JP2015525218A - Method for improving the yield of 2,4-D resistant crops - Google Patents

Abstract

The present invention relates to a method for improving plant height and / or crop yield that is resistant to the herbicide 2,4-D by treating the plant with an application rate of 2,4-D that is not harmful to the plant. About. More particularly, a method of using 2,4-D application to increase the yield of crops expressing AAD-12 gene for 2,4-D resistance is provided. The provided method is of particular interest for the treatment of crop plants including corn, soybean, spring and winter rape (canola), sugar beet, wheat, sunflower, barley, and rice.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Application No. 61 / 656,546, filed June 7, 2012, the entire disclosure of which is expressly incorporated herein by reference. To do.

INCORPORATION BY REFERENCE TO ELECTRONIC SUBMITTED SAMPLE The computer readable sequence listing filed concurrently with this application and identified as follows is incorporated by reference in its entirety: a 11,342 byte ASCII (text) file One file name “72747_ST25.txt”, created on May 13, 2013.

  Weeds can rapidly deplete valuable nutrients required by crops and other desirable plants from the soil. There are many types of herbicides currently used for weed control. One very popular herbicide is glyphosate.

  Crop crops such as corn, soybean, canola, cotton, sugar beet, wheat, lawn, and rice that are resistant to glyphosate have been developed. Thus, for example, weeds can be controlled by spraying onto fields with actively growing glyphosate-resistant corn without causing significant damage to the corn plants.

  With the introduction of genetically engineered glyphosate-tolerant crops (GTCs) in the mid-1990s, growers have a simple, convenient, flexible and inexpensive tool to control a wide range of broadleaf and grass weeds that are unmatched in agriculture Became possible. As a result, producers have adopted GTC quickly and are often accepted, such as rotation, herbicide rotation, tank mixing, mechanical weed control and chemical and cultivated weed control. Has abandoned many of the best agricultural practices. Currently, glyphosate-tolerant soybeans, cotton, corn, and canola are sold elsewhere in the United States and the Western Hemisphere. Additional GTCs (eg, wheat, rice, sugar beet, lawn, etc.) are ready to be introduced with global market acceptance. Many other glyphosate resistant species are in experimental to developmental stages (for example, alfalfa, sugarcane, sunflower, beet, pea, carrot, cucumber, lettuce, onion, strawberry, tomato, and tobacco, poplar and maple buffalo, etc. Forest species, and horticultural species such as marigold, petunia, and begonia, see the website "isb.vt.edu/cfdocs/fieldtests1.cfm, 2005"). Furthermore, in recent years, the cost of glyphosate has dropped dramatically to the extent that only a few conventional weed control programs can effectively compete with the glyphosate GTC system for price and performance.

  Glyphosate has been successfully used in burndown and other non-crop areas for whole vegetation management for over 15 years. Like GTC, in many cases glyphosate is used 1-3 times a year for 3, 5, 10, 15 years continuously. These situations have resulted in undue reliance on glyphosate and GTC technology and are heavy on native weed species against plants that are naturally more resistant to glyphosate or that have developed mechanisms that resist glyphosate herbicidal activity. Imposing selective pressure.

  The widespread use of glyphosate-only weed control programs has led to the selection of glyphosate-resistant weeds, and the selection of weed species that are inherently resistant to glyphosate over most target species (ie weeds). shift). (Ng et al., 2003, Simarmata 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 around the world for over 15 years, only a handful of weeds have been reported to develop resistance to glyphosate (Heap, 2005). However, most of these have been identified in the past 3-5 years. Resistive weeds include Gramineae and broadleaf species, Lolium rigidum, Lolium multiflorum, Eleusine indica, Ragweed (Ambrosia artemisiifolia), Conyza canadensis, Conyza canadensis Both Bonyariensis (Conyza bonariensis) and Plantago lanceolata are included. In addition, weeds that were not an agricultural issue prior to widespread use of GTC are now more prevalent, including over 80% of the US cotton and soybean area and over 20% of the US corn area Control is becoming more difficult in the context of GTC (Gianessi, 2005). These weed shifts occur mainly in broadleaf weeds that are difficult to control (but not exclusively). Some examples include species of the genus Ipomoea, Amaranthus, Chenopodium, Taraxacum, and Commelina.

  In areas where growers face a shift to glyphosate-resistant weeds or weed species that are more difficult to control, growers can use glyphosate by mixing with tanks or alternating with other herbicides that control missed weeds. You can make up for the shortcomings. One popular and effective tank mixing partner for controlling broadleaf avoidance in many cases is 2,4-diclorophenoxyacetic acid (2,4-D). 2,4-D has been used for a broad range of broadleaf weed control for more than 60 years in agronomical and non-crop conditions. Although individual cases of more resistant species have been reported, 2,4-D remains one of the most widely used herbicides worldwide. A limitation to the further use of 2,4-D is that its selectivity in dicot crops such as soybeans or cotton is very poor and therefore 2,4-D is typically a sensitive dicot crop. Against (and generally in the vicinity). Furthermore, the use of 2,4-D in gramineous crops is somewhat limited by the nature of crop damage that can occur. 2,4-D in combination with glyphosate has been used to provide a more robust slash-and-burn treatment prior to planting no-till soybeans and cotton. However, due to the sensitivity of these dicotyledonous species to 2,4-D, these slash-and-burn processes must occur at least 14-30 days prior to planting (Agriliance, 2003).

  2,4-D belongs to the herbicide class of phenoxy acid and is MCPA. 2,4-D is used in many monocotyledonous crops (such as corn, wheat, and rice) to selectively control broadleaf weeds without severe damage to the desired crop. 2,4-D is a synthetic auxin derivative that acts to deregulate hormonal homeostasis in normal cells and interfere with balanced controlled growth, but the exact mechanism of action is still unknown. Absent. Triclopyr and fluroxypyr are pyridyloxyacetic acid herbicides whose mechanism of action is similar to that of synthetic auxin.

  These herbicides have different levels of selectivity for certain plants (eg, dicotyledonous plants are more sensitive than Gramineae). Metabolism that varies across different plants is one explanation for the variable selectivity levels. In general, since plants metabolize 2,4-D slowly, the variable plant response to 2,4-D can be more appropriately explained by different activities at the target site (s) (WSSA, 2002). ). 2,4-D plant metabolism typically occurs via a two-step mechanism, typically hydroxylation followed by conjugation with amino acids or glucose (WSSA, 2002).

  Over time, microbial populations generate alternative and efficient pathways for degrading this particular xenobiotic, which results in 2,4-D complete mineralization. The continuous application of herbicides selects for microorganisms that can utilize the herbicide as a carbon source for growth, giving them a competitive advantage in the soil. Therefore, the currently formulated 2,4-D has a relatively short soil half-life and has not encountered a significant carry over effect on subsequent crops. This increases the herbicidal utility of 2,4-D.

  One organism that has been extensively studied for its ability to degrade 2,4-D is Ralstonia eutropha (Streber et al., 1987). The gene encoding the first enzymatic step of the mineralization pathway is tfdA. See U.S. Patent No. 6,153,401 and GENBANK Accession No. M16730. TfdA catalyzes the conversion of 2,4-D acid to dichlorophenol (DCP) via an α-ketoglutarate-dependent dioxygenase reaction (Smejkal et al., 2001). DCP has little herbicidal activity compared to 2,4-D. TfdA has been used in transgenic plants to confer 2,4-D resistance to dicotyledonous plants (eg cotton and tobacco) that are normally sensitive to 2,4-D (Streber et al. ( 1989), Lyon et al. (1989), Lyon (1993), and US Pat. No. 5,608,147).

A 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 homologues are similar to tfdA (greater than 85% amino acid identity) and have similar enzymatic properties to tfdA. However, there are several homologs that have significantly lower identity to tfdA (25-50%) but still have characteristic residues related to α-ketoglutarate dioxygenase Fe ⁺² dioxygenase To do. It is therefore not clear what the underlying specificity of these diverse dioxygenases is.

  One unique example of a low homology to tfdA (31% amino acid identity) is sdpA from Delftia acidovorans (Kohler et al., 1999, Westendorf et al., 2002, Westendorf et al. , 2003). This enzyme has been shown to catalyze the first step of mineralization of (S) -dichloroprop (and other (S) -phenoxypropionic acid) and 2,4-D (phenoxyacetic acid) ( Westendorf et al., 2003). The transformation of this gene into plants has not been reported to date.

  The success, development of new herbicide tolerant crop (HTC) technology has been limited largely by the effectiveness, low cost and convenience of GTC. As a result, the adoption rate of GTC is very high among producers. As a result, there was little incentive to develop new HTC technology.

  Aryloxyalkanoate chemistry substructures include phenoxyacetate auxin (such as 2,4-D and dichloroprop), pyridyloxyacetate auxin (such as fluroxypyr and triclopyr), aryloxyphenoxypropionate (AAPP) acetyl coenzyme A carboxylase ( ACCase) is a common part of many commercially available herbicides including inhibitors (such as haloxyhop, quizalofop, and diclohop) and pentasubstituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as pyraflufen and full microlac) . However, all of these herbicide classes are very unique and there is no evidence of a degradation pathway common between these chemical classes in the current literature. A multifunctional enzyme for breaking down herbicides that span multiple mechanisms of action has recently been described (PCT US / 2005/014737, filed May 2, 2005).

  The present invention relates to a method for improving plant height and / or crop yield that is resistant to the herbicide 2,4-D by treating the plant with an application rate of 2,4-D that is not harmful to the plant. About. More particularly, a method of using 2,4-D application to increase the yield of crops expressing AAD-12 gene for 2,4-D resistance is provided. The invention further relates to the use of 2,4-D to improve the yield of crops that are 2,4-D resistant. The provided method is of particular interest for the treatment of crop plants including corn, soybean, spring and winter rape (canola), sugar beet, wheat, sunflower, barley, and rice.

  In some embodiments, the 2,4-D resistant crop is a transgenic crop transformed with an aryloxyalkanoate dioxygenase (AAD). In a further embodiment, the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD-12. AAD-1 was previously disclosed in US2009 / 0093366 and AAD-12 was previously disclosed in WO2007 / 053482, the entire contents of which are incorporated by reference.

  The yield improvement effect of 2,4-D treatment is the application rate of 25 g ae / ha to 5000 g / ha, or 100 g ae / ha to 2500 g ae / ha, or specifically 1000 g ae / ha to 2000 g ae / ha. Can be observed. In one embodiment, 2,4-D from 1000 g ae / ha to 1500 g ae / ha is used. In another embodiment, 2000 g ae / ha to 2500 g ae / ha is used. Furthermore, the yield improvement effect of the 2,4-D treatment is particularly evident when 2,4-D is applied at the 2-8 leaf stage before the crops are flowering. However, the required application rate and / or the leaf stage of the crop varies as a function of the plant, its height and climatic conditions.

  The term increase in yield means an increase in plant yield by 50% or more. In one embodiment, the increase in yield is at least 10%. In another embodiment, the increase in yield is at least 20%. In another embodiment, the increase in yield is between 10% and 60%. In another embodiment, the increase in yield is between 20% and 50%. In another embodiment, the increase in yield is statistically significant. The growth enhancing activity of 2,4-D against 2,4-D resistant crops can be measured in a field test or a pot test. Herbicides with different mechanisms of action are generally known to either have an adverse effect on yield or have no effect on yield.

  In one aspect, a method is provided for improving the yield of 2,4-D resistant crops comprising treating a plant with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety.

  In one embodiment, the 2,4-D resistant crop is a transgenic plant transformed with an aryloxyalkanoate dioxygenase (AAD). In a further embodiment, the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD-12. In another embodiment, the herbicide comprising an aryloxyalkanoate moiety is a phenoxy herbicide or a phenoxyacetic acid herbicide. In a further embodiment, the herbicide comprising an aryloxyalkanoate moiety is 2,4-D. In a further embodiment, 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA).

  In one embodiment, the transgenic plant transformed with aryloxyalkanoate dioxygenase (AAD) is selected from cotton, soybean, and canola. In another embodiment, the treatment is performed at least once with a 2,4-D application rate that is also used in weed control. In another embodiment, the treatment is performed twice, with a 2,4-D application rate that is also used in weed control. In a further embodiment, 2,4-D is applied at the V3 and R2 growth stages of soybean with 2,4-D tolerance. In another embodiment, the treatment is performed at least three times with a 2,4-D application rate that is also used in weed control. In another embodiment, herbicides comprising aryloxyalkanoate moieties reach 2,4-D resistant crops via root absorption.

  In another embodiment, 2,4-D resistant crops are also treated with a different herbicide than 2,4-D for weed control. In a further embodiment, the herbicide different from 2,4-D is a phosphorus herbicide or an aryloxyphenoxypropionic acid herbicide. In further embodiments, the phosphorus herbicide comprises glyphosate, glufosinate, a derivative thereof, or a combination thereof. In further embodiments, the phosphorus herbicide is in the form of an ammonium salt, isopropylammonium salt, isopropylamine salt, or potassium salt. In another embodiment, the phosphorus herbicide reaches 2,4-D resistant crops via root absorption. In another embodiment, the aryloxyphenoxypropionic acid herbicide comprises chlorazihop, phenoxaprop, fluazihop, haloxyhop, quizalofop, a derivative thereof, or a combination thereof. In a further embodiment, the aryloxyphenoxypropionic acid herbicide reaches 2,4-D resistant crops via root absorption.

  In one embodiment, a 2,4-D resistant crop is treated at least once with 2,4-D from 25 g ae / ha to 5000 g ae / ha. In another embodiment, the 2,4-D resistant crop is treated at least once with 2,4-D from 100 g ae / ha to 2000 g ae / ha. In another embodiment, the 2,4-D resistant crop is treated at least once with 2,4-D from 100 g ae / ha to 2500 g ae / ha. In another embodiment, a 2,4-D resistant crop is treated at least once with 2,4-D from 1000 g ae / ha to 2000 g ae / ha. In a further embodiment, 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA).

In one embodiment, a method for improving the yield of 2,4-D resistant crops is provided. This method
(A) transforming a plant cell with a nucleic acid molecule comprising a nucleotide sequence encoding an aryloxyalkanoate dioxygenase (AAD);
(B) selecting transformed cells;
(C) regenerating the plant from the transformed cells;
(D) treating the plant with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety.

  In one embodiment, the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD-12. In another embodiment, the nucleic acid molecule comprises a selectable marker that is not an aryloxyalkanoate dioxygenase (AAD). In further or alternative embodiments, the selectable marker is a phosphinothricin acetyltransferase gene (pat) or a bialaphos resistance gene (bar). In another embodiment, the nucleic acid molecule is optimized for plants.

  In another aspect, there is provided the use of a herbicide comprising an aryloxyalkanoate moiety in the manufacture of a transgenic plant having 2,4-D resistance with increased yield compared to its non-transgenic parent plant. . In one embodiment, the herbicide comprising an aryloxyalkanoate moiety is 2,4-D. In a further embodiment, 2,4-D is applied at least once with 2,4-D from 25 g ae / ha to 5000 g / ha. In another embodiment, 2,4-D is applied at least once with 2,4-D from 100 g ae / ha to 2000 g ae / ha. In another embodiment, 2,4-D is applied at least once with 2,4-D from 100 g ae / ha to 2500 g ae / ha. In another embodiment, 2,4-D is applied at least once with 2,4-D from 1000 g ae / ha to 2000 g ae / ha. In a further embodiment, 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA). In a further embodiment, 2,4-D resistant crops are treated with 2,4-D at least twice before flowering. In another embodiment, the 2,4-D resistant crop is a transgenic plant transformed with an aryloxyalkanoate dioxygenase (AAD). In a further embodiment, the aryloxyalkanoate dioxygenase (AAD) is AAD-1 or AAD-12.

FIG. 2 illustrates a general chemical reaction catalyzed by the AAD-12 enzyme of the present invention. FIG. 5 shows a representative map of plasmid pDAB4468. FIG. 3 shows a representative map of plasmid pDAS1740.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the nucleotide sequence of AAD-12 from Delftia acidovorans.
SEQ ID NO: 2 is the translated protein sequence encoded by SEQ ID NO: 1.
SEQ ID NO: 3 is a nucleotide sequence optimized for AAD-12 plants (v1).
SEQ ID NO: 4 is the sequence of the translated protein encoded by SEQ ID NO: 3.
SEQ ID NO: 5 is the nucleotide sequence optimized for E. coli of AAD-12 (v2).
SEQ ID NO: 6 is the sequence of the M13 forward primer.
SEQ ID NO: 7 is the sequence of the M13 reverse primer.
SEQ ID NO: 8 is the sequence of the forward AAD-12 (v1) PTU primer.
SEQ ID NO: 9 is the sequence of reverse AAD-12 (v1) PTU primer.
SEQ ID NO: 10 is the sequence of the PCR primer encoding forward AAD-12 (v1).
Sequence number 11 is the arrangement | sequence of the PCR primer which codes reverse direction AAD-12 (v1).
SEQ ID NO: 12 shows the sequence of the “sdpacodF” AAD-12 (v1) primer.
SEQ ID NO: 13 shows the sequence of the “sdpacodR” AAD-12 (v1) primer.
SEQ ID NO: 14 shows the sequence of the “Brady Nco1” primer.
SEQ ID NO: 15 shows the sequence of the “Brady Sac1” primer.

Detailed Description of the Invention As used herein, the phrase "transformed" or "transformation" refers to the introduction of DNA into a cell. The phrase “transformants” or “transgenic” refers to plant cells, plants, etc. that have been transformed or have undergone a transformation procedure. Usually, the introduced DNA is in the form of a vector containing the inserted piece of DNA.

  As used herein, the phrase “selectable marker” or “selectable marker gene” refers to a plant, for example, to protect plant cells from a selective agent or to provide resistance / resistance to a selective agent. A gene that is optionally used in transformation. Only cells or plants that have received a functional selectable marker can divide or grow under conditions that have a selective agent. Examples of selective agents can include antibiotics including, for example, spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and hygromycin. These selectable markers include the gene for neomycin phosphotransferase (npt II), which expresses an enzyme that confers resistance to the antibiotic kanamycin, and the genes for the related antibiotics neomycin, paromomycin, gentamicin, and G418, or hygro The gene for hygromycin phosphotransferase (hpt) expressing an enzyme that confers resistance to mycin is included. Other selectable marker genes include Bar (resisting to BASTA® (glufosinate ammonium), or phosphinothricin (PPT)), acetolactate synthase (ALS, the first in the synthesis of branched chain amino acids). Resistance to inhibitors such as sulfonylurea (SU), imidazolinone (IMI), triazolopyrimidine (TP), pyrimidinyloxybenzoic acid (POB), and sulfonylaminocarbonyltriazolinone), glyphosate, , 4-D, as well as genes encoding herbicide resistance, including metal resistance or sensitivity. The phrase “marker positive” refers to a plant that has been transformed to contain a selectable marker gene.

  Various selectable or detectable markers can be incorporated into selected expression vectors to allow identification and selection of transformed plants, or transformants. For example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, proteins expressed from vectors, eg, precipitated proteins that mediate phosphinothricin resistance, or the reporter gene β-glucuronidase (GUS) ), Luciferase, green fluorescent protein (GFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT), including immunological methods for detecting other proteins such as alkaline phosphatase A number of methods are available to confirm the expression of selectable markers in plants (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, the entire contents of which are incorporated herein by reference) Cold sp see ring Harbor Press, N.Y., 2001).

  The selectable marker gene is used to select transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and resistance to herbicidal compounds. The gene to give is included. Herbicide resistance genes generally encode a modified target protein that is insensitive to the herbicide or an enzyme that degrades or detoxifies the herbicide in the plant before it can act. DeBlock et al. (1987) EMBO J., 6: 2513-2518, DeBlock et al. (1989) Plant Physiol., 91: 691-704, Fromm et al. (1990) 8: 833-839, Gordon-Kamm et al. (1990) 2: See 603-618). For example, resistance to glyphosate or sulfonylurea herbicides uses genes encoding mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS) It is obtained by doing. Resistance to glufosinate ammonium, bromoxynyl, and 2,4-dichlorophenoxyacetate (2,4-D) is due to the phosphinothricin acetyltransferase, nitrilase, or 2,4-dichlorophenoxyacetic acid monotoxin detoxifying the respective herbicide. It can be obtained by using a bacterial gene encoding oxygenase. Enzymes / genes for 2,4-D resistance have been previously disclosed in US 2009/0093366 and WO 2007/053482, the entire contents of which are hereby incorporated by reference.

  Other herbicides including imidazolinones or sulfonylureas can inhibit growth points or meristems. Exemplary genes in this class are mutations, for example as described by Lee et al., EMBO J. 7: 1241 (1988) and Miki et al., Theon. Appl. Genet. 80: 449 (1990), respectively. Encodes ALS and AHAS enzymes.

  The glyphosate resistance gene is mediated by the mutated 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) gene (introduction of recombinant nucleic acids and / or various forms of in vivo mutagenesis of the native EPSPs gene. ), AroA gene, and glyphosate acetyltransferase (GAT) gene, respectively). Other phosphono resistance genes include genes from Streptomyces species, including glufosinate (Streptomyces hygroscopicus and Streptomyces viridichromogenes). Phosphinothricin acetyltransferase (PAT) gene), and pyridineoxy or phenoxyproprionic acid and cyclohexone (a gene encoding an ACCase inhibitor), for example for plants against glyphosate See Shah et al., US Pat. No. 4,940,835 and Barry et al., US Pat. No. 6,248,876, which disclose nucleotide sequences in the form of EPSPs that can confer resistance. A DNA molecule encoding a mutated aroA gene can be obtained under ATCC accession number 39256, the nucleotide sequence of the mutated gene is a glutamine synthase that confers resistance to herbicides such as L-phosphinothricin Disclosed in US Pat. No. 4,769,061 to Comai, European Patent Application 0 333 033 to Kumada et al., And US Pat. No. 4,975,374 to Goodman et al., Which discloses the nucleotide sequence of the gene. ing. The nucleotide sequence of the PAT gene is provided in Leemans et al., European Patent Application 0 242 246. DeGreef et al., Bio / Technology 7:61 (1989) also describes the generation of transgenic plants that express a chimeric bar gene encoding PAT activity. Examples of genes that confer resistance to phenoxyproprionic acid and cyclohexone, including cetoxydim and haloxyhops, are described in Acc1-S1, described by Marshall et al., Theon. Appl. Genet. 83: 435 (1992). Acc1-S2 and Acc1-S3 genes. A GAT gene capable of conferring glyphosate resistance is described in Castle et al., WO2005012515. Genes that confer resistance to 2,4-D, fop and pyridyloxyauxin herbicides are described in WO2005107437 and US patent application Ser. No. 11 / 587,893.

  Other herbicides including triazine (psbA and 1s + gene) or benzonitrile (nitrilase gene) can inhibit photosynthesis. Przibila et al., Plant Cell 3: 169 (1991), describes the transformation of Chlamydomonas with a plasmid encoding a mutated psbA gene. The nucleotide sequence of the nitrilase gene is disclosed in Stalker US Pat. No. 4,810,648, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA encoding glutathione S-transferase has been described by Hayes et al., Biochem. J. 285: 173 (1992).

  For the purposes of the present invention, selectable marker genes include, but are not limited to, genes encoding the following: neomycin phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in Plant Science, 4 : 1-25), cyanamide hydrolase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 4250-4264), aspartate kinase, dihydrodipicolinate synthase (Perl et al. (1993) Bio / Technology, 11: 715-718), tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Bio., 22: 907-912), dihydrodipicolinate synthase and desensitized aspartade kinase (Perl et al. (1993) Bio / Technology, 11: 715-718), bar gene (Toki et al. (1992) Plant Physiol., 100: 1503-1507 and Meagher et al. (1996) and Crop Sci., 36: 1367), tryptophan decarboxylation. Enzyme (Goddijn et al. (1993) Plant Mol. Biol., 22: 907-912), Neo Isin phosphotransferase (NEO) (Southern et al. (1982) J. Mol. Appl. Gen., 1: 327, hygromycin phosphotransferase (HPT or HYG) (Shimizu et al. (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. 13D: 330), acetohydroxy acid synthase (Anderson et al., US Pat. No. 4,761,373, Haughn et al. (1988) Mol. Gen. Genet. 221 : 266), 5-enolpyruvyl-shikimate-phosphate synthase (aroA) (Comai et al. (1985) Nature 317: 741), haloaryl nitrilase (Stalker et al., Published PCT application WO 87/04181), acetyl complement Enzyme A carboxy Ase (Parker et al. (1990) Plant Physiol. 92: 1220), dihydropteroic acid synthase (sul I) (Guerineau et al. (1990) Plant Mol. Biol. 15: 127), and 32 kD photosystem II polypeptide (psbA) (Hirschberg et al. (1983) Science, 222: 1346).

  In addition, chloramphenicol (Herrera-Estrella et al. (1983) EMBO J., 2: 987-992), methotrexate (Herrera-Estrella et al. (1983) Nature, 303: 209-213, Meijer et al. (1991) Plant Mol Bio 16: 807-820 (1991), hygromycin (Waldron et al. (1985) Plant Mol. Biol., 5: 103-108, Zhijian et al. (1995) Plant Science, 108: 219-227 and Meijer et al. (1991). Plant Mol. Bio. 16: 807-820), streptomycin (Jones et al. (1987) Mol. Gen. Genet., 210: 86-91), spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res., 5: 131-137), bleomycin (Hille et al. (1986) Plant Mol. Biol., 7: 171-176), sulfonamides (Guerineau et al. (1990) Plant Mol. Bio., 15: 127-136), bromoxynil (Stalker et al. (1988) Science, 242: 419-423), 2,4-D (Streber et al. (1989) Bio / Technology, 7: 811-816), glyphosate (Shaw et al. (1986) Science, 233: 478-481), And phosphinothricin (DeBlock et al. (1987) EMBO J., 6: 2513-2518) Gene encoding resistance against also included. Unless otherwise stated, all references cited in this disclosure are incorporated herein by reference in its entirety.

  The above list of selectable markers and reporter genes is not intended to be limiting. Any reporter or selectable marker gene is encompassed by the present invention. If necessary, such genes can be sequenced by methods known in the art.

  Reporter and selectable marker genes are synthesized for optimal expression in plants. That is, the coding sequence of the gene has been modified to enhance expression in plants. Synthetic marker genes are designed to be expressed at higher levels in plants, resulting in higher transformation efficiency. Methods for gene synthesis optimization are available in the art. In fact, several genes have been optimized to increase the expression of gene products in plants.

  The marker gene sequence can be optimized for expression in a particular plant species or can be modified for optimal expression in a plant family. A preferred codon for a plant can be determined from the most frequent codon in the protein that is most abundantly expressed in a particular target plant species. See, for example, EPA 0359472, EPA 0385962, WO 91/16432, Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 3324-3328, and Murray et al. (1989), which are incorporated herein by reference. ) Nucleic Acids Research, 17: 477-498, US Pat. No. 5,380,831, and US Pat. No. 5,436,391. In this way, the nucleotide sequence can be optimized for expression in any plant. It should be understood that the entire gene sequence or any portion thereof may be optimized or synthetic. That is, fully optimized or partially optimized sequences can also be used.

  In addition, several transformation strategies have been developed that utilize an Agrobacterium-mediated transformation system. For example, the binary vector strategy is based on a two-plasmid system where the T-DNA is in a different plasmid than the rest of the Ti plasmid. In the co-integration strategy, a small portion of T-DNA is placed in the same vector as the foreign gene, which is then recombined with the Ti plasmid.

  As used herein, the phrase “plant” includes dicotyledonous and monocotyledonous plants. Examples of dicotyledonous plants include tobacco, Arabidopsis, soybean, tomato, papaya, canola, sunflower, cotton, alfalfa, potatoes, grapes, bean, pea, Brassica, chickpea, sugar beet, rapeseed, Includes watermelon, melon, pepper, peanuts, pumpkin, radish, spinach, pumpkin, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber, eggplant, and lettuce. Examples of monocotyledonous plants include corn, rice, wheat, sugar cane, barley, rye, sorghum, orchid, bamboo, banana, cattail, lily, oats, onion, millet, and triticale.

Targeted development of 2,4-D resistance genes and subsequent resistant crops offers excellent options for controlling broad-leaf glyphosate resistant (or highly resistant and shifted) weed species for in-crop applications provide. 2,4-D is a broad, relatively inexpensive, and robust broad-leaf herbicide that has excellent utility for growers if it can provide higher crop resistance in dicotyledonous and monocotyledonous crops as well. Provided. 2,4-D resistant transgenic dicotyledonous crops also have greater flexibility in application timing and quantity. 2,4-D target herbicide tolerance traits Further usefulness includes 2,4-D drift, volatilization, reversal (or other off-site migration phenomenon), misapplication, vandalism, etc. for normally sensitive crops Is its usefulness to prevent damage from. A further advantage of the AAD-12 gene is that, unlike all tfdA homologues that have been characterized to date, AAD-12 is achiral phenoxyauxin (eg 2,4-D, MCPA, 4-chlorophenoxyacetic acid). ) In addition to pyridyloxyacetic acid auxins (for example, triclopyr, fluoroxypyr). See Table 1. A general illustration of a chemical reaction catalyzed by the subject AAD-12 enzyme is shown in FIG. (The addition of O ₂ is stereospecific and the degradation of the intermediate to phenol and glyoxylic acid is spontaneous). The chemical structure in FIG. 1 illustrates the molecular main chain, and even though various R groups (such as those shown in Table 1) are included, they are not necessarily specifically illustrated in FIG. I want you to understand. Multiple mixtures of various phenoxy auxin combinations are used worldwide to address specific weed ranges and environmental conditions in various regions. The use of the AAD-12 gene in plants provides protection against a much wider range of auxin herbicides, thereby increasing the flexibility and range of weeds that can be controlled.

  A single gene (AAD-12) has now been identified that has never had innate tolerance when genetically engineered for expression in plants, or weeding these It has properties that allow the use of phenoxyauxin herbicides in plants that were not high enough to allow the use of the agent. In addition, AAD-12 can also provide protection against pyridyloxyacetic acid herbicides in plants where natural tolerance was not sufficient to allow selectivity, and the potential usefulness of these herbicides. Sex is expanding. Currently, plants containing AAD-12 alone can be treated sequentially or in tanks mixed with one, two or several phenoxyauxin herbicide combinations. The amount of each phenoxy auxin herbicide can range from 25 to 4000 g ae / ha, more typically from 100 to 2000 g ae / ha to control a wide range of dicotyledonous weeds. Similarly, a mixture of one, two or several pyridyloxyacetate auxin compounds may be applied to plants expressing AAD-12, reducing the risk of damage from the herbicide. The amount of each pyridyloxyacetic acid herbicide can range from 25 to 2000 g ae / ha, more typically from 35 to 840 g ae / ha to control additional dicotyledonous weeds.

  Glyphosate is used on a large scale to control a very wide range of broadleaf and grass weed species. However, repeated use of glyphosate in GTC and non-crop applications selects and continues to select weed shifts to naturally more resistant species or glyphosate-resistant biotypes. Tank mixed herbicide partners used in effective amounts that provide the same species of control but with different mechanisms of action are prescribed by most herbicide resistance management strategies as a way to delay the emergence of resistant weeds Yes. Stacking AAD-12 with glyphosate tolerance traits (and / or other herbicide tolerance traits) is the use of glyphosate, phenoxyauxin (eg 2,4-D) and pyridyloxyacetic acid auxin herbicides (eg triclopyr) May be able to provide a mechanism to allow control of glyphosate-resistant dicotyledonous weed species in GTC by selectively enabling in the same crop. The application of these herbicides can be simultaneous in a tank mixture containing two or more herbicides with different mechanisms of action, from sequential application as before planting, before emergence, or after emergence and from about 2 hours. It can be an individual application of a single herbicidal composition at a split application timing of about 3 months, or any combination of any number of herbicides representing the respective chemical class can be about It can be applied at any time from 7 months to crop harvest (or individual herbicide pre-harvest interval, whichever is shorter).

