapyr tolerant clones were expressing AAD-12 protein, were AAD-12 PTU PCR positive, and AHAS positive region coding. Expression levels ranged from 2.3 to 1.092.4 ppm total soluble protein.

Plant tolerance field pDAB4101 for 2,4-D and Triclopir Herbicides: A field level tolerance study was conducted with AAD-12 (v1) case pDAB4101 [20] and wild-type rice (Clearfold 131). ) in Wayside, Mississippi (the non-transgenic imidazoline-resistant variety). The experimental design was in randomized blocks with a single replication. Herbicidal treatments were 2 x rates of 2,4-D (dimethylamine salt) at 2,240 g ea / ha and triclopyr at 560 g ea / ha plus one untreated control. Within each herbicide treatment, two T1 generation pDAB4101 rows [20] and two rows of Clearfield rice were planted using a small 8-inch plot plot. PDAB4101 [20] rice contained the AHAS gene as a selectable marker for the gene AAD-12 (V1). Imazetapyr was applied at the leaf stage as a screening agent to remove any AAD-12 (v1) and null plants from the plots. Herbicide treatments were applied when the rice reached stage 2 of the leaf using costal spray compressed air delivering 187 L / ha carrier volume at 130-200 kPa. Visual lesion ratings were taken at 7, 14 and 21 days after application. AAD-12 (v1) and case response for 2,4-D and triclopyr are shown in Table 23. Unprocessed rice line (Clearfield) was severely injured (30% on 7DAT and 35% on 15DAT ) of 2,4-D at 2240 g ea / ha, which is considered to be the commercial utilization rate 4. The (v1) case A-AD-12 demonstrated excellent tolerance to 2,4-D, without prejudice to 7 or 15DAT. Unprocessed rice was significantly damaged (15% at 7DAT and 25% at 15DAT) at the rate of 2 x triclopyr (560 g ea / ha). Case (v1) AAD-12 demonstrated excellence tolerance at 2 x triplopyrin rates without injury observed at 7 or 15DAT.

These results indicate that AAD-12 (v1) and processed rice exhibited a high level of resistance to 2,4-D and triclopyr at rates that caused severe visual damage to Clearfield rice. It also demonstrates the ability to stack various herbicide tolerance genes with AAD-12, several species to provide resistance to a broad spectrum of effective chemicals.

Example 14 AAD-12 (v1) in Canola Canola Transformation: AAD-12 (v1) and the 2,4-D resistance gene was used to transform Brassica napus var. Use * 710 with Agrobacterium and pDAB3759 plasmid-mediated transformation. The construct contained the AAD-12 (V1) of the CsVMV promoter-driven gene and the promoter-driven Pat AtUbilO gene and the EPSPS glyphosate resistance trait driven by the AtUbilO promoter.

The seeds were surface sterilized with 10% commercial bleach for 10 minutes and washed 3 times with sterile distilled water.

The seeds were placed in one half of the MS medium concentration (Murashige and Skoog, 1962) and maintained at 25 ° C, 16 hours light / 8 hours dark photoperiod.

Hypocotyl segments (3-5 mm) were removed 5-7 days old from seedlings and placed in K1D1 callus induction medium (MS medium with 1 mg / L kinetin and 1 mg / L 2,4-D ) for 3 days as pre-treatment. The segments were then transferred to a petri dish, treated with Z707S or Agrobacterium strain LBA4404 containing pDAB3759. Agrobacterium was grown overnight at 28 ° C in the dark on a shaker at 150 rpm and subsequently resuspended in the culture medium.

After 30 min of treatment with the Agrobacterium hypocotyl segments, they were placed back in the callus induction medium for 3 days. After co-cultivation, the segments were placed in K1D1TC (callus-inducing medium containing 250 mg / l carbenicillin and 300 mg / l Timentin) for one week or two weeks of recovery.

Alternatively, the segments were placed directly on the K1D1H1 selection medium (above the medium with 1 mg / L Herbiace). Carbenicillin and Timentin were the antibiotics used to kill Agrobacterium. The selection agent Herbiace allowed the growth of transformed cells.

The callused hypocotyl segments were then placed in JB3Z1H1 (MS medium, 3 mg / l benzylamino purine, 1 mg / l zeatin, 0.5 g / l MES [2- (N-morpholino) ethanesulfonic], 5 mg / l L silver nitrate, 1 mg / L Herbiace, Carbenicillin and Timentin) by firing the regeneration medium. After 2-3 weeks, the shoots began to regenerate. Hypocotyl segments along with the shoots are transferred to B3Z1H3 medium (MS medium, 3 mg / l benzylamino purine, 1 mg / l zeatine, 0.5 g / l MES [2- (N-morpholino) ethanesulfonic] , 5 mg / L silver nitrate, 3 mg / L Herbiace, carbenicillin and Timentin) for a further 2-3 weeks.

The buds were excised from the hypocotyl segments and transferred to trigger MESH5 or MES10 elongation medium (MS, 0.5 g / L MES, 5 or 10 mg / L Herbiace, Carbenicillin, Timentin) for 2-4 weeks. The elongated shoots are grown for root induction in MSI.1 (MS with 0.1 mg / l indolbutyric acid). Since the plants had a well-established root system, they were transplanted into the soil. The plants were acclimated under controlled environmental conditions in Conviron for 1-2 weeks before transfer to the greenhouse effect.

