NZ615200B2 - Methods of weed control involving aad-1 plants, and re-plant and/or pre-emergence herbicide applications - Google Patents

Abstract

Disclosed is a method of controlling weeds, said method comprising planting seed in an area, and applying an aryloxyalkanoate herbicide to said area within 30 days before planting seed in said area, said seed comprising a polynucleotide that encodes an AAD-l protein having at least 75% identity with the AAD-l protein encoded by nucleotides 4265-5152 of SEQ ID NO: 29. the AAD-l protein encoded by nucleotides 4265-5152 of SEQ ID NO: 29.

Description

METHODS OF WEED CONTROL INVOLVING AAD-l PLANTS AND PRE—PLANT AND/OR PRE—EMERGENCE HERBICIDE APPLICATIONS BACKGROUND OF THE INVENTION Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in New d or any other jurisdiction.

The aad—l gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD—l) protein. The trait confers tolerance to 2,4— dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as “fop” ides such as quizalofop) herbicides and may be used as a selectable marker during plant transformation and in breeding nurseries. The clad-J gene, itself, for ide nce in plants was first disclosed in (see also US 2009—0093366).

The sion of heterologous or foreign genes in plants is influenced by where the n gene is inserted in the chromosome. This could be due to chromatin structure (eg, heterochromatin) or the proximity of riptional regulation elements (eg, enhancers) close to the integration site (Weising cl 0]., Ann. Rev. Gene] 22:421—477, 1988), for example. The same gene in the same type of transgenic plant (or other organism) can exhibit a wide variation 2O in expression level amongst different events. There may also be differences in spatial or al patterns of expression. For example, ences in the relative expression of a transgcnc in various plant tissues may not correspond to the patterns expected from transcriptional regulatory elements present in the uced gene construct.

Thus, large numbers of events are often created and screened in order to identify an event that expresses an introduced gene of interest to a satisfactory level for a given purpose. For cial purposes, it is common to produce hundreds to thousands of different events and to screen those events for a single event that has desired transgene sion levels and patterns.

An event that has desired levels and/or patterns of transgene expression is useful for ressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions. 1001302214 US. Patent Apps. 20020120964 Al and 20040009504 A1 relate to cotton event PV— GHGT07(1445) and compositions and methods for the detection thereof. WO 02/100163 relates to cotton event MONIS985 and compositions and methods for the detection thereof. WO 2004/011601 s to corn event MON863 plants and compositions and methods for the detection thereof. relates to cotton event MON 88913 and compositions and methods for the detection thereof. relates to corn event 3272. relates to corn event MIRI62.

US. Patent No. 7,179,965 relates to cotton having a crylF event and a crylAc event. 1O AAD—l corn having the c event disclosed herein has not previously been disclosed.

BRIEF SUMMARY OF THE INVENTION The subject ion includes pre—plant and/or pre—emergcnce applications ofa herbicide to an area or field that is planted with seed comprising an AAD-l event. In some preferred embodiments, the seed comprises corn event DAS—40278—9. In some red embodiments, the herbicide can be a formulation comprising a 2,4—D active ingredient. Such herbicides and ations can also be used in pre—plant applications. Additional herbicides, such as glyphosate, can be used in combination, including in the pre—plant applications.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a plasmid map of pDASl740.

Figure 2 shows ents ofthe insert for 278—9 (pDASl740).

Figure 3 shows a restriction map and components of the insert for DAS—40278—9 (pDASI740).

Figure 4 shows amplicons, primers, and a cloning strategy for the DNA insert and borders for DAS9.

Figure 5 illustrates a diagram of the primers used in PCR amplification for confirmation of flanking border regions of the corn event DAS—40278—9. The schematic diagram depicts the primer locations for confirming the full length sequencing of the AAD—l corn event DAS—40278— 9 from 5’ to 3’ borders.

Figure 6 illustrates a DNA sequence insertion in the junction s of corn event DAS— 9. A 2lbp insertion (Italics) at 5’-integration junction caused 2 bp on (underlined) 1001302214 from the original genome locus. A 1 bp insertion (Italics) was found at 3’-integration on as well.

Figure 7 is a breeding diagram for 40278 corn and generations used for analyses, as referenced in Example 7. 1001302214 BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NOS: 1—28 are s as bed herein.

SEQ ID NO:29 provides insert and g sequences for the subject event DAS SEQ ID NOs:30-33 are s for flanking markers as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "comprise" and variations of the term, such as "comprising", 1O "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

The subject invention includes pre—plant and/or pie—emergence applications of a herbicide to an area or field that is later planted with seed comprising an AAD—l event. In some preferred embodiments, the seed comprises corn event DAS—40278—9. In some red embodiments, the herbicide can be a formulation comprising a 2,4—D active ingredient. Such ides and fonnulations can also be used in pre—plant applications. Additional herbicides, such as glyphosate, can be used in combination, including in the pre—plant applications. The t invention is not limited to corn but can include use of cotton and/or soybeans, for example, comprising an aad—l gene.

Examples included herein are directed in part to pre—plant and/0r pre-emergence applications of herbicides. Such uses are not limited to the "278" event. One could expound further on the utility of the tolerance provided by the subject AAD—l genes with regard to shortened plant—back interval. This gives growers a great deal more flexibility in scheduling their planting relative to burndown. Without using the subject invention, waiting 7—30 days or so after burndown before planting could cause significant yield loss. Thus, the t invention provides advantages in this regard. See, for example, Example 13. Any planting / herbicidal application als, and any concentration ranges / use rates of ide(s) exemplified or suggested herein can be used in ance with the subject invention.

Thus, the subject invention includes novel methods of applying herbicides. Such applications can include tank mixes of more than one herbicide. Some preferred herbicides for use according to the subject invention e phenoxy auxin herbicide such as 2,4—D; 2,4-DB; MCPA; MCPB.

These can be stacked with one or more additional herbicide tolerance gene(s) and a corresponding herbicide (e.g. glyphosate and/or glufosinate). One, two, three, or more herbicides can be used in advantageous combinations that would be apparent to one skilled in the art having the benefit of the subject disclosure. One or more of the subject herbicides can be d to a field/area prior to planting it with seeds of the subject invention. Such applications can be within 14 days, for example, of planting. One or more of the t herbicides can also be applied nt and/or post-plant but pre-emergence. One or more of the subject herbicides can also be applied to the ground (for controlling weeds) or over the top of the weeds and/or transgenic plants of the subject invention. The subject three herbicides can be rotated or used in combination to, for example, l or prevent weeds that might to tolerant to one herbicide but not another. Various application times for the subject three types of herbicides can be used in various ways as would be known in the art.

Thus, the subject invention also includes pre-plant applications of a herbicide to an area or field that is later planted with seed comprising an AAD-1 event. In some preferred embodiments, the seed comprises corn event DAS9. In some preferred ments, the ide can be a ation comprising a 2,4-D active ingredient. Such herbicides and formulations can be used in pre-plant applications. Additional herbicides, such as glyphosate, can be used in combination in the pre-plant applications. Corn, cotton, and soybeans, for example, can be used in any such embodiments.

The aad- 1 gene can be combined with, for example, traits encoding glyphosate resistance (e.g., resistant plant or bacterial EPSPS, GOX, GAT), glufosinate resistance (e.g., Pat, bar), acetolactate synthase (ALS)-inhibiting herbicide resistance (e.g., imidazolinones [such as imazethapyr], ylureas, triazolopyrimidine sulfonanilide, pyrmidinylthiobenzoates, and other chemistries [Csrl, SurA, et al. ]), bromoxynil resistance (e.g. ,Bxn), resistance to inhibitors of HPPD (4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitors of phytoene rase (PDS), resistance to photosystem II inhibiting herbicides (e.g.,psbA), resistance to photosystem I inhibiting ides, resistance to protoporphyrinogen oxidase IX inhibiting herbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g. , CYP76B1), a-degrading enzymes (see, e.g., US 20030135879), and others could be stacked alone or in multiple combinations to provide the ability to ively control or prevent weed shifts and/or resistance to any herbicide of the aforementioned classes.

Regarding onal ides, some additional preferred ALS (also known as AHAS) inhibitors include the triazolopyrimidine sulfonanilides (such as cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, and penoxsulam), pyrimidinylthiobenzoates (such as bispyribac and pyrithiobac), and flucarbazone. Some preferred HPPD tors e mesotrione, isoxaflutole, and rione. Some preferred PPO inhibitors include flumiclorac, flumioxazin, flufenpyr, pyraflufen, fluthiacet, butafenacil, carfentrazone, sulfentrazone, and the diphenylethers (such as acifluorfen, fomesafen, lactofen, and oxyfluorfen).

AAD-1 genes for use according to the subject invention can also provide resistance to compounds that are converted to phenoxyacetate auxin herbicides (e.g. , MCPB, etc.). The butyric acid moiety present in the 2,4-DB herbicide is ted through b-oxidation to the phytotoxic 2,4-dichlorophenoxyacetic acid. Likewise, MCPB is converted through b-oxidation to the phytotoxic MCPA. The butanoic acid herbicides are themselves nonherbicidal. They are converted to their respective acid from by b-oxidation within susceptible plants, and it is the acetic acid form of the herbicide that is phytotoxic. Plants incapable of rapid b-oxidation are not harmed by the butanoic acid herbicides. However, plants that are capable of rapid b-oxidation and can convert the butanoic acid ide to the acetic form are uently protected by AAD-1.

Included in this disclosure is the AAD-1 corn event designated DAS9 having seed deposited with American Type Culture Collection (ATCC) with Accession No. PTA- 10244, and progeny derived thereof. Other aspects comprise the progeny plants, seeds and grain or rable parts of the plants and seeds and progeny of corn event DAS9, as well as food or feed products made from any thereof. This disclosure also includes plant parts of corn event DAS 9 that include, but are not limited to, , ovule, flowers, shoots, roots, and leaves, and nuclei of vegetative cells, pollen cells, and egg cells. Further disclosed are corn plants having tolerance to phenoxy auxinic and/or aryloxyalkanoate herbicides, novel genetic itions of corn event DAS9, and aspects of agronomic performance of corn plants sing corn event DAS- 40278-9.

Included herein are methods of plant breeding and herbicide tolerant plants, including an aad-1 transformation event in corn plants comprising a polynucleotide sequence, as described herein, ed into a specific site within the genome of a corn cell.

In some ments, said event / polynucleotide ce can be "stacked" with other , including, for e, other herbicide tolerance gene(s) and/or insect-inhibitory proteins.

Plants having the single event are also described herein.

The onal traits may be stacked into the plant genome via plant breeding, retransformation of the transgenic plant ning corn event DAS9, or addition of new traits through targeted integration via homologous recombination.

Other embodiments include the excision of polynucleotide sequences which comprise corn event DAS9, including for e, the pat gene expression cassette. Upon excision of a polynucleotide sequence, the modified event may be re-targeted at a specific chromosomal site wherein additional polynucleotide ces are stacked with corn event DAS9.

In one embodiment is a com chromosomal target site located on chromosome 2 at approximately 20 c between SSR s UMC1265 (see SEQ ID N():30 and SEQ ID O:31) and M CO (see SEQ ID NO:32 and SEQ D NO:33) at approximately 20 cM on the 2008 DAS corn iinakge map, wherein the target site comprises a heterologous nucleic acid. In another embodiment is a corn chromosomal target site comprising a location defined in or by SEQ ID NO:29 and the residues thereof as described herein, as would be recognized by one skilled in the art n one embodiment is a method of making a enic com plant comprising inserting a heterologous nucleic acid at a position on chromosome 2 at imately 20 cM betwee SSR markers MC 265 see SEQ D :30 and SEQ ID N :3 ) and MMCOl (see SEQ ID NO:32 and SEQ ID O:33) at approximately 20 cM on the 2008 DAS corn Hnakge map. In still another embodiment, the inserted heterologous nucleic acid is flanked 5' by all or part of the 5 flanking sequence as defined herein with refernce to SEQ D NO:29, and flanked 3 by a l or part of the 5 flanking sequence as defined herein with refernce to SEQ ID NO:29.