  With regard to timing of application, the amount of individual herbicides, and the ability to control difficult or resistant weeds, it is important to have flexibility in controlling a wide range of gramineous and broadleaf weeds. Application of glyphosate in crops with the glyphosate resistance gene / AAD-12 stacking can range from about 250-2500 g ae / ha and the phenoxy auxin herbicide (s) is about 25-4000 g ae. The pyridyloxyacetic acid auxin herbicide (s) can be applied at 25-2000 g ae / ha. The optimal combination (s) and timing of these applications will depend on the specific situation, species, and environment, and are best determined by engineers in the field of weed control with the benefit of this disclosure.

  Plantlets are typically resistant throughout the growth cycle. Transformed plants are typically resistant to new herbicide application at any point where the gene is expressed. Here, constitutive promoters tested so far (mainly CsVMV and AtUbi 10) are used to show resistance to 2,4-D throughout the life cycle. This is typically expected, but this is an improvement over other non-metabolic activities where, for example, tolerance can be significantly affected by reduced expression at the site of resistance mechanisms of action. is there. An example is Round Ready cotton, where plants were resistant when sprayed early, but when sprayed too slowly, glyphosate was concentrated in meristems (because it was not metabolized and moved) . The viral promoter Monsanto used is not well expressed in flowers. The present invention provides improvements in these respects.

  Herbicide formulations (eg, ester, acid or salt formulations, or soluble concentrates, emulsion concentrates, or soluble liquids) and tank mix additives (eg, adjuvants, surfactants, drift retarders, or adaptations) Agent) may significantly affect weed control from a given herbicide or a combination of one or more herbicides. Any combination of these with any of the foregoing herbicide chemistries is within the scope of the present invention.

  Those skilled in the art will also see the benefits of combining two or more mechanisms of action to increase the range of weeds to be controlled and / or the control of naturally more resistant or resistant weed species. This can also be extended beyond GTC to chemistry that enabled herbicide tolerance in crops, with human involvement (either transgenic or non-transgenic). Indeed, glyphosate resistance (eg resistant plant or bacteria EPSPS, glyphosate oxidoreductase (GOX), GAT), glufosinate resistance (eg Pat, bar), acetolactate synthase (ALS) inhibitory herbicide resistance (Eg, imidazolinones, sulfonylureas, triazolopyrimidinesulfonanilides, pyrmidinyl thiobenzoates, and other compounds = AHAS, Csr1, SurA, etc.), bromoxynyl resistance (eg, Bxn), HPPD (4-hydroxyl (hydroxl ) Phenyl-pyruvate dioxygenase) resistance to inhibitors of enzymes, resistance to inhibitors of phytoene desaturase (PDS), resistance to photosystem II inhibitor herbicides (eg psbA), photosystem I inhibition Herbicide Resistance to protoporphyrinogen oxidase IX (PPO) -inhibiting herbicides (eg PPO-1), resistance to phenylurea herbicides (eg CYP76B1), dicamba-degrading enzymes (see eg US200301535879), etc. Can be stacked alone or in combination to provide the ability to effectively control or prevent weed shift and / or resistance to any herbicide of the aforementioned class. In vivo modified EPSPS and class I, class II, and class III glyphosate resistance genes can be used in some preferred embodiments.

  With respect to additional herbicides, some additional preferred ALS inhibitors include, but are not limited to, sulfonylureas (such as chlorsulfuron, halosulfuron, nicosulfuron, sulfomethulone, sulfosulfuron, trifloxysulfuron), Includes imidazoloninones (such as imazamox, imazetapyr, imazaquin), triazolopyrimidine sulfonanilide (such as chloransrammethyl, diclosram, flurothram, flumethram, methotram, and penoxsulam), pyrimidinylthiobenzoates (such as bispyribac and pyrithiobac), and full carbazones It is. Some preferred HPPD inhibitors include, but are not limited to, mesotrione, isoxaflutole, and sulcotrione. Some preferred PPO inhibitors include, but are not limited to, full microlac, flumioxazin, flufenpyr, pyraflufen, flutiaceto, butaphenacyl, carfentrazone, sulfentrazone, and diphenyl ether (acifluorfen, fomesafen, Lactofen, and oxyfluorfen).

  In addition, AAD-12 alone, or AAD-12 stacked with one or more additional HTC traits, may include one or more additional inputs (eg, insect resistance, fungal resistance, or stress resistance) or It can be stacked with traits of output (eg, increased yield, improved oil profile, improved fiber quality, etc.). Thus, the present invention can be used to provide a complete agricultural package with improved crop quality that has the ability to control any number of agricultural aggression in a flexible and cost-effective manner. .

  The present invention is not only partially capable of degrading 2,4-D, but surprisingly, for example, an enzyme that also possesses novel properties that distinguish the enzyme of the present invention from previously known tfdA proteins. Related to identification. Although this enzyme has very low homology to tfdA, it is still possible to generally classify the genes of the present invention into the same whole family of α-ketoglutarate-dependent dioxygenases. This protein family is characterized by three conserved histidine residues in the “HX (D / E) X23-26 (T / S) X114-183HX10-13R” motif that includes the active site. Histidine coordinates Fe + 2 ions in the active site essential for catalytic activity (Hogan et al., 2000). The preliminary in vitro expression experiments described herein are tailored to help select new attributes. These experiments also show that the AAD-12 enzyme is another essence of the same class, disclosed in a previously filed patent application (PCT US / 2005/014737 filed May 2, 2005). It is unique compared to different enzymes. The AAD-1 enzyme of that application shares only about 25% sequence identity with the subject AAD-12 protein.

  More particularly, the present invention relates in part to the use of an enzyme that can degrade not only 2,4-D but also pyridyloxyacetic acid herbicides. To date, there are no α-ketoglutarate-dependent dioxygenase enzymes that have been reported to have the ability 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 AAD-12 (aryloxyalkanoate dioxygenase) genes and proteins.

  The protein of interest gave a positive test result for conversion from 2,4-D to 2,4-dichlorophenol ("DCP", herbicidal inactive) in the analytical assay. The partially purified protein of the present invention can rapidly convert in vitro 2,4-D to DCP. A further advantage provided by plants transformed with AAD-12 is the potential for parental herbicide (s) to be metabolized to an inactive form, thereby harvesting herbicide residues in the grain or fodder. Is to lower

  The present invention also includes a method for controlling weeds, comprising applying a pyridyloxyacetic acid herbicide and / or a phenoxyauxin herbicide to a plant containing the AAD-12 gene.

  In view of these discoveries, new plants are now provided that contain polynucleotides encoding this type of enzyme. So far, there has been no motive to produce such plants, and such plants effectively produce this enzyme, which not only gives phenoxy acid herbicides (such as 2,4-D) but also pyridyloxy. There was no expectation that resistance to acetic herbicides could be conferred. Thus, the present invention provides many advantages that have not previously been considered possible in the art.

  Publicly available strains (deposited in culture collections such as ATCC or DSMZ) can be obtained and screened for new genes using the techniques disclosed herein. For further screening and testing according to the present invention, the sequences disclosed herein can be used to amplify and clone homologous genes into recombinant expression systems.

  As discussed above in the background section, one organism that has been extensively studied for its ability to degrade 2,4-D is Ralstonia eutropha (Streber et al., 1987). The gene encoding the first enzyme in the degradation pathway is tfdA. See U.S. Patent No. 6,153,401 and GENBANK Accession No. M16730. TfdA catalyzes the conversion of 2,4-D acid to herbicidally inactive DCP via an α-ketoglutarate-dependent dioxygenase reaction (Smejkal et al., 2001). TfdA has been used in transgenic plants to confer 2,4-D resistance to dicotyledonous plants (eg cotton and tobacco) that are normally sensitive to 2,4-D (Streber et al., 1989, Lyon et al., 1989, Lyon et al., 1993). A 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 homologues are very similar to tfdA (greater than 85% amino acid identity) and have similar enzymatic properties to tfdA. However, a small collection of α-ketoglutarate-dependent dioxygenase homologs with a low level of homology to tfdA has now been identified.

  The present invention is in part a novel use of the distantly related enzyme sdpA from Delftia acidivorans (Westendorf et al., 2002, 2003), which has low homology to tfdA (31% amino acid identity). And regarding the amazing discovery of the function. This α-ketoglutarate-dependent dioxygenase enzyme purified in its native form has been previously shown to degrade 2,4-D and S-dichloroprop (Westendorf et al., 2002 and 2003). However, to date, there is no α-ketoglutarate-dependent dioxygenase enzyme that has been reported to have the ability to degrade pyridyloxyacetic acid chemical classes of herbicides. SdpA has never been expressed in plants and is partly due to the limited development of new HTC technology, largely due to the effectiveness, low cost and convenience of GTC. And there was no motivation to do that (Devine, 2005).

  In view of the new activity, the proteins and genes of the present invention are referred to herein as AAD-12 proteins and genes. AAD-12 is currently identified to degrade various phenoxyacetate auxin herbicides in vitro. However, it was surprisingly found that this enzyme can also degrade additional substrates of the class of aryloxyalkanoate molecules, as reported for the first time herein. Substrates of significant agricultural importance include pyridyloxyacetate auxin herbicides. This highly novel discovery underpins significant herbicide-tolerant crops (HTC) and the opportunity for selectable marker traits. This enzyme is unique in its ability to deliver herbicide degrading activity to a wide variety of broadleaf herbicides (phenoxyacetic acid and pyridyloxyacetate auxin).

  Accordingly, the present invention is based in part on recombinantly expressed aryloxyalkanoate dioxygenase enzyme (AAD-12), 2,4-dichlorophenoxyacetic acid, other phenoxyacetic auxin herbicides, and pyridyloxy It relates to the degradation of acetic acid herbicides. The present invention is also directed, in part, to the identification and use of a gene (AAD-12) encoding an aryloxyalkanoate dioxygenase degrading enzyme capable of degrading phenoxy and / or pyridyloxyauxin herbicides. Related.

  The enzyme of interest allows for transgenic expression and provides resistance to a combination of herbicides that control almost all broadleaf weeds. AAD-12 is, for example, other HTC traits [eg glyphosate resistance, glufosinate resistance, ALS inhibitor (eg imidazolinone, sulfonylurea, triazolopyrimidinesulfonanilide) resistance, bromoxynyl resistance, HPPD inhibitor Resistance, PPO inhibitor resistance, etc.] and excellent herbicide resistant crops for stacking with insect resistance traits (such as Cry1F, Cry1Ab, Cry34 / 45, other Bt proteins, or insecticidal proteins of non-gonococcal origin) HTC) can serve as a trait. Furthermore, AAD-12 can serve as a selectable marker to help select primary transformants of plants genetically engineered with a second gene or group of genes.

  Furthermore, the microbial gene of interest has been redesigned such that the protein is encoded by codons that are also biased in frequency of use in both monocotyledonous and dicotyledonous plants (hemicot). Arabidopsis, corn, tobacco, cotton, soybean, canola, and rice have been transformed with constructs containing AAD-12, demonstrating high levels of resistance to both phenoxy and pyridyloxyauxin herbicides doing. Accordingly, the present invention also relates to “plant optimized” genes encoding the proteins of the present invention.

  Oxyalkanoate groups are useful for introducing stable acid functional groups into herbicides. Acidic groups are a desirable attribute of herbicidal action and can therefore impart phloem mobility by “acid trapping” that can be incorporated into new herbicides for mobility purposes. Aspects of the invention also provide a mechanism for creating an HTC. There are many potential commercial and experimental herbicides that can serve as substrates for AAD-12. Thus, the use of the gene of interest may also lead to herbicide resistance to these other herbicides.

  The HTC traits of the present invention can be used in novel combinations with other HTC traits, including but not limited to glyphosate resistance. The combination of these traits creates a new method for controlling weed (etc.) species due to newly acquired or innate resistance to herbicides (eg glyphosate). Accordingly, within the scope of the present invention is a novel method for controlling weeds using herbicides, in addition to HTC traits, whose herbicide tolerance has been created by the enzyme in transgenic crops.

  The present invention can be applied, for example, in the context of commercializing the current glyphosate resistance trait and the stacked 2,4-D resistance trait in soybean. Thus, the present invention provides a tool for combating the selection of broadleaf weed species shifts and / or herbicide-resistant broadleaf weeds, resulting from growers' very high dependence on glyphosate for weed control of various crops To do.

  Transgenic expression of the subject AAD-12 gene is exemplified in, for example, Arabidopsis, tobacco, soybean, cotton, rice, corn and canola. Soybean is a preferred crop for transformation according to the present invention. However, the present invention can be utilized in several other monocotyledonous plants (such as grass or lawn) and dicotyledonous crops such as alfalfa, clover, tree species. Similarly, 2,4-D (or other AAD-12-substrate) can be used more actively by gramineous crops with moderate tolerance, and the increased resistance caused by this trait is It provides growers the opportunity to use these herbicides in a more effective amount and over a wider application timing without risk.

  Furthermore, the present invention provides a single gene that can provide resistance to herbicides that control broadleaf weeds. This gene can be utilized in multiple crops to allow the use of a wide range of herbicide combinations. The present invention can also control weeds that are resistant to current chemicals and also helps control the shifting weed range arising from current agricultural practices. The subject AAD-12 can also be used in an attempt to effectively detoxify additional herbicide substrates into a non-herbicidal form. Thus, the present invention provides for the development of additional HTC traits and / or selectable marker technology.

  In addition to or in addition to using the gene of interest to generate HTC, the gene of interest is also used as a selectable marker for successful selection of transformants in cell cultures, greenhouses, and fields. be able to. In biotechnology projects, there is a high inherent value as simply a selectable marker for the gene of interest. The promiscuity of AAD-12 in other aryloxyalkanoate auxin herbicides offers many opportunities to utilize this gene for HTC and / or selectable marker purposes.

  It is not easy to discuss the term “resistance” without using the verb “tolerant” or the adjective “tolerant”. The industry has taken countless hours to discuss herbicide-tolerant crops (HTC) versus herbicide-tolerant crops (HRC). HTC is the preferred term in the industry. However, the official definition of resistance by the American Weed Science Society of America is “the inheritance of plants that survive and reproduce after exposure to doses of herbicides that are normally lethal to wild-type. Sexual abilities.In plants, resistance can exist naturally or can be induced by techniques such as selection of mutants produced by genetic engineering or tissue culture or mutagenesis. Unless otherwise specified, the herbicide “resistant” as used herein is heritable as suggested by the current version of the Herbicide Handbook at the time of filing of this disclosure. And allows plants to grow and propagate in the presence of a typical effective herbicidal treatment with a given plant herbicide. As will be appreciated by those skilled in the art, plants may still be considered “resistant” even though some degree of plant damage from herbicidal exposure is evident. As used herein, the term “tolerance” is broader than the term “resistance” and is typically used at the same herbicidal dose in wild-type plants of the same genotype and “resistance” as defined herein. In particular, the improved ability of certain plants to withstand the damage induced by varying degrees of herbicides is included.

  Transfer of functional activity to a plant or bacterial system can include a nucleic acid sequence encoding the amino acid sequence of the protein of the invention incorporated into a protein expression vector appropriate for the host in which the vector resides. One method for obtaining a nucleic acid sequence that encodes a protein with functional activity is to use information deduced from the amino acid sequence of the protein, as disclosed herein, to derive native genetic material. To isolate from the bacterial species producing the protein of interest. The native sequence can be optimized for expression in plants, eg, as described in more detail below. Optimized polynucleotides can also be designed based on protein sequences.

  There are several ways to obtain a protein for use in accordance with the present invention. For example, antibodies against the proteins disclosed herein can be used to identify and isolate other proteins from a mixture of proteins. Specifically, antibodies can be raised against a portion of the protein that is most conservative or most distinctly different compared to other related proteins. These antibodies can then be used to specifically identify equivalent proteins with characteristic activities by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or immunoblotting. Antibodies against the proteins disclosed herein or equivalent proteins, or fragments of these proteins, can be readily prepared using standard procedures. Such antibodies are an aspect of the present invention. Antibodies of the present invention include monoclonal and polyclonal antibodies, preferably those produced in response to the exemplified or suggested protein.

  One skilled in the art will readily appreciate that the proteins (and genes) of the present invention can be obtained from a variety of sources. Since the entire herbicide-degrading operon is known to be encoded and integrated on a transposable element such as a plasmid, the proteins of the invention include, for example, recombinant and / or wild-type bacteria. It can be obtained from various microorganisms.

  Mutants of bacterial isolates can be made by procedures well known in the art. For example, a spore intangible mutant can be obtained by isolating ethyl methane sulfonate (EMS) mutagenesis. Mutant strains can also be generated by procedures well known in the art using ultraviolet light and nitrosoguanidine.

  A protein “from” or “obtainable from” any of the subject isolates mentioned or suggested herein refers to a protein (or similar protein), an isolate or another bacterium. It can be obtained from some other source such as a strain or plant. “Derived from” also has this implicit meaning and includes, for example, proteins obtainable from certain types of bacteria that have been modified for expression in plants. One skilled in the art will readily appreciate that given the disclosure of bacterial genes and proteins, plants can be manipulated to produce proteins. The polynucleotide and / or amino acid sequences disclosed herein are used to prepare antibody preparations, nucleic acid probes (eg, DNA, RNA, or PNA), and other related genes from other (natural) sources Can be used for screening and recovery.

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

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

  The polynucleotides of the invention can be used to form complete “genes” for encoding proteins or peptides in a desired host cell. For example, as will be readily understood by those skilled in the art, the polynucleotide of interest can be suitably placed under the control of a promoter in the target host, as is readily known in the art. The level of gene expression and temporal / tissue specific expression can greatly affect the usefulness of the present invention. In general, higher levels of protein expression of degraded genes result in faster and more complete degradation of the substrate (in this case the target herbicide). A promoter is desired to express the target gene at a high level, as long as high expression does not have a negative impact on plant health. Typically, it is desired that the AAD-12 gene be constitutively expressed in all tissues in order to fully protect the plant at all stages of growth. However, a plant-expressed resistance gene can be used instead. This makes it possible to use the target herbicide in the crop for weed control and subsequently to control the sexual reproduction of the target crop by application during the flowering stage. Furthermore, the desired level and timing of expression may also depend on the type of plant and the level of tolerance desired. In some preferred embodiments, a strong constitutive promoter or the like combined with a transcription enhancer is used to increase expression levels and enhance resistance to a desired level. Some such applications are described in more detail below before the Examples section.

  As is known to those skilled in the art, DNA is typically present in double-stranded form. In this arrangement, one strand is complementary to the other and vice versa. As DNA is replicated in plants (for example), additional complementary strands of DNA are produced. “Coding strand” is often used in the art to refer to the strand that binds to the antisense strand. mRNA is transcribed from the “antisense” strand of DNA. The “sense” or “coding” strand is read as a series of codons (a codon is read as a unit of 3 residues) that can be read as an open reading frame (ORF) to form the protein or peptide of interest. Specific amino acids can be specified). In order to produce a protein in vivo, typically a strand of DNA is transcribed into a complementary strand of mRNA, which is used as a template for the protein. Accordingly, the present invention includes the use of the exemplified polynucleotides shown in the accompanying sequence listing and / or equivalent, 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 can be identified and obtained using, for example, oligonucleotide probes. These probes can be detectable nucleotide sequences that can be detected by a suitable label or can be essentially fluorescent as described in International Application WO 93/16094. The probe (and the polynucleotide of the present invention) can be DNA, RNA, or PNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U, for RNA molecules), the synthetic probes (and polynucleotides) of the present invention contain inosine (all four And may also be used in place of a mixture of all four bases in synthetic probes) and / or other synthetic (unnatural) bases Can do. Thus, where reference is made herein to a synthetic degenerate oligonucleotide and “N” or “n” is generally used, “N” or “n” is G, A, T, C, or inosine. Can be. The ambiguity of the codes used herein conforms to the standard IUPAC nomenclature at the time of filing of this application (eg, R means A or G, Y means C or T) Such).

  As is well known in the art, when a probe molecule hybridizes with a nucleic acid sample, it is reasonably assumed that the probe and sample have substantial homology / similarity / identity. Preferably, polynucleotide hybridization is performed first, followed by techniques well known in the art as described, for example, in Keller, GH, MM Manak (1987) DNA Probes, Stockton Press, New York, NY, pages 169-170. Wash under low, moderate or high stringency conditions. For example, as described therein, low stringency conditions can be achieved using 2 × SSC (standard citrated saline) /0.1% SDS (sodium dodecyl sulfate) for 15 minutes at room temperature. This can be achieved by first washing. Typically two washes are performed. Thereafter, high stringency can be achieved by lowering the salt concentration and / or raising the temperature. For example, following the above wash, 0.1 × SSC / 0.1% SDS is used and washed twice at room temperature for 15 minutes each, followed by 0.1 × SSC / 0.1% SDS. Can be washed at 55 ° C. for 30 minutes each. These temperatures can be used with those described herein, and other hybridization and wash protocols known to those skilled in the art (eg, using SSPE instead of SSC as a salt). it can). 2 × SSC / 0.1% SDS can be prepared by adding 50 ml of 20 × SSC and 5 ml of 10% SDS to 445 ml of water. 20 × SSC was combined with NaCl (175.3 g / 0.150 M), sodium citrate (88.2 g / 0.015 M), and water, adjusted to pH 7.0 with 10 N NaOH, then volume Can be adjusted to 1 liter. 10% SDS can be prepared by dissolving 10 g SDS in 50 ml autoclave water and then diluting to 100 ml.

  Detection of the probe provides a means for determining in a known manner whether hybridization has been maintained. Such probe analysis provides a rapid method for identifying the genes of the present invention. Nucleotide segments used as probes according to the present invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers for amplifying the gene of the present invention.

  The hybridization characteristics of molecules can be used to define the polynucleotides of the invention. Accordingly, the present invention includes polynucleotides (and / or their complements, preferably their full complements) that hybridize to the polynucleotides exemplified herein. That is, one method for defining a gene (and the protein it encodes) is, for example, hybridizing to a known or specifically exemplified gene (among the conditions specifically disclosed herein). Due to its ability (under any conditions).

  As used herein, hybridization “stringency” conditions refer to conditions that achieve the same or approximately the same degree of hybridization specificity as those used by the Applicants. Specifically, hybridization of immobilized DNA on Southern blots using a probe specific for a gene labeled with 32P can be performed by standard methods (see, eg, Maniatis et al., 1982). In general, hybridization and subsequent washing can be performed under conditions that allow detection of the target sequence. For double-stranded DNA gene probes, hybridization is performed overnight at 20-25 ° C. below the melting temperature (Tm) of the DNA hybrid, 6 × SSPE, 5 × Denhardt's solution, 0.1% SDS, 0. It can be carried out in 1 mg / ml denatured DNA.

  Washing can typically be performed as follows: (1) 2 times, 15 minutes at room temperature, 1 × SSPE, in 0.1% SDS (low stringency wash), and (2 ) Once at Tm-20 ° C. for 15 minutes in 0.2 × SSPE, 0.1% SDS (moderate stringency wash).

  For oligonucleotide probes, hybridization is performed overnight at 10-20 ° C. below the melting temperature (Tm) of the hybrid, 6 × SSPE, 5 × Denhardt's solution, 0.1% SDS, 0.1 mg / ml denaturation. It can be performed in DNA.

  Washing can typically be performed as follows: (1) 2 × 15 minutes at room temperature, 1 × SSPE, 0.1% SDS (low stringency wash), (2) 1 1 × SSPE in 0.1% SDS (moderate stringency wash) for 15 minutes at hybridization temperature.

  In general, the stringency can be changed by changing the salt and / or temperature. For labeled DNA fragments longer than about 70 bases, the following conditions can be used: (1) Low: 1 or 2 × SSPE, room temperature, (2) Low: 1 or 2 × SSPE, 42 ° C, (3) moderate: 0.2 x or 1 x SSPE, 65 ° C, or (4) high: 0.1 x SSPE, 65 ° C.

  Duplex formation and stability depend on substantial complementarity between the two strands of the hybrid, and as noted above, a certain degree of mismatch is tolerated. Accordingly, the probe sequences of the present invention include mutations (both single and multiple), deletions, insertions, and combinations of the described sequences, wherein the mutations, insertions and deletions are of interest for the target Allows formation of stable hybrids with the polynucleotide. Many methods can cause mutations, insertions, and deletions in a given polynucleotide sequence, and these methods are known to those skilled in the art. Other methods may also be known in the future.

  PCR technology: Polymerase chain reaction (PCR) is a repetitive, enzymatic, primed synthesis of nucleic acid sequences. This procedure is well known and commonly used by those skilled in the art (Mullis, US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, (See Saiki et al., 1985). PCR is based on enzymatic amplification of a DNA fragment of interest flanked by two oligonucleotide primers that hybridize with opposite strands of the target sequence. The primers are preferably oriented so that the 3 'ends are pointing towards each other. Thermal cycling of the template, annealing of the primer and its complementary sequence, and repeated cycles of extension of the annealed primer with DNA polymerase result in amplification of the segment defined by the 5 'end of the PCR primer. Since each primer extension product can serve as a template for other primers, each cycle essentially doubles the amount of DNA fragments produced in the previous cycle. This results in an exponential accumulation of specific target fragments up to millions of times within hours. By using a thermostable DNA polymerase such as Tag polymerase isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be made fully automatic. Other enzymes that can be used are known to those skilled in the art.

  The exemplified DNA sequence or a segment thereof can be used as a primer for PCR amplification. In performing PCR amplification, a certain degree of mismatch between the primer and the template can be tolerated. Accordingly, mutations, deletions and insertions of the exemplified primers (especially addition of nucleotides at the 5 'end) are within the scope of the invention. Mutations, insertions and deletions can be made in a given primer by methods known to those skilled in the art.

  Gene and protein modification: The gene and protein of interest can be fused with other genes and proteins to produce chimeric or fusion proteins. Genes and proteins useful according to the present invention include not only the full-length sequences specifically exemplified, but also those sequences, variants, mutants, chimeras, and portions, segments and / or fragments (consecutive) of fusions thereof. Including internal and / or terminal deletions compared to fragments and full-length molecules). The protein of the present invention can have a substituted amino acid as long as it retains the desired functional activity. A “mutant” gene has a nucleotide sequence that encodes the same protein as the exemplified protein, or an equivalent protein having equivalent or similar activity.

  The top two results of the BLAST search using the native aad-12 nucleotide sequence show a reasonable level of homology (about 85%) over the 120 base pair sequence. Hybridization under specific conditions is expected to include these two sequences. See GENBANK accession numbers DQ406818.1 (893329742, Rhodoferax) and AJ6288601.1 (44903451, Sphingomonas). Rhodoferax is very similar to Delftia, but Sphingomonas is a completely different phylogenetic class.

  The terms “mutant protein” and “equivalent protein” refer to a protein having the same or essentially the same biological / functional activity as the exemplified protein for the target substrate and equivalent sequence. As used herein, reference to an “equivalent” sequence refers to a sequence having amino acid substitutions, deletions, additions or insertions that improve activity or do not adversely affect to a significant extent. Fragments that retain activity are also included in this definition. Fragments and other equivalents that retain the same or similar function or activity as the corresponding fragment of the exemplified protein are within the scope of the invention. Changes, such as amino acid substitutions or additions, to increase (or decrease) the protease stability of the protein (without significantly or substantially reducing the functional activity of the protein), to remove or add restriction sites Etc. for various purposes. Genetic variants can be readily constructed using, for example, standard techniques for creating point mutations.

  Further, for example, US Pat. No. 5,605,793 describes a method for generating additional molecular diversity by using DNA reconstruction after random or intensive fragmentation. This can be referred to as gene “shuffling” and typically involves mixing two or more fragments (of the desired size) of different DNA molecules and then performing repetitive rounds of regeneration. This can improve the activity of the protein encoded by the initiation gene. The result is a chimeric protein with improved activity, altered substrate specificity, increased enzyme stability, altered stereospecificity, or other characteristics.

  “Shuffling” can be designed and targeted after obtaining and examining atomic 3D (three-dimensional) coordinates and crystal structure of the protein of interest. Thus, “focused shuffling” preferably identifies proteins that are ideal for modification, such as segments exposed to the surface, rather than internal segments involved in protein folding and essential 3D structural integrity. Can be directed to the segment.

  Specific changes to the “active site” of an enzyme can be made to affect the intrinsic function of activity or stereospecificity. Muller et al. (2006) .The known tauD crystal structure was used as a model dioxygenase to determine active site residues while bound to its inherent substrate taurine., Elkins et al. (2002) `` X-ray crystal structure of Escerichia coli taurine / alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates ", Biochemistry 41 (16): 5185-5192. See Chakrabarti et al., PNAS, (August 23, 2005), 102 (34): 12035-12040 for optimization and design possibilities of the enzyme active site sequence.

  Full-length gene fragments can be made according to standard procedures using commercially available exonucleases or endonucleases. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cleave nucleotides from the ends of these genes. In addition, a gene encoding an active fragment may be obtained using various restriction enzymes. Proteases may be used to directly obtain active fragments of these proteins.

  It is within the scope of the invention disclosed herein that a protein can still be cleaved to retain functional activity. “Cleaved protein” means that even if a portion of the protein is cleaved, the remaining cleaved protein can retain and exhibit the desired activity after cleavage. Cleavage can be achieved by various proteases. In addition, the effectively cleaved protein may be a molecular biological technique that removes the DNA base encoding the protein either by digestion with restriction endonucleases or other techniques available to those skilled in the art. Can be generated using After cleavage, the protein is expressed in heterologous systems such as E. coli, baculoviruses, plant-based virus systems, yeast, and then placed in the insect assay disclosed herein for activity. Can be determined. It is well known in the art that it has been successful to generate cleaved proteins to retain functional activity while having less than the full length full length sequence. For example, B.I. t. The protein can be used in a truncated (core protein) form (see, eg, Hofte et al. (1989) and Adang et al. (1985)). As used herein, the term “protein” can include functionally active fragments.

  Unless otherwise specified, the percent sequence identity and / or similarity of two nucleic acids used herein is determined using the algorithm of Karlin and Altschul, 1990, modified as described in Karlin and Altschul 1993. To decide. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. The BLAST nucleotide search is performed using the NBLAST program, score = 100, word length = 12. Gapped BLAST can be used as described in Altschul et al., 1997. When using BLAST and a BLAST program with a gap, initial setting parameters of the respective programs (NBLAST and XBLAST) are used. See NCBI / NIH website. To obtain a gapped alignment for comparison purposes, the AlignX function of Vector NTI Suite 8 (InforMax, Inc., North Bethesda, MD, USA) was used with default parameters. These had a gap opening penalty of 15, a gap extension penalty of 6.66, and a gap separation penalty range of 8.

  It is also possible to change various properties and three-dimensional features of a protein without adversely affecting the activity / functionality of the protein. Conservative amino acid substitutions can be tolerated / can be done without adversely affecting the activity and / or three-dimensional configuration of the molecule. Amino acids can be assigned to the following classes: nonpolar, uncharged polar, basic, and acidic. Conservative substitutions that replace one class of amino acids with another of the same type are within the scope of the invention, so long as the substitution is not detrimental to the biological activity of the compound. Table 1 provides a list of examples of amino acids belonging to each class. In some examples, non-conservative substitutions can also be made. However, preferred substitutions do not significantly impair the functional / biological activity of the protein.

  As used herein, references to “isolated” polynucleotides and / or “purified” proteins refer to these molecules when they are not associated with other molecules found together in nature. Thus, reference to “isolated” and / or “purified” refers to the involvement of “human hands” as described herein. For example, a bacterial “gene” of the present invention placed in a plant for expression is an “isolated polynucleotide”. Similarly, proteins derived from bacterial proteins and produced by plants are “isolated proteins”.

  Due to the degeneracy / redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of one of ordinary skill in the art to create alternative DNA sequences that encode the same or essentially the same protein. These mutant DNA sequences are within the scope of the present invention. This is described in more detail in the section entitled "Optimizing sequences for expression in plants" below.