Molecular Analysis - Canola Materials and Methods: Harvesting DNA isolation tissue and quantification. Fresh tissue was tubeed and lyophilized at 4 ° C for 2 days. After the tissue was completely dried, a tungsten bead (Valenite) was placed in the tube and the samples were subjected to 1 minute dry milling using a Kelco ball mill. The standard DNeasy DNA isolation process was followed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA was then stained with Pico Green (Molecular Probes P7589) and read at the fluorometer (BioTek) with known standards to obtain the concentration in ng / mL.

Polymerase Chain Reaction: A total of 100 ng of total DNA was used as a template. 20 mM of each primer was used with the Ex Taq Polymerase Takara PCR Kit (Mirus TAKRR001A). Primers for the AAD-12 (v1) PCR coding region were (SEQ ID NO: 10) (forward) and (SEQ ID NO: 11) (reverse). The PCR reaction was performed on the GeneAmp 9700 thermocycler (Applied Biòsystems) 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 an EtBr stained 1% agarose gel. 35 samples from 35 plants with AAD-12 cases (v1) tested positive. Three negative negative control samples. ELISA: Using the established ELISA described in the previous section, AAD-12 protein was detected in 5 different cases of canola plant transformation. Expression levels ranged from 14 to over 700 ppm total soluble protein (TSP). Three samples of untransformed plants were tested in parallel with no signal detected, indicating that the antibodies used in the assay have minimal cross-reactivity with the canola cell matrix. These samples were also confirmed by western positive analysis. A summary of the results is given in Table 24. AAD-12 (v1) Transformed TO Canola Herbicide Tolerance: Forty-five TO cases of the transformed with the pDAB3759 construct were sent to the greenhouse by time, and were allowed to acclimate in the greenhouse. Plants were grown to 2-4 young, normal looking leaves had emerged (ie, if the transition from tissue culture plants to greenhouse growing conditions). The plants were then treated with a lethal dose of three commercial formulations of 2,4-D amine 4 at a rate of 560 g ea / ha. Herbicide applications were made with a track sprayer in a spray volume of 187 L / ha, spray height 50 cm. A lethal dose is defined as the rate that causes injury> 95% for unprocessed controls.

Twenty-four of the cases were tolerant to the application of the 2,4-D DMA herbicide. Some cases caused minor injuries, but recovered in 14 DAT. Cases were advanced to T1 (and T2 generation) through self-pollination under control of conditions. AAD-12 (v1) Heritability in Canola: A 100 plant progeny test was also performed on 11 AAD-12 (v1) T1 lines. The seeds were planted and transplanted into 3 inch pots filled with Metro Mix media. All plants were then sprayed with 560 g ea / ha of 2,4-D DMA as described above. After 14 DAT, resistant and sensitive plants were counted. Seven of the 11 lines tested segmented as a single dominant Mendelian trait (3R: 1S) as determined by Chi-square analysis. AAD-12 is hereditary as a robust auxin aryloxyalkanoate resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes. AAD-12 (v1) Heritability in Canola: A 100 plant progeny test was also performed on 11 AAD-12 (v1) T1 lines. The seeds were planted and transplanted into 3 inch pots filled with Metro Mix media. All plants were then sprayed with 560 g ea / ha of 2,4-D DMA as described above. After 14 DAT, resistant and sensitive plants were counted. Seven of the 11 lines tested segmented as a single dominant Mendelian trait (3R: 1S) as determined by Chi-square analysis. AAD-12 is hereditary as a robust auxin aryloxyalkanoate resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes.

High-Tolerance 2.4-D Verification on T1 Canola: For T1 AAD-12 (v1), 5-6 mg of seeds were stratified, seeded, and a thin layer of Sunshine Mix # 5 media was added as an upper layer. from soil. Emerging plants were selected at 560 g ea / ha jde 2,4-D at 7 and 13 days after planting. The surviving plants were transplanted to 3 inch pots containing the Metro Mix medium. Surviving T1 progeny plants, which were screened at 560 g æa / ha of 2,4-D, were also transplanted to 7.62 cm (3 inch) pots filled with Metro solo Mix. At leaf stage 2-4 plants were sprayed with either 280, 560, 1120, or 2240 g ea / ha of 2,4-D DMA. Plants were classified at 3 and 14 DAT and compared with non-transformed control plants.

A sampling of T1 14DAT injury case data can be seen in Table 25. The data suggest that several cases are robustly resistant to 2240 g ea / ha of 2.4-D, while other cases demonstrated less robust tolerance up to 1120 g ea / ha. ha of 2,4-D. Surviving plants were transplanted to 51/4 pots containing "Media Metro Mix" and placed under the same growth conditions as before and self-pollinated to produce only homozygous seeds.

PDAB3759 canola tolerance field for 2,4-D, Dicloprop, Triclopir and Fluoroxipir Herbicides: Field level tolerance judgment was performed in two AAD-12 (v1) cases 3759 (20) 018,001 and 3,759 (18 ) 030,001 and a wild type canola (Nex710) in Fowler, Ind. The experimental design was a randomized block design with three replications. Herbicidal treatments were 2,4-D (dimethylamine salt) at 280, 560, 1,120, 2,240 and 4,480 g ea / ha, triclopyr at 840 g ea / ha, fluoroxipir at 280 g ea / ha and a control. Within each herbicide treatment, a single 20 foot line / case for case 3759 (18) 030.0011, 3759 (18) 018.001 and a wild type line (Nex710) were planted with a 4-8 inch drill line. of spacing between flares. Iforam herbicide treatments applied when canola reached the 4-6 leaf stage using costal spray compressed air delivering 187 L / ha carrier volume at 130-200 kPa. Visual Lesions were classified 7, 14, and 21 days after application. ______________________________ Canola response to 2,4-D, triclopyr and fluoroxipir are shown in Table 26. Wild-type canola (Nex710) was severely injured (72% at 14DAT) by 2,4-D at 2240 g ea / ha, which is considered the rate x 4.