Additionally, disclosed herein are assays for detecting the presence of the subject event in a sample (of com grain, for example). The assays can be based on the DNA sequence of the recombinant construct, inserted into the com genome, and on the genomic sequences flanking the insertion site. Kits and ions useful in conducting the assays are also provided.

Also sed herein is cloning and analysis of the DNA sequences of a whole AAD-1 insert, and the border regions thereof (in transgenic corn lines). These sequences are unique. Based on these insert and border sequences, event-specific primers were ted. PCR is demonstrated that these events can be identified by analysis of the PCR amplicons generated with these event-specific primer sets. Thus, these and other related procedures can be used to uniquely identify corn lines comprising this event.

This disclosure includes the use of plant breeding and ide tolerant plants. This invention includes novel uses of transformation events of corn plants (maize) comprising a subject aad-1 polynucleotide sequences, as described herein, inserted into specific site within the genome of a corn cell. In some embodiments, said polynucleotide sequence can be "stacked" with other traits (such as other herbicide tolerance ) and/or gene(s) that encode insect-inhibitory proteins, for example. In some embodiments said polynucleotide ces can be excised and subsequently r e targeted with onal polynucleotide sequences. However, the subject invention includes plants having a single event, as described herein.

Additionally, this disclosure provides assays for detecting the presence of the subject event in a sample. Aspects include methods of ing and/or producing any diagnostic nucleic acid molecules exemplified or suggested herein, particularly those based wholly or partially on the subject flanking sequences.

More specifically, the subject invention relates in part to transgenic corn event DAS9 (also known as pDAS 1740-278), the use of plant lines comprising these , and the cloning and analysis of the DNA sequences of this insert, and/or the border regions thereof. Plant lines for use according to the subject invention can be detected using sequences disclosed and suggested herein.

In some embodiments, this invention relates to herbicide -tolerant corn lines, and the identification thereof. The subject ion relates in part to detecting the presence of the subject event in order to determine whether progeny of a sexual cross contain the event of interest. In addition, a method for detecting the event is included and is helpful, for e, for complying with regulations ing the rket approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of the subject event by any well-known nucleic acid detection method such as polymerase chain on (PCR) or DNA hybridization using nucleic acid . An event-specific PCR assay is discussed, for example, by Windels et al. (Med. Fac.

Landbouww, Univ. Gent 64/5b:459462, 1999). This related to the identification of glyphosate tolerant soybean event 402 by PCR using a primer set spanning the junction between the insert and flanking DNA. More specifically, one primer included sequence from the insert and a second primer included sequence from flanking DNA.

Corn was modified by the insertion of the aad-l gene from Sphingobium herbicidovorans which encodes the aryloxyalkanoate dioxygenase ) protein. The trait s tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate nly referred to as "fop" herbicides such as quizalofop) herbicides and may be used as a selectable marker during plant transformation and in breeding nurseries. Transformation of corn with a DNA fragment from the plasmid pDAS1740 was carried forward, through ng, to e event DAS9.

Genomic DNA samples extracted from twenty individual corn plants derived from five generations and four plants per generation of event DAS9 were selected for molecular characterization of the AAD-1 corn event DAS9. AAD-1 protein expression was tested using an AAD-1 specific rapid test strip kit. Only plants that tested positive for AAD-1 protein expression were selected for subsequent molecular characterization. Southern hybridization confirmed that the aad-l gene is present in corn plants that tested positive for AAD-1 protein sion, and the aad-l gene was inserted as a single intact copy in these plants when hybridized with an aad-l gene probe.

Molecular terization of the inserted DNA in AAD-1 corn event DAS9 is also described herein. The event was produced via Whiskers transformation with the Fsp I fragment of plasmid pDAS1740. Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and ine insert/copy number of the aad-l gene in event 278-9.

Data were generated to trate the integration and integrity of the aad-l transgene ed into the corn genome. Characterization of the integration of noncoding regions (designed to regulate the coding regions), such as promoters and terminators, the matrix attachment regions RB7 Mar v3 and RB7 Mar v4, as well as stability of the ene insert across generations, were evaluated. The stability of the inserted DNA was demonstrated across five distinct generations of plants.

Furthermore, absence of transformation plasmid backbone sequence including the llin resistance gene (Apr) region was demonstrated by probes covering nearly the whole backbone region flanking the restriction sites (Fsp I) of plasmid pDAS 1740. A detailed physical map of the insertion was drawn based on these Southern blot analyses of event DAS9.

Levels of AAD-1 protein were determined in corn tissues. In addition, compositional analysis was performed on corn forage and grain to investigate the lency between the isogenic non-transformed corn line and the transgenic corn line 278-9 (unsprayed, sprayed with 2,4- D, sprayed with quizalofop, and sprayed with 2,4-D and quizalofop). Agronomic characteristics of the isogenic non-transformed corn line were also compared to the DAS9 corn.

Field expression, nutrient composition, and agronomic trials of a non-transgenic control and a hybrid corn line containing Aryloxyalkanoate Dioxygenase- 1 (AAD-1) were conducted in the same year at six sites located in Iowa, Illinois (2 sites), Indiana, Nebraska and Ontario, .

Expression levels are summarized herein for the AAD-1 n in leaf, pollen, root, forage, whole plant, and grain, the results of mic determinations, and compositional analysis of forage and grain samples from the control and DAS9 AAD-1 corn.

The soluble, extractable AAD- 1protein was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) method in corn leaf, pollen, root, forage, whole plant, and grain.

Good average expression values were observed in root and pollen tissue, as sed in more detail . Expression values were r for all the sprayed treatments as well as for the plots sprayed and unsprayed with 2,4-D and quizalofop herbicides.

Compositional analyses, including proximates, minerals, amino acids, fatty acids, vitamins, anti-nutrients, and secondary metabolites were conducted to investigate the equivalency of DAS- 9 AAD-1 corn (with or without herbicide treatments) to the control. Results for DAS 9 AAD- 1composition samples were all as good as, or better than (biologically and agronomically), based on control lines and/or conventional corn, is of agronomic data ted from l and DAS9 AAD-1 corn plots.

As alluded to above in the Background section, the introduction and integration of a ene into a plant genome involves some random events (hence the name "event" for a given insertion that is expressed). That is, with many transformation techniques such as Agrobacterium transformation, the "gene gun," and WHISKERS, it is unpredictable where in the genome a transgene will become ed. Thus, identifying the flanking plant genomic DNA on both sides of the insert can be important for identifying a plant that has a given insertion event. For example, PCR s can be designed that generate a PCR amplicon across the junction region of the insert and the host genome. This PCR amplicon can be used to identify a unique or distinct type of ion event.

As "events" are originally random events, as part of this disclosure at least 2500 seeds of a corn line comprising the event have been ted and made available to the public without restriction (but subject to patent rights), with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 201 10. The deposit has been designated as ATCC Deposit No. PTA-10244 (Yellow Dent maize hybrid seed (Zea Mays L.):DAS9; ted on behalf of Dow AgroSciences LLC; Date of receipt of seeds/strain(s) by the ATTC: July 10, 2009; viability confirmed August 17, 2009). This deposit was made and will be maintained in accordance with and under the terms of the Budapest Treaty with respect to seed deposits for the purposes of patent procedure. The deposit will be ined without restriction at the ATCC depository, which is a public depository, for a period of 30 years, or five years after the most recent request, or for the effective life of the patent, whichever is , and will be replaced if it s nonviable during that period.

The deposited seeds are part of the subject disclosure. Clearly, corn plants can be grown from these seeds, and such plants are part of the subject invention. The t invention also relates to DNA sequences contained in these corn plants that are useful for detecting these plants and progeny f. Detection methods and kits of the subject invention can be directed to identifying any one, two, or even all three of these , depending on the ultimate purpose of the test.

Definitions and examples are provided herein to help describe the present invention and to guide those of ordinary skill in the art to practice the invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term "progeny" denotes the offspring of any generation of a parent plat which comprises AAD-1 corn evend DAS9.

A transgenic "event" is produced by transformation of plant cells with heterologous DNA, i.e., a c acid construct that es a transgene of interest, regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. The term "event" refers to the original transformant and progeny of the transformant that include the heterologous DNA.

The term "event" also refers to progeny produced by a sexual ss between the transformant and another variety that includes the c/transgene DNA. Even after repeated rossing to a recurrent , the inserted transgene DNA and flanking genomic DNA (genomic/transgene DNA) from the transformed parent is present in the progeny of the cross at the same chromosomal location.

The term "event" also refers to DNA from the original transformant and progeny thereof comprising the ed DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g. , the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.

A "junction sequence" spans the point at which DNA inserted into the genome is linked to DNA from the corn native genome flanking the insertion point, the identification or detection of one or the other junction sequences in a plant's genetic material being sufficient to be diagnostic for the event. Included are the DNA sequences that span the insertions in herein-described corn events and similar lengths of flanking DNA. Specific examples of such diagnostic sequences are provided herein; however, other sequences that overlap the junctions of the insertions, or the junctions of the insertions and the genomic sequence, are also diagnostic and could be used according to the subject ion.

The subject disclosure includes the identification of such flanking, junction, and insert sequences. Related PCR primers and amplicons are ed. PCR analysis methods using ons that span across inserted DNA and its borders can be used to detect or identify commercialized transgenic corn varieties or lines derived from the t proprietary transgenic corn lines.

The entire sequences of each of these inserts, together with ns of the tive flanking sequences, are provided herein as SEQ ID NO:29. The coordinates of the insert and flanking sequences for this event with respect to SEQ ID NO:29 (8557 basepairs total) are printed below. This is discussed in more detail in Example 3.8, for e. Pre-plant embodiments of the subject invention e use of the AAD-1 protein encoded by residues 1874-6689 of SEQ ID NO:29.

' Flanking Insert king residue #s (SEQ:29): 1-1873 1874-6689 6690-8557 length (bp): 1873 bp 4816 bp 1868 bp This insertion event, and further components thereof, are further illustrated in s 1 and 2. These sequences (particularly the ng sequences) are unique. Based on these insert and border sequences, event-specific primers were generated. PCR analysis demonstrated that these corn lines can be identified in different corn genotypes by analysis of the PCR ons generated with these specific primer sets. Thus, these and other related procedures can be used to uniquely identify these corn lines. The sequences identified herein are . For example, BLAST searches aginst GENBANK databases did not reveal any significant homology between the cloned border sequences and sequences in the database. ion techniques are especially useful in conjunction with plant breeding, to determine which progeny plants comprise a given event, after a parent plant sing an event of interest is crossed with another plant line in an effort to impart one or more additional traits of interest in the progeny. These PCR analysis methods benefit corn breeding programs as well as quality control, especially for commercialized transgenic cornseeds. PCR detection kits for these enic corn lines can also now be made and used. This can also benefit product registration and product stewardship.

Furthermore, flanking corn/genomic sequences can be used to specifically identify the genomic location of each insert. This information can be used to make molecular marker systems ic to each event. These can be used for accelerated breeding strategies and to establish linkage data.

Still further, the flanking sequence information can be used to study and characterize transgene integration processes, genomic integration site characteristics, event sorting, stability of transgenes and their flanking sequences, and gene expression (especially related to gene silencing, transgene methylation patterns, position s, and potential expression-related elements such as MARS x attachment regions], and the like).

The subject disclosure includes seeds available under ATCC Deposit No. PTA- 10244. The subject invention also includes use of a herbicide-resistant corn plant grown from a seed deposited with the ATCC under accession number PTA- 10244. The subject invention further includes use of parts of said plant, such as , tissue samples, seeds ed by said plant, pollen, and the like.