  Optimization of sequences for expression in plants: In order to obtain high expression of heterologous genes in plants, the genes can be engineered to be expressed more efficiently in (the cytoplasm of) plant cells. Generally preferred. Maize is one such plant where it may be preferable to redesign the heterologous gene (s) prior to transformation in order to increase its expression level in the plant. Thus, an additional step in the design of genes encoding bacterial proteins uses codon bias closely matched to the target plant sequence, whether dicotyledonous or monocotyledonous species. Re-engineering heterologous genes for optimal expression. The sequences can also be optimized for expression in any of the more specific types of plants described elsewhere in this specification.

  Transgenic host: The gene encoding the protein of the invention can be introduced into a variety of microbial or plant hosts. The present invention includes transgenic plant cells and transgenic plants. Preferred plants (and plant cells) are corn, Arabidopsis, tobacco, soybean, cotton, canola, rice, wheat, lawn, legumes (eg alfalfa and clover), grass and the like. Also, other types of transgenic plants such as fruits, vegetables, ornamental plants, and trees can be made according to the present invention. More generally, dicotyledonous plants and / or monocotyledonous plants can be used in various aspects of the present invention.

  In preferred embodiments, gene expression results in intracellular production (and maintenance) of the protein (s) of interest, either directly or indirectly. In this manner, plants can be given herbicide resistance. Such hosts can be referred to as transgenic, recombinant, transformed, and / or transfected hosts and / or cells. In some aspects of the invention (eg, when cloning and preparing a gene of interest), microbial (preferably bacterial) cells can be generated and used according to standard techniques, with the benefit of this disclosure.

  Plant cells transfected with the polynucleotide of the present invention can be regenerated into whole plants. The invention includes cell cultures including tissue cell cultures, liquid cultures, and plate cultures. Also included within the scope of the present invention are seeds produced by and / or used to produce the plants of the present invention. Other plant tissues and parts are also included in the present invention. Similarly, the present invention includes a method for producing a plant or cell comprising a polynucleotide of the present invention. One preferred method of producing such plants is by planting the seeds of the present invention.

  Although plants may be preferred, the invention also includes the production of highly active recombinant AAD-12 in, for example, a Pseudomonas fluorescens (Pf) host strain. The present invention includes preferred growth temperatures for maintaining soluble active AAD-12 in this host, fermentation conditions where AAD-12 is produced above 40% of total cellular protein, or at least 10 g / L produced, Pf A purification process that results in a high recovery of active recombinant AAD-12 from the host, a purification scheme that yields at least 10 g of active AAD-12 per kg of cells, a purification scheme that can yield 20 g of active AAD-12 per kg of cells, Formulation processes that can store and re-store AAD-12 activity in solution and lyophilization processes that can retain AAD-12 activity for long-term storage and shelf life are included.

  Insertion of genes to form transgenic hosts: One aspect of the present invention is the transformation / trait of plants, plant cells, and other host cells using the polynucleotides of the invention that express the proteins of the invention. It is an import. Plants transformed in this way can be given resistance to various herbicides having various mechanisms of action.

  A variety of methods are available for introducing a gene encoding a desired protein into a target host under conditions that allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in US Pat. No. 5,135,867.

  Vectors comprising AAD-12 polynucleotides are included within the scope of the present invention. For example, a number of cloning vectors containing a replication system in E. coli and markers that allow selection of transformed cells are available for preparing insertions of foreign genes into higher plants. Vectors include, for example, pBR322, pUC series, M13 mp series, pACYC184, and the like. Thus, the protein coding sequence can be inserted into an appropriate restriction site in the vector. The resulting plasmid is used for transformation into E. coli. E. coli cells are cultured in a suitable nutrient medium and then harvested and lysed. The plasmid is recovered by purification and removal from the genomic DNA. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are commonly performed as analytical methods. After each manipulation, the used DNA sequence can be digested by restriction and combined with the next DNA sequence. Each plasmid sequence can be cloned into the same or another plasmid. Depending on the method of inserting the desired gene into the plant, other DNA sequences may be required. For example, when a Ti or Ri plasmid is used for transformation of plant cells, at least the right border (in many cases, the left and right borders) of Ti or Ri plasmid T-DNA is bound as a flanking region of the gene to be inserted. It is necessary to let The use of T-DNA for plant cell transformation has been intensively studied and is described in EP 120 516, Hoekema (1985), Fraley et al. (1986), and An et al. (1985).

  A number of techniques are available for inserting DNA into plant host cells. These techniques include transformation, fusion, injection, and particle gun (microparticle irradiation) using T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformant. ), Silicon carbide soot crystals, aerosol beams, PEG, or electroporation, and other possible methods. When Agrobacterium is used for transformation, the DNA to be inserted must be cloned into a special plasmid, ie either in an intermediate vector or in a binary vector. The intermediate vector can be integrated into the Ti or Ri plasmid by homologous recombination thanks to a sequence homologous to the sequence in T-DNA. The Ti or Ri plasmid also contains a vir region necessary for T-DNA transfer. An intermediate vector cannot replicate itself in Agrobacterium. The intermediate vector can be transferred into Agrobacterium tumefaciens by a helper plasmid (conjugation). Binary vectors can replicate themselves in both E. coli and Agrobacterium. These include a selectable marker gene and a linker or polylinker surrounded by left and right border regions of T-DNA. They can be transformed directly into Agrobacterium (Holsters, 1978). The Agrobacterium used as a host cell should contain a plasmid carrying the vir region. The vir region is necessary for the transfer of T-DNA into plant cells. Additional T-DNA can be included. The bacteria transformed in this way are used for transformation of plant cells. Plant explants can be advantageously cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes in order to transfer the DNA into plant cells. Thereafter, the whole plant can be loaded with antibiotics or biocides for selection from infected plant material (eg, leaf pieces, stem segments, roots, but also protoplasts or suspension cultured cells). Can be regenerated in fresh medium. The plants thus obtained can then be tested for the presence of the inserted DNA. In the case of injection and electroporation no special requirements are made on the plasmid. For example, a normal plasmid such as a pUC derivative can be used.

  Transformed cells grow in plants in the normal manner. They can form germ cells and transmit the transformed trait (s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants having the same transformed genetic factor or other genetic factors. The resulting hybrid individuals have corresponding phenotypic characteristics. In some preferred embodiments of the invention, the gene encoding the bacterial protein is expressed from a transcription unit inserted into the plant genome. Preferably, the transcription unit is a recombinant vector capable of stable integration into the plant genome and enabling selection of transformed plant lines that express mRNA encoding the protein.

  After the inserted DNA is integrated into the genome, it is relatively stable there (and does not come out again). This usually contains a selectable marker that confers resistance to biocides or antibiotics, such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol, amongst transformed plant cells. Also, plant selectable markers typically include glufosinate (eg PAT / bar), glyphosate (EPSPS), ALS inhibitors (eg imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide, etc.), bromoxynil, HPPD inhibition Resistance to various herbicides such as drug resistance, PPO inhibitors, ACC-ase inhibitors can also be provided. Thus, the marker used individually should allow selection of transformed cells rather than cells that do not contain the inserted DNA. The gene of interest (s) is preferably expressed by either a constitutive or inducible promoter in the plant cell. After being expressed, the mRNA is translated into a protein, thereby incorporating the target amino acid into the protein. The gene encoding a protein expressed in plant cells can be under the control of a constitutive promoter, tissue-specific promoter, or inducible promoter.

  There are several techniques for introducing foreign recombinant vectors into plant cells and for obtaining plants that stably maintain and express the introduced gene. Such techniques include the direct introduction of genetic material coated on microparticles into cells (Cornell US Pat. No. 4,945,050 and DowElanco, currently Dow AgroSciences, LLC No. 5, 141, 131). In addition, plants may be transformed using Agrobacterium technology, US Patent No. 5,177,010 from Toledo University, US Patent No. 5,104,310 from Texas A & M University, European Patent Application No. 0131624B1, Schipperpert European Patent Applications No. 120516, 159418B1 and 176,112, Schipperpert US Pat. Nos. 5,149,645, 5,469,976, 5,464,763 No. 4,940,838 and No. 4,693,976, all of Max Planck's European Patent Applications No. 116718, No. 290799, No. 320500, Japanese Tobacco Inc. European Patent Application No. 604662, No. 627752, and US Pat. No. 5,591,616, Ciba Geigy, now Syngenta European Patent Applications 0267159, 0292435, and US Pat. No. 5,231,019, both of which are Calgene US Pat. Nos. 5,463,174 and 4,762. No. 785, as well as Agracetus, US Pat. Nos. 5,004,863 and 5,159,135. Other transformation techniques include the soot crystal technique. See both Zeneca, currently Syngenta US Pat. Nos. 5,302,523 and 5,464,765. Other direct DNA delivery transformation techniques include aerosol beam techniques. See US Pat. No. 6,809,232. Electroporation techniques have also been used to transform plants. See Boyce Thompson Institute's WO 87/06614, both of Dekalb US Pat. Nos. 5,472,869 and 5,384,253, both of which are Plant Genetic Systems WO 92/09696 and WO 93/21335. . Furthermore, a transgenic plant expressing the protein of interest can also be generated using a viral vector. For example, Mycogen Plant Science and Ciba-Geigy (now Syngenta) U.S. Pat. No. 5,569,597, and both Biosource, now Large Scale Biology U.S. Pat. Nos. 5,589,367 and 5,316. , 931 can be used to transform monocotyledonous plants with viral vectors.

  As already mentioned, the manner in which the DNA construct is introduced into the plant host is not critical to the invention. Any method that results in efficient transformation can be used. For example, various methods for transformation of plant cells are described herein, including performing Agrobacterium-mediated transformation using Ti or Ri plasmids or the like. It is. In many cases, it is desirable that one or both sides of the construct used for transformation, more specifically the right border, border the T-DNA border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as the mode of transformation, but the T-DNA border is not compatible with other transformations. Use can also be found in the form. When Agrobacterium is used for transformation of plant cells, vectors that can be introduced into the host for homologous recombination with T-DNA or Ti or Ri plasmids present in the host can be used. . Introduction of the vector can be done by electroporation, triparental mating and other techniques known to those skilled in the art for transforming gram negative bacteria. The mode of vector transformation into the Agrobacterium host is not critical to the present invention. Ti or Ri plasmids containing T-DNA for recombination may or may not cause goal formation, but are not critical to the present invention so long as the vir gene is present in the host.

  In some cases where Agrobacterium is used for transformation, expression constructs that are within the T-DNA boundary may be expressed as pRK2 or its derivatives as described in Ditta et al. (1980) and EPO 0 120 515, etc. Insert into a wide range of vectors. Within the expression construct and T-DNA are included one or more markers as described herein that allow for selection of transformed Agrobacterium and transformed plant cells. The particular marker used is not critical to the invention, and the preferred marker will depend on the host and construct used.

  Transforming plant cells using Agrobacterium requires a sufficient amount of time to allow the explants to be combined with the transformed Agrobacterium to allow the transformation. Incubate between. After transformation, Agrobacterium is killed by selection with an appropriate antibiotic and plant cells are cultured using an appropriate selection medium. After callus formation, shoot formation can be promoted by using appropriate plant hormones according to methods well known in the field of plant tissue culture and plant regeneration. However, the callus intermediate stage is not always necessary. After shoot formation, plant regeneration can be completed by transferring the plant cells to a medium that promotes root formation. Thereafter, the plant may be grown into seeds, which can be used to establish the next generation. Regardless of the transformation technique, the gene encoding the bacterial protein is preferably expressed in the plant cell by including in the vector a plant promoter regulatory element, a 3 ′ untranslated transcription termination region such as Nos, etc. It is incorporated into a gene transfer vector adapted to express the gene.

  In addition to numerous techniques for transforming plants, the type of tissue contacted with foreign genes can also be varied. Such tissues include, but are not limited to, embryogenic tissues, callus tissue types I, II, and III, hypocotyls, meristems, root tissues, tissues for expression in the phloem, etc. . Using the appropriate techniques described herein, almost all plant tissue can be transformed during dedifferentiation.

  As mentioned above, various selectable markers can be used if desired. Specific marker priorities are at the discretion of one of ordinary skill in the art, but any of the following selectable markers can function as selectable markers, any other not listed herein. Can be used with other genes. Such selectable markers include, but are not limited to, transposon Tn5 aminoglycoside phosphotransferase gene (Aph II), hygromycin resistance, methotrexate resistance, encoding resistance to the antibiotics kanamycin, neomycin and G41. Sex, genes encoding resistance to or resistance to glyphosate, phosphinothricin (bialaphos or glufosinate), ALS-inhibiting herbicides (imidazolinones, sulfonylureas and triazolopyrimidine herbicides), ACC-ase inhibitors ( Examples include aylyloxypropionates or cyclohexanediones) and bromoxynyl and HPPD inhibitors (eg mesotrione).

  In addition to a selectable marker, it may be desirable to use a reporter gene. In some examples, the reporter gene may be used with or without a selectable marker. A reporter gene is a gene that typically encodes a protein that is not present in the recipient organism or tissue and that typically results in some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al., 1988. Preferred reporter genes include E. coli uidA locus beta-glucuronidase (GUS), E. coli Tn9 chloramphenicol acetyltransferase gene, bioluminescent jellyfish Aequorea the green fluorescent protein from victoria), the luciferase gene from the firefly Photinus pyralis. Thereafter, an assay may be performed to detect the expression of the reporter gene at the appropriate time after the gene has been introduced into the recipient cell. A preferred such assay is a gene encoding beta-glucuronidase (GUS) at the uidA locus of E. coli as described in Jefferson et al. (1987) to identify transformed cells. With the use of.

  In addition to plant promoter regulatory elements, promoter regulatory elements from various sources can be efficiently used in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin such as octopine synthase promoter, nopaline synthase promoter, mannopine synthase promoter, and cauliflower mosaic virus (35S and 19S), 35T (this is a re-engineered 35S promoter, US Pat. No. 6,166,302, especially see Example 7E) and other promoters of viral origin can be used. Plant promoter regulatory elements include, but are not limited to, ribulose-1,6-diphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat shock Promoters and tissue specific promoters are included. Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences may be present, which may improve transcription efficiency or DNA integration. Such elements may be necessary or unnecessary for the function of the DNA, but they can provide better expression or function of the DNA by affecting transcription, mRNA stability, etc. Such elements can be included in the DNA as desired to obtain optimal performance of the transformed DNA in plants. Exemplary elements include, but are not limited to, Adh-intron 1, Adh-intron 6, alfalfa mosaic virus coat protein leader sequence, osmotin UTR sequence, corn streak virus coat protein leader sequence, and available to those of skill in the art Other things are included. A constitutive promoter regulatory element may also be used, thereby directing constant and continuous gene expression in any cell type (eg, actin, ubiquitin, CaMV 35S, etc.). Tissue specific promoter regulatory elements are responsible for gene expression in specific cells or tissue types such as leaves or seeds (eg, zein, oleosin, napin, ACP, globulin, etc.) and these may also be used.

  Promoter regulatory elements can also be active (or inactive) during certain stages of plant development and active in plant tissues and organs. Examples of such include, but are not limited to, pollen specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed-endosperm specific, or vegetative phase specific promoter regulation. Elements etc. are included. Under certain circumstances, expression of genes in response to specific signals such as physical stimuli (heat shock genes), light (RUBP carboxylase), hormones (Em), metabolites, compounds (tetracycline responsiveness), and stress In some cases, it may be desirable to use inducible promoter regulatory elements. Other desirable transcription and translation elements that function in plants may be used. A number of plant-specific gene transfer vectors are known in the art.

  Bacterial proteins can also be expressed using systems based on plant RNA viruses. In that case, the gene encoding the protein can be inserted into the coat promoter region of an appropriate plant virus that infects the target host plant. The protein can then be expressed, thus providing protection from plant herbicide damage. Plant RNA virus-based systems are available from Mycogen Plant Sciences, Inc. U.S. Pat. Nos. 5,500,360 and Biosource, currently Large Scale Biology U.S. Pat. Nos. 5,316,931 and 5,589,367.

  A means of further increasing resistance or resistance levels. It has been shown herein that novel herbicide resistance traits can be imparted to plants of the present invention without observable adverse effects on phenotypes including yield. Such plants are within the scope of the present invention. The plants exemplified and suggested herein can tolerate typical application levels of 2 ×, 3 ×, 4 ×, and 5 ×, for example, of at least one target herbicide. These improvements in tolerance levels are within the scope of the present invention. For example, various techniques are known in the art and are expected to be optimized and further developed to increase the expression of a given gene.

  One such method includes increasing the copy number of the subject AAD-12 gene (such as in an expression cassette). It is also possible to select transformation events for those with multiple copies of the gene.

  Strong promoters and enhancers can be used to “supercharge” expression. Examples of such promoters include the preferred 35T promoter using the 35S enhancer. 35S, corn ubiquitin, Arabidopsis ubiquitin, A. t. Actin, and the CSMV promoter are included for such use. Other strong viral promoters are also preferred. Enhancers include 4 OCS and 35S double enhancers. Matrix attachment regions (MARs) can also be used to increase transformation efficiency and transgene expression.

  Shuffling (directed evolution) and transcription factors can also be used in embodiments according to the present invention.

  It is also possible to design mutant proteins that differ at the sequence level but retain the same or similar overall essential three-dimensional structure, surface charge distribution, and the like. For example, US Pat. No. 7,058,515, Larson et al., Protein Sci. 2002 11: 2804-2813, “Thoroughly sampling sequence space: Large-scale protein design of structural ensembles”, Crameri et al., Nature Biotechnology 15, 436- 438 (1997), “Molecular evolution of an arsenate detoxification pathway by DNA shuffling”, Stemmer, WPC 1994. “DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution” Proc. Natl. Acad. Sci. USA 91: 10747-10751, Stemmer, WPC 1994. "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370: 389-391, Stemmer, WPC 1995. Searching sequence space. Bio / Technology 13: 549-553, Crameri, A. 1996. “Construction and evolution of antibody-phage libraries by DNA shuffling” Nature Medicine 2: 100-103, and Crameri, A. et al. 1996. “Improved green fluorescent protein by molecular evolution using DNA shuffling” Nature Biotechnology 14: See 315-319.

  The activity of a recombinant polynucleotide inserted into a plant cell may depend on the influence of endogenous plant DNA adjacent to the insert. Thus, another option is to take advantage of events that are known to be excellent places for insertion in the plant genome. See, for example, WO 2005/103266 A1 regarding the cry1F and cry1Ac cotton events. At these genomic loci, the subject AAD-12 gene can be substituted for the cry1F and / or cry1Ac inserts. Thus, for example, targeted homologous recombination can be used according to the present invention. This type of technology is the subject of, for example, WO 03/080809 A2 and the corresponding published US patent application 20030232410 relating to the use of zinc fingers for targeted recombination. The use of recombinases (such as cre-10x and flp-frt) is also known.

  AAD-12 detoxification is thought to occur in the cytoplasm. Thus, means for further stabilizing the protein and mRNA (including blocking of mRNA degradation) are included within embodiments of the invention, and techniques known in the art can be applied accordingly. The protein of interest can be designed to resist degradation by proteases and the like (protease cleavage sites can be effectively removed by re-engineering the amino acid sequence of the protein). Such embodiments include the use of 5 'and 3' stem loop structures such as UTR from osmotin, and per5 (AU-rich untranslated 5 'sequences). A 5 'cap such as a 7-methyl or 2'-O-methyl group, such as a 7-methylguanylate residue, can also be used. See, for example, Proc. Natl. Acad. Sci. USA, Vol. 74, No. 7, page 2734-2738 (July 1977) Importance of 5'-terminal blocking structure to stabilize mRNA in eukaryotic protein synthesis. Protein complexes or ligand blocking groups can also be used.

  Also, 5 'or 3' UTR computer designs most suitable for AAD-12 (synthetic hairpin) can be implemented within the scope of the present invention. Computer modeling in general, and gene shuffling and directed evolution are described elsewhere in this document. More specifically, with respect to computer modeling and UTR, computer modeling techniques for use in the prediction / evaluation of the 5 ′ and 3 ′ UTR derivatives of the present invention are not limited, but from the Genetics Corporation Group, Madison, Wis. Available MFold version 3.1 (Zucker et al., Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In RNA Biochemistry and Biotechnology, 11-43, edited by J. Barciszewski & BFC Clark, NATO ASI Series, Kluwer Academic Publishers , Dordrecht, NL, (1999), Zucker et al., Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure.J. Mol. Biol. 288, 911-940 (1999), Zucker et al., RNA Secondary Structure Prediction, Current Protocols in Nucleic Acid Chemistry, S. Beaucage, DE Bergstrom, G. D. Glick, and RA Jones, John Wiley & Sons, New York, 11.2.1-11.2.10, (2000)), distributed free of charge as source code, website genetics.wust1.edu/ COVE (RNA structure analysis using covariance model (grammar method without probabilistic context)) that can be downloaded by accessing eddy / software / v. 2.4.2 (Eddy and Durbin, Nucl. Acids Res., 1994, 22: 2079-2088) and FOLDALIGN, which is also distributed free of charge and can be downloaded from the website bioinf.au.dk. FOLDALIGN / (Finding the most significant common sequence and structure motifs in a set of RNA sequences. J. Gorodkin, LJ Heyer and GD Stormo. Nucleic Acids Research, Vol. 25, No. 18, pages 3724-3732, 1997, Finding Common Sequence and Structure Motifs in a set of RNA Sequences. See J. Gorodkin, LJ Heyer, and GD Stormo. ISMB 5; 120-123, 1997).

  Embodiments of the invention can be used in conjunction with naturally evolved or chemically induced mutants (mutants are selected by screening techniques and then AAD-12 and optionally Can be transformed with other genes). The plants of the present invention can be combined with ALS resistance and / or evolved glyphosate resistance. Also, for example, aminopyralide resistance can be combined or “stacked” with the AAD-12 gene.

  Traditional breeding techniques can also be combined with the present invention to strongly combine, permeate and improve desired traits.

  Further improvements also include the use of suitable mitigation agents to further protect the plant and / or add cross resistance to additional herbicides. Mitigating agents typically act to increase the immune system of the plant by activating / expressing cP450. A mitigating agent is a chemical that reduces the herbicide's phytotoxicity to crops by a physiological or molecular mechanism without compromising the effectiveness of weed control.

  Herbicidal agents include benoxacol, croquintoset, ciomethrinyl, dichlormide, dicyclonone, dietolate, fenchlorazole, fenchlorim, flurazole, flxophenim, flirazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, and oxavetrinyl. Plant activators (a novel class of compounds that protect plants by activating their defense mechanisms) can also be used in embodiments of the present invention. These include acibenzoral and probenazole.

  Commercial mitigating agents are for herbicides in the thiocarbamate and chloroacetanilide families that have been incorporated before planting or applied before emergence of large-seed gramineous crops such as corn, grain sorghum, and paddy rice. Can be used to protect. Also, mitigants have been developed to protect winter cereals such as wheat against postemergence application of aryloxyphenoxypropionic acid and sulfonylurea herbicides. The use of emollients to protect corn and rice against sulfonylureas, imidazolinones, cyclohexanediones, isoxazoles, and triketone herbicides is also well established. Relaxant-induced enhancement of herbicide detoxification in mitigant-treated plants is widely accepted as the primary mechanism involved in mitigant action. Mitigating agents induce cofactors such as glutathione and herbicide detoxifying enzymes such as glutathione S-transferase, cytochrome P450 monooxygenase, and glucosyltransferase. Hatzios K K, Burgos N (2004) “Metabolism-based herbicide resistance: regulation by safeners”, Weed Science: Vol. 52, No. 3, pp. 454-467.

  The use of a cytochrome p450 monooxygenase gene stacked with AAD-12 is one preferred embodiment. P450 is involved in the metabolism of herbicides. For example, cP450 can be of mammalian or plant origin. In higher plants, it is known that cytochrome P450 monooxygenase (P450) performs secondary metabolism. This also plays an important role in the oxidative metabolism of xenobiotics in cooperation with NADPH-cytochrome P450 oxidoreductase (reductase). Resistance to some herbicides has been reported as a result of metabolism by P450 and glutathione S-transferase. Several microsomal P450 species involved in xenobiotic metabolism in mammals have been characterized by molecular cloning. Some of these have been reported to efficiently metabolize some herbicides. Thus, transgenic plants with plant or mammalian P450 can exhibit resistance to several herbicides.

  One preferred embodiment of the foregoing is acetochlor (products based on acetochlor include Surpass (R), Keystone (R), Keystone LA, FulTime (R) and TopNotch (R) herbicides. And / or the use of cP450 for resistance to trifluralin (such as Treflan®). Such resistance in soybean and / or corn is included in some preferred embodiments. Further guidance regarding such embodiments includes, for example, Inui et al., “A selectable marker using cytochrome P450 monooxygenases for Arabidopsis transformation”, Plant Biotechnology 22, 281-286 (2005) (Human cytochrome P450 monooxygenase metabolizing herbicides. A selection system for the transformation of Arabidopsis thaliana via Agrobacterium tumefaciens using Agrobacterium tumefaciens, transforming herbicide-tolerant seedlings, herbicides acetochlor, amiprophos-methyl, chlorprofam , Chlorsulfuron, norflurazon, and pendimethalin), Siminszky et al., “Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides”, PNAS, Vol. 96, No. 4. No. 1750-1755, February 16, 1999, Sheldon et al., Weed Scien ce: Volume 48, No. 3, pp. 291-295, `` A cytochrome P450 monooxygenase cDNA (CYP71A10) confers resistance to linuron in transgenic Nicotiana tabacum '', and `` Phytoremediation of the herbicides atrazine and metolachlor by transgenic rice plants expressing human CYP1A1 , CYP2B6, and CYP2C19 ", J Agric Food Chem. April 19, 2006; 54 (8): 2985-91 (for testing human cytochrome p450 monooxygenase in rice. Rice plants are chloroacetomides (chloroacetomides) ( Acetochlor, alachlor, metoachlor, pretilachlor, and tenylchlor), oxyacetamide (mefenacet), pyridazinone (norflurazon), 2,6-dinitroanalines (trifluralin and pendimethalin), phosphamidate ( phosphamidates (amiprofos) -methi See thiocarbamate (Pyributicarb), and urea have been reported to exhibit high resistance to (chlortoluron)).

  There is also the possibility of changing 2,4-D chemistry or using a different 2,4-D chemistry to make the subject AAD-12 gene more efficient. Such possible changes include the creation of better substrates and better leaving groups (higher electronegativity). Auxin transport inhibitors (eg, diflufenzopyr) can also be used to increase herbicidal activity using 2,4-D.

  Unless specifically indicated or implied, the terms “a”, “an”, and “the” refer to “at least one” as used herein. All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety to the extent that they do not conflict with the clear teachings of this specification.

The following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise noted, all percentages are weight percentages and all solvent mixing ratios are volume percentages.
(Example)

Method for identifying genes that give resistance to 2,4-D in plants As a method for identifying genes possessing herbicide degrading activity in plants, NCBI (National Center for Biotechnology Information) and other current publications It is possible to search the database. In order to initiate this process, it is necessary that a functional gene sequence encoding a protein having the desired characteristics (ie α-ketoglutarate dioxygenase activity) has already been identified. This protein sequence is then used as an input to the BLAST (Basic Local Alignment Search Tool) (Altschul et al., 1997) algorithm to compare against the available NCBI protein sequences deposited. Using the default settings, this search returns over 100 homologous protein sequences at variable levels. These range from high identity (85-98%) to very low identity (23-32%) at the amino acid level. Conventionally, only sequences with high homology are expected to retain similar properties as the input sequence. In this case, only sequences with a homology of gtoreq 50% were selected. Using cloning and recombinant expression of homologues having as low as 31% amino acid conservation (as opposed to tfdA from Ralstonia eutropha), as exemplified herein Not only the intended herbicides, but also substrates that have never been tested with these enzymes before can provide commercial levels of resistance.

  A single gene (sdpA) was identified from the NCBI database (ncbi.nlm.nih.gov website, see accession number AF516752) as a homolog with only 31% amino acid identity to tfdA. Percent identity was first determined by translating both the sdpA and tfdA DNA sequences deposited in the database into proteins, followed by multiple sequence alignments using ClustalW in the VectorNTI software package. .

Optimization of sequences for expression in plants and bacteria In order to obtain higher levels of expression of heterologous genes in plants, the protein coding sequences of the genes are regenerated so that they are more efficiently expressed in plant cells. It may be preferable to operate. Maize may be preferred to redesign the heterologous protein coding region prior to transformation to increase the expression level of the gene and the level of the encoded protein in the plant. One. Thus, an additional step in the design of genes encoding bacterial proteins is to reengineer heterologous genes for optimal expression.

  One reason for re-engineering bacterial proteins for expression in corn is due to the non-optimal G + C content of the native gene. For example, the very low G + C content of many native bacterial genes (and the resulting slope to a high A + T content) mimics plant gene regulatory sequences known to be very rich in A + T or This results in the creation of an array to be copied. The presence of some A + T rich sequences in the DNA of the gene introduced into the plant (eg, the TATA box region normally found in gene promoters) can lead to abnormal transcription of the gene. On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (eg, a polyadenylation signal sequence (AAUAAA), or a sequence complementary to a small nuclear RNA involved in mRNA precursor splicing) Can lead to RNA instability. Thus, one goal in designing a gene encoding a bacterial protein for maize expression, more preferably referred to as a plant optimized gene, is to encode a higher G + C content, preferably a metabolic enzyme. It is to create a DNA sequence that has something close to that of the corn gene that is present. Another goal in the design of plant-optimized genes encoding bacterial proteins is to create DNA sequences whose sequence modifications do not interfere with translation.

  Table 2 illustrates how high the G + C content is in corn. For the data in Table 2, the coding region of the gene was extracted from the GenBank (Release 71) entry and the base composition was calculated using the MacVector ™ program (Accelerys, San Diego, CA). Intron sequences were ignored during the calculation.

  Due to the flexibility afforded by the redundancy / degeneracy of the genetic code (ie some amino acids are specified by multiple codons), the evolution of genomes in different organisms or classes of organisms results in the use of redundant codons Is different. This “codon bias” is reflected in the average base composition of the protein coding region. For example, organisms with relatively low G + C content utilize codons with A or T at the third position of the redundant codon, while organisms with higher G + C content have G or C at the third position. Use the codon you have. It is believed that the presence of a “minor” codon in an mRNA can reduce the absolute translation rate of that mRNA, particularly when the relative abundance of charged tRNA corresponding to the minor codon is low. An extension of this is that the reduction in translation rate by individual minor codons is at least additive for multiple minor codons. Thus, mRNA with a high relative content of minor codons has a correspondingly low translation rate. This translation rate is reflected by the low level of the encoded protein later.

  In manipulating genes encoding bacterial proteins for expression of corn (or other plants such as cotton or soybean), plant codon bias has been determined. Corn codon bias is the statistical codon distribution that plants use to encode the protein, and preferred codon usage is shown in Table 3. After determining the bias, the percent frequency of codons in the gene of interest is determined. The first codon preferred by the plant and the second, third and fourth choices of preferred codons are determined if there are multiple choices. A new DNA sequence encoding the amino acid sequence of the bacterial protein can then be designed, but the new DNA sequence is the plant's (first preferred, second preferred, third preferred, or fourth preferred The amino acid at each position is specified within the protein amino acid sequence, unlike the native bacterial DNA sequence (which encodes the protein) by preferred (codon substitution). The new sequence is then analyzed for restriction enzyme sites that may have been created by the modification. The identified site is further modified by replacing the codon with the preferred first, second, third, or fourth preferred codon. Other sites in the sequence that may affect transcription or translation of the gene of interest are exon: intron junctions (5 'or 3'), poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to doublets, G or C sequence blocks having more than about 4 identical residues may affect the transcription of the sequence. Thus, these blocks are also modified by replacing codons such as the first or second option with the next preferred codon option.