The AAD-12 cases (v1) all demonstrated excellent tolerance to 2,4-D at 14DAT with an average lesion of 2, 3 and 2% observed at 1,2 and 4 x rates, respectively. Wild canola was severely injured (25% at 14DAT) at the rate of 2 x triclopyr (840 g ea / ha). AAD-12 cases (v1) demonstrated tolerance at 2 x doses of triclopyr, with an average of 6% 14DAT injury between the two cases. Fluoroxipir at 280 g ea / ha caused serious injury (37%) to the 14DAA unprocessed line. AAD-12 cases (v1) demonstrated greater tolerance with an average lesion 8% at 5DAT.

These results indicate that AAD-12 (v1) and transformed cases exhibited a high level of resistance to 2,4-D, triclopyr and fluoroxipir at rates that were lethal or caused severe epinastic malformations for unprocessed canola. AAD-12 has been shown to have a relative efficacy of 2,4-D> triclopyr> fluoroxipir.

Example 15 1. Transformation and Selection of Soybean AAD-12 from Case DAS-68416-4 Transgenic soybean (Glycine max) from Case DAS-68416-4 was generated by Agrobacterium-mediated transformation of soybean cotyledon explants. at the. The disarmed Agrobacterium EHA101 strain (Hood et al, 2006) carrying the binary vector pDAB4468 (Figure 2) with the selectable marker (PAT) and the gene of interest (AAD-12) within the T-DNA strand region. , was used to initiate the transformation. Agrobacterium-mediated transformation was performed. Briefly, cv soybean seeds (Maverick) were germinated in basal medium and cotyledonary nodes were isolated and infected with Agrobacterium. Shoot initiation, shoot lengthening and rooting medium were supplemented with cefotaxime, timentin and vancomycin for Agrobacterium removal. Glufosinate selection was employed to inhibit the growth of unprocessed sprouts. The selected shoots were transferred to rooting medium for root development and then transferred to soil mix for seedling acclimatization.

The terminal foliage of selected seedlings was leaf glued with web glufosinate for putative transformants. The seedlings were screened transferred to the greenhouse, allowed to acclimate and painted with glufosinate to reconfirm tolerance and considered putative transformants.

Plants were collected and screened for molecular analysis to confirm the selection marker gene and / or the gene of interest were performed. TO plants were allowed to self-fertilize in the greenhouse to yield T1 seeds.

T1 plants were backcrossed and introgressed in the elite germplasm (Maverick). This case, the DAS-68416-4 soybean case, was generated from an independent transformed isolate organization. The case 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 of broad-based and multi-site genotypes. environment. Further Description of Soy Case DAS-68416-4 has been described in WO 2011/066384, which is incorporated by reference in its entirety by reference.

Example 16 Generation of 2008 Agronomic Data An agronomic study with the DAS-68416-4 soybean case and a non-transgenic control (var. Maverick) was conducted in 2008 at six sites located in Iowa, Illinois, Indiana, Nebraska and Ontario, Canada. (2 locations).

Agronomic determinants, including stand / Population Count, seedlings / plant vigor, plant height, bedding, disease incidence, insect damage, and days for flowering were evaluated to investigate the equivalence of the DAS-Soybean Case. 68416-4 (with and without herbicide treatments) compared to the independent control line.

This study is referred to as Agronomic Experiment S1. Soybean seed testing and control was planted at a sowing density of approximately 112 seeds per 25 feet of row with a spacing of about 30 inches (75 cm). At each site, three identical plots of each treatment were established, with each plot consisting of lines 2-25 feet. The plots were arranged in randomized block (RCB) design, with a unique randomization at each site. Each soybean plot was limited by two rows of a non-GMO soybean of similar maturity. The entire trial site was surrounded by a minimum of 10 feet of non-transgenic soybean of similar relative maturity.