Still further, the subject invention includes use of dant and/or progeny plants of plants grown from the deposited seed, preferably a herbicide-resistant corn plant wherein said plant has a genome comprising a detectable wild-type c DNA/insert DNA junction sequence as described herein. As used herein, the term "corn" means maize (Zea mays) and includes all varieties thereof that can be bred with corn.

This invention can further include processes of making crosses using a plant of the subject ion as at least one parent. For example, the subject invention es use of an Fi hybrid plant having as one or both parents any of the plants exemplified . Also within the subject invention is use of seed produced by such Fi hybrids of the subject invention. This invention includes a method for producing an F1hybrid seed by crossing an exemplified plant with a different (e.g. in-bred parent) plant and harvesting the resultant hybrid seed. The subject invention includes use of an exemplified plant that is either a female parent or a male parent. Characteristics of the resulting plants may be improved by careful consideration of the parent plants.

A herbicide-tolerant corn plant can be bred by first sexually crossing a first parental corn plant consisting of a corn plant grown from seed of any one of the lines referred to herein, and a second parental corn plant, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to a herbicide (or that possesses at least one of the events of the subject ion); and selfing the first progeny plant, thereby producing a plurality of second progeny plants; and then ing from the second progeny plants a plant that is resistant to a herbicide (or that possesses at least one of the events of the subject invention). These steps can further include the back-crossing of the first y plant or the second progeny plant to the second parental corn plant or a third parental corn plant. A corn crop comprising corn seeds of the subject invention, or y thereof, can then be d.

It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, ous genes. Selfing of appropriate progeny can produce plants that are gous for both added, exogenous genes.

Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Other breeding methods commonly used for different traits and crops are known in the art. Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line, which is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is ed to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait erred from the donor . After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting parent is expected to have the attributes of the recurrent parent (e.g. , cultivar) and the ble trait erred from the donor parent.

The DNA molecules disclosed herein can be used as molecular markers in a marker assisted breeding (MAB) method. DNA molecules of the present invention can be used in methods (such as, AFLP markers, RFLP markers, RAPD markers, SNPs, and SSRs) that identify genetically linked mically useful traits, as is known in the art. The herbicide-resistance trait can be tracked in the y of a cross with a corn plant of the subject ion (or progeny thereof and any other corn cultivar or variety) using the MAB methods. The DNA molecules are markers for this trait, and MAB methods that are well known in the art can be used to track the hebicide-resistance trait(s) in corn plants where at least one corn line of the subject invention, or progeny thereof, was a parent or ancestor. Methods of the present invention can be used to fy any corn variety having the t event.

Methods of the subject invention include a method of producing a herbicide-tolerant corn plant wherein said method comprises breeding with a plant for use with the subject invention. More specifically, said methods can comprise crossing two plants of the subject ion, or one plant of the subject invention and any other plant. Preferred methods further comprise selecting progeny of said cross by ing said progeny for an event detectable according to the subject invention. For example, the subject invention can include tracking the subject event through breeding cycles with plants comprising other desirable traits, such as agronomic traits such as those tested herein in various Examples. Plants comprising the subject event and the desired trait can be detected, identified, selected, and y used in further rounds of breeding, for example. The subject event / trait can also be combined h breeding, and tracked according to the subject invention, with an insect resistant trait(s) and/or with further herbicide nce traits. One preferred embodiment of the latter is a plant comprising the subject event combined with a gene encoding resistance to the herbicide dicamba.

Additionally, AAD-1 alone or stacked with one or more additional HTC traits can be d with one or more onal input (e.g. , insect resistance, fungal resistance, or stress tolerance, et al.) or output (e.g. , increased yield, improved oil profile, improved fiber y, etal.) traits. Thus, the subject invention can be used to provide a complete agronomic e of improved crop quality with the ability to ly and cost effectively control any number of agronomic pests. s to integrate a polynucleotide sequence within a specific chromosomal site of a plant cell via homologous recombination have been described within the art. For instance, site specific integration as described in US Patent Application Publication No. 2009/01 11188 A l describes the use of recombinases or integrases to e the introduction of a donor polynucleotide sequence into a chromosomal target. In addition, International Patent Application No. describes zinc finger ed-homologous recombination to integrate one or more donor polynucleotide ces within specific locations of the genome. The use of recombinases such as FLP/FRT as described in U.S. Patent No. 6,720,475 or CRE/LOX as described in U.S. Patent No. ,658,772 can be utilized to integrate a polynucleotide sequence into a specific chromosomal site.

Finally the use of meganucleases for targeting donor polynucleotides into a specific chromosomal location was described in Puchta et al, PNAS USA 93 (1996) pp. 5055-5060.

Other s methods for site specific integration within plant cells are generally known and applicable (Kumar et al, Trands in Plant Sci. 6(4) (2001) pp. 155-159). Furthermore, site-specific recombination s which have been identified in several prokaryotic and lower eukaryotic organisms may be applied to use in plants. Examples of such systems include, but are not limited too: the R/RS recombinase system from the pSRl plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J . Mol. Biol. 182: 191-203), and the Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 6).

In some embodiments of the present invention, it can be desirable to integrate or stack a new transgene(s) in proximity to an existing transgenic event. The transgenic event can be considered a red genomic locus which was selected based on unique characteristics such as single insertion site, normal Mendelian segregation and stable expression, and a superior combination of efficacy, including herbicide tolerance and agronomic performance in and across multiple environmental locations. The newly integrated transgenes should maintain the transgene expression characteristics of the existing ormants. Moreover, the development of assays for the detection and confirmation of the newly integrated event would be me as the genomic flanking sequences and chromosomal on of the newly integrated event are already identified. y, the integration of a new ene into a specific chromosomal location which is linked to an existing transgene would expedite the introgression of the transgenes into other genetic backgrounds by sexual out-crossing using conventional breeding methods.

In some embodiments, it can be desirable to excise polynucleotide sequences from a transgenic event. For instance transgene excision as described in Provisional US Patent ation No. 61/297,628 describes the use of zinc finger nucleases to remove a polynucleotide sequence, consisting of a gene expression cassette, from a chromosomally integrated transgenic event. The polynucleotide sequence which is d can be a selectable . Upon excision and removal of a polynucleotide sequence the ed transgenic event can be retargeted by the insertion of a polynucleotide sequence. The excision of a polynucleotide sequence and subsequent retargeting of the modified transgenic event provides advantages such as re-use of a selectable marker or the ability to me unintended changes to the plant transcriptome which results from the expression of specific genes.

Disclosed herein is a specific site on chromosome 2 in the com genome that is excel lent for insertion of heterologous nucleic acids. Also disclosed is a ' molecular marker, a 3' molecular marker, a 5' flanking sequence, and a 3' flanking sequence useful in identifying the location of a targeting site on chromosome 2. Thus, this disclosure provides methods to introduce heterologous c acids of st into this pre-established target site or in the vicinity of this target site. The subject invention also encompasses use of a corn seed and/or a corn plant comprising any heterologous nucleotide sequence inserted at the disclosed target site or in the general vicinity of such site. One option to accomplish such targeted integration is to excise and/or substitute a different insert in place of the pat expression te exemplified . In this l regard, targeted homologous recombination, for example and without tion, can be used according to the subject invention.

As used herein gene, event or trait "stacking" is combi i g desired traits into one transgenic line. Plant breeders stack transgenic traits by making crosses between parents that each have a desired trait and then identifying ing that have both of these desired traits. Another way to stack genes is by erring two or more genes into the cell s of a plan at the same time during transformation. Another way to stack genes is by re-trans r ing a transgenic plant with another gene of interest. For example gene stacking can be used to combine two or more different , including for example, two or more different insect traits, insect resistance trait(s) an disease resistance trait(s), two or more herbicide resistance , and/or insect resistance trait(s) and herbicide resistant trait(s). The use of a selectable marker in addition to a gene of interest can also be considered gene stacking.

''Homologousrecombination" refers to a reaction between any pair of nucleotide sequences having corresponding sites containing a similar nucleotide sequence through which the two nucleotide sequences can interact (recombine) to form a new recombinant DNA seq uence. The sites of r nucleotide sequence are each referred to herein as a "homology sequence." Generally, the frequency of gous recomb at n ses as the length of the homology sequence increases.

Thus, while homologous recombination can occur between two nucleotide sequences that are less than identical, the recombination frequency (or efficiency) declines as the divergence between the two sequences increases. Recombination may be accomplished using one homology sequence on each of the donor and target les, thereby generating a "single-crossover" recombination product. Alternatively, two homology sequences may be placed on each of the target and donor nucleotide sequences. Recombination n two gy sequences o the donor with two homology sequences on the target generates a "double-crossover" recombination product. f the homology sequences on the donor molecule flank a sequence that is to be manipulated (e.g., a sequence of interest), the double-crossover recombination with the target molecule will result in a recombination product wherein the ce of interest replaces a DNA sequence that was originally between the gy seq uences on the target molecule. The exchange of DNA ce between the target and donor through double-crossover recombination event is termed "sequence replacement." The subject AAD-1 enzyme enables transgenic expression resulting in tolerance to combinations of ides that would control nearly all broadleaf and grass weeds. AAD-1 can serve as an ent herbicide tolerant crop (HTC) trait to stack with other HTC traits (e.g., sate resistance, glufosinate resistance, imidazolinone resistance, bromoxynil resistance, et al. ), and insect resistance traits (CryIF, CrylAb, Cry 34/45, et al.) for example. Additionally,AAD- 1 can serve as a selectable marker to aid in selection of primary transformants of plants genetically engineered with a second gene or group of genes.

HTC traits of the t ion can be used in novel combinations with other HTC traits (including but not limited to glyphosate tolerance). These combinations of traits give rise to novel methods of controlling weed (and like) species, due to the newly acquired resistance or inherent tolerance to ides (e.g., glyphosate). Thus, in addition to the HTC , novel methods for controlling weeds using herbicides, for which ide tolerance was created by said enzyme in transgenic crops, are within the scope of the invention. onally, glyphosate tolerant crops grown worldwide are prevalent. Many times in rotation with other glyphosate tolerant crops, control of sate-resistant eers may be difficult in rotational crops. Thus, the use of the subject transgenic traits, stacked or transformed individually into crops, provides a tool for controlling other HTC volunteer crops.

A preferred plant, or a seed, for use with the subject invention comprises in its genome the insert sequences, as identified herein, together with at least 20-500 or more uous flanking tides on both sides of the insert, as identified herein. Unless indicated otherwise, reference to flanking sequences refers to those identified with respect to SEQ ID NO:29 (see the Table above).

Again, SEQ ID NO:29 includes the heterologous DNA ed in the original transformant and illustrative flanking genomic sequences immediately adjacent to the inserted DNA. All or part of these flanking sequences could be expected to be transferred to progeny that receives the inserted DNA as a result of a sexual cross of a parental line that includes the event.

The subject ion includes use of tissue cultures of regenerable cells of a plant of the subject invention. Also included is use of a plant regenerated from such tissue culture, particularly where said plant is capable of expressing all the morphological and physiological properties of an exemplified variety. Preferred plants for use with the subject invention can have all the physiological and morphological characteristics of a plant grown from the deposited seed. This invention further comprises use of progeny of such seed and seed possessing the quality traits of interest.

Manipulations (such as mutation, further transfection, and further breeding) of plants or seeds, or parts thereof, may lead to the creation of what may be termed "essentially derived" varieties. The International Union for the Protection of New Varieties of Plants (UPOV) has provided the following ine for determining if a variety has been essentially d from a protected variety: [A] variety shall be deemed to be essentially derived from another variety ("the initial variety") when (i) it is predominantly derived from the initial variety, or from a variety that is itself predominantly derived from the initial y, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety; (ii) it is clearly distinguishable from the initial variety; and (iii) except for the differences which result from the act of derivation, it ms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the l variety.