  A plant-optimized gene encoding a bacterial protein comprises about 63% first option codons, about 22% to about 37% second option codons when the total percentage is 100%, and about Preferably it contains 15% to about 0% of the third or fourth option codon. Most preferably, the plant-optimized gene is about 63% first option codon, at least about 22% second option codon, and about 7.5% first codon when the total percentage is 100%. Contains 3 choice codons and about 7.5% 4th choice codons. The methods described above allow one skilled in the art to modify a gene that is foreign to a particular plant so that the gene is optimally expressed in the plant. This method is further exemplified in PCT application WO 97/13402.

  Therefore, to design a plant-optimized gene encoding a bacterial protein, a redundant genetic code established from a codon bias table compiled from the gene sequence of a particular plant or plants Utilizing this, a DNA sequence is designed to encode the amino acid sequence of the protein. The resulting DNA sequence has a higher degree of codon diversity, desirable base composition, can contain strategically placed restriction enzyme recognition sites, and can prevent gene transcription or translation of mRNA products Lacks an array. 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 regarding the production of synthetic genes can be found, for example, in US Pat. No. 5,380,831.

  AAD-12 plant reconstruction analysis: A large-scale analysis of 876 base pairs (bp) of the DNA sequence of the native AAD-12 coding region (SEQ ID NO: 1) reveals some that are considered detrimental to optimal plant expression. The presence of sequence motifs and non-optimal codon composition were revealed. The protein (AAD-12) encoded by SEQ ID NO: 1 is presented as SEQ ID NO: 2. A protein that is the same as native SEQ ID NO: 2 except that an alanine residue is added at position 2 (underlined in SEQ ID NO: 4) to improve production of recombinant proteins in monocotyledonous and dicotyledonous plants A “plant-optimized” DNA sequence AAD-12 (v1) (SEQ ID NO: 3) encoding (SEQ ID NO: 4) was developed. An additional alanine codon (GCT, underlined in SEQ ID NO: 3) encodes a portion of the NcoI restriction enzyme recognition site (CCATGG) that spans the ATG translation initiation codon. This thus serves the dual purpose of facilitating subsequent cloning operations while improving the sequence organization around the ATG start codon to optimize translation initiation. The protein encoded by the native and plant optimized (v1) coding region is 99.3% identical and differs only at amino acid number 2. In contrast, the DNA sequence of the native and plant optimized (v1) coding region is only 79.7% identical.

  Table 4 shows the difference in codon composition between native (columns A and D) and plant-optimized sequences (columns B and E), with theoretical plant-optimized sequences (columns C and C). Allows comparison with F).

  From the examination in Table 4, it is clear that the native and plant-optimized coding regions encode substantially the same protein while differing substantially from each other. The plant-optimized form (v1) closely mimics the codon composition of the theoretical plant-optimized coding region that encodes the AAD-12 protein.

  Reconstruction for E. coli expression: Specially engineered strains of Escherichia coli and related vector systems have been developed to produce relatively large amounts of protein for biochemical and analytical studies. Often used. Sometimes the native gene encoding the desired protein is not well suited for high level expression in E. coli, even if the source organism of the gene can be another bacterial genus . In such cases, it is possible and desirable to reengineer the protein coding region of the gene to make it suitable for expression in E. coli. An E. coli class II gene is defined as being highly and continuously expressed during the exponential growth phase of E. coli cells. (Henaut, A. and Danchin, A. (1996), Escherichia coli and Salmonella typhimurium cellular and molecular biology, Volume 2, pages 2047-2066. Neidhardt, F., Curtiss III, R., Ingraham, J., Lin E., Low, B., Masasanik, B., Reznikoff, W., Riley, M., Schaechter, M. and Umbarger, H. (eds.) American Society for Microbiology, Washington, DC). By examining the codon composition of the coding regions of the E. coli class II gene, an average codon composition of these E. coli class II gene coding regions can be assumed.

  A protein coding region with an average codon composition that mimics that of a class II gene is considered convenient for expression during the exponential growth phase of E. coli. Using these guidelines, a new DNA sequence encoding the AAD-12 protein (SEQ ID NO: 4) (containing an additional alanine at position 2 as described above) can be transferred to E. coli. Designed according to the average codon composition of the class II gene coding region. The initial sequence, whose design was based solely on codon composition, was further manipulated to include specific restriction enzyme recognition sequences suitable for cloning into E. coli expression vectors. Harmful sequence features such as a highly stable stem-loop structure were avoided, and an intragenic sequence homologous to the 3 'end of 16S ribosomal RNA (Le. Shine-Dalgarno sequence) was also avoided. The sequence (v2) optimized for E. coli is disclosed as SEQ ID NO: 5 and encodes the protein disclosed in SEQ ID NO: 4.

  Native and E. coli optimized (v2) DNA sequence is 84.0% identical, while plant optimized (v1) DNA sequence and E. coli are optimal The homologous (v2) DNA sequence is 76.0% identical. Table 5 shows the codon composition of native AAD-12 coding region (columns A and D), AAD-12 coding region optimized for expression in E. coli (v2, columns B and E), and Figure 5 represents the codon composition of the theoretical coding region (columns C and F) of the AAD-12 protein with the optimal codon composition of the E. coli class II gene.

  From the tests in Table 6, it is clear that the coding regions optimized for native and E. coli encode substantially identical proteins while differing substantially from each other. The type (v2) optimized for E. coli closely mimics the codon composition of the coding region optimized for the theoretical E. coli encoding the AAD-12 protein. ing.

  Design of a DNA sequence biased towards soybean codons encoding soybean EPSPS with a mutation conferring glyphosate resistance. This example encodes a mutated soybean 5-enolpyruvoyl shikimate 3-phosphate synthase (EPSPS), but is a new DNA optimized for expression in soybean cells. Teaches the design of sequences. The amino acid sequence of triple mutated soybean EPSPS is disclosed as SEQ ID NO: 5 of WO2004 / 009761. The mutated amino acids in the sequence so disclosed are residue 183 (the native protein threonine is replaced with isoleucine), residue 186 (arginine in the native protein is replaced with lysine), And residue 187 (proline in the native protein is replaced by serine). Therefore, the amino acid sequence of the native soybean EPSPS protein can be deduced by replacing the substituted amino acid of SEQ ID NO: 5 of WO2004 / 009761 with a native amino acid at an appropriate position. Such a native protein sequence is disclosed as SEQ ID NO: 20 of PCT / US2005 / 014737 (filed May 2, 2005). Doubly mutated soybean EPSPS containing a mutation at residue 183 (the native protein threonine is replaced by isoleucine) and residue 187 (proline in the native protein is replaced by serine) This protein sequence is disclosed as SEQ ID NO: 21 of PCT / US2005 / 014737.

  A table of codon usage of the protein coding sequence of soybean (Glycine max) calculated from 362,096 codons (about 870 coding sequences) was obtained from the world-wide website “kazusa.or.jp/codon”. These data were reformatted as shown in Table 6. Columns D and H in Table 6 represent the distribution of synonymous codons for each amino acid found in the protein coding region of the soybean gene (% of all codon usage for that amino acid). It is clear that some synonymous codons for some amino acids (amino acids can be specified by 1, 2, 3, 4, or 6 codons) are relatively rarely present in the soy protein coding region. Yes (eg, comparison of GCG and GCT codon usage to specify alanine)

A table of biased soybean codon usage was calculated from the data in Table 6. Codons that were found in less than about 10% of all soybean genes for a particular amino acid were ignored. In order to balance the distribution of the remaining codon choices of amino acids, the weighted average representation of each codon was calculated using the following formula:
C1 weighted% = 1 / (% C1 +% C2 +% C3 + etc.) ×% C1 × 100
[Where C1 is the codon being queried, C2, C3, etc. represent the remaining synonymous codons, and the% values for the relevant codons are taken from columns D and H in Table 6 (ignoring rare codon values in bold) Yes)].

  The weighted% values for each codon are shown in columns C and G of Table 6. TGA was arbitrarily chosen as a translation terminator. The biased codon usage was then entered into a special genetic code table for use by the OptGene ™ gene design program (Ocimum Biosolutions LLC, Indianapolis, IN).

  To derive a soybean optimized DNA sequence encoding a double mutated EPSPS protein, the protein sequence of SEQ ID NO: 21 from PCT / US2005 / 014737 was obtained by the OptGene ™ program. Reverse translation was performed using the soybean-derived genetic code derived above. The so-derived initial DNA sequence is then highly stable intra-strand secondary, reducing the number of CG and TA doublets between adjacent codons, increasing the number of CT and TG doublets between adjacent codons. To remove structure, remove or add restriction enzyme recognition sites, and other sequences that can be detrimental to the expression or cloning operations of the engineered genes (while maintaining the overall weighted average representation of the codons) ) Modified by compensating for codon changes. To eliminate potential plant intron splicing sites, long runs of A / T or C / G residues, and other motifs that can interfere with RNA stability, transcription, or translation of the coding region in plant cells, Further improvements to the sequence were made. Other changes were made to eliminate long internal open reading frames (frames other than +1). All of these changes were made within the constraint of preserving the amino acid sequence disclosed as SEQ ID NO: 21 of PCT / US2005 / 014737, while maintaining the above-mentioned soy-biased codon composition.

  The soybean-biased DNA sequence encoding the EPSPS protein of SEQ ID NO: 21 is disclosed as bases 1-1575 of SEQ ID NO: 22 of PCT / US2005 / 014737. The synthesis of a DNA fragment comprising SEQ ID NO: 22 of PCT / US2005 / 014737 was performed by a commercial supplier (PicoScript, Houston, TX).

Cloning of Expression and Transformation Vectors Construction of E. coli pET expression vector: AAD-12 (Xba 1, Xho 1) was used using restriction enzymes (Xba 1, Xho 1) corresponding to the added site using an additional cloning linker. v2) was excised from the picoscript vector and ligated into the pET280 streptomycin / spectinomycin resistant vector. The ligation product was then transformed into TOP10F ′ E. coli and plated on luria broth + 50 μg / ml streptomycin and spectinomycin (LB S / S) agar plates.

  To distinguish the ligation of AAD-12 (v2): pET280 and pCR2.1: pET280, about 20 isolated colonies were picked into 6 ml LB-S / S and stirred at 37 ° C. for 4 hours. Grown up. Each culture was then spotted onto a 50 μg / ml plate of LB + kanamycin, which was incubated at 37 ° C. overnight. Colonies that grew on LB-K were presumed that the pCR2.1 vector had been ligated inside and discarded. Plasmids were isolated from the remaining cultures as before and verified for accuracy using digestion with XbaI / XhoI. The final expression construct was named pDAB3222.

  Construction of Pseudomonas expression vector: The AAD-12 (v2) open reading frame was first cloned as an XbaI-XhoI fragment into the modified pET expression vector (Novagen) “pET280 S / S”. The resulting plasmid pDAB725 was confirmed by restriction enzyme digestion and sequencing reactions. The AAD-12 (v2) open reading frame from pDAB725 was transferred as an XbaI-XhoI fragment into pMYC1803, a Pseudomonas expression vector. Positive colonies were confirmed by restriction enzyme digestion. The completed construct pDAB739 was transformed into MB217 and MB324 Pseudomonas expression strains.

  Completion of binary vector: Received plant-optimized gene AAD-12 (v1) from Picscript (completed gene reconstruction design (see above), outsourced to Picscript for construction) and confirmed sequence internally (SEQ ID NO: 3), confirming the absence of the expected sequence modification. Sequencing reactions were performed using Meck forward (SEQ ID NO: 6) and M13 reverse (SEQ ID NO: 7) primers, as before, using Beckman Coulter's "Dye Terminator Cycle Sequencing with Quick Start Kit" reagent. did. Sequence data was analyzed and the results showed that there were no abnormalities in the plant-optimized AAD-12 (v1) DNA sequence. The AAD-12 (v1) gene was cloned into pDAB726 as an Nco I-Sac I fragment. The resulting construct was named pDAB723 (using PvuII and Not I restriction digests) containing [AtUbi10 promoter: Nt OSM 5′UTR: AAD-12 (v1): Nt OSM 3′UTR: ORF1 poly A 3′UTR]. Confirmation). Subsequently, the Not I-Not I fragment containing the described cassette was cloned into the Not I site of the binary vector pDAB3038. The resulting binary containing the following cassette [AtUbi10 promoter: Nt OSM 5′UTR: AAD-12 (v1): Nt OSM 3′UTR: ORF1 poly A 3′UTR: CsVMV promoter: PAT: ORF25 / 26 3′UTR] Vector pDAB724 was restriction digested to confirm correct orientation (using BamHI, NcoI, NotI, SacI, and XmnI). The confirmed finished construct (pDAB724) was used for transformation into Agrobacterium.

  Cloning Additional Transformation Constructs: All other constructs made for transformation into the appropriate plant species are subject to similar procedures already described herein, and other standard molecular cloning methods. (Maniatis et al., 1982).

Transformation and selection into Arabidopsis Arabidopsis thaliana Growth conditions: Wild Arabidopsis seeds suspended in 0.1% agarose (Sigma Chemical Co., St. Louis, MO) solution I let you. The suspended seeds were stored at 4 ° C. for 2 days to complete the dormancy requirement and to ensure simultaneous seed germination (stratus storage).

  Sunshine Mix LP5 (Sun Gro Horticuture, Bellvue, WA) was covered with fine vermiculite and the Hoagland liquid passed under it until wet. The soil mixture was drained for 24 hours. Layered stored seeds were sown on vermiculite and covered with a humidity dome (KORD Products, Bramarea, Ontario, Canada) for 7 days.

Seed germination and plants in Conviron (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 hours light / 8 hours dark), 120-150 μmol / m ² sec. Grow under light intensity, constant temperature (22 ° C.) and humidity (40-50%). Plants were watered with Hogland liquid followed by deionized water to keep the soil moist but not wet.

  Agrobacterium transformation: LB + containing erythromycin (Sigma Chemical Co., St. Louis, MO) (200 mg / L) or spectinomycin (100 mg / L) containing streaked DH5α colonies Agar plates were used to provide colonies for inoculation with 4 ml of mini prep broth (liquid LB + erythromycin). The culture was incubated overnight at 37 ° C. with constant agitation. Plasmid DNA was purified using Qiagen (Valencia, Calif.) Spin Mini Preps performed according to the manufacturer's instructions.

  Electrocompetent Agrobacterium tumefaciens (strains Z707, EHA101, and LBA4404) cells were prepared using the protocol from Weigel and Glazebrook (2002). Competent Agrobacterium cells were transformed using electroporation methods adapted from Weigel and Glazebrook (2002). 50 μl of competent agro cells were thawed on ice and 10-25 ng of the desired plasmid was added to the cells. The DNA and cell mixture was added to a pre-chilled electroporation cuvette (2 mm). An Eppendorf electroporator 2510 was used for transformation under the following conditions: voltage: 2.4 kV, pulse length: 5 msec.

  After electroporation, 1 ml of YEP broth (per liter: 10 g yeast extract, 10 g bactopeptone, 5 g NaCl) was added to the cuvette and the cell-YEP suspension was transferred to a 15 ml culture tube. Cells were incubated for 4 hours in a water bath at 28 ° C. with constant agitation. Following incubation, cultures were plated on YEP + agar containing erythromycin (200 mg / L) or spectinomycin (100 mg / L) and streptomycin (Sigma Chemical Co., St. Louis, Mo.) (250 mg / L). Plates were incubated at 28 ° C. for 2-4 days.

  Colonies were selected and streaked on fresh YEP + agar plates containing erythromycin (200 mg / L) or spectinomycin (100 mg / L) and streptomycin (250 mg / L) and incubated at 28 ° C. for 1-3 days. . Colonies were selected for PCR analysis to confirm the presence of the gene insert by using vector specific primers. Plasmid DNA was purified from selected Agrobacterium colonies using Qiagen Spin Mini Preps performed according to the manufacturer's instructions with the following exception: 15 ml overnight mini prep culture (liquid 4 ml aliquots of YEP + erythromycin (200 mg / L) or spectinomycin (100 mg / L)) and streptomycin (250 mg / L)) were used for DNA purification. An alternative to using Qiagen Spin Mini Prep DNA was to lyse transformed Agrobacterium cells suspended in 10 μl of water at 100 ° C. for 5 minutes. Plasmid DNA from the binary vector used in the transformation of Agrobacterium was included as a control. The PCR reaction was performed by Takara Miras Bio Inc. Completed using Taq DNA polymerase (Madison, Wis.) At 0.5 × concentration according to manufacturer's instructions. The PCR reaction was performed on an MJ Research Peltier 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. 29 cycles, followed by 1 cycle at 72 ° C. for 10 minutes. The reaction was kept at 4 ° C. after cycling. Amplification was analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Colonies whose PCR product was identical to the plasmid control were selected.

  Transformation of Arabidopsis: Arabidopsis was transformed using the floral dip method. Using selected colonies, pre-cultures of one or more 15-30 ml YEP broth containing erythromycin (200 mg / L) or spectinomycin (100 mg / L) and streptomycin (250 mg / L) Vaccinated. The culture was incubated overnight at 28 ° C. with constant stirring at 220 rpm. Each preculture is 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) Incubate overnight at 28 ° C. with constant stirring. Thereafter, the cells were pelleted at about 8700 × g for 10 minutes at room temperature, and the resulting supernatant was discarded. Cell pellets were ½ × Murashige & Skoog salt / Gamborg B5 vitamin, 10% (w / v) sucrose, 0.044 μM benzylaminopurine (10 μl / liter, 1 mg / ml stock in DMSO) And gently resuspended in 500 ml invasion medium containing 300 μl / liter Silwet L-77. Approximately one month old plants were immersed in the medium for 15 seconds to ensure that the newest inflorescences were immersed. The plants were then laid down and covered (clear or opaque) for 24 hours, then washed with water and allowed to stand upright. Plants were grown using a photoperiod of 22 ° C, 16 hours light / 8 hours dark. The seeds were harvested after about 4 weeks of soaking.

  Selection of transformed plants: Freshly harvested T1 seed [AAD-12 (v1) gene] was dried at room temperature for 7 days. T1 seeds are sown in a 26.5 × 51 cm germination tray (TO Plastics Inc., Clearwater, Minn.), Each suspended in advance in 40 ml 0.1% agarose solution to meet dormancy requirements. A 200 mg aliquot of stratified T1 seeds (approximately 10,000 seeds) stored for 2 days at 4 ° C. was placed to ensure complete and concurrent seed germination.

  Sunshine Mix LP5 (Sun Gro Horticuture Inc., Bellvue, WA) was covered with fine vermiculite, and hoeland liquid was passed under it until wet, and then drained by gravity. A 40 ml aliquot of each of the layered seeds was pipetted on vermiculite evenly and covered with a humidity dome (KORD Products, Bramale, Ontario, Canada) for 4-5 days. The dome was removed one day prior to initial transformant selection (selection of co-transformed PAT gene) using postemergence spray of glufosinate.

Seven days after planting (DAP) and again at 11 DAP, T1 plants (cotyledon and 2-4-1f stages, respectively) were subjected to 0.2% solution of Liberty herbicide (200 g ai / L glufosinate, Bayer Crop Sciences, MO). Kansas City) was sprayed using a DeVilbiss compressed air spray tip at a spray volume of 10 ml / tray (703 L / ha) to deliver an effective amount of 280 g ai / ha glufosinate / application. Survivors (actively growing plants) were identified 4-7 days after the final spray and individually transplanted into 3 inch pots prepared with potted medium (Metro Mix 360). The transplanted plants were covered with a humidity dome for 3-4 days and placed in a 22 ° C. growth chamber as before or moved directly to the greenhouse. Subsequently, the dome was removed and the plants were grown in the greenhouse (22 ± 5) at least one day before testing for the ability of AAD-12 (v1) (plant-optimized gene) to confer phenoxyauxin herbicide resistance. C, 50 ± 30% RH, 14 hours light: 10 hours dark, minimum 500 μE / m ² s ¹ natural light + auxiliary light)

  Subsequently, T1 plants were randomly assigned to various amounts of 2,4-D. In Arabidopsis, 50 g ae / ha 2,4-D is an effective amount to distinguish sensitive plants from those with significant levels of resistance. Also, increased amounts were applied (50, 200, 800, or 3200 g ae / ha) to determine the relative level of resistance.

  All auxin herbicide applications were applied at a spray volume of 703 L / ha (0.4 ml solution / 3 inch bowl) using the DeVilbiss sprayer described above or at a spray volume of 187 L / ha by a track sprayer. Applied. The 2,4-D used was dissolved in DMSO and diluted with water (final concentration of less than 1% DMSO) technical grade (Sigma, St. Louis, MO) or commercially available dimethylamine salt formulation (456 g ae). / L, NuFarm, St Joseph, MO). The dichloroprop used was a commercial grade formulated as the potassium salt of R-dichloroprop (600 g ai / L, AH Marks). As the amount of herbicide increases beyond 800 g ae / ha, the pH of the spray solution becomes excessively acidic, causing the leaves of young soft Arabidopsis plants to burn, complicating the evaluation of the primary effects of the herbicide I made it. Application of these large amounts of herbicide in 200 mM HEPES buffer, pH 7.5 became standard practice.

  Some T1 individuals were subjected to alternative commercial herbicides instead of phenoxyauxin. One concern was to determine whether the pyridyloxyacetate auxin herbicides triclopyr and fluoroxypyr could be effectively degraded in plants. The herbicide was applied to the T1 plants using a track sprayer at a spray volume of 187 L / ha. T1 plants that showed resistance to 2,4-D DMA were further accessed in the T2 generation.

  Results of selection of transformed plants: The first Arabidopsis transformation was performed using AAD-12 (v1) (plant optimized gene). Initially, T1 transformants were selected from a background of non-transformed seeds using a glufosinate selection scheme. Screening more than 300,000 T1 seeds, 316 glufosinate resistant plants were identified (PAT gene), which is equivalent to 0.10% transformation / selection frequency, using PAT + Liberty for selection Within the normal range of construct selection frequencies. Subsequently, the selected T1 plants were transplanted into individual pots and sprayed with various amounts of commercially available aryloxyalkanoate herbicides.

  Table 7 compares the response of AAD-12 (v1) and the control gene, which confers resistance to 2,4-D on Arabidopsis T1 transformants. Responses are expressed in terms of% visual damage 2WAT. Data are presented as a histogram of individuals showing slight or no injury (less than 20%), moderate injury (20-40%), or severe injury (greater than 40%). Since each T1 is an independent transformation event, significant variations in individual T1 responses within a given rate can be predicted. Arithmetic mean and standard deviation are presented for each treatment. The range of individual responses is also shown in the last column for each quantity and transformation. Arabidopsis transformed with PAT / Cry1F served as an auxin-sensitive transformation control. The AAD-12 (v1) gene conferred resistance to herbicides on individual T1 Arabidopsis plants. Within a given treatment, the level of plant response varies greatly, which can be attributed to each plant representing an independent transformation event.

  Importantly, for each 2,4-D dose tested, there were individuals that were not affected while some were severely affected. To demonstrate significant differences between plants transformed simply with AAD-12 (v1) versus wild type or PAT / Cry1F transformed, the overall population damage average by dose is presented in Table 7. At increased amounts up to 3,200 g ae / ha (or up to about 6 × field volume), the damage level was higher and the frequency of uninjured plants tended to be lower. Also, at these high amounts, the spray solution is highly acidic unless buffered. Arabidopsis, which has grown almost in the growth chamber, has a very thin cuticle, and severe burn effects can complicate testing with these increased amounts. Nevertheless, many individuals survived 3,200 g ae / ha 2,4-D with little or no damage.

  Table 8 shows a similar dose response of T1 Arabidopsis to phenoxypropionic acid, dichloroprop. The data show that the (R-) dichloroprop isomer with active herbicidal activity does not serve as a suitable substrate for AAD-12 (v1). It is one distinguishing feature that separates the two genes that AAD-1 metabolizes R-dichloroprop sufficiently to confer commercially acceptable tolerance. (Table 8). AAD-1 and AAD-12 are considered R and S specific α-ketoglutarate dioxygenases, respectively.

  AAD-12 (v1) as a selectable marker: The ability to use 2,4-D as a selection agent and AAD-12 (v1) as a selectable marker was compared to the Arabidopsis transformed as described above. ) First analyzed. About 50 T4 generation Arabidopsis seeds (homozygous for AAD-12 (v1)) were spiked into about 5,000 wild-type (sensitive) seeds. Several treatments were compared and each plant tray received either one of the following treatment schemes, one of 2,4-D one or two application timings: 7DAP, 11DAP, Or 7, then 11 DAP. Since all individuals also contained the PAT gene in the same transformation vector, AAD-12 selected with 2,4-D could be directly compared to PAT selected with glufosinate.

  The treatment was applied using a DeVilbiss spray tip as previously described. Plants were identified as resistant or sensitive to 17DAP. Optimal treatment is 75 g ae / ha 2,4-D applied 7 and 11 days after planting (DAP), equally effective in selection frequency, and herbicidal damage to transformed individuals is a Liberty selection scheme It was lower than. These results indicate that it can be used effectively as an alternative selectable marker for the Arabidopsis population transformed with AAD-12 (v1).

  Heritability: Various T1 events were self-pollinated to produce T2 seeds. These seeds were progeny tested by applying 2,4-D (200 g ae / ha) to 100 random T2 siblings. Each individual T2 plant was transplanted into a 7.5 cm square pot prior to spray application (track sprayer at 187 L / ha application rate). As determined by chi-square analysis (P> 0.05), 75 percent of the T1 family (T2 plants) separated in a model of resistance 3: sensitivity 1 predicted for a single locus of dominant inheritance with Mendelian inheritance It was.

  Seeds were collected from 12 to 20 T2 individuals (T3 seeds). Twenty-five T3 siblings from each of eight randomly selected T2 families were progeny tested as previously described. Approximately one third of the T2 family (non-segregating population) predicted to be homozygous was identified in each line. These data indicate that AAD-12 (v1) is stably integrated and is inherited in a Mendelian manner for at least 3 generations.

  Additional foliar herbicide resistance in AAD-12 Arabidopsis: The ability of AAD-12 (v1) to confer resistance to other aryloxyalkanoate auxin herbicides in transgenic Arabidopsis, Determined by foliar application of various substrates. T2 generation Arabidopsis seeds were layered and stored in selection trays very similar to those of Arabidopsis. Transformed control lines containing PAT and the insect resistance gene Cry1F were planted in a similar manner. Seedlings were transferred to individual 3-inch pots in the greenhouse. All plants were sprayed using a track sprayer set at 187 L / ha. Plants were sprayed with a range of pyridyloxyacetic acid herbicides: 280-2240 g ae / ha triclopyr (Garlon 3A, Dow AgroSciences) and 280-2240 g ae / ha fluoroxypyr (Starane, Dow AgroSciences), and 2,4-Dichlorophenol (DCP, Sigma), a 2,4-D metabolite resulting from AAD-12 activity (molar concentration equivalent to 2,4-D of 280-2240 g ae / ha, technical grade DCP use). All applications were formulated in water. Each treatment was repeated 3-4 times. Plants were evaluated after 3 and 14 days of treatment.

  There is no effect of the 2,4-D metabolite 2,4-dichlorophenol (DCP) on the transgenic non-AAD-12 control Arabidopsis (Pat / Cry1F). Plants transformed with AAD-12 were also clearly protected from triclopyr and fluoroxypyr herbicide damage seen in transformed non-resistant controls (see Table 9). These results confirm that AAD-12 (v1) in Arabidopsis provides resistance to the tested pyridyloxyacetic acid auxins. This is the first report of an enzyme with significant activity against pyridyloxyacetic acid herbicides. Other 2,4-D degrading enzymes with similar activity have not been reported.

  Molecular analysis of AAD-12 (v1) Arabidopsis: Invader assay for PAT gene copy number analysis to determine stable integration of transforming units of plants containing PAT and AAD-12 (v1) ( The Third Wave Agbio Kit Procedures) was performed on multiple AAD-12 (v1) homozygous lines using total DNA obtained from the Qiagen DNeasy kit. Since these genes were contained on the same plasmid, the analysis assumed that they were directly physically linked.

  The results indicate that all 2,4-D resistant plants assayed contained PAT (and thus AAD-12 (v1) by speculation). Copy number analysis indicated that the total insert was in the range of 1-5 copies. This also correlates with AAD-12 (v1) protein expression data indicating that the presence of the enzyme confers a significantly higher level of resistance to all commercially available phenoxyacetic acid and pyridyloxyacetic acid.

  Arabidopsis transformed with molecular stacking of AAD-12 (v1) and glyphosate resistance gene: contains pDAB3759 plasmid (AAD-12 (v1) + EPSPS) encoding a putative glyphosate resistance trait T1 Arabidopsis seeds were generated as previously described. T1 transformants were selected using AAD-12 (v1) as a selectable marker as described. T1 plants (individually transformed events) were recovered from the first selection attempt and transferred to 3 inch pots in the greenhouse as previously described. Three different control Arabidopsis lines were also tested: wild type Columbia-0, AAD-12 (v1) + PAT T4 homozygous line (transformed with pDAB724), and PAT + Cry1F homozygous line (transformation control). Plants transformed with pDAB3759 and pDAB724 were preselected for 2,4-D resistance at the seedling stage. Four days after transplanting, the plants were subjected to foliar treatment with a track sprayer using 0, 26.25, 105, 420, or 1680 g ae / ha glyphosate in water (Glyphomax Plus, Dow AgroSciences) as previously described. Divided evenly. All treatments were repeated 5-20 times. Plants were evaluated after 7 and 14 days of treatment.

  Initial resistance assessment showed that plants resistant to 2,4-D were later resistant to glyphosate when compared to the responses of the three control lines. These results can confer resistance to two herbicides with different modes of action, including 2,4-D and glyphosate tolerance, allowing post-emergence application of either herbicide It is shown that. In addition, AAD-12 + 2,4-D was effectively used as a selectable marker for true resistance selection.

  AAD-12 Arabidopsis gene-stacked with AAD-1 to confer a wider range of herbicide tolerance: AAD-12 (v1) (pDAB724) and AAD-1 (v3) (pDAB721) plants reciprocally crossed, F1 seed was collected. Eight F1 seeds were planted and grown to produce seeds. Thereafter, tissue samples were collected from 8 F1 plants and subjected to Western analysis to confirm the presence of both genes. It was concluded that all 8 plants tested expressed both AAD-1 and AAD-12 proteins. Seeds were bulked and dried for 1 week prior to planting.

  100 F2 seeds were sown and 280 g ai / ha glufosinate was applied. Ninety-six F2 plants survived glufosinate selection and met the expected separation ratio (15R: 1S) of two independent assorted loci that were glufosinate-resistant. The glufosinate resistant plants were then treated with 560 g ae / ha R-dichloroprop + 560 g ae / ha triclopyr applied to the plants under the same spray regime as used in other tests. Plants were graded to 3 and 14 DAT. Of the 96 plants that survived glufosinate selection, 63 survived herbicide application. These data show that the predicted segregation pattern of two independent categorical dominant traits in which each gene confers resistance to only one of the auxinic herbicides (either R-dichlroprop or triclopyr) It is consistent with (9R: 6S). The result is successful stacking of AAD-12 (pDAB724) and AAD-1 (pDAB721), thus increasing the range of herbicides that can be applied to the target crop [each (2,4-D + R-di Chloroprop) and (2,4-D + fluoroxypyr + triclopyr)]. The conventional stacking of two separate 2,4-D resistance genes may be useful to bring 2,4-D resistance to highly sensitive species. Furthermore, if either gene was used as a selectable marker for the third and fourth genes of interest by independent transformation activities, each gene pair was brought together by conventional breeding activities, It can be selected in the F1 generation by spraying with herbicides exclusive between AAD-1 and AAD-12 enzymes (with R-dichloroprop and triclopyr for AAD-1 and AAD-12 respectively) It is shown).