The herbicides were applied with a spray volume of approximately 20 liters per hectare (187 'L / ha). These applications are designed to replicate maximum label rate business practices. 2,4-D was applied as three diffusion over the top applications for a total of £ 3 seasonal ae / A. Individual applications of £ 1.0 ae A (1,120 g / ha) were made pre-emergence and growth stages of approximately V4 and R2. Glufosinate was applied as two transmission across the top of applications for a seasonal total of £ 0.74 ai / A (828 g ai / ha). Individual applications of £ 0.33 ai / A and £ 0.41 ai / A (374 and 454 g ai / ha) were made at about V6 and R1 growth stages. Analysis of variance was performed across field sites for agronomic data using a mixed model (SAS version 8, SAS Institute, 1999). Input was considered a fixed effect, and the location, within position block, input position, and e-block input on site were designated as random effect. The significance of an overall treatment effect was estimated using an F-test. Paired contrasts were made between the control and the unpulped case. DAS-68416-4 (unpulped) soybean, Sprayed DAS-68416-4 soybean case with glufosinate (soybean case DAS-68416-4 + glufosinate), soybean case DAS-68416-4 sprayed with 2,4-D (DAS-soybean Case 68.416-4 + 2,4-D) and soybean Case DAS- 68.416-4 sprayed with both glufosinate and 2,4-D (DAS-soy Case 68.416-4 + both) from the transgenic inputs using t-tests. Adjusted p values were also calculated using the false discovery rate (FDR) to control multiplicity (Benjamini and Hochberg, 1995). Analysis of data collected from control agronomics, Case DAS-68.416-4 soybeans sprayed, Case DAS-68416-4 + 2.4-D soybeans, Case DAS-68.416-4 soybeans + Glufosinate and Case DAS soybeans -68.416-4 + both herbicides was conducted. No statistically significant differences were observed for stand count, initial population, seedling life, lesions after application, housing, final stand count or days for flowering (Table 28). For height, a significant paired t-test was observed between the control and the DAS-68416-4 + 2,4-D soybean spray case. However, there was no significant overall treatment effect, the differences were very small between the DAS-68416-4 soybean treatment and control case, and the differences were not shared between the different DAS- soybean case. 68416-4 treatments.

Based on these results, Case DAS-68416-4 soybean was agronomically equivalent to almost isogenic non-transgenic control.

• f * V- * F The overall treatment effect estimated by an F. test. B Comparison of sprayed and non-sprayed treatments for the control using a t-test. 0 P-values adjusted using a false discovery procedure rate (FDR). d 0-100% scale; (support count divided by the number of seeds planted.) * 100. and visual estimate on scale 1-10; 10 = growth equivalent to unprocessed plants. visual festival scale of 0-100%, 0% = no damage. f The number of days from the time of planting to flowering.

P values in bold are significant (p <0.05).

Example 17 Generation of 2009 Agronomic Data An agronomic study with the DAS-68416-4 soybean case and a non-transgenic control (var. Maverick) was conducted in 2009 at eight sites located in Arkansas, Iowa, Illinois, Indiana, Missouri and Nebraska. . Agronomic determinants including stand / Population Count, seedlings / plant vigor, plant height, disease incidence, insect damage, and days for flowering were evaluated to investigate soybean equivalence case DAS-68416 -4 soy '(with and without herbicides) for the control (Table 29). * Β - Spray and non-spray tests, S - Spray only tests A randomized complete block design was used for the tests. Entries were DAS-68416-4 soybean case, a Maverick control line and non-GMO soybean lines available on the market. The test, control and reference soybean seeds were planted at a sowing density of approximately 112 seeds per row with a spacing of approximately 30 inches (75 cm). At each site, four identical plots of each treatment were established, with each plot consisting of 2-25 ft lines. Each soybean plot was limited by two non-transgenic soybean lines (Maverick). The entire trial site was surrounded by a minimum of 4 lines (or 10 feet) of non-transgenic soybean (Maverick). Proper insects, weeds, and disease control practices have been applied to produce an agronomically acceptable crop.

Herbicides have been applied to replicate maximum label rate commercial practices. The treatments consisted of a non-sprayed witness and applications of 2,4-D, glufosinate, 2,4-D / glufosinate herbicides applied at specific growth stages. For 2,4-D applications, the herbicide was applied at a rate of 1.0 £ aE / D (1.120 g ae / ha) at stages V4 and R2. For the glufosinate treatments, applications were made to plants in stages V4 and V6-R2. For both applications, glufosinate was applied at a rate of 0.33 µ ai / A (374 g ai / ha) and £ 0.41 ai / A (454 g ai / ha) for V4 and V6-R2 in a respective manner. Applications for both herbicide applications were DAS-68416-4 soybean and controls including non-transgenic Maverick. Maverick plots were expected to die after herbicide application. Analysis of variance was performed across field sites for agronomic data using a mixed model (SAS version 8, SAS Institute, 1999). Input was considered a fixed effect, and location within position block, position-to-input, and block-by-place input were designated as random effect. Analysis at individual sites was done analogously with input, such as a fixed effect, and input block and block as random effects. Data were not rounded for statistical analysis. Significant differences were stated at the 95% confidence level, and the significance of an overall treatment effect was estimated using an F-test. Unpunched AAD-12 (unpowdered) peer contrasts were made, 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 transgenic inputs 2,4- D (AAD-12 + 2,4-D + glufosinate) and the control input using t-tests.

Due to the large number of contrasts made in this study, multiplicity was a problem. Multiplicity is a problem when large numbers of comparisons are made in a single study to analyze unexpected effects. Under these conditions, the probability of falsely stating differences based on wise comparison p values is very high (1- 0.95nuber de comparisoils). In this study, there were four comparisons per analyte (16 observation types analyzed for agronomy), resulting in 64 comparisons for agronomy. Therefore, the probability of declaring one or more false differences based on unadjusted p values was 99% for agronomy (1-0.9564). Analysis of the data collected from control agronomics, unpowdered AAD-12, AAD-12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + 2,4-D + glufosinate inputs was analyzed. conducted. For the analysis through the site, no statistically significant differences were observed for seedling vigor, final plant vigor / injury population (V4, RI), lodging, disease incidence, insect damage, days for flowering, days for maturity, number of pods, number of seeds, yield and plant height. For support and initial population counts, a significant paired t-test was observed between control and AAD-12 + glufosinate entry, but was not accompanied by an overall significant treatment effect or adjusted FDR. p value For plant vigor / injury (R2), significant t-tests and a significant overall treatment effect paired were observed between the control and both AAD-12 + glufosinate and AAD-12 + 2,4-D + glufosinate inputs, but were not accompanied by a significant adjusted FDR p value. The mean results for all these variables are also within the range found for the reference lines tested in this study.