UPOV, Sixth Meeting with International Organizations, Geneva, Oct. 30, 1992; document prepared by the Office of the Union.

As used herein, a "line" is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of ollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.

As used herein, the terms "cultivar" and ty" are synonymous and refer to a line which is used for commercial tion.

"Stability" or "stable" means that with respect to the given component, the component is maintained from generation to generation and, ably, at least three generations at substantially the same level, e.g., preferably ±15%, more preferably ± 10% , most preferably ±5%. The stability may be affected by temperature, location, stress and the time of planting. Comparison of uent generations under field conditions should produce the component in a similar manner.

"Commercial Utility" is defined as having good plant vigor and high fertility, such that the crop can be produced by s using conventional farming equipment, and the oil with the described components can be extracted from the seed using conventional ng and extraction equipment. To be commercially useful, the yield, as ed by seed weight, oil content, and total oil produced per acre, is within 15% of the average yield of an otherwise able commercial canola variety without the premium value traits grown in the same region.

"Agronomically elite" means that a line has desirable mic characteristics such as yield, maturity, disease resistance, and the like, in addition to the insect resistance due to the t event(s). Agronomic traits, taken individually or in any combination, as set forth in Examples, below, in a plant comprising an event of the subject invention, are within the scope of the t invention. Any and all of these agronomic characteristics and data points can be used to identify such plants, either as a point or at either end or both ends of a range of chracteristics used to define such plants.

As one skilled in the art will recognize in light of this disclosure, preferred embodiments of detection kits, for example, can include probes and/or primers directed to and/or sing "junction sequences" or "transition sequences" (where the corn c flanking sequence meets the insert sequence). For e, this includes a polynucleotide probes, primers, and/or amplicons designed to identify one or both on sequences (where the insert meets the flanking sequence), as indicated in Table 1. One common design is to have one primer that hybridizes in the flanking region, and one primer that hybridizes in the insert. Such primers are often each about at least -15 residues in length. With this arrangement, the primers can be used to generate/amplify a detectable amplicon that indicates the ce of an event of the subject invention. These primers can be used to generate an amplicon that spans (and includes) a junction sequence as indicated above.

The primer(s) "touching down" in the flanking ce is typically not designed to hybridize beyond about 200 bases or beyond the junction. Thus, typical ng primers would be designed to comprise at least 15 residues of either strand within 200 bases into the flanking sequences from the beginning of the insert. That is, primers comprising sequence of an appropriate size in residues 1873 and/or 6890 of SEQ ID NO:29 are within the scope of the subject invention. Insert primers can likewise be ed anywhere on the , but residues 2074 and 6689, can be used, for example, non-exclusively for such primer design.

One skilled in the art will also recognize that primers and probes can be designed to hybridize, under a range of standard hybridization and/or PCR conditions, to a segment of SEQ ID NO:29 (or the complement), and complements thereof, wherein the primer or probe is not perfectly complementary to the exemplified sequence. That is, some degree of mismatch can be tolerated.

For an approximately 20 nucleotide primer, for example, typically one or two or so tides do not need to bind with the opposite strand if the mismatched base is internal or on the end of the primer that is opposite the amplicon. Various appropriate hybridization conditions are provided below. Synthetic nucleotide analogs, such as e, can also be used in probes. Peptide nucleic acid (PNA) , as well as DNA and RNA probes, can also be used. What is important is that such probes and primers are diagnostic for (able to uniquely identify and distinguish) the presence of an event of the subject invention.

It should be noted that errors in PC amplification can occur which might result in minor cing errors, for e. That is, unless otherwise indicated, the sequences listed herein were determined by generating long amplicons from corn genomic DNAs, and then cloning and sequencing the amplicons. It is not unusual to find slight differences and minor pancies in ces generated and determined in this manner, given the many rounds of amplification that are necessary to generate enough amplicon for sequencing from genomic DNAs. One skilled in the art should recognize and be put on notice than any adjustments needed due to these types of common sequencing errors or discrepancies are within the scope of the subject invention.

It should also be noted that it is not uncommon for some genomic sequence to be d, for example, when a sequence is inserted during the creation of an event. Thus, some differences can also appear n the subject flanking sequences and genomic sequences listed in K, for example. Some of these difference(s) are discussed below in the Examples section. Adjustments to probes and primers can be made accordingly.

Thus, use of a plant comprising a polynucleotide having some range of identity with the subject flanking and/or insert sequences is within the scope of the subject invention. Identity to the sequence of the present ion can be a polynucleotide sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity, and more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ce identity with a sequence exemplified or described herein. Hybridization and hybridization conditions as provided herein can also be used to define such plants and polynculeotide sequences of the subject invention. The sequence of the flanking sequences plus insert sequence can be confirmed with reference to the deposited seed.

The components of each of the "inserts" are illustrated in Figures 1 and 2 and are discussed in more detail below in the Examples. The DNA polynucleotide sequences ofthese components, or fragments f, can be used as DNA primers or probes in the methods of the present invention.

In some ments, itions and methods are provided for detecting the presence of the transgene/genomic insertion region, in plants and seeds and the like, from a corn plant. DNA sequences are provided that comprise the subject transgene/genomic insertion region junction sequence ed herein (between residues 1873-1874 and 6689-6690 of SEQ ID NO:29), segments thereof, and complements of the ified sequences and any segments thereof. The insertion region junction sequence spans the junction between heterologous DNA inserted into the genome and the DNA from the corn cell ng the insertion site. Such sequences can be diagnostic for the given event.

Based on these insert and border sequences, event-specific primers can be generated. PCR analysis demonstrated that corn lines of the subject invention can be identified in different corn genotypes by analysis of the PCR amplicons generated with these event-specific primer sets. These and other d procedures can be used to uniquely identify these corn lines. Thus, PCR amplicons derived from such primer pairs are unique and can be used to identify these corn lines.

In some embodiments, DNA ces that comprise a contiguous fragment of the novel transgene/genomic insertion region are an aspect of this invention. Included are DNA ces that comprise a sufficient length of polynucleotides of transgene insert sequence and a sufficient length of polynucleotides of corn genomic sequence from one or more of the three entioned corn plants and/or sequences that are useful as primer sequences for the production of an amplicon product diagnostic for one or more of these corn plants.

Related embodiments pertain to DNA sequences that comprise at least 2, 3, 4, 5, 6, 7, 8, 9, , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more contiguous nucleotides of a transgene portion of a DNA sequence identified herein (such as SEQ ID NO:29 and segments thereof), or complements thereof, and a similar length of ng corn DNA sequence from these ces, or complements thereof. Such sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for any of the corn events ed to herein. Therefore, the invention also includes the amplicons produced by such DNA primers and homologous primers.

This invention can also includes methods of detecting the presence of DNA, in a sample, that corresponds to the corn event referred to herein. Such methods can comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic acid amplification reaction with DNA from at least one of these corn events, produces an amplicon that is stic for said event(s); (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.

Further ion methods can include a method of detecting the presence of a DNA, in a sample, corresponding to at least one of said events, wherein said method comprises: (a) contacting the sample comprising DNA with a probe that hybridizes under stringent hybridization conditions with DNA from at least one of said corn events and which does not hybridize under the stringent hybridization conditions with a control corn plant (non-event-of-interest DNA); (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.

Some embodiments include methods of producing a corn plant comprising the aad-1 event of the subject invention, wherein said method comprises the steps of: (a) sexually crossing a first parental corn line (comprising an expression cassettes of the t invention, which confers said herbicideresistance trait to plants of said line) and a second al corn line (that lacks this herbicide tolerance trait) y ing a plurality of progeny plants; and (b) selecting a progeny plant by the use of molecular markers. Such s may optionally comprise the further step of back-crossing the progeny plant to the second parental corn line to producing a true-breeding corn plant that comprises said insect tolerance trait.

According to another aspect of the ion, methods of determining the zygosity of progeny of a cross with any one (or more) of said three events are provided. Said methods can comprise ting a sample, comprising corn DNA, with a primer set of the subject invention.

Said primers, when used in a nucleic-acid amplification reaction with genomic DNA from at least one of said corn , es a first amplicon that is diagnostic for at least one of said corn events. Such methods r comprise performing a nucleic acid amplification reaction, thereby producing the first amplicon; detecting the first amplicon; and contacting the sample comprising corn DNA with said primer set (said primer set, when used in a nucleic-acid amplification reaction with genomic DNA from corn plants, es a second amplicon comprising the native corn genomic DNA homologous to the corn genomic region; and performing a nucleic acid amplification reaction, thereby producing the second amplicon. The methods further se detecting the second amplicon, and comparing the first and second amplicons in a sample, wherein the presence of both amplicons indicates that the sample is heterozygous for the transgene insertion.

DNA detection kits can be developed using the compositions sed herein and methods well known in the art of DNA detection. The kits are useful for identification of the subject corn event DNA in a sample and can be applied to methods for breeding corn plants containing this DNA.

The kits contain DNA sequences homologous or complementary to the amplicons, for example, disclosed herein, or to DNA sequences homologous or complementary to DNA contained in the transgene genetic elements of the subject events. These DNA sequences can be used in DNA ication reactions or as probes in a DNA ization method. The kits may also contain the reagents and als ary for the performance of the detection method.

A "probe" is an isolated nucleic acid molecule to which is attached a conventional detectable label or reporter molecule (such as a radioactive isotope, , chemiluminescent agent, or enzyme). Such a probe is complementary to a strand of a target c acid, in the case of the present invention, to a strand of genomic DNA from one of said corn events, whether from a corn plant or from a sample that includes DNA from the event. Probes ing to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA ce. rs" are isolated/synthesized nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g. , a DNA polymerase. Primer pairs of the present invention refer to their use for amplification of a target nucleic acid ce, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 polynucleotides or more in length. Such probes and s hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete ce similarity with the target sequence, although probes differing from the target sequence and that retain the y to hybridize to target sequences may be designed by conventional methods.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. PCR-primer pairs can be d from a known sequence, for example, by using computer programs intended for that purpose.

Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g. , by re-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain stances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they t complete complementarity. As used herein, les are said to t "complete complementarity" when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions.

Similarly, the molecules are said to be "complementary" if they can hybridize to one another with sufficient stability to permit them to remain ed to one another under conventional tringency" conditions. Conventional stringency conditions are described by Sambrook etal, 1989.

Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be iently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a c acid sequence that will specifically hybridize to the complement of the nucleic acid ce to which it is being compared under high stringency ions. The term "stringent conditions" is functionally d with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular c-acid ce of interest) by the specific hybridization procedure discussed in Sambrook et al. , 1989, at 9.52-9.55. See also, ok et al., 1989 at 9.47-9.52 and .58. Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.

Depending on the ation envisioned, one can use g conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically employ relatively stringent conditions to form the hybrids, e.g. , one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C to about 70° C. Stringent ions, for example, could involve washing the hybridization filter at least twice with high-stringency wash buffer (0.2X SSC, 0.1% SDS, 65° C). Appropriate ency conditions which promote DNA hybridization, for example, 6 .OX sodium chloride/sodium citrate (SSC) at about 45° C, followed by a wash of 2 .OX SSC at 50° C are known to those skilled in the art, 6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2 .OX SSC at 50° C to a high stringency of about 0.2X SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C, to high stringency conditions at about 65° C . Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other le is changed. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand.

Detection of DNA sequences via hybridization is well-known to those of skill in the art, and the teachings of U.S. Patent Nos. 4,965,188 and 5,176,995 are exemplary of the methods of hybridization analyses.