  Other AAD stacking is also within the scope of the present invention. For example, the TfdA protein described elsewhere herein (Streber et al.) Can be used in conjunction with the subject AAD-12 gene to confer herbicide resistance ranges to the transgenic plants of the invention. .

Maize crystal-mediated transformation of corn using imazetapil selection Cloning of AAD-12 (v1): The AAD-12 (v1) gene was excised from the intermediate vector pDAB3283 as an Nco1 / Sac1 fragment. This was ligated in one direction into a similarly cut pDAB3403 vector containing the ZmUbi1 monocotyledonous promoter. The two fragments were ligated together using T4 DNA ligase and transformed into DH5α cells. The resulting colonies were miniprepped using Qiagen's QIA Spin mini prep kit and the colonies were digested to confirm orientation. This first intermediate construct (pDAB4100) contains the ZmUbi1: AAD-12 (v1) cassette. This construct was digested with Not1 and Pvu1 to release the gene cassette and digest the unwanted backbone. This was ligated with Not1 cut pDAB2212, containing an AHAS selectable marker driven by the rice actin promoter OsAct1. The final construct is named pDAB4101 or pDAS1863 and contains ZmUbi1 / AAD-12 (v1) / ZmPer5 :: OsAct1 / AHAS / LZmLip.

  Initiation of callus / suspension culture: To obtain immature embryos to initiate callus culture, an F1 cross between Hi-II parents A and B (Armstrong et al., 1991) grown in a greenhouse was performed. When the embryo size reaches 1.0-1.2 mm (about 9-10 days after pollination), the ears are harvested and the surface is cleaned by rubbing with Liqui-Nox (registered trademark) soap. Sterilized and soaked in 70% ethanol for 2-3 minutes, then soaked in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes.

The ears are rinsed with sterile distilled water and immature zygotic embryos are aseptically excised, 15Ag10 medium (N6 medium (Chu et al., 1975), 1.0 mg / L 2,4-D, 20 g / L sucrose, 100 mg / L casein hydrolyzate (enzymatic digest), 25 mM L-proline, 10 mg / L AgNO ₃ , 2.5 g / L Gelrite, pH 5.8) for 2-3 weeks, the scutella is removed from the medium The cells were cultured so as to face away from each other. Tissues exhibiting the correct morphology (Welter et al., 1995) were selectively transferred on fresh 15Ag10 medium for about 6 weeks at biweekly intervals, after which four mediums (N6 medium, 1.0 mg / L 2,4 -D, transferred to 20 g / L sucrose, 100 mg / L casein hydrolyzate (enzymatic digest), 6 mM L-proline, 2.5 g / L Gelrite, pH 5.8) at biweekly intervals for about 2 months .

  To initiate embryogenic suspension culture, approximately 3 ml of packed cell volume (PCV) of callus tissue from a single embryo was transferred to approximately 30 ml of H9CP + liquid medium (MS basal salt mixture (Murashige and Skog, 1962). ) Modified MS vitamin containing 10 times less nicotinic acid and 5 times more thiamine-HCl, 2.0 mg / L 2,4-D, 2.0 mg / L α-naphthalene acetic acid (NAA), 30 g / L Sucrose, 200 mg / L casein hydrolyzate (acid digestion), 100 mg / L myo-inositol, 6 mM L-proline, 5% v / v coconut water (added just before subculture), pH 6.0 ). The suspension culture was maintained in a 125 ml Erlenmeyer flask under dark conditions on a temperature-controlled shaker set at 125 rpm and 28 ° C. Cell lines were typically established within 2-3 months of initiation. During establishment, the suspension was subcultured every 3.5 days by adding 3 ml PCV cells and 7 ml conditioned medium to 20 ml fresh H9CP + liquid medium using a wide caliber pipette. When tissue growth began to double, the suspension was scaled up and maintained in a 500 ml flask by transferring 12 ml PCV cells and 28 ml conditioned medium into 80 ml H9CP + medium. After suspensions were fully established, they were stored frozen for future use.

  Cryopreservation and thawing of suspension: After 2 days of subculture, 4 ml PCV suspension cells and 4 ml conditioned medium were added to 8 ml cryoprotectant (H9CP + medium without coconut water, 1 M glycerol, 1 M DMSO, 2 M And sterilized by filtration, and shaken in a 125 ml flask at 125 rpm and 4 ° C. for 1 hour. After 1 hour, 4.5 ml was added to a chilled 5.0 ml Corning cryovial. After filling, individual vials were kept in a freezer with controlled cooling rate at 4 ° C for 15 minutes and then frozen at a rate of -0.5 ° C / min until the final temperature of -40 ° C was reached. After reaching the final temperature, the vial was transferred to a box in a rack in a Cryoplus 4 storage unit (Form a Scientific) filled with liquid nitrogen vapor.

  For thawing, the vials were removed from the storage unit, placed in a sealed dry ice container, and submerged in a water bath maintained at 40-45 ° C. until “boiling” was weakened. After thawing, the contents were poured onto a stack of approximately 8 sterile 70 mm Whatman filter papers (No. 4) in a 100 × 25 mm Petri dish with a lid. The liquid is allowed to absorb on the filter paper for a few minutes, after which the top filter paper containing the cells is GN6 medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 2.5 g / L Of Gelrite, pH 5.8) for 1 week. After 1 week, only tissues with promising morphology were removed from the filter paper and transferred directly onto fresh GN6 medium. The tissue was subcultured every 7-14 days in a 125 ml Erlenmeyer flask in approximately 30 ml H9CP + medium until 1-3 grams were available for initiation of suspension culture. Three milliliters of PCV were subcultured every 3.5 days in fresh H9CP + medium until a total of 12 ml PCV was obtained, at which point subculture was performed as previously described.

  Stable transformation: Approximately 24 hours prior to transformation, 12 ml PCV cryopreserved embryogenic corn suspension cells + 28 ml conditioned medium in 80 ml GN6 liquid medium (GN6 medium lacking Gelrite), 500 ml Erlenmeyer Subcultured in a flask and placed on a shaker at 125 rpm and 28 ° C. This was repeated twice using the same cell line so that a total of 36 ml PCV was distributed across the three flasks. After 24 hours, 72 ml GN6 S / M osmotic medium (N6 medium, 2.0 mg / L 2,4-D, 30 g) per flask to remove GN6 liquid medium and allow protoplast separation of cells. / L sucrose, 45.5 g / L sorbitol, 45.5 g / L mannitol, 100 mg / L myo-inositol, pH 6.0). Place the flask on a shaker and shake at 125 RPM for 30-35 minutes in the dark at 28 ° C., during which time an appropriate volume of 8.1 ml of GN6 S / M liquid medium is added to about 405 mg pre-autoclaved. A suspension of 50 mg / ml silicon carbide agate crystals was prepared by adding to the prepared sterile silicon carbide agate crystals (Advanced Composite Materials, Inc.).

  After incubation in GN6 S / M, the contents of each flask were pooled in a 250 ml centrifuge bottle. After all cells settled to the bottom, other than about 44 ml of GN6 S / M liquid was removed and collected in a sterile 1 L flask for future use. Vortex the pre-wet suspension of agate crystals for 60 seconds at maximum speed, after which 8.1 ml was added to the bottle, to which 170 μg of DNA was added as a final step. The bottle was immediately placed in a modified Red Devil 5400, a commercial paint mixer, and stirred for 10 seconds. After agitation, a cocktail of cells, media, agate crystals and DNA was added to the contents of the 1 L flask along with 125 ml of fresh GN6 liquid media to reduce osmotic regulators. Cells were allowed to recover on a shaker at 125 RPM for 2 hours at 28 ° C. and then filtered onto Whatman # 4 filter paper (5.5 cm) using a glass cell collector unit connected to a house vacuum line. .

  About 2 ml of the dispersion suspension was pipetted onto the surface of the filter paper while applying a vacuum. The filter paper was placed on a 60 × 20 mm plate of GN6 medium. Plates were incubated for 1 week at 28 ° C. in a dark box.

  After one week, filter paper was washed with GN6 (3P) medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 100 mg / L myo-inositol, 3 μM Pursuit® DG imazetapill. , 2.5 g / L Gelrite, pH 5.8) 60 × 20 mm plate. Plates were placed in boxes and incubated for an additional week.

  Two weeks after transformation, all cells on the plate were washed with 3.0 ml of melted GN6 agarose medium (N6 medium, 2.0 mg / L 2,4 containing 3 μM Pursuit® DG imazetapyr. The tissue was embedded by scraping onto -D, 30 g / L sucrose, 100 mg / L myo-inositol, 7 g / L Sea Plaque agarose, pH 5.8, autoclaving only at 121 ° C. for 10 minutes). The tissue was crushed and 3 ml of agarose and tissue were evenly poured on the surface of a GN6 100 × 15 mm plate (3P). This was repeated for all remaining plates. Post-embedding plates were individually sealed with Nescofilm® or Parafilm M® and then cultured until a putative isolate appeared.

  Isolation recovery and regeneration protocol: Pursuit®-containing by transferring putatively transformed events to fresh selection media at the same concentration in 60 × 20 mm plates approximately 9 weeks after transformation. Isolated and removed from the embedding plate. If growth maintenance was evident after about 2-3 weeks, the event was considered resistant and subjected to molecular analysis.

Regeneration consists of callus tissue, 3 μM Pursuit® DG imazetapil, MS salts and vitamins, 30.0 g / L sucrose, 5 mg / L BAP, 0.25 mg / L 2,4-D, 2 Started by transferring to 28 (3P), an induction medium based on cytokinin containing 0.5 g / L Gelrite, pH 5.7. Cells were grown for 1 week in low light (13 μEm ⁻² s ⁻¹ ) and then for an additional week in stronger light (40 μEm ⁻² s ⁻¹ ), then 28 (3P) except lacking plant growth regulators And transferred to regeneration medium 36 (3P), which is identical to Small (3-5 cm) plantlets are removed and unselected SHGA 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). ) In a 150 × 25 mm culture tube. After the plantlets had developed sufficient root and shoot systems, they were transferred to soil in the greenhouse.

  From four experiments, complete plantlets consisting of shoots and roots were formed under dark conditions on embedded selection plates in vitro without experiencing the traditional callus period. Leaf tissue from 9 of these “early regenerants” was subjected to coding region PCR and plant transcription unit (PTU) PCR for the AAD-12 gene and gene cassette, respectively. All had intact AAD-12 coding regions, while 3 did not have full-length PTU (Table 11). These “initial regenerators” were identified as 4101 events to distinguish them from traditionally induced events identified as “1283” events. Plants from 19 additional events obtained by standard selection and regeneration were sent to the greenhouse, grown to maturity, and cross-pollinated with proprietary inbred lines to produce T1 seeds. Due to the similar band pattern after the Southern blot, only 14 unique events are represented because some events are considered to be clones of each other. TO plants from the event were resistant to 70 g / ha imazetapil. Invader analysis (AHAS gene) showed insertion complexity ranging from 1 to over 10 copies. Thirteen events contained the AAD-12 compete coding region, but further analysis showed that nine events did not incorporate complete plant transformation units. None of the compromised 1863 events progressed beyond the T1 stage, and 4101 events were utilized for further characterization.

  Molecular analysis-maize materials and methods: tissue harvest, DNA isolation and quantification. Fresh tissue is placed in a tube and lyophilized at 4 ° C. for 2 days. After the tissue is completely dry, tungsten beads (Vallite) are placed in the tube and the sample is subjected to 1 minute dry grinding using a Kelco bead mill. A standard DNeasy DNA isolation procedure is then performed (Qiagen, DNeasy 69109). Subsequently, an aliquot of the extracted DNA is stained with Pico Green (Molecular Probes P7589) and read on a fluorimeter (BioTek) using known standards to obtain a concentration of ng / μl.

Invader assay analysis: DNA samples are diluted to 20 ng / μl and then denatured by incubation in a thermocycler at 95 ° C. for 10 minutes. A signal probe mixture is then prepared using the provided oligo mixture and MgCl ₂ (Third Wave Technologies). 7.5 μl aliquots, then 7.5 μl of controls, standards, and 20 ng / μl of diluted unknown sample aliquots are placed in each well of the Invader assay plate. Place 15 μl of mineral oil (Sigma) in each well. The plates are then incubated for 1 hour at 63 ° C. and read on a fluorimeter (Biotek). The ratio is calculated by calculating the% signal above the background of the target probe divided by the% signal above the background internal control probe. The ratio of known copy number standards developed and validated by Southern blot analysis is used to identify the estimated copy number of the unknown event.

  Polymerase chain reaction: A total of 100 ng of total DNA is used as a template. 20 mM of each primer is used with Takara Ex Taq PCR polymerase kit (Mirus TAKRR001A). The primers for AAD-12 (v1) PTU are forward-GAACAGTTTAG ACATGGTCTA AAGG (SEQ ID NO: 8) and reverse-GCTGCAACAC TGATAAAGC CAACTGG (SEQ ID NO: 9). The PCR reaction was performed in a 9700 Geneamp thermocycler (Applied Biosystems) with samples being run at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 63 ° C for 30 seconds, 72 ° C for 1 minute and 45 seconds, and then at 72 ° C. Perform by subjecting to 10 minutes.

  Primers for AAD-12 (v1) coding region PCR are forward-ATGGCTCAGA CCACTCTCCA AA (SEQ ID NO: 10) and reverse-AGCTGCATCC ATGCCAGGGGA (SEQ ID NO: 11). The PCR reaction was carried out in a 9700 Geneamp thermocycler (Applied Biosystems) with samples run at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 1 minute 45 seconds, and then at 72 ° C. Perform by subjecting to 10 minutes. PCR products are analyzed by electrophoresis on a 1% agarose gel stained with EtBr.

  Southern blot analysis: Southern blot analysis is performed using genomic DNA obtained from the Qiagen DNeasy kit. A total of 2 μg genomic leaf DNA or 10 μg genomic callus DNA is subjected to overnight digestion using BSM I and SWA I restriction enzymes to obtain PTU data.

  After overnight digestion, approximately 100 ng aliquots are run on a 1% gel to ensure complete digestion. After this belief, the sample is run on a large 0.85% agarose gel overnight at 40 volts. The gel is then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gel is then neutralized in 0.5 M Tris HCl, 1.5 M NaCl, pH 7.5 for 30 minutes. The gel apparatus containing 20 × SSC is then set up to obtain overnight transfer from the gel by gravity to a nylon membrane (Millipore INYC00010). After overnight transfer, the membrane is subjected to UV light at 1200 × 100 microjoules via a cross-linking agent (Stratagene UV stratalinker 1800). The membrane is then washed with 0.1% SDS, 0.1 SSC for 45 minutes. After washing for 45 minutes, the membrane is baked for 3 hours at 80 ° C. and then stored at 4 ° C. until hybridization. Hybridization template fragments are prepared using the above coding region PCR using plasmid DNA. The product is run on a 1% agarose gel, cut and then gel extracted using the Qiagen (28706) gel extraction procedure. The membrane is then subjected to prehybridization for 1 hour at 60 ° C. in Perfect Hyb buffer (Sigma H7033). The Prime it RmT dCTP labeling reaction (Stratagene 300392) procedure is used to develop a p32 based probe (Perkin Elmer). Clean up the probe using Probe Quant. G50 column (Amersham 27-5335-01). Southern blots are hybridized overnight using 2 million CPM. After overnight hybridization, the blot is subjected to two 20 minute washes in 65 ° C., 0.1% SDS, 0.1 SSC. The blot is then exposed to film overnight with incubation at −80 ° C.

  Postemergence herbicide resistance in TO corn transformed with AAD-12: until 4 TO events have been acclimatized in the greenhouse until 2-4 new normal-looking leaves emerge from the rotifera (ie, (Until the plant transitioned from tissue culture to greenhouse growth conditions). Plants were grown in a greenhouse at 27 ° C., 16 hours light: 8 hours dark. The plants were then treated with either Pursuit® (Imazetapyr) or 2,4-Damine 4 commercial formulation. Pursuit® was sprayed to demonstrate the function of the selectable marker gene present in the event tested. The herbicide application was performed using a truck sprayer with a spray volume of 187 L / ha and a spray height of 50 cm. Plants were sprayed with either a lethal dose of imazetapil (70 g ae / ha) or an amount of 2,4-D DMA salt (2240 g ae / ha) that could cause significant damage to untransformed corn lines. . A lethal dose is defined as an amount that causes more than 95% damage to the Hi-II inbred line. Hi-II is the genetic background of the transformant of the present invention.

  Several individuals were treated with a mitigating agent from the herbicide where each gene provided resistance. However, the individual clone “001” from event “001” (ie, 4101 (0) -001-001) was slightly damaged but recovered by 14 DAT. Three of the four events proceeded and the individual was crossed with 5XH751 to become the next generation. Each herbicide-tolerant plant was positive for the presence of the AAD-12 coding region (PCR assay) or the presence of the AHAS gene (Invader assay) for 2,4-D and imazetapil-tolerant plants, respectively. AAD-12 protein was detected in all 2,4-D resistant TO plant events containing an intact coding region. The transgene copy number (AHAS, and speculatively AAD-12) varied significantly from 1 to 15 copies. Individual T0 plants were grown to maturity and cross-pollinated with proprietary inbred lines to produce T1 seeds.

  Verification of high 2,4-D tolerance in T1 corn: 70 g at the two leaf stage to plant T1 AAD-12 (v1) seeds in 3 inch pots containing Metro Mix medium and to eliminate nulls Ae / ha imazetapil was sprayed. Surviving plants were transplanted into 1 gallon pots containing Metro Mix media and placed under the same growth conditions as before. At stage V3-V4, plants were sprayed at 187 L / ha in a track sprayer set to either 560 or 2240 g ae / ha 2,4-D DMA. Plants were graded to 3 and 14 DAT and compared to 5XH751xHi II control plants. A rating scale of 0-10 (no damage to extreme auxin damage) was developed to identify strut root damage. Post root grading was performed at 14 DAT to indicate 2,4-D resistance. 2,4-D causes strut root malformation and is a consistent indicator of auxin herbicide damage in maize. The strut root data (found in the table below) demonstrates that 2 of the 3 events tested were robustly resistant to 2,240 g ae / ha of 2,4-D DMA. doing. Event “pDAB4101 (0) 001.001” was clearly unstable, but the other two events were robust against 2,4-D and 2,4-D + imazetapill or 2,4-D + glyphosate (See Table 12).

  Heritability of AAD-12 (v1) in maize: Progeny tests were also performed on 7 AAD-12 (v1) T1 families crossed with 5XH751. Seeds were planted in 3 inch pots as described above. At the three-leaf stage, all plants were sprayed with 70 g ae / ha imazetapill in a track sprayer as previously described. After 14 DAT, resistant and sensitive plants were counted. As determined by chi-square analysis, 4 out of the 6 lines tested were isolated as single loci dominant Mendelian traits (1R: 1S). The surviving plants were sprayed with 2,4-D, and all plants were considered resistant to 2,4-D (amounts above 560 g ae / ha). AAD-12 is hereditary as a robust aryloxyalkanoate auxin resistance gene in multiple species when reciprocally crossed with a commercial hybrid.

  Stacking of AAD-12 (v1) to increase herbicide coverage: AAD-12 (v1) (pDAB4101) and the superior Roundup Ready inbred line (BE1146RR) were reciprocally crossed and F1 seeds were collected. Seeds from two F1 lines were planted and treated with 70 g ae / ha imazetapill at the V2 stage to eliminate nulls. For surviving plants, isolated representatives (reps), and track sprayer calibrated to 187 L / ha, 1120 g ae / ha 2,4-D DMA + 70 g ae / ha imazetapil (AHAS gene present) Or 1120 g ae / ha 2,4-D DMA + 1680 g ae / ha glyphosate (to confirm the presence of the Round Up Ready gene). Plants were graded to 3 and 16 DAT. The spray data show that AAD-12 (v1) is conventionally stacked with glyphosate tolerance genes (such as Roundup CP4-EPSPS gene) or other herbicide tolerance genes to increase the range of herbicides that can be safely applied to corn. It was shown that the agent can be provided. Similarly, imidazolinone + 2,4-D + glyphosate tolerance was observed in F1 plants, and no negative phenotype was shown by a combination of these multiple transgene molecules or breeding stacking.

  Field resistance of maize plants transformed with pDAB4101 to 2,4-D, triclopyr and fluoroxypyr herbicides: A field level tolerance test was performed using two AAD-12 (v1) pDAB4101 events (4101 (0) 003.R). .003.AF and 4101 (0) 005.R001.AF) and one Roundup Ready (RR) control hybrid (2P782) were performed in Fowler, Indiana and Wayside, Mississippi. Using a corn seeder, seeds were planted at a row spacing of 40 inches on Wayside and 30 inches on Fowler. The experimental design was a complete random block method, with 3 iterations. Herbicide treatments were 1120, 2240 and 4480 g ae / ha 2,4-D (dimethylamine salt), 840 g ae / ha triclopyr, 280 g ae / ha fluoroxypyr, and an untreated control. The AAD-12 (v1) event contained the AHAS gene as a selectable marker. The F2 corn event was isolated so that AAD-12 (v1) plants were treated with 70 g ae / ha imazetapy to remove null plants. The herbicide treatment was applied using a compressed air-back sprayer that delivered a carrier volume of 187 L / ha at a pressure of 130-200 kpa when the corn reached stage V6. Visual damage assessments were made 7, 14, and 21 days after treatment. Strut root damage assessment is performed on 28 DAT on a scale of 0-10, 0-1 is slight strut root fusion, 1-3 are moderate strut root swelling / wandering and root growth, 3-5 are moderate strut root fusion, 5-9 are severe strut root fusion and malformations, and 10 is complete inhibition of strut roots.

  The response of AAD-12 (v1) events to 2,4-D, triclopyr, and fluoroxypyr after 14 days of treatment is shown in Table 14. The crop damage was most severe at 14 DAT. The RR control corn (2P782) was severely damaged (44% to 14 DAT) by 4480 g ae / ha of 2,4-D, which is 8 times the normal field usage (8 ×). All AAD-12 (v1) events demonstrated excellent resistance to 2,4-D at 14 DAT, with 0% damage in 1, 2 and 4 × amounts, respectively. Control corn (2P782) was severely damaged (31% at 14 DAT) by 2 × amount of triclopyr (840 g ae / ha). The AAD-12 (v1) event demonstrated resistance with 2 × amount of triclopyr, with an average of 3% damage over 2 events at 14 DAT. 280 g ae / ha of fluoroxypy caused 11% visual damage to wild-type corn at 14 DAT. The AAD-12 (v1) event demonstrated increased resistance with an average of 8% damage to 5DAT.

  Application of auxin herbicides to V6 growing corn can cause strut root malformation. Table 15 shows the severity of strut root injury caused by 2,4-D, triclopyr, and fluoroxypyr. 840 g ae / ha of triclopyr caused the most severe strut root fusion and dysplasia, resulting in an average strut root damage score of 7 in 2P782 control corn.

  Neither AAD-12 (v1) corn event showed strut root damage from triclopyr treatment. The strut root damage of 2P782 corn increased with increasing amount of 2,4-D. At 4480 g ae / ha 2,4-D, the AAD-12 event showed no strut root damage, while the 2P782 hybrid showed severe strut root fusion and dysplasia. Fluoroxypyr caused only moderate strut root swelling and migration in wild-type corn, and the AAD-12 (v1) event showed no strut root damage.

  This data shows that AAD-12 (v1) exceeds the amount commercially used in corn and is an amount of 2,4 that causes severe visual and strut root damage in non-AAD-12 (v1) corn. It clearly shows that it conveys a high level of resistance to -D, triclopyr and fluoroxypyr.

Tobacco Transformation Tobacco transformation using Agrobacterium tumefaciens was performed by a method similar to, but not identical to, the published method (Horsch et al., 1988). TOB, a hormone-free Murashige & Skoog medium (Murashige and Skoog, 1962) obtained by surface sterilizing tobacco seeds (Nicotiana tabacum cv. KY160) and solidifying with agar to provide a source tissue for transformation. Planted on the surface of the medium. Plants were grown for 6-8 weeks in a lighted incubator room at 28-30 ° C. and leaves were collected aseptically for use in transformation protocols. From these leaves, a small piece of about 1 square centimeter was cut aseptically, except for the midrib. A culture of Agrobacterium strain (EHA101S containing pDAB3278, ie pDAS1580, AAD-12 (v1) + PAT) grown on a shaker set at 250 rpm and 28 ° C. overnight in a flask. Pelleted by centrifugation, resuspended in sterile Murashige & Skoog salt and adjusted to a final optical density of 0.5 at 600 nm. The leaf pieces are immersed in this bacterial suspension for about 30 seconds, then blotted on a sterile paper towel and dried, with the correct orientation up and TOB + medium (1 mg / L indoleacetic acid and 2.5 mg / L benzyl). (Murashige & Skoog medium containing adenine) and incubated at 28 ° C. in the dark. Two days later, the leaf pieces were transferred to TOB + medium containing 250 mg / L cefotaxime (Agri-Bio, North Miami, FL) and 5 mg / L glufosinate ammonium (active ingredient in Basta, Bayer Crop Sciences) Incubated in the light at 30 ° C. Leaf pieces were transferred to fresh TOB + medium containing 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, small plants resulting from transformed foci were removed from the tissue preparation and 250 mg / L cefotaxime and 10 mg / L in a Phytray ™ II container (Sigma). Planted in TOB medium containing 1 of Basta. These plantlets were grown in a lighted incubator room. After 3 weeks, the stems were taken out and rooted again in the same medium. The plants were ready to be sent to the greenhouse after a further 2-3 weeks.

The plant washes the agar from the roots, transplants the soil into a 13.75 cm square pot, puts the pot in a Ziploc® bag (SC Johnson & Son, Inc.), puts tap water at the bottom of the bag, and indirectly It was moved into the greenhouse by placing it in a 30 ° C. greenhouse for 1 week in the light. After 3-7 days, the bag was opened and the plants were fertilized and grown in open bags until the plants were acclimated to the greenhouse, at which point the bags were removed. Plants were grown under normal warm greenhouse conditions (30 ° C., 16 hours day, 8 hours night, lowest natural light + auxiliary light = 500 μE / m ² s ¹ ).

  Prior to breeding, T0 plants were sampled for DNA analysis to determine the copy number of the insert. For convenience, the PAT gene molecularly linked to AAD-12 (v1) was assayed. Fresh tissue was placed in a tube and lyophilized at 4 ° C. for 2 days. After the tissue was completely dried, tungsten beads (Vallite) were placed in the tube and the sample was subjected to 1 minute dry grinding using a Kelco bead mill. A standard DNeasy DNA isolation procedure was then performed (Qiagen, DNeasy 69109). Subsequently, aliquots of the extracted DNA were stained with Pico Green (Molecular Probes P7589) and read with a fluorometer (BioTek) using known standards to obtain a concentration of ng / μl.

The DNA sample was diluted to 9 ng / μl and then denatured by incubation in a thermocycler at 95 ° C. for 10 minutes. A signal probe mixture was then prepared using the provided oligo mixture and MgCl ₂ (Third Wave Technologies). 7.5 μl aliquots, then 7.5 μl of controls, standards, and 20 ng / μl of diluted unknown sample aliquots were placed in each well of the Invader assay plate. Each well was loaded with 15 μl of mineral oil (Sigma). The plates were then incubated for 1.5 hours at 63 ° C. and read on a fluorimeter (Biotek). The ratio is calculated by calculating the% signal above the background of the target probe divided by the% signal above the background internal control probe. The ratio of known copy number standards developed and validated by Southern blot analysis was used to identify the estimated copy number of unknown events.

  All events were also assayed for the presence of the AAD-12 (v1) gene by PCR using the same extracted DNA sample. A total of 100 ng of total DNA was used as a template. 20 mM of each primer was used with Takara Ex Taq PCR polymerase kit. The primers for the plant transcription unit (PTU) PCR AAD-12 were (SdpacodF: ATGGCTCATG CTGCCCTCAG CC) (SEQ ID NO: 12) and (SdpacodR: CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 13). The PCR reaction was carried out in a 9700 Geneamp thermocycler (Applied Biosystems) with samples run at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 64 ° C for 30 seconds, 72 ° C for 1 minute 45 seconds, then at 72 ° C. Performed by subjecting to 10 minutes. PCR products were analyzed by electrophoresis on a 1% agarose gel stained with EtBr. 4-12 from each of 18 PCR positive events with 1-3 copies of the PAT gene (and presumably AAD-12 (v1), because these genes are physically linked) Clone lines were regenerated and moved to the greenhouse.

  Postemergence herbicide resistance in T0 tobacco transformed with AAD-12 (v1): A broad range of 2,4-sprayed TO plants from each of the 19 events were sprayed onto plants that were 3-4 inches in height. Challenged with D, triclopyr, or fluoroxypyr. The spray application was performed using a track sprayer with a spray volume of 187 L / ha, as already described. 2,4-D dimethylamine salt (Riverside Corp) was applied to representative clones from each event mixed with deionized water at 0, 140, 560, or 2240 g ae / ha. Fluoroxypyr was similarly applied at 35, 140, or 560 g ae / ha. Triclopyr was applied at 70, 280, or 1120 g ae / ha. Each treatment was repeated 1-3 times. Damage ratings were recorded at 3 and 14 DAT. All the events tested were more resistant to 2,4-D than the untransformed control line KY160. In some events, upregulation associated with some early auxinic herbicides occurred at doses of 2,4-D below 560 g ae / ha. Some events were undamaged with 2,4-D (equivalent to 4 × field volume) applied at 2240 g ae / ha. Overall, the AAD-12 (v1) event was more sensitive to fluoroxypy then triclopyr and was the least affected by 2,4-D. The quality of the event on the magnitude of resistance was identified using the response of the TO plant to 560 g ae / ha fluoroxypyr. Events were classified as “low” (more than 40% damage at 14 DAT), “moderate” (20-40% damage), and “high” (less than 20% damage). Some events were considered “variable” with inconsistent responses between repeated iterations.

  Verification of high 2,4-D tolerance in T1 tobacco: 2 to 4 T0 individuals that survived large amounts of 2,4-D and fluoroxypyr are stored from each event and self-pollinated in a greenhouse T1 seeds were produced. T1 seeds are stored in layers and seeded in a selection tray very similar to that of Arabidopsis, and then 560 g ai / ha glufosinate (PAT gene selection) is used to untransform nulls in this segregating population. Was selectively removed. Survivors were transferred to individual 3-inch pots in the greenhouse. These lines provided a high level of resistance to 2,4-D in the T0 generation. Because it is not derived directly from tissue culture, an improved consistency of response is expected in T1 plants. These plants were compared to wild type KY160 tobacco. All plants were sprayed with a track sprayer set at 187 L / ha. Plants were sprayed in the range of 140-2240 g ae / ha 2,4-D dimethylamine salt (DMA), 70-1120 g ae / ha triclopyr or 35-560 g ae / ha fluoroxypyr. All applications were formulated in water. Each treatment was repeated 2-4 times. Plants were evaluated after 3 and 14 days of treatment. Plants were assigned damage ratings for atrophy, whitening, and necrosis. Since the T1 generation is separated, some variability response is expected due to the difference in bonding state.

  No damage was observed at 2,4-D below 4 × field volume (2240 g ae / ha). In one event line, some damage was observed with triclopyr treatment, but the greatest damage was observed with fluoroxypyr. Fluoroxypyr damage was short lived and by 14 DAT, new growth in one event was almost indistinguishable from untreated controls (Table 17). Importantly, note that non-transformed tobacco is overly sensitive to fluoroxypyr. These results indicate that AAD-12 (v1) can provide commercial levels of 2,4-D resistance even in very auxin-sensitive dicotyledonous crops such as tobacco. These results also indicate that resistance to pyridyloxyacetic acid herbicides, triclopyr and fluoroxypyr can also be imparted. It is very useful for growers to have the ability to formulate treatments in herbicide-tolerant crops that are protected by AAD-12 using a variety of active ingredients with a variable range of weed control.