Example 18 Transformation and Selection of the AADL Case PDAs 1740-278 The AADL Case, PDAs 1740-278 was produced by WHISKER - Hi-ll maize row mediated transformation. The transformation method used is described in US Patent Application # 20090093366. An Fspl fragment pDAS1740 of the plasmid (Figure 3), also referred to as pDAB3812, was transformed into the maize lineage. This plasmid construct contains the plant expression cassette containing the RB7 MARv3: Zea mays Ubiquitin a promoter v2 // AADL v3 // Zea mays PER5 3'UTR: RB 7 MARv4 plant transcription unit (PTU).

Numerous cases have been produced. Those cases that survived and produced healthy, resistant haloxifop callus tissue were assigned unique identification codes that represent putative transformation cases, and continuously transferred to fresh selection medium.

Plants were regenerated from tissue derived from each single case and transferred to the greenhouse.

Leaf samples were taken for molecular analysis to verify the presence of Southern Blot transgene AAD-I, DNA boundary confirmation, and genomic marker assisted confirmation. Positive for plants were pollinated with pure lines to obtain IT seeds. IT plants from PDAs cases 1470-278-9 (DAS-40278-9) was selected, self-pollination and characterized by five generations. Meanwhile, the IT plants have been backcrossed and introgressed into the elite germplasm (XHH 13) through multi-generation marker-assisted selection. This case was generated from an independent transformed organization isolate. This case 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 of broad and multi-bottom genotypes. locations of the environment. Further description regarding the case of maize PDAS-1740-278-9 has been described in WO 2011/022469, which is hereby incorporated by reference in its entirety.

Example 19 Herbicide application and agronomic data Herbicides were applied with a spray volume of approximately 20 liters per hectare (187 L / ha).

These applications are designed to replicate maximum label rate business practices. Weedar 64 (026491-0006) at a concentration of 39%, 3.76 ae Ib / gal, 451 g ae / l Assure and II (106155) at a concentration of 10.2%, £ 0.87 ai / gal, 104 g ia / g were used. 2,4-D (Weedar 64) was applied in three top-loading applications to test inputs 4 and 5 (seasonal total of 3 Ib ae / A).

Individual applications were in pre-emergency and about V4 and V8-V8.5 steps. Individual target application rates were £ 1.0 ae / A for Weedar 64, or 1,120 g ea / ha. Actual application rates ranged from 1096 - 1231 g ae / A.

Quizalofope (Ensure II) was applied as a single top-down transmission to test inputs 3 and 5. Application season was approximately V6 growth phase. The application rate was 0.0825 µ ai / A for Assure II, or 92 g ai / ha. Actual application rates ranged from 90.8-103 g ai / ha. Agronomic characteristics were recorded on all test inputs within blocks 2, 3 and 4 at each location. Table 30 lists the characteristics that were measured. Analysis of the data collected from control agronomics, unpulped AAD-I, aad-1 + 2,4-D, AAD-\ + quizalofope, and AAD- \ + both inputs was conducted. For the analysis through the site, no statistically significant differences were observed for the initial population (VI and V4), vigor, final population, crop injury, flowering time, pollination time, stalking housing, lodging, disease incidence, insect damage, days to maturation, plant height and pollen viability (shape and color) values in the summary analysis. To turn green and ear height, significant paired t-tests were observed between the control and the aad-L + quizalofope entries, but were not accompanied by significant global treatment effects or false Rate Findings (FDR) adjusted values. of p (Table 31). a global treatment effect estimated by a F. b test. Comparison of sprayed and unpowdered treatments for control using a t-test. c P-values adjusted using a false discovery procedure rate (FDR). d Visual estimate on scale 1-9, 9 = tall plants with large and stubby leaves. and 0-100% scale, with 0 = no injury and 100 = dead plant. f The number of heat units that have accumulated since the time of planting. 9 0-100% of scale, with% of pollen grains with collapsed walls. h 0-100% of scale, with% intense yellow pollen grains, 'visual estimate on scale 1-9 with visible green nonwoven. J visual estimate on scale 1-9 with 1 being the resistance to poor diseases. k 1-9 scale visual estimate with a poor insect resistance. NA = statistical analysis I not performed since no variability in repetitions or treatment. m statistical difference indicated by P-value <0.05.

Example 20 Additional Argon Testing Agronomic characteristics of the 40278 maize line compared to an almost isoline maize line were evaluated across different environments. Treatments included four genetically distinct hybrids and their appropriate near-isoline control hybrids tested in a total of 21 locations.

The four erarVi test hybrids averaged to final maturity | from hybrids ranging from 99 to relative maturity 113 days. Experiment A case tested DAS-40278-9 on the genetic background Congenital C x BC3S1 conversion. This hybrid has a relative maturity of 109 days and was tested at 16 sites (Table 32). Experiment B tested the hybrid background E x conversion BC3S1 Congenital, one day hybrid relative maturity 113. This hybrid was tested at 14 sites using a slightly different set of A sites. Experiment C and D tested hybrid backgrounds BC2S1 con version x D and Inborn BC2S1 conversion x Inbred F, in a respective way.