In some embodiments, a nucleic acid for use with the present ion will specifically hybridize to one or more of the primers (or amplicons or other sequences) exemplified or suggested , ing complements and fragments thereof, under high stringency conditions. In one aspect, a marker nucleic acid molecule of the present invention has the nucleic acid sequence set forth in SEQ ID NOS:3-14, or complements and/or fragments f.

In r aspect, a marker nucleic acid le of the t invention shares between 80% and 100% or 90% and 100% sequence identity with such nucleic acid sequences. In a further aspect, a marker nucleic acid le for use with the present invention shares between 95% and 100% sequence identity with such sequence. Such sequences may be used as markers in plant breeding methods to identify the progeny of genetic crosses. The hybridization of the probe to the target DNA le can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody based tags, and chemiluminescent tags. ing the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon.

The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.

As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic-acid amplification of a target nucleic acid ce that is part of a nucleic acid template. For example, to determine whether the corn plant resulting from a sexual cross contains transgenic event genomic DNA from the corn plant of the present invention, DNA extracted from a corn plant tissue sample may be subjected to nucleic acid amplification method using a primer pair that includes a primer derived from flanking ce in the genome of the plant adjacent to the insertion site of inserted heterologous DNA, and a second primer d from the inserted heterologous DNA to e an on that is diagnostic for the presence of the event DNA. The amplicon is of a length and has a sequence that is also diagnostic for the event. The amplicon may range in length from the combined length of the primer pairs plus one tide base pair, and/or the combined length of the primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250, 1500, 1750, 2000, or more nucleotide base pairs (plus or minus any of the increments listed above).

Alternatively, a primer pair can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence. A member of a primer pair derived from the plant genomic ce may be located a distance from the inserted DNA sequence. This distance can range from one nucleotide base pair up to about twenty thousand tide base pairs. The use of the term "amplicon" ically excludes primer dimers that may be formed in the DNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent No. 4,683,195 and U.S. Patent No. 4,683,202. PCR ication s have been developed to amplify up to 22 kb of genomic DNA. These methods as well as other methods known in the art of DNA amplification may be used in the practice of the t invention. The sequence of the heterologous transgene DNA insert or ng genomic sequence from a subject corn event can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequences provided herein followed by rd DNA sequencing of the PCR amplicon or of the cloned DNA.

The amplicon ed by these methods may be detected by a plurality of techniques.

Agarose gel electrophoresis and staining with ethidium bromide is a common well known method of detecting DNA amplicons. Another such method is Genetic Bit Analysis where an DNA oligonucleotide is designed which overlaps both the nt flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate.

Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the lized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and ed ddNTPs ic for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base ion.

Another method is the Pyrosequencing technique as described by Winge (Innov. Pharma.

Tech. 00:18-24, 2000). In this method an oligonucleotide is ed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to -stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic ce) and ted in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5'phosphosulfate and luciferin. DNTPs are added individually and the incorporation results in a light signal that is measured. A light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.

Fluorescence Polarization is r method that can be used to detect an amplicon of the present invention. ing this method, an ucleotide is designed which overlaps the genomic flanking and inserted DNAjunction. The oligonucleotide is hybridized to single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescentlabeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single base extension.

TAQMAN (PE Applied tems, Foster City, Calif.) is a method of detecting and quantifying the presence of a DNA ce. Briefly, a FRET ucleotide probe is designed that overlaps the genomic flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. During specific amplification, Taq DNA polymerase cleans and releases the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.

Molecular Beacons have been described for use in sequence detection. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close ity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the ce of a thermostable rase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking c/transgene insert ce due to successful amplification and ization.

Having disclosed a location in the corn genome that is excellent for an insertion, the subject invention also comprises use of a corn seed and/or a corn plant comprising at least one dl insert in the general vicinity of this genomic location. One option is to substitute a different insert in place of the aad-l insert exemplified herein. In these generally regards, targeted homologous recombination, for example, can be used ing to the t invention. This type of technology is the subject of, for example, WO 03/080809 A2 and the corresponding published U.S. application (US 20030232410). Thus, the t invention includes use of plants and plant cells comprising a heterologous insert (in place of or with multi-copies oiaad-l), flanked by all or a recognizable part ofthe flanking sequences identified herein (e.g. residues 1-1873 and 6690-8557 of SEQ IDNO:29).

Following are some items / embodiments of plants, seeds, polynucleotides, and detection s that can be used according to some ments of the subject ion: 1. A transgenic corn plant comprising a , said genome comprising SEQ ID NO:29. 2. A corn seed comprising a genome comprising AAD- 1event DAS9 as present in seed deposited with American Type Culture Collection (ATCC) under ion No.

PTA- 10244. 3. The corn seed of item 2, said seed comprising a genome, said genome comprising SEQ ID NO:29. 4. A corn plant produced by growing the seed of item 2, said plant comprising SEQ ID NO:29.

. A progeny plant of the corn plant of item 4, said progeny plant comprising AAD- 1 event DAS9. 6. A herbicide-tolerant progeny plant of the corn plant of item , said progeny plant comprising SEQ ID NO:29. 7. A transgenic corn plant comprising a transgene insert in corn chromosomal target site located on some 2 at approximately 20 c between SSl markers UMC1265, amplifiable in pari by SEQ ID G:30 and SEQ D O:3 , and MC0 1, ampltfiahle in part by SEQ ID NO:32 and SEQ ID NO:33, wherein the target site comprises a heterologous nucleic acid. 8. A method of making the transgenic com plant of item Ί said method comprising inserting a heterologous nucleic acid at a position on chromosome 2 at approximately 20 cM n SSRmarkers MC 265, amplifiab!e in part by SEQ ID NO:30 and SEQ ID 0 :3 and MMCGi 1, amplifiable in part by SEQ ID NO:32 and SEQ ID O:33. 9. A part of the plant of item 4 wherein said part is selected from the group consisting of , ovule, flowers, bolls, lint, shoots, roots, and leaves, said part comprising SEQ ID NO:29.

. The transgenic corn plant of item 7, said plant comprising a transgene insert in, or flanked by, a genomic sequence selected from the group consisting of residues 1-1873 of SEQ ID NO:29 and residues 6690-8557 of SEQ ID NO:29. 11. An ed cleotide molecule wherein said molecule comprises at least 1 nucleotides and maintains hybridization under stringent wash conditions with a nucleic acid ce selected from the group ting of residues 1-1873 of SEQ ID NO:29, residues 6690-8557 of SEQ ID NO:29, and complements thereof; comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:l-33; and/or hybridizes under stringent wash conditions with a nucleotide sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO:29, residues 6679 to 6700 of SEQ ID NO:29, and complements thereof. 12. The isolated polynucleotide of item 1 1 wherein said cleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-33. 13. The isolated polynucleotide of item ein said polynucleotide hybridizes under stringent wash conditions with a nucleotide sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO:29, residues 6679 to 6700 of SEQ ID NO:29, and complements thereof. 14. The polynucleotide of item 13 wherein said polynucleotide is an amplicon generated by polymerase chain reaction. 1 . A method of detecting a corn event in a sample comprising corn DNA wherein said method comprises ting said sample with at least one polynucleotide that is diagnostic for AAD-1 corn event DAS9 as present in seed deposited with American Type Culture Collection (ATCC) under Accession No. PTA- 10244. 16. The method of item 1 wherein said method comprises contacting said sample with a . a first primer that binds to a flanking sequence selected from the group consisting of residues 1-1873 of SEQ ID NO:29, es 6690-8557 of SEQ ID NO:29, and complements f; and b . a second primer that binds to an insert sequence comprising residues 1874- 6689 of SEQ ID NO:29 or the complement f; subjecting said sample to polymerase chain reaction; and assaying for an amplicon ted between said primers. 17. The method of item 16 wherein said primers are selected from the group consisting of SEQ ID NOs:l-28. 18. The method of item 15 wherein said polynucleotide comprising at least 30 nucleotides and hybridizes under stringent conditions with a sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO:29, residues 6679 to 6700 of SEQ ID NO:29, and complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent ization conditions; and ng said sample for hybridization of said polynucleotide to said DNA. 19. A DNA detection kit comprising a first primer and a second primer according to item . A DNA detection kit for performing the method of item 18. 2 1. A DNA detection kit comprising a cleotide as defined in item 13. 22. The polynucleotide of item 12, said polynucleotide comprising SEQ ID NO:29. 23. A method of ing the polynucleotide of item 22. 24. A method of producing the transgenic plant of item 10, said method comprising inserting a transgene into a DNA segment of a corn genome, said DNA segment comprising a 5' end comprising nucleotide residues 1-1873 of SEQ ID NO:29 and a 3' end comprising nucleotide residues 6690-8557 of SEQ ID NO:29.

. A method comprising ng a first corn plant comprising SEQ ID NO:29 with a second corn plant to produce a third corn plant comprising a , and assaying said third corn plant for presence of SEQ ID NO:29 in said genome. 26. The method of item 25 wherein said method is used for breeding a corn plant and/or for introgressing a herbicide tolerance trait into a corn plant.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

The following examples are included to illustrate procedures for cing the invention and to demonstrate certain red embodiments of the invention. These examples should not be construed as limiting. It should be iated by those of skill in the art that the techniques disclosed in the following examples represent specific approaches used to illustrate preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in these specific embodiments while still obtaining like or similar results without departing from the spirit and scope of the invention. Unless otherwise indicated, all percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

The following Examples include pre-plant and/or pre-emergence uses. Such uses are not limited to the "278" event. One could expound r on the utility of the tolerance provided by the subject AAD-1 genes with regard to shortened plant-back interval. This gives growers a great deal more ility in scheduling their planting relative to bumdown. Without using the subject invention, waiting 7-30 days or so after bumdown before planting could cause significant yield loss.

Thus, the subject invention provides advantages in this regard. See, for example, Example 13. Any planting / herbicidal application intervals, and any concentration ranges / use rates of herbicide(s) ified or suggested herein can be used in accordance with the subject invention.

The following iations are used unless otherwise indicated.

AAD-1 aryloxyalkanoate enase-1 bp base pair °C degrees Celcius DNA deoxyribonucleic acid DIG digoxigenin EDTA ethylenediaminetetraacetic acid kb se microgram iter mL milliliter M molar mass OLP overlapping probe PCR polymerase chain reaction PTU plant transcription unit SDS sodium dodecyl sulfate SOP standard ing procedure ssc a buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 a buffer solution containing a mixture of Tris base, boric acid and EDTA, pH 8.3 volts Example 1. Transformation and Selection of the AADl Event pDAS 1740-278 The AADl event, pDAS 1740-278, was produced by WHISKER - mediated transformation of maize line Hi-II. The transformation method used is described in US Patent Application # 20090093366. An Fspl fragment of d pDAS1740, also referred to as pDAB3812, (Figure 1) was transformed into the maize line. This plasmid construct contains the plant expression cassette containing the RB7 MARv3 :: Zea mays Ubiquitin 1 promoter v2 // AADl v3 // Zea mays PER5 3'UTR : : RB 7 MARv4 plant transcription unit (PTU).

Numerous events were produced. Those events that survived and produced healthy, haloxyfop-resistant callus tissue were assigned unique identification codes representing putative ormation events, and continually transferred to fresh selection . Plants were regenerated from tissue derived from each unique event and transferred to the greenhouse.

Leaf samples were taken for molecular analysis to verify the presence of the AAD-1 transgene by Southern Blot, DNA border confirmation, and genomic marker assisted confirmation. Positive T O plants were pollinated with inbred lines to obtain Tl seed. Tl plants of Event pDAS 14709 (DAS9) was selected, ollinated and characterized for five generations. Meanwhile, the Tl plants were backcrossed and introgressed into elite germplasm (XHH13) through marker-assisted selection for several generations. This event was generated from an independent transformed e. The event was selected based on its unique teristics such as single insertion site, normal Mendelian segregation and stable expression, and a superior combination of efficacy, including herbicide tolerance and agronomic performance in broad genotype backgrounds and across multiple nmental locations. The following examples n the data which were used to terize event pDAS278-9.