  Heritability of AAD-12 (v1) in tobacco: A progeny test of 100 plants was also performed on 7 T1 lines of the AAD-12 (v1) line. With the exception of not being removed by Liberty selection, seeds were stored in layers, sown and transplanted in compliance with the above procedure. Thereafter, all plants were sprayed with 560 g ae / ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. As determined by chi-square analysis, 5 out of the 7 lines tested were isolated as single loci dominant Mendelian traits (3R: 1S). AAD-12 is hereditary as a robust aryloxyalkanoate auxin resistance gene in multiple species.

  Field Tolerance of pDAS1580 Tobacco Plants to 2,4-D, Dichloprop, Triclopyr and Fluoroxypyr Herbicide: Field Level Tolerance Test was performed on three AAD-12 (v1) lines (event pDAS1580- [1]- 018.001, pDAS1580- [1] -004.001 and pDAS1580- [1] -020.016) and one wild type strain (KY160) were performed at Indiana and Mississippi field stations. Tobacco transplants were grown in a greenhouse by planting T1 seeds in 72-well transplant flats (Hummert International) containing Metro360 medium according to the growth conditions described above. Null plants were selectively removed by Liberty selection as previously described. Transplanted plants were transported to a field station and planted using an industrial plant seeder at either 14 or 24 inch intervals. To maintain vigorous plant growth, drip irrigation was used at the Mississippi site and overhead irrigation was used at the Indiana site.

  The experimental design was 4 iterations in the partition method. The main plot was herbicide treatment and the secondary plot was a tobacco line. Herbicide treatment included 280, 560, 1120, 2240 and 4480 g ae / ha 2,4-D (dimethylamine salt), 840 g ae / ha triclopyr, 280 g ae / ha fluoroxypyr, and untreated control Met. The compartment was one row of 25-30 ft. Herbicide treatment was applied 3 to 4 weeks after implantation using a compressed air-backed nebulizer delivering a 187 L / ha carrier volume at a pressure of 130-200 kpa. Visual assessment of injury, growth inhibition, and top growth was performed 7, 14, and 21 days after treatment.

  Responses of AAD-12 (v1) events to 2,4-D, triclopyr, and fluoroxypyr are shown in Table 18. Untransformed tobacco lines were severely damaged by 560 g ae / ha 2,4-D, which is considered 1 × field application (63% at 14 DAT). All AAD-12 (v1) strains demonstrated excellent resistance to 2,4-D at 14 DAT, with an average damage of 1, 4, and 4% damage observed at 2, 4, and 8 × doses, respectively. It was. Untransformed tobacco lines were severely damaged by 2 × amount of triclopyr (840 g ae / ha) (53% in 14 DAT), while AAD-12 (v1) lines were 3 in 14 DAT Tolerance with an average of 5% damage was demonstrated across the line. 280 g ae / ha of fluoroxypy caused severe damage (99%) to lines not transformed into 14 DAT. The AAD-12 (v1) line demonstrated increased resistance with an average of 11% damage to 14DAT.

  These results indicate that the event lines transformed with AAD-12 (v1) are lethal to tobacco that has not been transformed under typical field conditions, or have severe upregulated dysplasia. It was shown that the multiple commercial usages caused showed a high level of resistance to 2,4-D, triclopyr and fluoroxypyr.

  AAD-12 (v1) protection against elevated amounts of 2,4-D: Results showing protection of AAD-12 (v1) against elevated amounts of 2,4-D DMA in the greenhouse are shown in Table 19. T1 AAD-12 (v1) plants from an event segregated to 3R: 1S when selected with a 560 g ai / ha Liberty using the same protocol as previously described. T1 AAD-1 (v3) seeds were also planted on transformed tobacco controls (see PCT / US2005 / 014737). Non-transformed KY160 served as a sensitivity control. Plants were sprayed using a track sprayer set to 2,4-D DMA of 187 L / ha, 140, 560, 2240, 8960, and 35840 g ae / ha and rated at 3 and 14 DAT.

  Both AAD-12 (v1) and AAD-1 (v3) effectively protected tobacco against 2,4-D injury at doses up to 4 × commercial use. However, AAD-12 (v1) clearly demonstrated significant advantages over AAD-1 (v3) by protecting up to 64 × standard field volumes.

  Stacking AAD-12 to increase herbicide coverage: homozygous AAD-12 (v1) (pDAS1580) and AAD-1 (v3) (pDAB721) plants (see PCT / US2005 / 014737 for the latter) Both were crossed and F1 seeds were collected. F1 seeds from two reciprocal crosses of each gene are layered and stored and four representatives (reps) processed for each cross are under the same spray regimine as used in the other studies. Treated with one of the following treatments: 70, 140, 280 g ae / ha fluoroxypyr (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 2,4-D resistance). Each gene homozygous T2 plant was also planted for use as a control. Plants were graded to 3 and 14 DAT. The results of spraying are shown in Table 20.

  The results show a range of herbicides that successfully stack AAD-12 (v1) and AAD-1 (v3) and can therefore be applied to target crops (phenoxyactetic acid for AAD-1 and AAD-12, respectively) It proves that + phenoxypropionic acid vs. penoxyacetic acid + pyridyloxyacetic acid) can be increased. The complementary nature of the herbicide cross-resistance pattern allows for convenient use of these two genes as complementary and stackable field-selectable markers. In crops where tolerance using a single gene can be marginal, the skilled person will appreciate that tolerance can be increased by stacking a second tolerance gene of the same herbicide. Such can be done using the same gene but with the same or different promoters, but as observed here, the stacking and tracking of two complementary traits is phenoxypropionic acid [AAD Can be facilitated by identifying cross-protection against -1 (from v3)] or pyidyloxyacetic acid [AAD-12 (v1)].

Soybean Transformation Soybean improvements through introgression techniques are due to traits such as herbicide resistance (Padgette et al., 1995), amino acid modifications (Falco et al., 1995), and insect resistance (Parrott et al., 1994). Has been achieved. Introduction of exogenous traits into crop species requires the method to allow routine generation of transgenic lines using selectable marker sequences containing simple inserts. The transgene should be heritable as a single functional locus to simplify breeding. Delivery of foreign genes into cultivated soybeans by microparticle irradiation of mating hypocotyls (McCabe et al., 1988) or somatic embryogenic cultures (Finer and McMullen, 1991), and cotyledon explants (Hinchee et al., 1988) or Agrobacterium-mediated transformation of zygotic embryos (Chee et al., 1989) has been reported.

  Transformants derived from Agrobacterium-mediated transformation tend to carry a simple insert with a low copy number (Birch, 1991). Three target tissues that have been investigated for gene transfer into soybean: mating hypocotyl (Chee et al., 1989, McCabe et al., 1988), cotyledons (Hinchee et al., 1988) and somatic embryogenic cultures (Finer and McMullen) , 1991), there are advantages and disadvantages. The latter has been extensively investigated as a target tissue for direct gene transfer. Embryogenic cultures tend to be very prolific and can be maintained for long periods of time. However, sterility and chromosomal abnormalities in primary transformants have been linked to the age of embryogenic suspensions (Singh et al., 1998), and so soybean transformation systems utilizing this tissue are not A continuous start is considered necessary. This system requires a high level of 2,4-D, 40 mg / L concentration to initiate embryogenic callus, which is transformed with 2,4-D present in the medium. Since the locus cannot be further developed, it poses a fundamental problem in the use of the AAD-12 (v1) gene. Therefore, meristem-based transformation is ideal for the development of 2,4-D resistant plants using AAD-12 (v1).

  Gateway cloning of binary constructs: AAD-12 (v1) coding sequence was cloned into 5 different Gateway donor vectors containing different plant promoters. Subsequently, the resulting AAD-12 (v1) plant expression cassette was cloned into the Gateway Destination binary vector via the LR Clonase reaction (Invitrogen Corporation, Carlsbad, CA, catalog # 11791-019).

  The NcoI-SacI fragment containing the AAD-12 (v1) coding sequence was digested from DASPICO12 and ligated into the corresponding NcoI-SacI restriction site in the following Gateway donor vector: pDAB3912 (attL1 // CsVMV promoter // AtuORF23 3′UTR // attL2), pDAB3916 (attL1 // AtUbi10 promoter // AtuORF23 3′UTR // attL2), pDAB4458 (attL1 // AtUbi3 promoter // AtuORF23 3′UTR // attL2), pDAB459Z Promoter // AtuORF23 3'UTR // attL2), and pDAB4460 (attL1 // AtAct2 pro Ta // AtuORF23 3'UTR // attL2). The resulting constructs containing the following plant expression cassettes were named: pDAB4463 (attL1 // CsVMV promoter // AAD-12 (v1) // AtuORF23 3′UTR // attL2), pDAB4467 (attL1 // AtUbi10 promoter // AAD-12 (v1) // AtuORF23 3′UTR // attL2), pDAB4471 (attL1 // AtUbi3 promoter // AAD-12 (v1) // AtuORF23 3′UTR // attL2), pDAB4475 (attL1 // ZmUbi1 promoter) // AAD-12 (v1) // AtuORF23 3′UTR // attL2), and pDAB4479 (attL1 // AtAct2 promoter // AAD-12 (v1) // A uORF23 3'UTR // attL2). These constructs were confirmed by restriction enzyme digestion and sequencing.

  The plant expression cassette is transferred to the Gateway Destination binary vector pDAB4484 (RB7 MARv3 // attR1-ccdB-chloramphenicol resistance-attR2 // CsVMV promoter // ATUORF3) via Gateway LR Clonase reaction. Recombined. Gateway technology uses site-specific recombination based on lambda phage instead of restriction endonucleases and ligases to insert the gene of interest into an expression vector. Invitrogen Corporation, Gateway Technology: A Universal Technology to Clone DNA Sequences for Functional Analysis and Expression in multipe Systems, technical manuals, catalogs # 12535-019 and 12535-027, Gateway technology version E, September 22, 2003, # 25- 022. DNA recombination sequences (attL and attR) and the LR Clonase enzyme mixture allow all DNA fragments flanked by recombination sites to be transferred to any vector containing the corresponding sites. The attL1 site of the donor vector corresponds to the attR1 of the binary vector. Similarly, the attL2 site of the donor vector corresponds to the attR2 of the binary vector. Using Gateway technology, plant expression cassettes (from donor vectors) flanked by attL sites can be recombined into the attR sites of binary vectors. The resulting construct containing the following plant expression cassette was designated as follows: pDAB4464 (RB7 MARv3 // CsVMV promoter // AAD-12 (v1) // AtuORF23 3′UTR // CsVMV promoter // PATv6 AtuORF1 3 'UTR), pDAB4468 (RB7 MARv3 // AtUbi10 promoter // AAD-12 (v1) // AtuORF23 3'UTR // CsVMV promoter // PATv6 // AtuORF1 3'UTR), pDAB4472 (RB7 MARv3 / tpro) / AAD-12 (v1) // AtuORF23 3′UTR // CsVMV promoter // PATv6 // AtuORF1 3′UTR), pDAB4476 (RB7) MARv3 // ZmUbi1 promoter // AAD-12 (v1) // AtuORF23 3'UTR // CsVMV promoter // PATv6 AtuORF1 3'UTR) and pDAB4480 (RB7 MARv3 // AtAct2 promoter // AAD-12v1) / AtuORF23 3′UTR // CsVMV promoter // PATv6 // AtuORF1 3′UTR). These constructs were confirmed by restriction enzyme digestion and sequencing.

  Transformation Method 1-Agrobacterium-mediated transformation: The first report of soybean transformation is the transformation of shoots from meristem cells (Hinchee et al., 1988) and apical meristems in the cotyledonary region. Targeting proliferation (McCabe et al., 1988). In the cotyledonary node method based on A. tumefaciens, explant preparation and culture medium composition stimulates proliferation of auxiliary meristems in the node (Hinchee et al., 1988). It is still unclear whether callus cultures that are truly dedifferentiated but totipotent are initiated by these treatments. Recovery of multiple clones of a single transformation event from a single explant and rare recovery of chimeric plants (Clemente et al., 2000, Olhoft et al., 2003) is a single cell origin followed by the growth of transgenic cells. Show that either a transgenic transgenic meristem culture or a uniformly transformed shoot undergoing further shoot growth is produced. Soybean shoot propagation methods originally based on microparticle irradiation (McCabe et al., 1988) and more recently adapted to Agrobacterium-mediated transformation (Martinell et al., 2002) Based on the successful identification of germline chimeras, it is believed not to undergo the same level or type of dedifferentiation as the cotyledonary node method. This is a protocol that is not based on 2,4-D and is ideal for 2,4-D selection systems. Thus, the cotyledon node method may be the optimal method for developing 2,4-D resistant soybean cultivars.

  Generation of AAD-12 (v1) resistant phenotype plant transformations. AAD-12 (v1) transgenic plants were generated using explants derived from “Maverick” seeds and a cotyledon node transformation protocol mediated by Agrobacterium.

  Preparation and inoculation of Agrobacterium: transformation using Agrobacterium strain EHA101 (Hood et al., 1986) carrying each of the five binary pDAB vectors (Table 8). Started. Each binary vector contains an AAD-12 (v1) gene and a plant selectable gene (PAT) cassette within the T-DNA region. The plasmid was mobilized by electroporation into the EHA101 strain of Agrobacterium. The selected colonies were then analyzed for gene integration and then soybean explants were treated with Agrobacterium. Maverick seeds were used in all transformation experiments and seeds were obtained from the University of Missouri, Columbia, Missouri.

  Agrobacterium-mediated transformation of soybean (Glycine max) was performed using the PAT gene as a selectable marker coupled with the herbicide glufosinate as a selection agent. Seeds were germinated on B5 basal medium (Gamborg et al., 1968) solidified with 3 g / L of Phytagel (Sigma-Aldrich, St. Louis, MO). The selected shoots were then transferred to the rooting medium. The optimal selection scheme was the use of 8 mg / L glufosinate during the first and second shoot initiation stages in the medium and 3-4 mg / L during shoot elongation in the medium.

  Before the elongated shoots (3-5 cm) were transferred to the rooting medium, the dissected ends of the internodes were immersed in 1 mg / L indole 3-butyric acid for 1-3 minutes to promote rooting (Khan et al. 1994). The shoots are rooted in a 25 × 100 mm glass culture tube containing rooting medium, after which they are placed in an open Magenta box in Conviron in Metro-mix 200 (Hummert International, Earth City, Mo.). Transferred to a soil mixture for climate adaptation of plantlets. Glufosinate, the active ingredient of the Liberty herbicide (Bayer Crop Science), was used for selection during shoot initiation and elongation. After rooted plantlets were acclimated for several weeks in the open Magenta box, they were screened and transferred to the greenhouse for further climatic acclimatization and establishment.

  Estimates of putatively transformed plantlets, and T0 plants in the greenhouse established in the analysis: to screen putative transformants, the terminal leaflets of selected leaves of these plantlets for 1 week Twice at intervals, 50 mg / L glufosinate was applied to the leaves and the results were observed. The screened plantlets are then transferred to the greenhouse, and after acclimatization, glufosinate is reapplied to the leaves to confirm the resistance state of these plantlets in the greenhouse and are considered putative transformants. It was done.

  Plants that have been transferred to the greenhouse have glufosinate solution [0.05-2% v / v Liberty herbicide, preferably 0.25-1.0% (v / V) = 500-2000 ppm glufosinate, Bayer Crop Science], can be further assayed for the presence of active PAT gene in a non-destructive manner. Depending on the concentration used, glufosinate damage assessment can be performed 1-7 days after treatment. Also, 2,4-D aqueous solution (0.25 to 1% v / v commercial 2,4-D dimethylamine salt formulation, preferably 0.5% v / v = 2280 ppm 2,4-D ae ) To the newly expanding three-leaflet terminal leaflet in one or two of the youngest budding trifolioate, preferably two nodes below, to make the plant non-destructive It can also be tested for 2,4-D resistance in a manner. This assay allows assessment of 2,4-D sensitive plants 6 hours to several days after application by assessing leaf reversal or rotation greater than 90 degrees from the plane of adjacent leaflets. Plants resistant to 2,4-D do not respond to 2,4-D. T0 plants are self-pollinated in a greenhouse to produce T1 seeds. T1 plants (to the extent that sufficient T0 plant clones are generated) are sprayed with various ranges of herbicide doses to protect the herbicide protection conferred by the AAD-12 (v1) and PAT genes in transgenic soybeans. Determine the level. The amount of 2,4-D used for T0 plants is typically one or two selective amounts in the range of 100-1120 g ae / ha using a track sprayer as previously described. Including. T1 plants are treated with wider herbicidal doses in the 2,4-D range of 50-3200 g ae / ha. Similarly, T0 and T1 plants can be screened for glufosinate resistance by postemergence treatment with 200-800 and 50-3200 g ae / ha glufosinate, respectively. Glyphosate resistance (in plants transformed with a construct containing EPSPS) or another glyphosate resistance gene is assessed in the T1 generation by postemergence application of glyphosate using a glyphosate dose range of 280-2240 g ae / ha be able to. Individual T0 plants were evaluated for the presence and copy number of the coding region of the gene of interest (AAD-12 (v1) or PAT v6). Determination of AAD-12 (v1) inheritance is performed using T1 and T2 progeny segregation for herbicide resistance, as described in previous examples.

  A subset of early transformants was evaluated in the T0 generation according to the method described above. All plants identified as having the AAD-12 (v1) coding region did not respond to 2,4-D leaf application regardless of the promoter driving the gene, whereas wild type Maberick soybean responded. Plants transformed with PAT alone responded to 2,4-D leaf application in the same way as wild type plants.

  2,4-D was applied at either 560 or 1120 g ae 2,4-D to a plant subset that was similar in size to the wild-type control plant. All AAD-12 (v1) containing plants were clearly resistant to herbicide application compared to wild type Maverick soybeans. A minor level of damage was observed in 2 AAD-12 (v1) plants (2DAT), but the damage was transient and no damage was observed with 7DAT. Wild-type control plants were severely damaged at 560 g ae / ha 2,4-D at 7-14 DAT and died at 1120 g ae / ha. These data are consistent with the fact that AAD-12 (v1) can confer high tolerance (over 2 × field volume) to sensitive crops such as soybeans. The screened plants were then sampled for molecular and biochemical analysis to confirm AAD12 (v1) gene integration, copy number, and gene expression levels.

  Molecular analysis-soybean: tissue harvest, DNA isolation and quantification. Fresh tissue is placed in a tube and lyophilized at 4 ° C. for 2 days. After the tissue is completely dry, tungsten beads (Vallite) are placed in the tube and the sample is subjected to 1 minute dry grinding using a Kelco bead mill. A standard DNeasy DNA isolation procedure is then performed (Qiagen, DNeasy 69109). Subsequently, aliquots of the extracted DNA are stained with Pico Green (Molecular Probes P7589) and read on a fluorimeter (BioTek) using known standards to obtain a concentration of ng / μL.

  Polymerase chain reaction: A total of 100 ng of total DNA is used as a template. 20 mM of each primer is used with Takara Ex Taq PCR polymerase kit (Mirus TAKRR001A). The primers for AAD-12 (v1) PTU are (forward direction-ATAATGCCCAG CCGTTAAAAC GCC) (SEQ ID NO: 8) and (reverse direction-CTCAAGCATA TGAATGACCT CGA) (SEQ ID NO: 9). The PCR reaction was performed in a 9700 Geneamp thermocycler (Applied Biosystems) with samples being run at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 63 ° C for 30 seconds, 72 ° C for 1 minute and 45 seconds, and then at 72 ° C. Perform by subjecting to 10 minutes. The primers for the coding region PCR AAD-12 (v1) are (forward-ATGGCTCATG CTGCCCTCAG CC) (SEQ ID NO: 10) and (reverse-CGGGCAGGCC TAACTCCACC AA) (SEQ ID NO: 11). The PCR reaction was carried out in a 9700 Geneamp thermocycler (Applied Biosystems) with samples run at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 1 minute 45 seconds, and then at 72 ° C. Perform by subjecting to 10 minutes. PCR products are analyzed by electrophoresis on a 1% agarose gel stained with EtBr.

  Southern blot analysis: Southern blot analysis is performed using total DNA obtained from the Qiagen DNeasy kit. A total of 10 μg of genomic DNA is subjected to overnight digestion to obtain integration data. After overnight digestion, approximately 100 ng aliquots are run on a 1% gel to ensure complete digestion. After this belief, the sample is run on a large 0.85% agarose gel overnight at 40 volts. The gel is then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gel is then neutralized in 0.5 M Tris HCl, 1.5 M NaCl, pH 7.5 for 30 minutes. The gel apparatus containing 20 × SSC is then set up to obtain overnight transfer from the gel by gravity to a nylon membrane (Millipore INYC00010). After overnight transfer, the membrane is subjected to UV light at 1200 × 100 microjoules via a cross-linking agent (Stratagene UV stratalinker 1800). The membrane is then washed with 0.1% SDS, 0.1 SSC for 45 minutes. After washing for 45 minutes, the membrane is baked for 3 hours at 80 ° C and then stored at 4 ° C until hybridization. Hybridization template fragments are prepared using the above coding region PCR using plasmid DNA. The product is run on a 1% agarose gel, cut and then gel extracted using the Qiagen (28706) gel extraction procedure. The membrane is then subjected to prehybridization for 1 hour at 60 ° C. in Perfect Hyb buffer (Sigma H7033). The Prime it RmT dCTP labeling reaction (Stratagene 300392) procedure is used to develop a p32 based probe (Perkin Elmer). Clean up the probe using Probe Quant. G50 column (Amersham 27-5335-01). Southern blots are hybridized overnight using 2 million CPM. After overnight hybridization, the blot is subjected to two 20 minute washes in 65 ° C., 0.1% SDS, 0.1 SSC. The blot is then exposed to film overnight with incubation at −80 ° C.

  Biochemical analysis-Soybean: Tissue sampling and extraction of AAD-12 (v1) protein from soybean leaves. About 50 to 100 mg of leaf tissue was sampled after 1 DAT from N-2 leaves coated with 2,4-D. The terminal N-2 leaflets were removed and cut into either small pieces or 2 single hole punch leaf pieces (approximately 0.5 cm in diameter) and snap frozen on dry ice. Protein analysis (ELISA and Western analysis) was completed accordingly.

  T1 progeny evaluation: T0 plants are self-pollinated to induce T1 family. The progeny assay (separation analysis) is assayed using 560 g ai / ha glufosinate applied as a selective agent in the V1-V2 growth stage. Surviving plants are further assayed for 2,4-D resistance at one or more growth stages from V2 to V6. Seeds are generated by self-pollination, allowing a broader herbicide test on transgenic soybeans.

  AAD-12 (v1) transgenic Maberick soybean plants have been produced by an Agrobacterium-mediated transformation system. The resulting T0 plants tolerated up to 2,4-D field application up to 2 × levels and generated reproductive seeds. The frequency of reproductive transgenic soybean plants was up to 5.9%. Integration of the AAD1-12 (v1) gene into the soybean genome was confirmed by Southern blot analysis. This analysis indicated that most of the transgenic plants contained low copy numbers. Plants screened with AAD-12 (v1) antibody showed positive for the appropriate bands in ELISA and Western analysis.

  Transformation Method 2—Aerosol Beam-Mediated Transformation of Embryogenic Soy Callus Tissue: Culture of embryogenic soy callus tissue and subsequent beam irradiation is accomplished as described in US Pat. No. 6,809,232 (Held et al.). , Transformants using the constructs provided herein can be made.

  Transformation Method 3-Soybean Particle Gun Irradiation: This can be achieved using hypocotyl meristems derived from mature seeds (McCabe et al. (1988)). According to established methods of particle gun irradiation, recovery of transformed soybean plants can be expected.

  Transformation Method 4-Agate Crystal-Mediated Transformation: Preparation of agate crystals and transformation of agate crystals can be performed according to the methods previously described by Terakawa et al. (2005). According to established methods of particle gun irradiation, recovery of transformed soybean plants can be expected.

  Maberick seeds were surface sterilized with 70% ethanol for 1 minute, then immersed in 1% sodium hypochlorite for 20 minutes, and then rinsed 3 times with sterile distilled water. Seeds were soaked in distilled water for 18-20 hours. The hypocotyl was excised from the seed and the apical meristem was exposed by removing the primary leaves. With the apical region facing upward, hypocotyls were irradiated medium (BM: MS (Murashige and Skoog 1962) basal salt medium, 3% sucrose and 0.8% in a 5 cm culture dish containing 12 ml culture medium. Phytagel, Sigma, pH 5.7].

  Transformation Method 5—Microparticle irradiation-mediated transformation of embryogenic callus tissue can be optimized according to previous methods (Khalafalla et al., 2005, El-Shemy et al., 2004, 2006).

AAD-12 (v1) in cotton
Cotton Transformation Protocol: Cottonseed (genotype Co310) is surface sterilized with 95% ethanol for 1 minute, rinsed, sterilized with 50% commercial bleach for 20 minutes, and then rinsed 3 times with sterile distilled water Then, germinate on G medium (Table 21) in Magenta GA-7 container and under high light intensity of 40-60 μE / m ² , set photoperiod to 16 hours light and 8 hours dark to 28 Maintain at ° C.

  Square pieces of cotyledon segments (about 5 mm) are isolated from 7-10 day old seedlings in Petri dishes (Nunc, item # 0875728) into liquid M liquid medium (Table 21). The cut segment is treated with an Agrobacterium solution (30 minutes), then transferred to a semi-solid M medium (Table 21) and co-cultured for 2-3 days. After co-culture, the segments are transferred to MG medium (Table 21). Carbenicillin is an antibiotic used to kill Agrobacterium and is a selective agent that allows growth of only cells containing the gene to which glufosinate-ammonium has been transferred.

Preparation of Agrobacterium: 35 ml of Y medium (Table 21) (containing streptomycin (100 mg / ml stock) and erythromycin (100 mg / ml stock)) inoculated with a platinum loop of bacteria, Grow overnight at 28 ° C. in the dark with shaking at 150 rpm. The next day, pour the Agrobacterium solution into a sterile Oak Ridge tube (Nalge-Nunc, 3139-0050) and centrifuge at 8,000 rpm for 5 minutes in Beckman J2-21. The supernatant is poured out and the pellet is resuspended in 25 ml of M liquid (Table 21) and vortexed. One aliquot is placed in a glass culture tube (Fisher, 14-961-27) for Klett reading (Klett-Summerson, model 800-3). Using M liquid medium, the new suspension is diluted to a Klett meter reading of 10 ⁸ colony forming units / ml, for a total volume of 40 ml.

  After 3 weeks, callus from the cotyledon segment is isolated and transferred to fresh MG medium. Callus is transferred onto MG medium for an additional 3 weeks. Dichlorprop is not a substrate for the AAD-12 enzyme, but dichlorprop is more active on cotton than 2,4-D, so in a side-by-side comparison, MG is used to compensate for 2,4-D degradation. Dichloroprop (added to the medium at concentrations of 0.01 and 0.05 mg / L) can be added to the medium. In another comparison, segments plated on MG medium without growth regulators showed reduced callus formation when compared to standard MG medium, but callus growth is still present. The callus is then transferred to CG medium (Table 21) and again after 3 weeks to fresh selection medium. After another 3 weeks, callus tissue is transferred to D medium lacking plant growth regulators (Table 21) for induction of embryogenic callus. After 4-8 weeks on this medium, embryogenic calli are formed, which can be distinguished from non-embryogenic calli by their yellowish white and granule cells. The embryo begins to regenerate soon after, and the color is clear green. Cotton can take time to regenerate and form embryos, and one way to accelerate this process is to stress the tissue. One common way to accomplish this is to use tissue culture and the microenvironment of the tissue and plate by using less culture media and / or employing various plate encapsulation formats (taping versus parafilm). Dessication through change.

  Larger, fully developed embryos are isolated and transferred to DK medium (Table 21) for embryo development. After 3 weeks (or when embryos develop), germinated embryos are transferred to fresh media for shoot and root development. After 4-8 weeks, all fully developed plants are transferred to the soil and grown to maturity. After several months, the plants are growing until they can be sprayed to determine if they have resistance to 2,4-D.

  Cell transformation: Several experiments were started in which the cotyledon segments were treated with Agrobacterium containing pDAB724. Over 2000 resulting segments were used for growth of pDAB724 cotton calli, either 0.1 or 0.5 mg / L R-dichloroprop, standard 2,4-D concentration and no auxin treatment. Processed using various auxin options. Callus was selected on glufosinate-ammonium because the PAT gene was included in the construct. Phylogenetic analysis of callus in the form of PCR and Invader is used to determine if the gene was present at the callus stage and to ensure it is present. Thereafter, callus lines that are embryogenic are subjected to Western analysis. In order to improve the quality and quantity of tissue recovered, dessication techniques were used to stress the embryogenic cotton callus. Nearly 200 callus events have been screened for intact PTU and expression using Western analysis for the AAD-12 (v1) gene.

  Plant regeneration: Send the AAD-12 (v1) cotton line that produced plants according to the above protocol to the greenhouse. In order to demonstrate that the AAD-12 (v1) gene confers resistance to 2,4-D in cotton, both AAD-12 (v1) cotton plants and wild type cotton plants were 560 g ae / ha 2 , 4-D is sprayed using a track sprayer delivering a spray volume of 187 L / ha. Plants are evaluated 3 and 14 days after treatment. Plants that survive a selective amount of 2,4-D are self-pollinated to produce T1 seeds, or crossed with excellent cotton lines to produce F1 seeds. The resulting seeds are then planted and evaluated for herbicide resistance as previously described. AAD-12 (v1) events can be combined with other desired HT or IR trants.

Agrobacterium Transformation of Other Crops In view of the present disclosure, additional crops can be transformed according to the present invention using techniques known in the art. See, for example, Popelka and Altpeter (2003) for Agrobacterium-mediated transformation of rye. See, for example, Hinchee et al., 1988 for Agrobacterium-mediated transformation of soybean. See, for example, Zhao et al., 2000 for Agrobacterium-mediated transformation of sorghum. See, for example, Tingay et al., 1997 for Agrobacterium-mediated transformation of barley. See, for example, Cheng et al., 1997 for Agrobacterium-mediated transformation of wheat. See, for example, Hiei et al., 1997 for Agrobacterium-mediated transformation of rice.

  The Latin names of these and other plants are shown below. Using these and other (non-Agrobacterium) transformation techniques, AAD-12 (v1) can be used, for example, but not limited to, Zea mays, wheat (Wheat species) (Triticum spp.)), Rice (Oryza spp.) And Zizania spp.), Barley (Hordeum spp.), Cotton (Abroma augusta) and cotton species (Gossypium spp.), soybean (Glycine max), sugar and table beet (Beta spp.), sugar cane (Arenga pinnata), tomato (Lycopersicon) esculentum and other species, Physalis ixocarpa, Solanum incanum and other species, Cyphomandra betacea ), Potato (Solanum tubersoum), sweet potato (Ipomoea betatas), rye (Secale spp.), Pepper (Capsicum annuum), capsicum sinense, and frutescens ), Lettuce (Lactuca sativa, Lactuca perennis, and pulchella), cabbage (Brassica spp), celery (Apium graveolens), eggplant (Solanum) melongena), peanuts (Arachis hypogea), sorghum (all Sorghum species), alfalfa (Medicago sativua), carrot (Daucus carota), legumes (Phaseolus spp.) and others Genus), oats (Avena sativa and strigosa), peas (Pisum, Vigna, and Tetragonolobus spp. Species), sunflower (Helianthus annuus), pumpkin (Cucurbita spp.), Cucumber (Cucumis sativa) ), Tobacco (Nicotiana spp.), Arabidopsis thaliana, lawn (Lolium), Agrostis, strawberry genus (Poa), cynodon (Cynadon), and others It is clear that it can be transformed into these and other plants, including the genus), clover (Tifolium), and oak (Vicia). For example, such plants having the AAD-12 (v1) gene are included in the present invention.