Both hybrids have a relative maturity of 99 days and were tested in the same 10 places.

For each trial, a randomized block design was used with two replications per location of two row plots. Row length was 20 feet, and each row was sown at 34 seeds per row. Regional agronomic practices of the standards were used for trial management.

Data were collected and analyzed by eight agronomic characteristics, plant height, ear height, stem accommodation, root lodging, final population, grain moisture, weight, test and yield. Plant height and ear height provide information on the appearance of hybrids. The agronomic characteristics of percent stem and root hosting hosting determine the harvestability of a hybrid. Final population count measures seed quality and seasonal growing conditions that affect yield. Percent grain moisture at harvest defines hybrid maturity, and yield (bushels / acre adjusted for moisture) and test weight (kilogram weight of a bushel of corn adjusted to 15.5% moisture) describe the ability of the hybrid. Analysis of variance was performed through field sites using a linear model. Input and location were included in the model as fixed effects. Mixed models, including localization and localization by input, such as random effects were explored, but the input localization explained only a small portion of variance and its variance component was often not significantly different from zero. For stock and root that presents a logarithmic transformation was used to stabilize the variance, however means and intervals are reported in the original scale. Significant differences were declared at the 95% confidence level. The significance of an overall treatment effect was estimated using a t-test.

The results of these agronomic characterization assays can be found in Table 32. No statistically significant differences were found for any of the four hybrids of 40,278 compared to isoline controls (p <0.05) for height parameters. ear, stem accommodation, root lodging, grain moisture, weight, testing and production. Last population count and plant height were statistically different in experiments A and B, respectively, but similar differences were not observed compared to the other 40,278 hybrids tested. Some of the observed variation may be due to the low levels of genetic variability remaining from DAS-40.278-9 backcrossing in the elite lines. The general range of values for the measured parameters are all within the range of values obtained for traditional maize hybrids and would not lead to an increase in the weediness conclusion. In summary, the data indicate that the agronomic characterization 40278 maize is biologically equivalent to conventional maize.

The agronomic characteristics of case-containing hybrid corn DAS-40278-9 compared to quasi-isoline maize were collected from various field tests in different geopark environments for the growing season. The results obtained for hybrid maize lines ^ containing case DAS-40278-9 compared to null plants are listed in Table 33.

Agronomic characteristics for hybrid maize lines containing case DAS-40278-9 and null plants sprayed with the herbicide quizalofop (280 g ea / ha), at stage V3 of development and 2,4-D (2,240 g ea / ha). ) applied at stage V6 of development are in Table 34.

Example 21 2,4-D increases the growth of 2,4-DA resistant transgenic soybean AAD-12 soybean provides protection for the soybean plant, while weeds are destroyed by the application of 2, 4-D. It was unexpectedly observed that 2,4-D also increase growth in tolerant soybean 2,4-D: This increase in growth resulted in increased plant height and / or yield of sprayed plots compared to non-plots. sprayed. Increased plant growth and / or yield resulting from the application of 2,4-D is described for genetically engineered soybean plants for 2,4-D tolerant. Trials were grown at various locations covering soybeans growing North American region. Entries included in elite lines that case DAS-68416-4 (whose tolerance conditions for 2,4-D) were introgred. Treatments consisted of non-spray and 2,4-D spray treatment applied at both V3 level and R2 growth stages. Plots were measured for various agronomic characteristics throughout the season, including plant height and grain yield. Weeds were controlled at all times in both sprayed and unpowdered lots to eliminate any effect of competition. At the conclusion of the study, analysis of the measured data showed a significant increase in both height and yield for the inlets that had been sprayed with 2,4-D, compared with those receiving no treatment. An increase in yield is one of the added benefits to weed control delivered by 2,4-D, 2,4-D resistant soybeans.

Field tests were performed in 2011 to compare the agronomic characteristics of the DAS-68416-4 soybean case (International Patent Application No. 2011/066384), which had been sprayed with 2,4-D, with the characteristics agronomics of non-pulverized soybean case DAS-68416-4 The field trials contained entries from four elite soybean strains in which case DAS-68416-4 soybean had been introgression, and the respective null isolines of the elite non-soybean lines containing DAS-68416-4 soybean. The trials were planted in different geographical locations (ten locations in total). The experiment was installed in modified split plots with two replications per site. Experimental plots were treatments and subplots were entries. Each plot consisted of two rows, 12.5 meters long, planted 30 centimeters apart. The sprayed plots were treated with 2,4-D (1,120 g ea / ha) sprayed at stages V3 and R2. Throughout the season, field plots were kept under normal agronomic practices and kept weed free. Several agronomic characteristics were measured for soybean plants to determine how the application of 2,4-D affected the performance of soybean agronomic characteristics. The agro- nomic characteristics tested and growth phase when data were collected are presented in Table 35.

At the end of the soybean growing period, data from all sites were combined and analysis was performed by location. Data analysis was performed using JMP ® Pro 9.0.3 (SAS, Carl, NC). The method of least squares analysis is presented in Table 28. The application of 2,4-D in the case of DAS-68,416-4 soybean containing the AAD-12 transgene resulted in an increase of the growth conditioning effect. . Higher growth culminated in significantly higher yield and plant height in 2,4-D sprayed field plots compared to non-sprayed 2,4-D field plots. These increases were determinable when data were cumulatively analyzed at all sites. In contrast, the increase in yield of the DAS-68.416-4 case sprayed with 2,4-D was decreased by an interaction treatment site. Both average height and yield were increased by about 5% in 2,4-D applications in Table 36.