Example 2. pDAS 17409 Event Characterization via Southern Blot Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and determine insert/copy number of the aad-1 gene in event pDAS- 17409 (DAS- 9). Data were generated to demonstrate the integration and integrity of the aad-l transgene ed into the corn genome. rn blot data suggested that the pDAS 17 /Fsp I fragment insert in corn event DAS9 occurred as a simple integration of a single, intact copy of the aad-1 PTU from plasmid pDAS1740. ed Southern blot analysis was conducted using probes specific to gene, er, terminator, and other regulation elements contained in the plasmid region and descriptive restriction enzymes that have cleavage sites located within the plasmid and e hybridizing fragments internal to the plasmid or fragments that span the junction of the plasmid with corn genomic DNA (border fragments). The molecular weights indicated from the Southern ization for the combination of the restriction enzyme and the probe were unique for the event, and ished its identification patterns. These analyses also showed that the plasmid fragment had been inserted into corn genomic DNA without rearrangements of the aad- 1 PTU. Identical hybridization nts were observed in five distinct generations of transgenic corn event DAS9 indicating stability of tance of the aad-1 PTU insertion across generations. Hybridization with a mixture of three backbone probes located e of the restriction site of Fsp I on plasmid pDAS1740 did not detect any ic DNA/gene fragments, indicating the absence of the Ampicillin resistance gene and the absence of the other vector backbone regions immediately adjacent to the Fsp I restriction sites of the plasmid pDAS1740 in transgenic corn event DAS9. The illustrated map of the insert in aad-1 corn event DAS9 is presented in Figures 2-3.

Example 2.1 Corn Leaf Sample Collection and Genomic DNA (gDNA) Isolation gDNA prepared from leaf of the individual plants of the aad-1 corn event DAS9. gDNA was extracted from leaf tissue harvested from individual plants carrying aad-1 corn event 278-9. Transgenic corn seeds from five distinct generations of event DAS9 were used. Twenty individual corn plants, derived from four plants per generation, for event DAS- 9 were selected. In addition, gDNA was isolated from a conventional corn plant, XHH13, which contains the genetic background that is entative of the substance line, absent the aad-l gene.

Prior to ing the gDNA, leaf punches were taken from each plant to test aad-l protein expression using a rapid test strip kit (American Bionostica, Swedesboro, NJ) ing to the manufacturer's recommended procedure. Each leaf punch sample was given a score of + or - for the presence or absence of aad-l, respectively. Only positive plants from the five generations of event DAS9 were subjected to further characterization.

Corn leaf samples were collected from the dual plants of the event DAS9 and the tional control XHH13. Leaf samples were quickly frozen in liquid nitrogen and stored at imately -80°C until usage.

Individual genomic DNA was ted from frozen corn leaf tissue following the standard CTAB method. When necessary, some of the genomic DNA was further purified with Qiagen Genomic-Tip n, Valencia, CA) following procedures recommended by the manufacturer. Following extraction, the DNA was quantified spectrofluorometrically using Pico Green reagent (Invitrogen, Carlsbad, CA). The DNA was then visualized on an agarose gel to confirm values from the Pico Green is and to determine the DNA quality.

Example 2.2 DNA Digestion and Separation For molecular characterization of the DNA, nine micrograms (9 mg) of genomic DNA from the corn event DAS9 DNA sample and the conventional control were digested by adding approximately five to eleven units of ed ction enzyme per mg of DNA and the corresponding reaction buffer to each DNA sample. Each sample was incubated at approximately 37 °C overnight. The restriction enzymes EcoR I, Nco I , Sac I, Fse I, and Hind III were used for the digests (New England Biolabs, Ipswich, MA). A positive hybridization control sample was prepared by ing plasmid DNA, pDAS1740 (pDAB3812), with genomic DNA from the conventional control at a ratio of approximately equivalent to 1 copy of transgene per corn genome, and digested using the same procedures and restriction enzyme as the test samples. DNA from the conventional corn control (XHH13) was digested using the same procedures and restriction enzymes as the test samples to serve as a negative control.

The digested DNA samples were precipitated with Quick-Precip (Edge tems, Gaithersburg, MD) and resuspended in l x Blue Juice (Invitrogen, Carlsbad, CA) to achieve the desired volume for gel loading. The DNA s and molecular size markers were then electrophoresed through 0.8% agarose gels with lxTBE buffer r Scientific, Pittsburgh, PA) at 55-65 volts for approximately 18-22 hours to achieve fragment separation. The gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA) and the DNA was visualized under ultraviolet (UV) light.

Example 2.3 Southern Transfer and Membrane Treatment Southern blot analysis was performed essentially as described by Memelink, et al. (1994) Southern, Northern, and Western Blot is. Plant Mol. Biol. Manual F l :1-23. y, following electrophoretic separation and visualization of the DNA fragments, the gels were depurinated with 0.25N HC1 (Fisher Scientific, Pittsburgh, PA) for imately 15 s, and then d to a denaturing solution (AccuGENE, Sigma, St. Louis, MO) for approximately 30 minutes followed by neutralizing solution (AccuGENE, Sigma, St. Louis, MO) for at least 30 minutes. Southern er was performed overnight onto nylon membranes (Roche Diagnostics, Indianapolis, IN) using a wicking system with lOxSSC (Sigma, St. Louis, MO). After transfer the membranes were washed in a 2x SSC solution and the DNA was bound to the membrane by UV crosslinking. This process resulted in Southern blot membranes ready for hybridization.

Example 2.4 DNA Probe Labeling and Hybridization The DNA fragments bound to the nylon ne were detected using a labeled probe.

Probes used for the study were generated by a PCR-based incorporation of a digoxigenin (DIG) labeled nucleotide, [DIG-1 l]-dUTP, from fragments generated by primers specific to gene elements and other regions from plasmid pDAS1740. tion of DNA probes by PCR synthesis was carried out using a PCR DIG Probe Synthesis Kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's recommended procedures. A list of probes used for the study is described in Table 1.

Table 1 . Location and Length of Probes used in Southern Analysis.

Labeled probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. A desired amount of labeled probe was then used for ization to the target DNA on the nylon membranes for detection of the ic fragments using the procedures described for DIG Easy Hyb Solution (Roche Diagnostics, Indianapolis, IN). Briefly, nylon ne blots with DNA fixed on were briefly washed in 2xSSC and prehybridized with 20-25 mL of prewarmed DIG Easy Hyb solution in hybridization bottles at approximately 50°C for a minimal of 30 minutes in a ization oven. The prehybridization solution were then decanted and replaced with 20 mL of prewarmed DIG Easy Hyb solution containing a desired amount of specific probes predenatured by boiling in water for 5 s. The hybridization step was then conducted at approximately 40-60°C overnight in the ization oven.

Example 2.5 Detection At the end of the probe hybridization, DIG Easy Hyb solutions containing the probes were decanted into clean tubes and stored at -20°C. These probes could be reused for 2-3 times according to the manufacturer's recommended procedure. The membrane blots were rinsed briefly and washed twice in clean plastic containers with low stringency wash buffer (2xSSC, 0.1%SDS) for imately 5 minutes at room temperature, followed by washing twice with high stringency wash buffer (O.lxSSC, 0.1% SDS) for 15 minutes each at approximately 65°C.

The membrane blots were then transferred to other clean plastic containers and briefly washed with 1xwashing buffer from the DIG Wash and Block Buffer Set (Roche Diagnostics, Indianapolis, IN) for approximately 2 minutes, proceeded to blocking in 1x blocking buffer for a minimum of 30 minutes, followed by incubation with anti-DIG-AP (alkaline phosphatase) antibody (1:5,000 dilution, Roche Diagnostics, apolis, IN) in l x blocking buffer for a minimum of 30 minutes. After 2-3 washes with l x washing buffer, specific DNA probes remain bound to the membrane blots and DIG-labeled DNA standards were visualized using CDP-Star uminescent Nucleic Acid Detection System (Roche Diagnostics, Indianapolis, IN) following the manufacturer's endation. Blots were exposed to chemiluminescent film (Roche Diagnostics, Indianapolis, IN) for one or more time points to detect hybridizing fragments and to visualize lar size rds. Films were then developed with an All-Pro 100 Plus film developer (Konica SRX-101) and images were scanned for report. The number and sizes of detected bands were documented for each probe. DIG-labeled DNA Molecular Weight Marker II (MWM DIG II), visible after DIG detection as described, was used to determine hybridizing fragment size on the rn blots.

Example 2.6 Probe Stripping DNA probes were stripped off the membrane blots after the Southern hybridization data were obtained, and the membrane blots could be reused for ization with a different DNA probe ing to the manufacturer's recommended ures (DIG Application Manual for Filter Hybridization, (2003). Roche Diagnostics). Briefly, after signal detection and film exposure, membrane blots were thoroughly rinsed with Milli-Q water and followed by washing twice in stripping buffer (0.2N NaOH, 0.1% SDS) for approximately 15 minutes at room temperature or at 37°C. The ne blots were then briefly washed in 2xSSC and were ready for prehybridization and hybridization with r DNA probe. The membrane blots were exposed to a new chemiluminescent film to ensure all the DNA probes were stripped of before ding to the next hybridization. The re-exposed films were kept along with the previous hybridization data package in the study file for record.

Example 2.7 Southern Blot Results Expected and observed fragment sizes with a ular digest and probe, based on the known restriction enzyme sites of the pDAS 1 0/Fsp I fragment, are given in Table 2 . Two types of fragments were identified from these digests and hybridizations: internal fragments, where known enzyme sites flank the probe region and are completely contained within the pDAS 1740/Fsp I fragment and border fragments where a known enzyme site is located at one end of the probe region and a second site is expected in the com genome. Border fragment sizes vary by event because, in most cases, DNA fragment integration sites are unique for each event.

The border fragments provide a means to locate a restriction enzyme site relative to the integrated DNA and to evaluate the number of DNA insertions. Based on the Southern blot analyses completed in this study, it was concluded that a single copy of an intact aad-\ PTU from plasmid pDAS 1740/Fsp I ed into the corn genome of event DAS9 as detailed in the insert map (Figures 2-3).

Table 2 . Predicted and ed izing Fragments in rn Blot Analysis.

WO 15968 —DAS9 >4389(b0rder) ~3800*,~16000 pDASI740 3361j ~6400* Eve 1 /Hmd 111 XIIH 13 ~6400* Note: * An asterisk after the observed fragment size indicates endogenous sequence hybridization that was ed across all samples (including negative controls) # Doublets in the conventional control, BC3S1, and some BC3S2 s 1. Expected fragment sizes are based on the plasmid map of the pDAS1740 (pDAB3812) as shown in Figure 1. 2. Observed nt sizes are considered approximately from these analyses and are based on the indicated sizes of the DIG-labeled DNA Molecular Weight Marker II fragments. Due to the incorporation of DIG molecules for ization, the marker fragments typically run approximately 5-10% larger than their actual ted molecular weight.

Restriction enzymes with unique restriction site in plasmid 40, EcoR I, Nco I, Sac I, Fse l/Hind III, were selected to characterize aad-l gene insert in event DAS9.