  AAD-12 (v1) has the potential to increase the applicability of major auxinic herbicides for use during the season in many deciduous and evergreen timber planting systems. Triclopyr, 2,4-D, and / or fluoroxypyr resistant timber species increase the flexibility of the use of these herbicides without fear of damage. These species include, but are not limited to, alder (Alnus spp.), Ash (Fraxinus spp.), Boxwood and poplar (Populus) species. spp.)), beech (Fagus spp.), birch (Betula spp.), cherry (Prunus spp.), eucalyptus (eucalyptus species) (Eucalyptus spp.)), Hickory (Carya spp.), Maple (Acer spp.), Oak (Quercus spp), and pine (pine) Species (Pinus spp)). Also within the scope of the invention is the use of auxin resistance for selective weed control in ornamental and fruiting species. Examples include, but are not limited to, roses (Rosa spp.), Broomfish (Euonymus spp.), Petunia (Petunia spp), begonia ( Begonia spp.), Azalea (Rhododendron spp), crabapple or apple (Malus spp.), Pear (Pyrus spp.) ), Peach (Sakura species ()), and marigold (Tagetes spp.).

Further evidence of surprising results: AAD-12 vs. AAD-2
Initial cloning of AAD-2 (v1): Another gene has only 44% amino acid identity to tfdA from the NCBI database (see ncbi.nlm.nih.gov website, accession # AP005940) Not identified as a homolog. For consistency, this gene is referred to herein as AAD-2 (v1). The percent identity is determined by first comparing the DNA sequences of both AAD-2 and tfdA (SEQ ID NO: 12 of PCT / US2005 / 014737 and GENBANK Accession No. M16730, respectively) to protein (SEQ ID NO: 13 and PCT / US2005 / 014737, respectively). GENBANK accession number M16730), followed by multiple sequence alignment using ClustalW in the VectorNTI software package.

  A strain of Bradyrhizobium japonicum containing the AAD-2 (v1) gene was obtained from Northern Regional Research Laboratory (NRRL, strain # B4450). The lyophilized strain was resuscitated according to the NRRL protocol and stored in Dow bacterial strain DB663 for internal use in -80 ° C., 20% glycerol. Subsequently, Tryptic Soy Agar plates were struck out from this freezer stock with platinum loops of cells for isolation and incubated at 28 ° C. for 3 days. A single colony was used to inoculate 100 ml Tryptic Soy Broth in a 500 ml Tribaffle flask, which was incubated overnight at 28 ° C., 150 rpm on a floor shaker. From this, total DNA was isolated using the Gram negative protocol of Qiagen's DNeasy kit (Qiagen catalog # 69504). The following primers were designed to amplify the target gene from genomic DNA: Forward: 5 ′ ACT AGT AAC AAA GAA GGA GAT ATA CCA TGA CGA T 3 ′ [(brjap 5 ′ (speI), PCT / US2005 / 0147737 SEQ ID NO: 14 (added Spe I restriction site and ribosome binding site (RBS))] and reverse direction: 5 ′ TTC TCG AGC TAT CAC TCC GCC GCC TGC TGC TGC 3 ′ [(br jap 3 ′ (xhoI), PCT / US2005 / 014737 SEQ ID NO: 15 (Xho I site added)].

A 50 microliter reaction was set up as follows: Fail Safe buffer 25 μl, each primer 1 μl (50 ng / μl), gDNA 1 μl (200 ng / μl), H ₂ O 21 μl, Taq polymerase 1 μl (2.5 units / liter) μl). Three Fail Safe buffers, A, B, and C were used in three separate reactions. PCR was then carried out under the following conditions: thermal denaturation cycle at 95 ° C for 3.0 minutes, 95 ° C for 1.0 minute, 50 ° C for 1.0 minute, 72 ° C for 1.5 minute for 30 cycles Then, use the FailSafe PCR System (Epicenter catalog # F599100) for a final cycle of 5 min at 72 ° C. To verify the nucleotide sequence, the resulting approximately 1 kb PCR product was purified using pCR2.1 (Invitrogen catalog #) using chemically competent TOP10F ′ E. coli as the host strain according to the included protocol. K4550-40).

  Ten of the resulting white colonies were picked into 3 μl luria broth + 1000 μg / ml ampicillin (LB Amp) and grown overnight at 37 ° C. with stirring. Plasmids were purified from each culture using the Nucleospin Plus Plasmid Miniprep kit (BD Biosciences catalog # K3063-2) according to 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 catalog # R0101S). Sequencing uses the Beckman CEQ Quick Start Kit (Beckman Coulter catalog # 608120), M13 forward [5 ′ GTA AAA CGA CGG CCA G 3 ′] (SEQ ID NO: 6) and reverse [5 ′ CAG GAA ACA GCT ATG AC 3 ′] (SEQ ID NO: 7) primer was used and performed according to the manufacturer's instructions. For internal consistency, this gene sequence and its corresponding protein were given the new generic name AAD-2 (v1).

  Completion of AAD-2 (v1) binary vector: The AAD-2 (v1) gene was PCR amplified from pDAB3202. During the PCR reaction, changes were made in the primers to introduce AflIII and SacI restriction sites into the 5 'and 3' primers, respectively. See PCT / US2005 / 014737. Primers “NcoI of Brady” [5 ′ TAT ACC ACA TGT CGA TCG CCA TCC GGC AGC TT 3 ′] (SEQ ID NO: 14) and “Brady SacI” [5 ′ GAG CTC CTA TCA CTC CGC CGC CGC CGC CGC CGC ] (SEQ ID NO: 15) and the DNA fragment was amplified using the Fail Safe PCR System (Epicentre). The PCR product was cloned into the pCR2.1 TOPO TA cloning vector (Invitrogen) and sequenced using the Beckman Coulter “Dye Terminator Cycle Sequencing with Quick Start Kit” sequencing reagent using M13 forward and M13 reverse primers. Verified. Sequence data identified a clone with the correct sequence (pDAB716). Subsequently, the AflIII / SacI AAD-2 (v1) gene fragment was cloned into the NcoI / SacI pDAB726 vector. The resulting construct (pDAB717), AtUbi10 promoter: Nt OSM 5'UTR: AAD-2 (v1): Nt OSM 3 'UTR: ORF1 poly A 3'UTR was verified by restriction digest (using NcoI / SacI). This construct was cloned into the binary pDAB3038 as a NotI-NotI DNA fragment. In order to verify the correct orientation, the resulting construct (pDAB767), AtUbi10 promoter: Nt OSM 5′UTR: AAD-2 (v1): Nt OSM 3′UTR: ORF1 poly A 3′UTR: CsVMV promoter: PAT: ORF 25/26 The 3′UTR was restriction digested (using Nod, EcoRI, HinDIII, NcoI, PvuII, and SalI). The completed construct (pDAB767) was then used for transformation into Agrobacterium.

  Evaluation of transformed Arabidopsis: Freshly harvested T1 seeds were transformed with plant-optimized AAD-12 (v1) or planted with native AAD-2 (v1) gene already Selected for resistance to glufosinate as described. The plants were then randomly assigned to varying amounts of 2,4-D (50-3200 g ae / ha). Herbicide application was applied with a truck sprayer at a spray volume of 187 L / ha. The 2,4-D used was a commercial dimethylamine salt formulation (456 g ae / L, NuFarm, MO) mixed in 200 mM Tris buffer (pH 9.0) or 200 mM HEPES buffer (pH 7.5). State St Joseph).

  AAD-12 (v1) and AAD-2 (v1) resulted in detectable 2,4-D resistance compared to transformed and non-transformed control lines. However, the individual constructs varied greatly in their ability to confer resistance to 2,4-D to individual T1 Arabidopsis plants. Surprisingly, AAD-2 (v1) and AAD-2 (v2) transformants are resistant to 2,4-D from both the frequency of high-tolerant plants and overall average damage. (V1) Much lower than the gene. None of the plants transformed with AAD-2 (v1) are relatively undamaged (less than 20% visual damage) will not survive 200 g ae / ha of 2,4-D, and the overall population damage is about 83% (see PCT / US2005 / 014737). Conversely, AAD-12 (v1) had a population damage average of about 6% when treated with 3,200 g ae / ha of 2,4-D. Tolerance was slightly improved with plant-optimized AAD-2 (v2) vs. native gene, but comparison of both AAD-12 and AAD-2 plant-optimized genes revealed that plant A significant advantage of AAD-12 (v1) is shown.

  In vitro comparison of AAD-2 (v1) (see PCT / US2005 / 014737) and AAD-12 (v2) both are highly effective in degrading 2,4-D, both chiral These results are unexpected considering that it has been shown to share S-type specificity for aryloxyalkanoate substrates. AAD-2 (v1) is expressed at variable levels in individual T1 plants. However, the protection afforded by this expressed protein from 2,4-D damage is negligible. Substantial differences in protein expression levels (in plants) were not evident with native and plant optimized AAD-2 genes (see PCT / US2005 / 014737). These data support early findings that unexpectedly indicate the functional expression of AAD-12 (v1) in plants and the resulting herbicide resistance to 2,4-D and pyridyloxyacetic acid herbicides.

In-crop use of phenoxyauxin herbicides in soybean, cotton, and other dicotyledonous plants transformed with AAD-12 (v1) alone AAD-12 (v1) is usually sensitive to 2,4-D Can enable the use of phenoxyauxin herbicides (eg, 2,4-D and MCPA) and pyridyloxyauxins (triclopyr and fluoroxypyr) to directly control a wide range of broadleaf weeds in crops . Application of 280-2240 g ae / ha of 2,4-D controls most broad-leaved weed species present in the agricultural environment. More typically 560-1120 g ae / ha are used. For triclopyr, the application rate will typically range from 70 to 1120 g ae / ha, more typically from 140 to 420 g ae / ha. For fluoroxypyr, application rates typically range from 35 to 560 g ae / ha, more typically from 70 to 280 ae / ha.

  One advantage of this additional tool is that the broad-leaf herbicide ingredients are very cheap and that non-residue herbicides such as glyphosate do not provide control of weeds that later germinate, while at higher levels. Higher amounts of 2,4-D, triclopyr, and fluoroxypyr, when used, result in potential short-lived residual weed control. The tool also provides a mechanism that combines the action mechanism of herbicides with the convenience of HTC as an integrated herbicide resistance and weed shift management strategy.

  A further advantage provided by this tool is the ability to tank mix a wide range of broadleaf weed control herbicides (eg, 2,4-D, triclopyr and fluoroxypyr) with commonly used residual weed control herbicides. It is. These herbicides are typically applied before or at planting, but are often less effective against budding, established weeds that may be present in the field prior to planting. Extending the usefulness of these aryloxyauxin herbicides to include application at planting, pre-emergence, or pre-planting increases the flexibility of the residual weed control program. Those skilled in the art will understand that persistent herbicide programs will vary based on the target crop, but typical programs include the chloracetmide and dinitroaniline herbicide family herbicides, Also included are herbicides such as clomazone, sulfentrazone, and various ALS inhibitory, PPO inhibitory, and HPPD inhibitory herbicides.

  Further advantages include, after application of aryloxyacetate auxin herbicide, tolerance to 2,4-D, triclopyr or fluoroxypyr required before planting (see previous examples), and previously 2,4 This may include reducing problems from contamination damage to dicot crops resulting from incompletely washed bulk tanks that contained -D, triclopyr or fluoroxypyr. Dicamba (and many other herbicides) can still be used for subsequent control of dicotyledonous native plants transformed with AAD-12 (v1).

  Those skilled in the art will also recognize that the above examples are any 2,4-D sensitive (or other aryloxyauxin herbicide) crop that is protected by the AAD-12 (v1) gene when stably transformed. It will be understood that it can be applied to. Here, it is understood by those in the field of weed control that AAD-12 (v1) transformation allows the use of various commercially available phenoxy or pyridyloxyauxin herbicides alone or in combination with herbicides. Like. Specific amounts of other herbicides typical of these chemistries can be obtained from any commercial or academic such as CPR (Crop Protection Reference) book or similar editorial book or Crop Protection Guide (2005) from Agility. It can be determined by herbicide labeling compiled in the crop protection reference. Each alternative herbicide made available for use in HTC by AAD-12 (v1) is considered to be within the scope of the present invention, whether used alone, in a tank mix or sequentially.

In-crop use of phenoxyauxin and pyridyloxyauxin herbicides in corn, rice, and other monocotyledonous plant species transformed only with AAD-12 (v1) AAD-12 (v1) was used in a similar manner. Transformation of grass species (such as, but not limited to, corn, rice, wheat, barley, or lawn and pasture) uses highly effective phenoxy and pyridyloxyauxins in crops that are usually uncertain Enable. Most grass species have a natural resistance to auxinic herbicides such as phenoxyauxin (ie 2,4-D). However, due to the shortened application timing window or the risk of unacceptable damage, relatively low levels of crop selectivity have resulted in a loss of usefulness in these crops. Therefore, monocotyledons transformed with AAD-12 (v1) have been described for dicotyledonous crops such as application of 280-2240 g ae / ha 2,4-D to control most broadleaf weed species. Allows the use of similar combinations of processing. More typically 560-1120 g ae / ha are used. For triclopyr, the application rate will typically range from 70 to 1120 g ae / ha, more typically from 140 to 420 g ae / ha. For fluoroxypyr, application rates typically range from 35 to 560 g ae / ha, more typically from 70 to 280 ae / ha.

  One advantage of this additional tool is that the broad-leaf herbicide ingredients are very inexpensive, and higher amounts of 2,4-D, triclopyr, or fluoroxypyr control potential short-lived residual weeds. Is to bring In contrast, non-residue herbicides such as glyphosate do not provide control of weeds that later germinate. This tool also demonstrates the mechanism of herbicide action as an integrated herbicide resistance and weed shift management strategy in a glyphosate-tolerant crop / AAD-12 (v1) HTC combination strategy, regardless of whether the crop species is rotated. It also provides a mechanism for circulating HTC with the convenience of HTC.

  A further advantage provided by this tool is the ability to tank mix a wide range of broadleaf weed control herbicides (eg, 2,4-D, triclopyr and fluoroxypyr) with commonly used residual weed control herbicides. It is. These herbicides are typically applied before or at planting, but are often less effective against budding, established weeds that may be present in the field prior to planting. Extending the usefulness of these aryloxyauxin herbicides to include application at planting, pre-emergence, or pre-planting increases the flexibility of the residual weed control program. Those skilled in the art will understand that persistent herbicide programs will vary based on the target crop, but typical programs include the chloracetmide and dinitroaniline herbicide family herbicides, Also included are herbicides such as clomazone, sulfentrazone, and various ALS inhibitory, PPO inhibitory, and HPPD inhibitory herbicides.

  Increased resistance of corn, rice, and other monocotyledonous plants to phenoxy or pyridyloxyauxin is due to limited growth stages or potential for crop tilt, spread phenomena such as “tapering”, crop tilt, growth on corn Allows the use of these herbicides in crops, without stem brittleness or malformed strut roots induced by regulators. Each alternative herbicide made available for use in HTC by AAD-12 (v1) is considered to be within the scope of the present invention, whether used alone, in a tank mix or sequentially.

AAD-12 in rice (v1)
Media description: The culture medium used was adjusted to pH 5.8 with 1 M KOH and solidified with 2.5 g / L of Phytagel (Sigma). Embryogenic calli were cultured in 100 x 20 mm Petri dishes containing 40 ml semi-solid medium. Rice plantlets were grown on 50 ml of medium in the Magenta box. The cell suspension was maintained in a 125 ml Erlenmeyer flask containing 35 ml of liquid medium and rotated at 125 rpm. Induction and maintenance of embryogenic cultures were performed in the dark at 25-26 ° C., plant regeneration and whole plant culture were performed with a 16 hour photoperiod (Zhang et al., 1996).

  Induction and maintenance of embryogenic callus was previously described (Li et al., 1993), but was performed on NB basal medium adapted to contain 500 mg / L glutamine. Suspension culture was initiated and maintained in SZ liquid medium (Zhang et al., 1998) containing 30 g / L sucrose instead of maltose. The osmotic medium (NBO) consisted of NB medium supplemented with 0.256 M mannitol and sorbitol, respectively. Hygromycin-B resistant callus was selected on NB medium supplemented with 50 mg / L hygromycin B for 3-4 weeks. Pre-regeneration does not contain 2,4-dichlorophenoxyacetic acid (2,4-D), but 2 mg / L 6-benzylaminopurine (BAP), 1 mg / L α-naphthaleneacetic acid (NAA), 5 mg / L The test was performed for 1 week on a medium (PRH50) consisting of NB medium supplemented with L abscisic acid (ABA) and 50 mg / L hygromycin B. Subsequently, on a regeneration medium (RNH50) that does not contain 2,4-D, and has an NB medium supplemented with 3 mg / L BAP, 0.5 mg / L NAA, and 50 mg / L hygromycin B. Plantlets were regenerated by culturing until the shoots were regenerated. The shoots were transferred to rooting medium (1 / 2MSH50) containing half strength Murashige & Skoog basal salt and Gamborg B5 vitamins supplemented with 1% sucrose and 50 mg / L hygromycin B.

  Generation of tissue culture: Oriza sativa L. Japonica cv. Mature dry seeds of Taipei 309 (Oryza sativa L. japonica cv. Taipei 309) were sterilized as described in Zhang et al., 1996. By culturing sterile mature rice seeds on NB medium in the dark. Embryogenic tissue was induced. Primary callus about 1 mm in diameter was removed from the scutellum and used to initiate cell suspension culture in SZ liquid medium. Thereafter, the suspension was maintained as described in Zhang 1995. Three to five days after the previous subculture, the embryogenic tissue derived from the suspension is removed from the liquid culture and placed on NBO osmotic medium to form a circle about 2.5 cm in diameter in a Petri dish. Irradiation after 4 hours (hous) culture. After 16-20 hours of irradiation, ensuring that the irradiated surface was facing up, the tissue was transferred from NBO medium onto NBH50 hygromycin B selective medium and incubated for 14-17 days in the dark. The newly formed callus was then separated from the original irradiated explant and placed close to the same medium. After an additional 8-12 days, relatively small opaque calli were visually identified and transferred to PRH50 pre-regeneration medium for 7 days in the dark. Thereafter, the growing callus, which became smaller and opaque, was subcultured on RNH50 regeneration medium with a photoperiod of 16 hours for a period of 14-21 days. Regenerating shoots were transferred to a Magenta box containing 1/2 MSH50 medium. Multiple plants regenerated from a single explant were considered siblings and treated as one independent plant line. Plants produced thick white roots and scored positive for the hph gene when actively growing on 1/2 MSH50 medium. After the plantlets reach the top of the Magenta box, they are transferred to soil in a 6 cm pot at 100% humidity for 1 week, followed by growth with a 14 hour light period, 30 ° C. and dark, 21 ° C. After moving to the chamber for 2-3 weeks, it was transplanted into a 13 cm pot in a greenhouse. Seeds were collected and stored after drying for 1 week at 37 ° C.

  Particle irradiation: All irradiations were performed using a particle gun PDS-1000 / He® 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 of water in a siliconized Eppendorf tube. 1: 6 molar ratio of pDOW3303 (Hpt-containing vector) to pDAB4101 (AAD-12 (v1) + AHAS) 5 micrograms of plasmid DNA, 20 μl spermidine (0.1 M) and 50 μl calcium chloride (2.5 M) Was added to the gold suspension. The mixture was incubated at room temperature for 10 minutes, pelleted at 10000 rpm for 10 seconds, resuspended in 60 μl 100% cold ethanol, and 8-9 μl was dispensed onto each macrocarrier. Tissue samples were irradiated at 1100 psi and 27 in Hg vacuum as described in Zhang et al. (1996).

  Postemergence herbicide resistance in T0 rice transformed with AAD-12 (v1): 3% single-leaf rice plantlets using 1% Sunit II (1) using a track sprayer calibrated to 187 L / ha nebulized with lethal dose of 0.16% (v / v) Pursuit solution (to confirm the presence of AHAS gene) containing v / v) and 1.25% UAN (v / v) . Sensitivity or resistance assessments were made 36 days after treatment (DAT). Ten of the 33 events sent to the greenhouse were robustly resistant to Pursuit and others suffered variable levels of herbicide damage. Plants were sampled and subjected to molecular characterization, whereby 7 of these 10 events were identified as containing both the AAD-12 (v1) PTU and the entire AHAS coding region.

  Heritability of AAD-12 (v1) in T1 rice: 100 plant progeny tests, 5 of AAD-12 (v1) lines that contained both AAD-12 (v1) PTU and AHAS coding regions It implemented with respect to T1 system | strain. Following the above procedure, seeds were planted and sprayed with 140 g ae / ha imazetapill using a track sprayer as previously described. After 14 DAT, resistant and sensitive plants were counted. As determined by chi-square analysis, two of the five lines tested were segregated as a single locus dominant Mendelian trait (3R: 1S). As determined by the following 2,4-D resistance test, AAD-12 was cosedated with the AHAS selectable marker.

  Verification of high 2,4-D resistance in T1 rice: The following T1 AAD-12 (v1) single segregated locus lines were planted in 3 inch pots containing Metro Mix medium: pDAB4101 (20) 003 and pDAB4101 (27) 002. 140 g ae / ha imazetapil was sprayed at the 2-3 sheet stage. Eliminating nulls, individuals in V3-V4 stage, 187 L / ha, 1120, 2240 or 4480 g ae / ha 2,4-D DMA (2 ×, 4 ×, and 8 of typical commercial usage, respectively) It sprayed with the track sprayer set to x). Plants were graded to 7 and 14 DAT and compared to the non-transformed commercial rice cultivar “Lamont” as a negative control plant.

  Damage data (Table 22) shows that lines transformed with AAD-12 (v1) are more resistant to higher amounts of 2,4-D DMA than untransformed controls. The pDAB4101 (20) 003 line is more resistant to high levels of 2,4-D than the pDAB4101 (27) 002 line. The data also demonstrates that 2,4-D resistance is stable for at least two generations.

  Tissue harvest, DNA isolation and quantification: Fresh tissue was placed in tubes and lyophilized at 4 ° C. for 2 days. After the tissue was completely dried, tungsten beads (Vallite) were placed in the tube and the sample was subjected to 1 minute dry grinding using a Kelco bead mill. A standard DNeasy DNA isolation procedure was then performed (Qiagen, Dneasy 69109). Subsequently, aliquots of the extracted DNA were stained with Pico Green (Molecular Probes P7589) and scanned with a florometer (BioTek) using known standards to obtain a concentration of ng / μl.

  Expression of AAD-12 (v1): All 33 T0 transgenic rice lines and one non-transgenic control were analyzed for AAD-12 expression using ELISA blots. AAD-12 was detected in 20 strains of clones but not in Taipai 309 strain control plants. Twelve of the 20 lines where some clones were resistant to imazetapill expressed AAD-12 protein, were positive for AAD-12 PCR PTU, and positive for the AHAS coding region. It was. Expression levels ranged from 2.3 to 1092.4 ppm total soluble protein.

  Field Tolerance of pDAB4101 Rice Plants to 2,4-D and Triclopyr Herbicide: A Field Level Tolerance Test was performed using AAD-12 (v1) event pDAB4101 [20] and one wild-type rice (Clearfield 131), Mississippi Wayside (Non-transgenic imidazolinone resistant variant). The experimental design was a complete random block method, with a single iteration. Herbicide treatment was 2240 g ae / ha 2 × amount of 2,4-D (dimethylamine salt) and 560 g ae / ha triclopyr and an untreated control. Within each herbicide treatment, two rows of T1 generation pDAB4101 [20] and two rows of Clearfield rice were planted at 8 inch row spacing using a small plot drill. pDAB4101 [20] rice contained the AHAS gene as a selectable marker for the AAD-12 (v1) gene. Imazetapill was applied at the single leaf stage as a selective agent to remove all AAD-12 (v1) null plants from the compartment. The herbicide treatment was applied using a compressed air back sprayer that delivered a carrier volume of 187 L / ha at a pressure of 130-200 kpa when the rice reached the two-leaf stage. Visual assessment of injury was made 7, 14, and 21 days after application.

  Responses of AAD-12 (v1) events to 2,4-D and triclopyr are shown in Table 23. Untransformed rice lines (Clearfield) were severely damaged (30% at 7 DAT and 35% at 15 DAT) by 2240 g ae / ha 2,4-D, considered as 4 × commercial use. The AAD-12 (v1) event demonstrated excellent resistance to 2,4-D and no damage was observed at 7 or 15 DAT. Untransformed rice was significantly damaged by 2 × amount of triclopyr (560 g ae / ha) (15% at 7 DAT and 25% at 15 DAT). The AAD-12 (v1) event demonstrated excellent resistance to 2 × amounts of triclopyr and no damage was observed with either 7 or 15 DAT.

  These results indicated that rice transformed with AAD-12 (v1) showed a high level of resistance to the amounts of 2,4-D and triclopyr that caused severe visual damage to Clearfield rice. It is shown that. It has also demonstrated the ability to stack multiple herbicide resistance genes with multiple species of AAD-12 I resulting in resistance to a wider range of effective chemistry.

AAD-12 in canola (v1)
Canola transformation: Brassica napus variant Nexera ^* 710 was transformed into Agrobacterium-mediated traits using the AAD-12 (v1) gene conferring resistance to 2,4-D. Transformation and transformation with plasmid pDAB3759. The construct contained the APS-12 (v1) gene driven by the CsVMV promoter and the Pat gene driven by the AtUbi10 promoter and the EPSPS glyphosate resistance trait driven by the AtUbi10 promoter.

  The seeds were surface sterilized with 10% commercial bleach for 10 minutes and rinsed 3 times with sterile distilled water. The seeds were then placed on half-concentrated MS basal medium (Murashige and Skog, 1962) and maintained under a growth regime set at 25 ° C. and a 16 hour light / 8 hour dark photoperiod.

  A hypocotyl segment (3-5 mm) was excised from 5-7 day old seedlings and pre-treated on callus induction medium K1D1 (MS medium containing 1 mg / L kinetin and 1 mg / L 2,4-D) I left for 3 days. The segment was then transferred into a Petri dish and treated with Agrobacterium Z707S or LBA4404 strain containing pDAB3759. Agrobacterium was grown overnight on a 150 rpm shaker in the dark at 28 ° C. and subsequently resuspended in culture medium.

  The hypocotyl segments were treated with Agrobacterium for 30 minutes and then returned to the callus induction medium for 3 days. After co-culture, the segments were placed on K1D1TC (callus induction medium containing 250 mg / L carbenicillin and 300 mg / L Timetin) and allowed to recover for 1 or 2 weeks. Alternatively, the segments were placed directly on selective medium K1D1H1 (above medium containing 1 mg / L Herbiace). Carbenicillin and Timentin were antibiotics used to kill Agrobacterium. The selective agent Herbiace allowed the growth of transformed cells.

  Thereafter, the callus-formed hypocotyl segment was transformed into B3Z1H1 (MS medium, 3 mg / L benzylaminopurine, 1 mg / L zeatin, 0.5 gm / L MES [2- (N-morpholino) ethanesulfonic acid], 5 mg / L silver nitrate, 1 mg / L Herbiace, carbenicillin and Timentin) on shoot regeneration medium. The shoots began to regenerate after 2-3 weeks. Hypocotyl segment with shoots, B3Z1H3 medium (MS medium, 3 mg / L benzylaminopurine, 1 mg / L zeatin, 0.5 gm / L MES [2- (N-morpholino) ethanesulfonic acid], 5 mg / L Of silver nitrate, 3 mg / L Herbiace, carbenicillin and Timentin) for a further 2-3 weeks.

  The shoots were excised from the hypocotyl segment and transferred to shoot elongation medium MES5 or MES10 (MS, 0.5 gm / L MES, 5 or 10 mg / L Herbiace, carbenicillin, Timentin) for 2-4 weeks. The shoots elongated for root induction were treated with MSI. 1 (MS containing 0.1 mg / L indolebutyric acid). After the plants had a well established root system, they were transplanted into the soil. Plants were acclimated for 1-2 weeks under controlled environmental conditions in Conviron and then transferred to the greenhouse.

  Molecular analysis-Canola materials and methods: tissue harvest, DNA isolation and quantification. Fresh tissue was placed in a tube and lyophilized at 4 ° C. for 2 days. After the tissue was completely dried, tungsten beads (Vallite) were placed in the tube and the sample was subjected to 1 minute dry grinding using a Kelco bead mill. A standard DNeasy DNA isolation procedure was then performed (Qiagen, DNeasy 69109). Subsequently, aliquots of the extracted DNA were stained with Pico Green (Molecular Probes P7589) and read with a fluorometer (BioTek) using known standards to obtain a concentration of ng / μl.

  Polymerase chain reaction: A total of 100 ng of total DNA was used as a template. 20 mM of each primer was used with Takara Ex Taq PCR polymerase kit (Mirus TAKRR001A). The primers for the coding region PCR AAD-12 (v1) were (SEQ ID NO: 10) (forward direction) and (SEQ ID NO: 11) (reverse direction). The PCR reaction was carried out in a 9700 Geneamp thermocycler (Applied Biosystems) with samples at 94 ° C for 3 minutes, 35 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 2 minutes, then 72 ° C. Performed by subjecting to 10 minutes. PCR products were analyzed by electrophoresis on a 1% agarose gel stained with EtBr. 35 samples from 35 plants with AAD-12 (v1) event gave a positive test result. Three negative control samples gave negative test results.

  ELISA: Using the established ELISA described in the previous section, AAD-12 protein was detected in 5 different canola transformed plant events. Expression levels ranged from 14 to over 700 ppm total soluble protein (TSP). Three different non-transformed plant samples were tested in parallel and no signal was detected, indicating that the antibody used in the assay has minimal cross-reactivity with the canola cell matrix. These samples were confirmed to be positive by Western analysis. A summary of the results is presented in Table 24.

  Postemergence herbicide resistance in T0 canola transformed with AAD-12 (v1): 45 T0 events of those transformed with construct pDAB3759 were sent to the greenhouse for a period of time to allow acclimation in the greenhouse It was. Plants were grown until 2-4 new normal-looking leaves emerged (i.e., until the plant transitioned from tissue culture to greenhouse growth conditions). The plants were then treated with a lethal dose of a commercial 2,4-Damine 4 formulation in an amount of 560 g ae / ha. The herbicide application was performed using a truck sprayer with a spray volume of 187 L / ha and a spray height of 50 cm. A lethal dose is defined as the amount that causes more than 95% damage to an untransformed control.

  24 of the events were resistant to 2,4-D DMA herbicide application. Some events received minor damage but recovered by 14 DAT. Events were allowed to progress to T1 (and T2 generations) by self-pollination under controlled bag conditions.

  Heritability of AAD-12 (v1) in canola: A progeny test of 100 plants was also performed on 11 T1 lines of AAD-12 (v1). Seeds were sown and transplanted into 3-inch pots filled with Metro Mix medium. Thereafter, all plants were sprayed with 560 g ae / ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. As determined by chi-square analysis, seven of the eleven lines tested were isolated as single loci dominant Mendelian traits (3R: 1S). AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes.

  Heritability of AAD-12 (v1) in canola: A progeny test of 100 plants was also performed on 11 T1 lines of AAD-12 (v1). Seeds were sown and transplanted into 3-inch pots filled with Metro Mix medium. Thereafter, all plants were sprayed with 560 g ae / ha 2,4-D DMA as previously described. After 14 DAT, resistant and sensitive plants were counted. As determined by chi-square analysis, seven of the eleven lines tested were isolated as single loci dominant Mendelian traits (3R: 1S). AAD-12 is heritable as a robust aryloxyalkanoate auxin resistance gene in multiple species and can be stacked with one or more additional herbicide resistance genes.

  Verification of high 2,4-D tolerance in T1 canola: For T1 AAD-12 (v1), 5-6 mg seeds are stored in layers, sowed, and a thin layer of Sunshine Mix # 5 medium added as top layer of soil It was. Emergence plants were selected 7 and 13 days after planting with 560 g ae / ha 2,4-D.