As shown in Table 37, at least one of the ten sites (Location # a3) reported significantly higher yield harvests for the DAS-68.416-4 unpowdered soybean case, compared to the 2.4-D sprayed soybean DAS case. -68,416-4 plants.

When the results for all sites were accumulated, the application of 2,4-D in the case of DAS-68,416-4 soybean containing the AAD-12 transgene indicated a conditioning effect, resulting in increased growth. For example, the yield of soybean case DAS-68.416-4 plants sprayed with the 2.4-D herbicide was 56.4 bu / acre, which is considerably higher than the case yield of unpulverized soybean DAS-68.416- 4 plants that was 53.7 bu / acre. Similarly, the height of the DAS-68.416-4 soybean case sprayed with the 2.4-D herbicide was 81 cm, which is considerably higher than the height of the DAS-68.416- sprayed soybean case. 4 plants that was 77 cm.

Example 22 2.4-D, Increases Growth of 2,4-D-Resistant Soybean to 2,4-D / Glyphosate Combination Similar field trials as in the previous example were performed in 2010, but with two applications of 2, 4-D in combination with glyphosate. The results show that the increase in growth of 2,4-D resistant soybean at plant height and / or yield of sprayed plots compared to non-sprayed plots is due to the application of 2.4-D.

Significant treatment effects were observed for a number of measured parameters. Both 2,4-D and glyphosate were sprayed at stages V3 and R2. The trials were planted in different geographical locations (six points in total). The agro- nomic characteristics tested and the growth phase when data were collected are presented in Table 30. The average height was increased by 6%, and the average yield increased by 17% for the pulverized soybean in Table 38. In addition In addition, the average seed weight increased by 6% for pulverized soybeans.

As shown in table 39, certain geographic variations were also observed in this example. The yield medium was increased to 21.6% for the pulverized soybean in Table 39.

Example 23 Experimental yield results comparing sprayed and unsprayed treatments 2,4-D resistant transgenic crop plants transformed with an aryloxyalkanoate dioxigenase (DAA) resulted in increased productivity when treated with a stimulating amount of herbicide comprising an aryloxyalkanoate unit. Soybean cases comprising an AAD-12 gene expression cassette were tested in yield assays under sprayed and non-sprayed replicate conditions. There was a series of experiments containing early soybeans adapted to northern latitudes and another series of experiments containing final soybeans adapted to southern latitudes. In previous experiments, there were cases where soybean starters comprising one AAD-12 gene expression cassette were treated with 2,4-D during the displayed growing season and increased yield over cheeses. - non-pulverized ones.

Modified subdivided plots with two replications were used for the trials. Each plot was 2 lines wide, spaced 30 inches and 12.5 meters long. There was a 2.5 to 3 foot pass between the end-to-end planted plots to allow movement within the trial during the season. Pulverized blocks were sprayed sequentially (twice) during the growing season with 2,4-D choline + glyphosate (premix) at 2,185 g ea / ha + AMS at 2 wt% .________________________________________________ A The first application was in growth phase V3 and the second application in growth phase R2. Both experimental and control field trials were kept weed free throughout the season by use of conventional herbicides or weeding. Data were collected on emergence, seedling vigor, harvest injury, flowering date, whether R2 count, disease incidence, insect plant damage, maturity date, housing, breaking 100-seed weight and productivity. . Data were analyzed using JMP ® Pro 9.0.3. Table 40 lists the locations that were used in the final analysis. Some locations that were planted were not included in the analysis due to within-frame variability.

Throughout the local analysis were performed for both initial and final assays. Tables 41 and 42 show the variance yield analysis for the initial and final assays, respectively.

In both early and late trials there was a significant (P = 0.05) name effect. This was expected since each elite soybean line in which a case had been introgred was from a different genetic background.

A significant treatment effect was measured for early judgment indicating that sprayed and non-sprayed treatments differed for yield. For the final judgment there was no significant treatment effect indicating that sprayed and non-sprayed plots did not differ for yield.

In both early and late name trials the treatment interaction effect was not significant, indicating that the treatment effect (or lack of an effect) was the same for each entry of a particular trial. Table 43 shows the average yield of each entry per treatment combination at the start of the trial, where HOMO means homozygote. Values followed by the same letter (within a given range) are no different according to Student's P t = 0.05. There were four entries that showed higher yield when sequentially sprayed on V3 and R3 with 2,4-D choline + glyphosate (premix) at 2,185 g ea / ha + AMS. Table 44 shows the average yield of each entry per treatment combination. Values followed by the same letter (within a given range) are no different according to Student's P t = 0.05. As reported above there was no significant treatment effect or the entry effect treatment for the final judgment for the mean separation was not performed. Letters in the table indicate that there was no difference between sprayed and non-sprayed treatments in the final trial.

The yield test results in this example show again that in some environments of some soybean genotypes there may be an increase in yield after application of 2,4-D. In the last two years this yield increase has been observed in yield trials that have been performed in MG 2 growing region.