Border fragment of > 3382 bp, >2764 bp, >4389 bp was predicted to hybridize with the aad-l gene probe following EcoR I, Nco I, and Sac I digest respectively (Table 2). Single aad-l hybridization band of -12000 bp, -4000 bp, and -16000 bp were observed when EcoR I, Nco I, and Sac I were used respectively, indicating a single site of aad-l gene insertion in the corn genome of event DAS9. Double digestion with Fse I and Hind III was selected to release a fragment of 3361 bp which contains the aad-l plant transcription unit (PTU, er/gene/terminator) (Table 2). The predicted 3361 bp nt was observed with the aad-l gene probe following Fse I/Hind III digestion. Results obtained with all four enzymes/enzyme combination digestion of the 278-9 sample followed by aad-l gene probe hybridization indicated that a single copy of an intact aad-l PTU from plasmid pDAS1740 was inserted into the corn genome of event DAS9. ction enzymes Nco I, Sac I and Fse l/Hind III were selected to characterize the promoter (ZmUbil) region for aad-l in event DAS9. Nco I and Sac I digests are expected to generate a border region fragment of >3472 bp and >4389 bp, respectively, when hybridized to DNA probes ically to the ZmUbil promoter region (Table 2). Two hybridization bands of -6300 bp and -3600 bp were detected with ZmUbil promoter probe ing Nco I digestion. The -3600 bp band, however, was present across all sample lanes including the conventional controls, suggesting that the -3600 bp band is a non-specific signal band resulting from the homologous binding of the corn-derived ubiquitin promoter (ZmUbil) probe to the corn endogenous ubi gene. On the contrary, the -6300 bp signal band was detected in the tested DAS9 s but not in the conventional controls, indicating that the -6300 bp band is specific to the ZmUbil promoter probe from plasmid pDAS1740 and therefore it is the expected Nco I/ZmUbil band indicated in Table 2 . Similarly, two hybridization bands of -3800 bp and -16000 bp were detected with ZmUbil promoter probe following Sac I digestion. The -3800 bp band appeared in all sample lanes including conventional controls and thus is considered as non-specific hybridization of ZmUbil promoter probe to the corn endogenous ubi gene. The -16000 bp hybridization band that is only present in 278-9 samples is considered the expected Sac 1/ ZmUbil band. Double ion with Fse 1/ Hind III is expected to release the aad-l PTU fragment of 3361 bp that hybridizes to the ZmUbil promoter probe (Table 2). This 3361 bp band and a non-specific ization band of -6400 bp were detected by ZmUbil promoter probe following Fse II Hind III digestion. The -6400 bp band is considered non-specific binding of the ZmUbil promoter probe to the corn endogenous ubi gene because this band is present in all sample lanes including the conventional controls. onally, another band very close to -6400 bp was ed in the conventional control, BC3S1, and some of the BC3S2 samples. This onal band very close to -6400 bp is also considered non-specific because it is present in the conventional control XHH13 sample lanes and is most likely associated with the genetic background of XHH13.

The same restriction enzymes/enzyme combination, Nco I, Sac I and Fse 1/Hind III were selected to characterize the ator (ZmPer5) region for aad-l in event DAS9. Nco I digest is expected to generate a border region fragment of >2764 bp when hybridized to DNA probes specifically to the ZmPer5 terminator region (Table 2). Two hybridization bands of -4000 bp and -3900 bp were detected with ZmPer5 terminator probe following Nco I digestion.

The -3900 bp band was present across all sample lanes including the conventional controls, suggesting that the -3900 bp band is a non-specific signal band probably due to the homologous g of the corn-derived peroxidase gene terminator (ZmPer5) probe to the corn endogenous per gene. On the contrary, the -4000 bp signal band was detected in the tested DAS9 samples but not in the conventional controls, indicating that the -4000 bp band is specific to the ZmPer5 terminator probe from plasmid pDAS 1740 and therefore it is the expected Nco I/ZmPer5 band indicated in Table 2. A >1847 bp border fragment is expected to hybridized to the ZmPer5 terminator probe ing Sac I digestion. Two hybridization bands of -1900 bp and -9000 bp were detected with ZmPer5 terminator probe following Sac I ion. The -9000 bp band appeared in all sample lanes including conventional controls and thus considered as non-specific hybridization of ZmPer5 terminator probe to the corn endogenous per gene. The -1900 bp hybridization band that was only present in DAS9 samples is considered the expected Sac 1/ ZmPer5 band. Double digestion with Fse 1/ Hind III is expected to release the aad-\ PTU fragment of 3361 bp that hybridizes to the ZmPer5 terminator probe (Table 2). This 3361 bp band and an additional non-specific hybridization band of -2100 bp were detected by ZmPer5 terminator probe following Fse 1/ Hind III digestion. The additional ~ 100 bp band is the non-specific binding of the ZmPer5 terminator probe to the corn endogenous gene since this band is present in all sample lanes including the negative controls. Results obtained with these digestions of the DAS9 sample followed by ZmUbil promoter and ZmPer5 terminator probe ization further confirmed that a single copy of an intact aad- 1 PTU from plasmid pDAS1740 was inserted into the corn genome of event DAS9.

Restriction enzymes, Nco I and Sac I, were selected to characterize the rest of the components from pDAS1740/i¾? I fragment in AAD-1 corn event 278-9 (Table 2).

DNA sequences of components RB7 Mar v3 and RB7 Mar v4 have over 99.7% identity, ore DNA probes ic for RB7 Mar v3 or RB7 Mar v4 were expected to hybridize to DNA fragments containing either version of the RB7 Mar. Two border fragments of >2764 bp and >3472 bp were ed to hybridize with RB7 Mar v4 and RB7 Mar v3 probes following Nco I digestion (Table 2). Two hybridization bands of -4000 bp and -6300 bp were observed with either RB7 Mar v4 or RB7 Mar v3 probe after Nco I digestion in DAS9 samples.

Similarly, two border fragments of >1847 bp and >4389 bp were predicted with RB7 Mar v4 and RB7 Mar v3 probes following Sac I digestion (Table 2). Hybridization bands of -1900 bp and -16000 bp were detected in DAS9 samples with RB7 Mar v4 or RB7 Mar v3 probe after Sac I digestion.

Taken together, the Southern hybridization s obtained with these element probes indicated that the DNA inserted in corn event DAS9 ns an intact aad-l PTU along with the matrix attachment regions RB7 Mar v3 and RB7 Mar v4 at the 5' and 3' ends of the insert, respectively. e 2.8 Absence of Backbone Sequences Equal molar ratio combination of three DNA fragments (Table 1) covering nearly the entire Fsp I backbone region (4867-7143 bp in d pDAS1740) of plasmid pDAS1740 were used as the backbone probe to characterize AAD-1 corn event DAS9. Plasmid pDAS sp I fragment was used to generate event DAS9, therefore, no specific hybridization signal was expected with the backbone probe combination (Table 2) following any restriction enzyme digestion. It was confirmed that no ic hybridization signal was detected with backbone probe following Nco I or Sac I digestion in all DAS9 samples. Positive control lanes ned the ed hybridizing bands demonstrating that the probes were capable of izing to any homologous DNA fragments if t in the samples. The data suggested that the insertion in corn event DAS9 did not include any vector backbone sequence outside of the Fsp I region from plasmid pDAS1740.

Leaf samples from five distinct generations of the event DAS9 were used to conduct the Southern blot analysis for molecular characterization. The integration pattern was investigated using ed restriction enzyme digest and probe combinations to characterize the ed gene, aad-1, as well as the non-coding regions including promoter, terminator of gene expression, and the matrix attachment regions.

Southern blot characterization of the DNA inserted into event DAS9 indicate that a single intact copy of the aad-1 PTU has been integrated into event DAS9. The molecular weights indicated by the Southern hybridization for the combination of the restriction enzyme and the probe were unique for the event, and established its identification patterns. The hybridization n is identical across all five generations, indicating that the insert is stable in the corn genome. Hybridization with probes covering the backbone region beyond the pDAS 1740/Fsp I transformation nt from plasmid pDAS 1740 confirms that no vector backbone sequences have been incorporated into the event DAS9.

Example 3. Cloning and terization of DNA Sequence in the Insert and the Flanking Border Regions of Corn Event DAS9 To characterize the inserted DNA and describe the genomic insertion site, DNA sequences of the insert and the border regions of event DAS9 were determined. In total, 8557 bp of event DAS9 genomic sequence were confirmed, comprising 1873 bp of 5' flanking border sequence, 1868 bp of 3' flanking border sequence, and 4816 bp of DNA insert.

The 4816 bp DNA insert ns an intact aad-l expression cassette, a 259 bp partial MAR v3 on the 5' terminus, and a 1096 bp l MAR v4 on the 3' terminus. Sequence analysis revealed a 2 1 bp insertion at '-integration junction and a two base pair deletion from the insertion locus of the com genome. A one base pair insertion was found at 3'-integration on between the com genome and the DAS9 insert. Also, a single base change (T to C) was found in the insert at position 5212 in the non-coding region of the 3' UTR. None of these s affect the open reading frame composition of the aad-l expression cassette.

PCR amplification based on the event DAS9 insert and border sequences confirmed that the border regions were of com origin and that the junction regions could be used for event-specific identification of 278-9. Analysis of the sequence spanning the junction regions indicated that no novel open reading frames (ORF>= 200 codons) resulted from the DNA insertion in event 278-9 and also no genomic open reading frames were upted by the DAS9 integration in the native com genome. Overall, characterization of the insert and border sequences of the AAD-1 com event DAS9 indicated that a single intact copy of the aad-l expression te was integrated into the native com genome.

Example 3.1. Genomic DNA Extraction and Quantification c DNA was extracted from lyophilized or freshly ground leaf tissues using a modified CTAB method. DNA samples were dissolved in l x TE (lOmM Tris pH8.0, lmM EDTA) (Fluka, Sigma, St. Louis, MO) and quantified with the Pico Green method according to manufacturer's instructions (Molecular , Eugene, OR). For PCR analysis, DNA samples were d with molecular biology grade water (5 PRIME, Gaithersburg, MD) to result in a concentration of 10-100 g m .

Example 3.2. PCR Primers Table 3 lists the primer sequences that were used to clone the DNA insert and the flanking border regions of event DAS9, with positions and descriptions marked in Figure 4 . Table 4 lists the primer sequences that were used to confirm the insert and border sequences. The primer positions were marked in Figures 4 and 5, respectively. All s were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Primers were dissolved in water (5 PRIME, Gaithersburg, MD) to a tration of 100 mM for the stock solution and diluted with water to a concentration of 10 mM for the working solution.