  Surviving plants were transplanted into 3 inch pots containing Metro Mix medium. Surviving plants from T1 progeny, selected at 560 g ae / ha 2,4-D, were also transplanted into 3 inch pots filled with Metro Mix soil. At the 2-4 leaf stage, plants were sprayed with either 280, 560, 1120, or 2240 g ae / ha of 2,4-D DMA. Plants were graded to 3 and 14 DAT and compared to non-transformed control plants. Sampling of damage data for T1 events at 14 DAT can be seen in Table 25. The data shows that multiple events are robustly resistant to 2,240 g ae / ha 2,4-D, while other events are less resistant to 1,120 g ae / ha 2,4-D It is suggested that Surviving plants were transplanted into 51/4 ″ pots containing Metro Mix medium, placed under the same growth conditions as before, and self-pollinated to produce only homozygous seeds.

  Field Tolerance of pDAB3759 Canola Plants to 2,4-D, Dichloprop, Triclopyr and Fluoroxypyr Herbicides: Field Level Tolerance Tests, Two AAD-12 (v1) Events 3759 (20) 018.001 and 3759 (18) 030.001 as well as wild type canola (Nex 710) in Fowler, IN. The experimental design was a complete random block method, with 3 iterations. Herbicide treatment was 280, 560, 1120, 2240 and 4480 g ae / ha 2,4-D (dimethylamine salt), 840 g ae / ha triclopyr, 280 g ae / ha fluoroxypyr, and untreated control Met. Within each herbicide treatment, events 3759 (18) 030.0011, 3759 (18) 018.001, and a single 20 ft row / event of the wild type strain (Nex710) were transferred to 8 using a 4 row drill. Planted at inch spacing. The herbicide treatment was applied using a compressed air-backed sprayer that delivered a carrier volume of 187 L / ha at a pressure of 130-200 kpa when the canola reached the 4-6 sheet stage. Visual damage assessments were made 7, 14, and 21 days after application.

  The response of canola to 2,4-D, triclopyr, and fluoroxypy is shown in Table 26. Wild-type canola (Nex 710) was severely damaged by 2240 g ae / ha 2,4-D considered as 4 × dose (72% at 14 DAT). All AAD-12 (v1) events demonstrated excellent resistance to 2,4-D at 14 DAT, with average damages of 2, 3 and 2% observed in 1, 2 and 4 × amounts, respectively. Wild type canola was severely damaged (25% at 14 DAT) by 2 × amount of triclopyr (840 g ae / ha). The AAD-12 (v1) event demonstrated resistance with 2 × amount of triclopyr and averaged 6% damage over 2 events at 14 DAT. 280 g ae / ha of fluoroxypy caused severe damage (37%) on lines not transformed into 14 DAA. The AAD-12 (v1) event demonstrated increased resistance with an average of 8% damage to 5DAT.

  These results indicate that the amount of 2,4-in the event that an event transformed with AAD-12 (v1) was lethal to untransformed canola or caused severe hypertrophic dysplasia. It shows a high level of resistance to D, triclopyr and fluoroxypyr. AAD-12 has been shown to have a relative effectiveness of 2,4-D> triclopyr> fluoroxypyr.

Transformation and selection of AAD-12 soybean event DAS-68416-4 Transgenic soybean (Glycine max) event DAS-68416-4 was transformed into Agrobacterium-mediated transformation of soybean cotyledon explants. Produced. A deprotected Agrobacterium strain EHA101 (Hood et al.) Carrying a binary vector pDAB4468 (FIG. 2) having a selectable marker (pat) and a gene of interest (AAD-12) in the T-strand DNA region. 2006) was used to initiate transformation.

  Agrobacterium-mediated transformation was performed. Briefly, soybean seeds (cv Mavick) were germinated on basal medium, cotyledon nodes were isolated and infected with Agrobacterium. To remove Agrobacterium, cefotaxime, timetin and vancomycin were added to the shoot initiation, shoot elongation, and rooting media. Glufosinate selection was used to inhibit the growth of untransformed shoots. Selected shoots were transferred to rooting media for root development and then transferred to a soil mixture for acclimation of plantlets.

  Glufosinate was applied to the terminal leaflets of selected plantlets and screened for putative transformants. The screened plantlets were transferred to a greenhouse and acclimated, and then glufosinate was leaf-applied to reconfirm resistance and considered to be putative transformants. The screened plants were sampled and molecular analysis was performed to confirm the selectable marker gene and / or the gene of interest. T0 plants were self-pollinated in a greenhouse to produce T1 seeds.

  T1 plants were backcrossed and osmotically crossed into a good germplasm. This event, soybean event DAS-68416-4, was made from an independent transformed isolate. Events include efficacy, including a single insertion site, its unique characteristics such as normal Mendelian isolation and stable expression, and herbicide tolerance and agronomic performance across a wide genotypic background and multiple environmental locations. Selected based on superior combinations. A further description of the soybean event DAS-68416-4 is disclosed in WO2011 / 066384, which is incorporated by reference in its entirety.

Creation of Agricultural Data for 2008 Agricultural studies using soybean and non-transgenic control (variant Maverick) of Event DAS-68416-4 were conducted in 2008 in Iowa, Illinois, Indiana, Nebraska. And 6 locations located in Ontario, Canada (2 locations). Agronomic determinants, including napped / population number, seedling / plant vitality, plant height, lodging, disease incidence, insect damage, and days to flowering, were evaluated and compared to the control strain Mavicick The equivalence of soybean event DAS-68416-4 (with and without herbicide treatment) was investigated. This study is referred to as agricultural experiment S1.

  Test and control soybean seeds were planted at a seed rate of about 112 seeds per 25 ft row, with a row spacing of about 30 inches (75 cm). At each location, three replicated sections of each treatment were established, each section consisting of 2-25 ft columns. The compartments were placed by the complete randomized block method (RCB) and were randomly randomized at each location. Each soybean compartment was bordered by two rows of similar mature non-transgenic soybeans. The entire test site was surrounded by a similar relatively mature non-transgenic soybean with a minimum of 10 ft.

  The herbicide treatment was applied at a spray volume of about 20 gallons / acre (187 L / ha). These applications were designed to replicate the commercial practice of maximum display rate. 2,4-D was applied as an application after 3 spraying times in total during the 3 lb ae / A season. Individual applications of 1.0 lb ae A (1,120 g / ha) were made before emergence and at about V4 and R2 growth stages. Glufosinate was applied as an application over the course of two sprays for a total of 0.74 lb ai / A (828 g ai / ha) season. Individual applications of 0.33 lb ai / A and 0.41 lb ai / A (374 and 454 g ai / ha) were made to about V6 and R1 growth stages.

  Analysis of variance across field sites was performed on agronomic data using a mixed model (SAS version 8, SAS Institute 1999). The entry was considered a fixed effect, and the location, the block within the location, the location by entry, and the entry by the block within location were designated as random effects. The significance of the overall treatment effect was estimated using the F test. Corresponding contrasts were sprayed with control and non-sprayed soy event DAS-68416-4 (non-sprayed), soy event DAS-68416-4 sprayed with glufosinate (soybean event DAS-68416-4 + glufosinate), 2,4-D Soybean event DAS-68416-4 (soybean event DAS-68416-4 + 2,4-D), and soybean event DAS-68416-4 (both soybean event DAS-68416-4 +) sprayed with both glufosinate and 2,4-D ) Using the t-test. An adjusted P value was also calculated using the false discovery rate (FDR) to control multiplicity (Benjamini and Hochberg, 1995).

  Agronomics collected from herbicides for control, soybean event DAS-68416-4 unsprayed, soybean event DAS-68416-4 + 2, 4-D, soybean event DAS-68416-4 + glufosinate, and soybean event DAS-68416-4 + Data analysis was performed. No statistically significant differences were observed in the number of napped, initial population, seedling vitality, post-application damage, lodging, number of final napped, or days to flowering (Table 28). At height, a significant paired significant t-test was observed between the control and the soybean event DAS-68416-4 + 2,4-D spray. However, no significant overall treatment effect was observed and the difference between the soybean event DAS-68416-4 treatment and the control was very small, and the difference was common between the various soybean event DAS-68416-4 treatments. It wasn't. Based on these results, the soybean event DAS-68416-4 was agronomically equivalent to the near-isogenic non-transgenic control.

Agricultural data generation for 2009 Agricultural studies using soybean event DAS-68416-4 and a non-transgenic control (variant Maverick) were conducted in 2009 in Arkansas, Iowa, Illinois, Indiana, The test was conducted at 8 locations in Missouri and Nebraska. Agricultural determinants including napped / population number, seedling / plant vitality, plant height, disease incidence, insect damage, and days to flowering were assessed and compared to the soybean event DAS- The equivalence of 68416-4 soybean (with and without herbicide treatment) was investigated (Table 29).

  The complete randomized method was used for the test. Entries were the soybean event DAS-68416-4, the Maverick control line, and a commercially available non-transgenic soybean line. Test, control and reference soybean seeds were planted at a seed rate of about 112 seeds per row, with a row spacing of about 30 inches (75 cm). At each location, four replicated sections of each treatment were established, each section consisting of 2-25 ft columns. Each soybean compartment was bordered by two rows of non-transgenic soybeans (Maverick). The entire test site was surrounded by a minimum of 4 rows (or 10 ft) of non-transgenic soybean (Maverick). Appropriate insect, weed, and disease control practices were applied to produce agronomically acceptable crops.

  Herbicide treatment was applied to recreate the commercial performance of maximum display rate. Treatment consisted of herbicide application of 2,4-D, glufosinate, 2,4-D / glufosinate applied to the non-spray control as well as the designated growth stage. For the 2,4-D application, the herbicide was applied to the V4 and R2 growth stages in an amount of 1.0 lb ae / A (1,120 g ae / ha). For glufosinate treatment, plants were applied during the V4 and V6-R2 growth stages. For both applications glufosinate was applied in amounts of 0.33 lb ai / A (374 g ai / ha) and 0.41 lb ai / A (454 g ai / ha) for application at V4 and V6-R2, respectively. Both herbicide application entries were controls including the soybean event DAS-68416-4 and non-transgenic Mavelick. The Maberick compartment was expected to die after herbicide application.

  Analysis of variance across field sites was performed on agronomic data using a mixed model (SAS version 8, SAS Institute 1999). The entry was considered a fixed effect, and the location, the block within the location, the location by entry, and the entry by the block within location were designated as random effects. Analysis at each location was done in a similar manner, with entry as a fixed effect and block and block entry as a random effect. Data were not rounded for statistical analysis. Significance was declared with a 95% confidence level and the significance of the overall treatment effect was estimated using the F test. Corresponding contrasts include non-sprayed AAD-12 (non-spray), AAD-12 sprayed with glufosinate (AAD-12 + glufosinate), AAD-12 sprayed with 2,4-D (AAD-12 + 2,4-D), and A t-test was used between the transgenic and control entries of AAD-12 sprayed with both glufosinate and 2,4-D (AAD-12 + 2,4-D + glufosinate).

Multiplicity was a problem because of the many contrasts in this study. Multiplicity is a problem when many comparisons are made in a single study to look for unexpected effects. Under these conditions, the probability of erroneously declaring a difference based on the P value for each comparison is very high ( ^{number of} 1-0.95 ^comparisons ). In this study, there are 4 comparisons per analyte (16 analyzed agricultural observation types), resulting in 64 agricultural comparisons. Therefore, the probability of declaring one or more false differences based on unadjusted P values was 99% agriculturally (1-0.95 ⁶⁴ ).

  Analysis of agricultural data collected from control, AAD-12 non-spray, AAD-12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + 2,4-D + glufosinate entries was performed. Analysis across sites included seedling vitality, final population, plant vitality / damage (V4, R1), lodging, disease incidence, insect damage, days to flowering, days to maturity, number of pods, seeds No statistically significant differences were observed in number, yield, and plant height. A significant paired significant paired t-test was observed between the control and the AAD-12 + glufosinate entry in the napped number and initial population, but without a significant overall treatment effect or FDR adjusted P value. In plant vitality / damage (R2), significant paired t-tests and significant overall treatment effects were observed between both controls and AAD-12 + glufosinate and AAD-12 + 2,4-D + glufosinate entries. Was not accompanied by a significant FDR adjusted P value. The average results for all these variables were also within the range found for the reference strains tested in this study.

Transformation and Selection of AAD1 Event pDAS 1740-278 The AAD1 event, pDAS 1740-278, was generated by anther crystal mediated transformation of the maize line Hi-II. The transformation method used is described in US Patent Application No. 20090093366. The Fspl fragment of plasmid pDAS1740 (FIG. 3), also called pDAB3812, was transformed into a maize line. This plasmid construct contains an expression cassette containing RB7 MARv3 :: maize (Zea mays) ubiquitin 1 promoter v2 // AAD1 v3 // maize (Zea mays) PER5 3′UTR :: RB7 MARv4 plant transcription unit (PTU) To do.

  Generated numerous events. Events that generated viable and healthy haloxyhop-resistant callus tissue were assigned a unique identification code representing a putative transformation event and were transferred continuously to fresh selective media. Plants were regenerated from tissues derived from each unique event and transferred to the greenhouse.

  Leaf samples were taken for molecular analysis and verified for the presence of the AAD-1 transgene by Southern blot, DNA border confirmation, and genomic marker assisted confirmation. Positive T0 plants were pollinated with inbred lines to obtain T1 seeds. T1 plants of event pDAS 1470-278-9 (DAS-40278-9) were selected, self-pollinated for 5 generations and characterized. On the other hand, T1 plants were backcrossed and penetrating into a good germplasm (XHH13) for several generations by marker-assisted selection. This event was generated from an independent transformed isolate. Events include efficacy, including a single insertion site, its unique characteristics such as normal Mendelian isolation and stable expression, and herbicide tolerance and agronomic performance across a wide genotypic background and multiple environmental locations. Selected based on superior combinations. Further description of the corn event pDAS-1740-278-9 is disclosed in WO2011 / 022469, which is incorporated by reference in its entirety.

Herbicide Application and Agronomic Data Herbicide treatment was applied at a spray volume of about 20 gallons / acre (187 L / ha).

  These applications were designed to replicate the commercial practice of maximum display rate. Uses Weedar 64 (026991-0006) at 39%, 3,76 lb ae / gal, 451 g ae / l, and Asse II (106155) at 10.7%, 0.87 lb ai / gal, 104 g ai / g did.

  2,4-D (Weedar 64) was applied to Test Entries 4 and 5 as the application after 3 sprays (total of 3lb ae / A during the season). Individual applications were pre-emergence and about V4 and V8-V8.5 stages. The individual target application rate was 1.0 lb ae / A for Weedar 64, ie 1120 g ae / ha. Actual application rates ranged from 1096 to 1231 g ae / A.

  Quizarohop (Assure II) was applied to Test Entries 3 and 5 as an application after a single application. The application timing was about the V6 growth stage. The target application rate was 0.0825 lb ai / A, or 92 g ai / ha for Assure II. Actual application rates ranged from 90.8 to 103 g ai / ha. Agronomic characteristics were recorded for all test entries in blocks 2, 3, and 4 at each location. Table 30 lists the measured features.

  Analysis of the agronomic data collected from both the control, aad-1 non-spray, aad-1 + 2,4-D, aad-1 + quizalofop, and aad-1 + entries was performed. Cross-site analysis includes initial population (V1 and V4), vitality, final population, crop damage, time to silk production, time to pollen, stem lodging, root lodging, No statistically significant differences were observed in disease incidence, insect damage, days to maturity, plant height, and pollen viability (shape and color) values. For green maintenance and spike height, a significant paired t-test was observed between the control and aad-1 + quizalofop entries, but with a significant overall treatment effect or false discovery rate (FDR) adjusted P-value. It was not accompanied (Table 31).

Further Argonomic Testing The agronomic characteristics of corn line 40278 compared to corn lines of near-isogenic lines were evaluated across a variety of environments. The treatment included 4 genetically distinct hybrids and their appropriate near-isogenic lineage hybrids tested over a total of 21 sites.

  The four test hybrids were mesophilic to late matured hybrids ranging from 99 to 113 days of relative maturity. Experiment A tested event DAS-40278-9 in the genetic background of inbred CxBC3S1 conversion. This hybrid had a relative maturity of 109 days and was tested at 16 locations (Table 32). Experiment B examined a hybrid background of 113-day relative maturation hybrids of self-breeding E × BC3S1 conversion. This hybrid was tested in 14 locations using a slightly different set of locations than in Experiment A. Experiments C and D tested a hybrid background of BC2S1 conversion x inbred D and BC2S1 conversion x inbred F, respectively. Both of these hybrids had a relative maturity of 99 days and were tested in the same 10 locations.

  For each test, the complete randomized block method was used with two replicates per location and two rows. The row length was 20 feet and each row was sown with 34 seeds per row. Standard regional agricultural practices were used in the management of the trial.

  Data were collected and analyzed for eight agronomic characteristics: plant height, ear height, stem lodging, root lodging, final population, kernel moisture, test weight, and yield. The plant height and ear height parameters provide information on the appearance of the hybrid. The agronomic characteristics of percent stem lodging and root lodging determine the harvest potential of the hybrid. The final population number measures seed quality and seasonal growth conditions that affect yield. Percentage grain moisture at harvest defines hybrid maturity, yield (bushel / acre, adjusted for moisture) and test weight (1 bushel corn weight in pounds, adjusted to 15.5% moisture) Explains the fertility of the hybrid.

  An analysis of variance over the field locations was performed using a linear model. Entry and location were included as fixed effects in the model. A mixed model was investigated in which location and location by entry were included as random effects, but location by entry only explained a small fraction of the variance, and its variance component was often not significantly different from zero. For stem and root lodging, logarithmic transformation was used to stabilize the variance, but the means and range are reported on the original scale. Significant differences were declared with a 95% confidence level. The significance of the overall treatment effect was estimated using a t-test.

  The results of these agricultural characterization tests can be found in Table 32. Statistically significant for all four 40278 hybrids in terms of ear height, stem lodging, root lodging, grain moisture, test weight, and yield parameters compared to isogenic line controls. No difference was found (p <0.05). Although the final population number and plant height were statistically different in Experiments A and B, respectively, similar differences were not seen in comparison with the other 40278 hybrids tested. Some of the variation seen can be attributed to low level gene variability remaining since the DAS-40278-9 event was backcrossed into a good inbred line. The overall range of measured parameter values is all within the range of values obtained with traditional maize hybrids and does not lead to the conclusion that weedability has increased. In summary, the agricultural characterization data show that 40278 corn is biologically equivalent to conventional corn.

  Agronomic characteristics of hybrid maize containing event DAS-40278-9 compared to near-isogenic line maize were collected during a single growing season from multiple field trials across diverse geographic environments. The results for the hybrid maize line containing event DAS-40278-9 compared to null plants are listed in Table 33.

  Hybrid maize line containing event DAS-40278-9 sprayed with herbicide quizalofop (280 g ae / ha) at V3 developmental stage and 2,4-D (2,240 g ae / ha) at V6 developmental stage The agronomic characteristics of the null plants are shown in Table 34.

2,4-D increases the growth of 2,4-D resistant soybeans While applying 2,4-D, transgenic soybeans with the AAD-12 transgene provide protection to soybean plants The weeds are destroyed. It was unexpectedly observed that 2,4-D also increased the growth of 2,4-D resistant soybeans. This increased growth has resulted in an increase in plant height and / or yield in the spray zone compared to the non-spray zone.

  The increase in plant growth and / or yield resulting from the application of 2,4-D has been described for soybean plants that have been genetically engineered to be resistant to 2,4-D. Specimens were grown across multiple locations covering the North American soybean growing area. Entries included good lines that were cross-bred with event DAS-68416-4 (adjusting resistance to 2,4-D). The treatment consisted of a non-spray treatment and a 2,4-D spray treatment applied at both the V3 and R2 growth stages. The parcels were measured for various agronomic characteristics including plant height and kernel yield over the season. To eliminate all competing effects, weeds were controlled throughout the season in both sprayed and non-sprayed sections. At the end of the study, data analysis measured a significant increase in both height and yield in entries sprayed with 2,4-D compared to those that received no treatment. Increased yield is an advantage in addition to the weed control delivered by 2,4-D against 2,4-D resistant noise.

  To compare the agricultural characteristics of soybean event DAS-68416-4 sprayed with 2,4-D (International Patent Application No. 2011/066384) with the agricultural characteristics of non-sprayed soybean event DAS-68416-4 A field test was carried out in 2011. The field trials consisted of four good soybean lines that were osmotically crossed with soybean event DAS-68416-4, as well as each null isogenic of four good soybean lines that did not contain soybean event DAS-68416-4. Contained strains. Specimens were planted over a variety of geographic locations (10 total). The experiment was set up as an improved partition method using 2 iterations per location. All parcels were processed and subpartitions were entries. Each compartment consisted of two rows of 12.5 feet long planted 30 inches apart. The spray compartment was treated with 2,4-D (1120 g ae / ha) sprayed in the V3 and R2 growth stages. Throughout the season, field plots were maintained under normal agricultural practices and kept free of weeds. Various agronomic characteristics were measured on soybean plants to determine how application of 2,4-D affected the performance of soybean agronomic characteristics. The agronomic characteristics tested and the growth stages for which data were collected are listed in Table 35.

  At the end of the soybean growing season, data from all locations were combined and analysis across various locations was performed. Data analysis was performed using hwJMP ™ Pro 9.0.3 (SAS, Cary, NC). The least mean square from the analysis is reported in Table 28. Application of 2,4-D to the soybean event DAS-68416-4 containing the AAD-12 transgene resulted in an increased growth regulating effect. Increased growth resulted in significantly higher yield and plant height measurements in field plots sprayed with 2,4-D than in field plots not sprayed with 2,4-D. These increases were confirmed when the data were cumulatively analyzed across all locations. In contrast, the increased yield of soybean event DAS-68416-4 sprayed with 2,4-D was attenuated at one location due to treatment interactions. In Table 36, both average height and yield increased by about 5% with 2,4-D application.

  As shown in Table 37, soybean event DAS-68416-4 plants sprayed with 2,4-D in non-sprayed soybean event DAS-68416-4 plants in at least one of ten locations (location # a3) A significantly higher yield was reported. Accumulating results from all locations, 2,4-D application to the soybean event DAS-68416-4 containing the AAD-12 transgene showed a modulating effect that resulted in increased growth. For example, the yield of soybean event DAS-68416-4 plants sprayed with 2,4-D is 56.4 bu / acre, which is the yield of unsprayed soybean event DAS-68416-4 plants, 53.7 bu / ac. Much higher than an acre. Similarly, the height of the soybean event DAS-68416-4 plant sprayed with 2,4-D is 81 cm, which is significantly higher than the height of the non-sprayed soybean event DAS-68416-4 plant, 77 cm. .

2,4-D increases the growth of 2,4-D resistant soybeans in the 2,4-D / glyphosate combination as in previous examples, but with 2,4-D 2 in combination with glyphosate A field test with a single application was carried out in 2010. The results show that the increased 2,4-D resistant soybean growth, plant height and / or yield in the spray section compared to the non-spray section is due to the 2,4-D application. .

  Significant treatment effects were observed with several parameters measured. Both 2,4-D and glyphosate were sprayed at the V3 and R2 growth stages. Specimens were planted over various geographic locations (6 locations total). The agronomic characteristics tested and the growth stages for which data was collected are listed in Table 30. In Table 38, sprayed soybeans had an average height increase of 6% and an average yield of 17%. Furthermore, the average seed weight increased by 6% in the sprayed soybean.

  As shown in Table 39, certain geographic variations were also observed in this example. In Table 39, the average yield increased by 21.6% for sprayed soybeans.

Yield test results comparing spray and non-spray treatments Transgenic crops transformed with 2,4-D-resistant aryloxyalkanoate dioxygenase (AAD) were stimulated with a stimulating amount of herbicide containing an aryloxyalkanoate moiety. It resulted in increased yield when treated. Soybean events containing the AAD-12 gene expression cassette were tested in repeated yield tests under spray and non-spray conditions. There was a series of experiments that contained early soybeans adapted to the northern hemisphere and another series that contained late soybeans adapted to southern latitudes. In previous experiments, there were cases where soybean entries containing AAD-12 gene expression cassettes treated with 2,4-D during the growing season showed increased yields compared to non-spray studies. .

  A modified partition method with two iterations was used for the test. Each section was 2 rows wide, 30 inches apart, and 12.5 feet long. There was a 2.5 to 3 foot passage between the sections planted from end to end to allow movement during the test during the season. The spray block was sprayed sequentially (twice) with 2185 g ae / ha 2,4-D choline + glyphosate (premix) + 2% weight / weight AMS during the growing season.

  The first application was at the V3 growth stage and the second application was at the R2 growth stage. Both experimental and control field trials were kept free of weed throughout the season by the use of conventional herbicides or by manual weeding. Data on budding, seedling vitality, crop damage, flowering date, number of raised hairs in R2, disease incidence, insect damage, plant height, maturity date, lodging, fall, 100 seed weight, and yield Collected. Data was analyzed using JMP ™ Pro 9.0.3. Table 40 lists the locations used in the final analysis. Some planted locations were not included in the analysis due to variations within the plot.

  Analysis across various locations was performed for both early and late life trials. Tables 41 and 42 show the yield variance analysis for the early and late trials, respectively.

  There was a significant (P = 0.05) name effect in both the early and late life trials. This was expected because each good soybean line that crossed the event was from a different genetic background.

  Significant treatment effects were measured in the premature test, indicating that the spray and non-spray treatment yields were different. In the late life test there was no significant treatment effect, indicating that the yield was not different in the sprayed and non-sprayed compartments.

  In both early and late trials, the name due to the interaction effect of treatment was not significant, indicating that the effect of treatment (or lack of effect) was the same for each entry in a particular trial.

  Table 43 shows the average yield of each entry by combination of treatments in the premature test, and HOMO indicates homozygosity. Values following the same letter (within a given variant) are not different at P = 0.05 according to Student's t test. There were 4 entries showing higher yields when 2185 g ae / ha of 2,4-D choline + glyphosate (premix) + AMS were sprayed sequentially on V3 and R3.

  Table 44 shows the average yield for each entry by treatment combination. Values following the same letter (within a given variant) are not different at P = 0.05 according to Student's t test. As reported above, no mean separation was performed because there was no significant treatment effect or treatment effect by entry for late life trials. The letters in the table indicate that there was no difference between spray treatment and non-spray treatment in the late life test.

  The results from the yield test in this example again show that the yield can increase after application of 2,4-D in some environments of some soybean genotypes. During the past two years, such yield increases have been observed in yield tests conducted in MG2 growing areas.

Comparison of Soybeans and Maize Field trial yield results in soybeans containing the AAD-12 transgene show that application of 2,4-D can increase the yield of soybeans of a particular soybean genotype in a particular environment . These results are surprising when compared to transgenic corn events that contain the AAD-1 transgene. The yield of AAD-1 transgenic corn plants did not consistently show a statistically significant increase in yield after spraying 2,4-D. These AAD-1 transgenic corn plants are biologically equivalent to conventional corn. Further field studies in diverse geographic locations have been completed for hybrid maize lines between 2010 and 2012. Throughout these field studies, the yield of corn lines sprayed with 2,4-D (2,185 g ae / ha and 4,370 g ae / ha) was measured using untreated control corn lines (eg, 2,4-D). Compared to those not sprayed). The results of these experiments further demonstrate that corn plants containing the AAD-1 transgene do not result in a significant yield increase as a result of treatment with 2,4-D spray. In comparison, some soybean genotypes show increased yields after 2,4-D application. The increase in yield observed in the soybean genotype, shown after application of 2,4-D, is an unexpected improvement that can be applied to increase crop yield. The disclosed method can be deployed in using 2,4-D treatment to increase the yield of transgenic crops that express, for example, the AAD-12 gene.

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

Claims (32)

  A method for improving the yield of 2,4-D resistant crops comprising treating a plant with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety.   The method according to claim 1, wherein the 2,4-D resistant crop is a transgenic plant transformed with aryloxyalkanoate dioxygenase (AAD).   The method of claim 2, wherein the aryloxyalkanoate dioxygenase (AAD) is AAD-12.   The method according to claim 1, wherein the herbicide containing an aryloxyalkanoate moiety is a phenoxy herbicide or a phenoxyacetic acid herbicide.   The method of claim 1, wherein the herbicide comprising an aryloxyalkanoate moiety is 2,4-D.   6. The method of claim 5, wherein 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA).   The method according to claim 1, wherein the treatment is carried out at least once at a 2,4-D application rate that is also used in weed control.   The method according to claim 1, wherein the treatment is carried out twice, at a 2,4-D application rate that is also used in weed control.   9. The method of claim 8, wherein 2,4-D is applied at the V3 and R2 growth stages of soybean having 2,4-D tolerance.   The method according to claim 1, wherein the treatment is carried out at least three times with a 2,4-D application rate that is also used in weed control.   The method of claim 1, wherein the 2,4-D resistant crop is under stress.   The method of claim 1, wherein the 2,4-D resistant crop is also treated with a different herbicide than 2,4-D for controlling weeds.   The method according to claim 12, wherein the herbicide different from 2,4-D is a phosphorus herbicide or an aryloxyphenoxypropionic acid herbicide.   14. The method of claim 13, wherein the phosphorus herbicide comprises glyphosate, glufosinate, a derivative thereof, or a combination thereof.   14. The method of claim 13, wherein the phosphorus herbicide is in the form of an ammonium salt, isopropylammonium salt, isopropylamine salt, or potassium salt.   14. The method of claim 13, wherein the aryloxyphenoxypropionic acid herbicide comprises chlorazihop, phenoxaprop, fluazihop, haloxyhop, quizalofop, derivatives thereof, or combinations thereof.   The method of claim 1, wherein the 2,4-D resistant crop is treated at least once with 2,4-D from 25 g ae / ha to 5000 g ae / ha.   The method of claim 1, wherein the 2,4-D resistant crop is treated at least once with 2,4-D from 100 g ae / ha to 2500 g ae / ha.   The method of claim 1, wherein the herbicide comprising an aryloxyalkanoate moiety reaches a 2,4-D resistant crop via root absorption.   14. The method of claim 13, wherein the phosphorus herbicide reaches a 2,4-D resistant crop via root absorption.   14. The method of claim 13, wherein the aryloxyphenoxypropionic acid herbicide reaches a 2,4-D resistant crop via root absorption.   The method of claim 2, wherein the transgenic plant transformed with aryloxyalkanoate dioxygenase (AAD) is selected from cotton, soybean, and canola. (A) transforming a plant cell with a nucleic acid molecule comprising a nucleotide sequence encoding an aryloxyalkanoate dioxygenase (AAD);
(B) selecting transformed cells;
(C) regenerating the plant from the transformed cells;
(D) treating the plant with a stimulating amount of a herbicide comprising an aryloxyalkanoate moiety to improve the yield of 2,4-D resistant crops.   24. The method of claim 23, wherein the aryloxyalkanoate dioxygenase (AAD) is AAD-12.   24. The method of claim 23, wherein the nucleic acid molecule comprises a selectable marker that is not an aryloxyalkanoate dioxygenase (AAD).   26. The method of claim 25, wherein the selectable marker is a phosphinothricin acetyltransferase gene (pat) or a bialaphos resistance gene (bar).   24. The method of claim 23, wherein the nucleic acid molecule is optimized for plants.   Use of 2,4-D in the production of transgenic plants with 2,4-D resistance with increased yield compared to its non-transgenic parent plant.   29. Use according to claim 28, wherein 2,4-D is applied at least once with 2,4-D from 25 g ae / ha to 5000 g / ha.   29. Use according to claim 28, wherein 2,4-D is applied at least once in 2,4-D from 100 g ae / ha to 2500 g ae / ha.   29. Use according to claim 28, wherein 2,4-D comprises 2,4-D choline or 2,4-D dimethylamine (DMA). 29. Use according to claim 28, wherein the 2,4-D resistant crop is treated with 2,4-D at least twice before flowering.