Example 24 Comparison between soybean and corn Yield from results of field trials on soybean comprising an AAD-12 transgene indicate that a 2,4-D application may increase soybean yield in certain environments for certain genotypes. These results are surprising when compared to transgenic maize cases comprising an AAD-1 transgene. The yield of transgenic AAD-1 maize plants did not consistently show a statistically significant increase in yield after spraying with 2,4-D. These transgenic AAD-1 maize plants are biologically equivalent to conventional maize. Additional field studies at various geographic locations were completed from 2010 to 2012 on hybrid maize lines. During these fields studies the yield of 2.4-D sprayed maize lines (2,185 g ea / ha and 4,370 g ea / ha) were compared to untreated control maize lines (eg 2-sprayed untreated maize lines). , 4-D). The results of these experiments further show that maize plants containing the AAD-1 transgene do not result in a significant increase in yield as a result of treatment with a 2,4-D sprayer. Comparatively, an increase in yield has been shown in some soybean genotypes after 2,4-D application. The observed increase in yield of soybean cultivars, which is shown following a 2,4-D application is an unexpected improvement that is applicable for the purpose of increasing crop yields. The described method can be implemented by using a 2,4-D treatment to increase the yield of transgenic crop plants, for example by expressing an AAD-12 gene.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be made within the scope of the appended claims.

CLAIM

Claims (32)

A method of improving the yield of 2,4-D-resistant crops comprising treating plants with a stimulating amount of an herbicide comprising an aryloxy alkanoate moiety. The method of claim 1, wherein the 2,4-D resistant crop plants are transgenic plants transformed with an aryloxy alkanoate dioxigenase (DAA). A method according to claim 2, wherein the aryloxy alkanoate dioxigenesis (DAA) is AAD-12. The method of claim 1, wherein the herbicide comprising an aryloxy alkanoate moiety is a phenoxyacetic phenoxy herbicide or herbicides, The method of claim 1, wherein the herbicide comprising an aryloxy alkanoate moiety is 2,4-D. The method of claim 5, wherein the 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA). The method of claim 1, wherein the treatment is performed at least once at a rate of application of 2,4-D as also employed for weed control. A method according to claim 1, wherein the treatment is performed twice at an application rate of 2,4-D as also employed for weed control. The method of claim 8, wherein 2,4-D is applied at the 2,4-D tolerant soybean growth stages V3 and R2. The method of claim 1, wherein the treatment is performed at least three times at a rate of application of 2,4-D as also employed for weed control. The method of claim 1, wherein plants of 2,4-D resistant crops are under stress. The method of claim 1, wherein plants of 2,4-D resistant crops are also treated with a different 2,4-D herbicide for weed control. The method of claim 12, wherein the herbicide other than 2,4-D is a phosphorus herbicide or aryloxyphenoxypropionic herbicide. The method of claim 13, wherein the phosphorus herbicide comprises glyphosate, glufosinate, derivatives thereof, or combinations thereof. The method of claim 13, wherein the herbicide is phosphorus in the form of ammonium salt, isopropylammonium salt, isopropylamine salt or potassium salt. The method according to claim 13, wherein the aryloxyphenoxypropionic herbicide comprises chlorazifope, fenoxaprop, fluazifope, haloxifope, quizalofope, or combinations thereof. The method of claim 1, wherein plants of 2,4-D resistant crops are treated at least once with 25 g ae / ha at 5000 g ae / ha of 2,4-D. The method of claim 1, wherein plants of 2,4-D resistant crops are treated at least once with 100 g ea / ha to 2500 g ea / ha of 2,4-D. The method of claim 1, wherein the herbicide comprising an aryloxy alkanoate moiety reaches 2,4-D resistant crop plants by root absorption. The method of claim 13, wherein the phosphorus herbicide reaches 2,4-D resistant crop plants by root absorption. The method of claim 13, wherein the aryloxyphenoxypropionic herbicide targets 2,4-D resistant crop plants by root absorption. The method of claim 2, wherein the transgenic plants transformed with an aryloxy alkanoate dioxigenase (DAA) are selected from cotton, soybean, and canola oil. 23. A method of improving the yield of 2,4-D resistant crop plants comprising (A) transforming plant cells with a nucleic acid molecule comprising a nucleotide sequence encoding an aryloxy dioxigenase alkanoate (DAA) ); (B) selection of transformed cells; (C) the regeneration of plants from transformed cells; and (D) treating the plants with a stimulating amount of a herbicide comprising an aryloxy alkanoate moiety. The method according to claim 23, wherein the aryloxy alkanoate dioxigenenase (DAA) is AAD-12. The method of claim 23, wherein the nucleic acid molecule comprises a selectable marker that is not an aryloxy alkanoate dioxigenase (DAA). The method of claim 25, wherein the selection marker is a phosphinothricin acetyltransferase (PAT) gene or bialaphos resistance (bar) gene. The method of claim 23, wherein the nucleic acid molecule is an optimized plant. 28. Use of 2,4-D in the production of higher yielding 2,4-D-resistant transgenic plants compared to their non-transgenic parent plants. Use according to claim 28, wherein 2,4-D is applied at least once with 25 g ae / ha to 5000 g / ha of 2,4-D. Use according to claim 28, wherein 2,4-D is applied at least once with 100 g ea / ha to 2500 g ea / ha of 2,4-D. Use according to claim 28, wherein 2,4-D comprising 2,4-D choline or 2,4-D dimethylamine (DMA). Use according to claim 28, wherein the resistant crop 2,4-D plants are treated with 2,4-D at least twice before flowering.