List of primer sequences used in the g of the insert in Corn Event DAS- 40278-9 and flanking border sequence Primer Name Size (bp) Location (bp) ce Purpose Seq ID o: 1: 5' - Primary PCR for 5End3812_A 26 223 1-2256 (-) TGCACTGCAGGTCGACTCTAGAGGAT - 3' 5' border sequence Secondary PCR Seq ID o: 2 : 5' - 5End3812_B 23 2 110-2132 (-) for 5' border GCGGTGGCCACTATTTTCAGAAG - 3' sequence Seq ID No: 3: 5' - Primary PCR for 3End3812_C 26 5535-5560 (+) TTGTTACGGCATATATCCAATAGCGG - 3' 3' border sequence Secondary PCR Seq ID o: 4 : 5' - 3End3812_D 26 5587-5612 (+) for 3' border CCGTGGCCTATTTTCAGAAGAAGTTC - 3' sequence Amplification of Seq ID No: 5: 5' - the insert, Amp IF 23 736-758 (+) ACAACCATATTGGCTTTGGCTGA - 3' Amplicon 1, used with Amp 1R ication of Seq ID o: 6 : 5' - the insert, Amp 1R 28 2475-2502 (-) CCTGTTGTCAAAATACTCAATTGTCCTT - 3' Amplicon 1, used with Amp IF Amplification of Seq ID o: 7 : 5' - the insert, Amp 2F 23 1696-1718 (+) CTCCATTCAGGAGACCTCGCTTG - 3' Amplicon 2, used with Amp 2R Amplification of Seq ID o: 8: 5' - the insert, Amp 2R 23 398 (-) GTACAGGTCGCATCCGTGTACGA - 3' Amplicon 2, used with Amp 2F ication of Seq ID o: 9 : 5' - the insert, Amp 3F 25 3254-3278 (+) CCCCCCCTCTCTACCTTCTCTAGAT - 3' on 3, used with Amp 3R Amplification of Seq ID No: 10: 5' - the insert, Amp 3R 23 493 1-4953 (-) GTCATGCCCTCAATTCTCTGACA - 3' Amplicon 3, used with Amp 3F Amplification of Seq ID No: 11: 5' - the insert, Amp 4F 23 4806-4828 (+) GTCGCTTCAGCAACACCTCAGTC - 3' Amplicon 4, used with Amp 4R Amplification of Seq ID No: 12: 5' - the insert, Amp 4R 23 6767-6789 (-) AGCTCAGATCAAAGACACACCCC - 3' Amplicon 4, used with Amp 4F Amplification of Seq ID No: 13: 5' - the insert, Amp 5F 28 6300-6327 (+) TCGTTTGACTAATTTTTCGTTGATGTAC - 3' Amplicon 5, used with Amp 5R Amplification of Seq ID No: 14: 5' - the insert, Amp 5R 23 7761-7783 (-) TCTCACTTTCGTGTCATCGGTCG - 3' Amplicon 5, used with Amp 5F (+): Direct ce; (-): Complementary sequence; Table 4 . List of primer ces used in the confirmation of corn genomic DNA Primer Size Name (bp) Location (bp) ce Purpose Seq ID o: 15: 5' - confirmation of 5'border genomic DNA, lF5End01 17 1816-1832 (+) CCAGCACGAACCATTGA - 3' used with ABEndOl Seq ID No: 16: 5' - CGTGTATATAAGGTCCAGAGGGTA- confirmation of 5'border genomic DNA, lF5End02 24 1629-1652 (+) 3' used with AI5End02 Seq ID No: 17: 5' - mation of 5'border genomic DNA, 01 17 428 1-4297 (-) TTGGGAGAGAGGGCTGA- 3' used with lF5End01 Seq ID No: 18: 5' - confirmation of 5'border genomic DNA, AI5End02 20 4406-4426 (-) TGGTAAGTGTGGAAGGCATC- 3' used with 02 Seq ID No: 19: 5' - confirmation of genomic DNA, used with lF3End03 20 8296-83 15 (-) GAGGTACAACCGGAGCGTTT- 3' lF5End03 lF3End04 Seq ID No: 20: 5' - confirmation of genomic DNA, used with 19 8419-8437 (-) CCGACGCTTTTCTGGAGTA- 3' lF5End04 Seq ID No: 2 1: 5' - confirmation of genomic DNA, used with 03 22 378-399 (+) TGTGCCACATAATCACGTAACA- 3' lF3End03 Seq ID No: 22: 5' - confirmation of genomic DNA, used with lF5End04 20 267-286 (+) GAGACGTATGCGAAAATTCG- 3' lF3End04 Seq ID No: 23: 5' - confirmation of 3'border c DNA, ABEndOl 22 4973-4994 (+) TTGCTTCAGTTCCTCTATGAGC- 3' used with lF3End05 Seq ID No: 24: 5' - confirmation of 3'border genomic DNA, lF3End05 19 7060-7078 (-) TCCACTCCTTTGT- 3' used with ABEndOl Seq ID No: 25: 5' - 278 specific sequence ication at 5' lF5EndTlF 22 2033-2054 (-) GCAAAGGAAAACTGCCATTCTT- 3' junction Seq ID No: 26: 5' - 278 specific sequence amplification at 5' lF5EndTlR 20 1765-1784 (+) TCTCTAAGCGGCCCAAACTT- 3' junction Seq ID No: 27: 5' - 278 specific sequence amplification at 5' Corn278-F 23 1884-1906 (-) ATTCTGGCTTTGCTGTAAATCGT- 3' junction Seq ID o: 28: 5' - TTACAATCAACAGCACCGTACCTT- 278 specific sequence amplification at 5' Corn278-R 24 1834-1 857 (+) 3' junction (+): Direct sequence; (-): Complementary sequence; e 3.3. Genome Walking The GenomeWalker™ Universal Kit (Clontech Laboratories, Inc., Mountain View, CA) was used to clone the 5' and 3' flanking border ces of corn event DAS9. ing to the manufacturer's instruction, about 2.5 g of genomic DNA from AAD-1 corn event DAS9 was digested overnight with EcoR V, Stu I (both provided by the kit) or Sea I (New d Biolabs, Ipswich, MA). Digested DNA was purified using the DNA Clean & trator™-25 (ZYMO Research,Orange, CA) followed by ligation to GenomeWalker™ adaptors to construct GenomeWalker™ libraries. Each GenomeWalker™ library was used as DNA template for primary PCR ication with the adaptor primer AP1, provided in the kit, and each construct-specific primer 5End38 12_A and 3End38 12 C. One microliter of 1:25 dilution of primary PCR reaction was then used as template for secondary PCR amplification with the nested adaptor primer AP2 and each nested construct-specific primer 5End3812_B and 3End3812_D. TaKaRa LA Taq™ HS (Takara Bio Inc., Shiga, Japan) was used in the PCR amplification. In a 50 m PCR reaction, 1 of DNA template, 8 of 2.5 mM of dNTP mix, 0.2 mM of each primer, 2.5 units of TaKaRa LA Taq™ HS DNA Polymerase, 5 mΐ of 10 LA PCR Buffer II (Mg2+ plus), and 1.5 m of 25 mM MgCl2 were used. Specific PCR conditions are listed in Table 5.

Table . Conditions for Genome Walking of the AAD- 1 Corn Event DAS9 to Amplify the Flanking Border s Example 3.4. Conventional PCR rd PCR was used to clone and confirm the DNA insert and border sequence in the corn event DAS9. TaKaRa LA Taq™ (Takara Bio Inc., Shiga, Japan), HotStarTaq DNA Polymerase (Qiagen, Valencia, CA), Expand High Fidelity PCR System (Roche Diagnostics, Inc., Indianapolis, IN), or the Easy-A® High-Fidelity PCR Cloning Enzyme & Master Mix (Stratagene, LaJolla, CA) was used for conventional PCR amplification according to the manufacturer's recommended procedures. ic PCR conditions and amplicon descriptions are listed in Table 6 .

Table 6. ions for Standard PCR Amplification of the Border Regions in the Corn Event DAS9 / 95/3 cycles ABEndOl Example 3.5 PCR Product Detection. Purification. Sub-cloning of PCR Products, and Sequencing PCR products were inspected by electrophoresis using 1.2 % or 2 % E-gel (Invitrogen, Carlsbad, CA) according to the product instruction. Fragment size was estimated by comparison with the DNA markers. If necessary, PCR fragments were purified by excising the fragments from 1% agarose gel in l x TBE stained with ethidium bromide, using the QIAquick Gel Extraction Kit n, Carlsbad, CA).

PCR fragments were sub-cloned into the pCR®4-TOPO® using TOPO TA Cloning® Kit for Sequencing rogen, Carlsbad, CA) according to the product instruction. Specifically, two to five microliters of the TOPO® cloning reaction was transformed into the One Shot chemically competent TOP 10 cells following the manufacturer's instruction. Cloned fragments were verified by eparation of the plasmid DNA (QIAprep Spin Miniprep Kit, Qiagen, Carlsbad, CA) followed by restriction digestion with EcoR I or by direct colony PCR using T3 and T7 primers, provided in the kit. Plasmid DNA or glycerol stocks of the selected colonies were then sent for sequencing.

After sub-cloning, the ve target PCR products were sequenced initially to confirm that the expected DNA fragments had been . The es containing appropriate DNA sequences were selected for primer walking to determine the complete DNA sequences.

Sequencing was performed by Cogenics (Houston, TX).

Final assembly of insert and border sequences was completed using Sequencher software on 4.8 Gene Codes Corporation, Ann Arbor, MI). Annotation of the insert and border ces of corn event DAS9 was performed using the Vector NTI (Version 10 and 11, Invitrogen, Carlsbad, CA).

Homology searching was done using the BLAST program against the GenBank se.

Open g frame (ORF) analysis using Vector NTI (Version 11, Invitrogen) was performed to fy ORFs (>= 200 ) in the full insert and flanking border sequences.

Example 3.6. 5' End Border Sequence A DNA fragment was amplified from each corn event DAS9 GenomeWalker™ library using the specific nested primer set for 5' end of the transgene. An approximately 800 bp PCR product was observed from both the event DAS9 EcoR V and Stu I GenomeWalker™ libraries. The Sea I Walker™ library generated a product around 2 kb. The fragments were cloned into pCR®4-TOPO® and six colonies from each library were ly picked for end sequencing to confirm the insert contained the expected sequences.

Complete sequencing by primer walking of the inserts ed that the fragments amplified from corn event DAS9 Stu I, EcoR V, and Sea I GenomeWalker™ libraries were 793, 822, and 2132 bp, respectively. The DNA fragments generated from the Stu I and EcoR V GenomeWalker™ libraries were a 100% match to the DNA fragment generated from Sea I GenomeWalker™ library, suggesting that these DNA fragments were amplified from the ' region of the transgene insert. BLAST search of the resultant 1873 bp corn genomic sequence indicated a high similarity to the ce of a corn BAC clone. er, sequence analysis of the insertion junction indicated that 917 bp of the MAR v3 at its 5' end region was truncated compared to the plasmid pDAS1740/i¾? I fragment, leaving a 259 bp partial MAR v3 at the 5' region of the aad-l expression cassette.

Example 3.7. 3' End Border Sequence A DNA fragment with size of approximately 3 kb was amplified from corn event DAS- 40278-9 Stu I GenomeWalker™ library using the specific nested primer set for the 3' end of the transgene. The DNA nt was cloned into pCR®4-TOPO® and ten colonies were randomly picked for end sequencing to m the insertion of the expected sequences. Three clones with the expected inserts were completely sequenced, ting a 2997 bp DNA fragment. Sequence analysis of this DNA fragment ed a partial MAR v4 t (missing 70 bp of its 5' region) and 1867 bp corn genomic sequence. BLAST search showed the 1867 bp genomic DNA sequence was a 100% match to sequence in the same corn BAC clone as was identified with the 5' border sequence.

Example 3.8. DNA Insert and Junction Sequence The DNA insert and the junction regions were cloned from corn event DAS9 using PCR based methods as previously described. Five pairs of primers were designed based on the 5' and 3' flanking border sequences and the expected transgene sequence. In total, five overlapping DNA nts (Amplicon 1 of 1767 bp, Amplicon 2 of 1703 bp, Amplicon 3 of 1700 bp, Amplicon 4 of 1984 bp, and Amplicon 5 of 1484 bp) were cloned and sequenced (Figure 4). The whole insert and ng border sequences were led based on pping sequence among the five fragments. The final sequence confirms the presence of 4816 bp of the DNA insert derived from pDAS1740/i¾? I, 1873 bp of the 5' ng border sequence, and 1868 bp of 3' flanking border sequence. The 4816 bp DNA insert contains an intact aad-l expression cassette, a 259 bp partial MAR v3 on the 5' terminus, and a 1096 bp partial MAR v4 on the 3' terminus (Seq ID No: 29).

At least two clones for each primer pair were used for primer walking in order to obtain the complete sequence information on the DNA insert and its border sequences. Sequence analysis indicated a 2 1 bp insertion at egration junction between corn genome DNA and the integrated partial MAR v3 from the pDAS1740/i¾t> I . BLAST search and Vector NTI analysis results indicated that the 2 1 bp insert DNA did not demonstrate homology to any plant species DNA or the 40 plasmid DNA. A single base pair insertion was found at the 3'- integration on between corn genome DNA and the partial MAR v4 from the pDAS 1lAQIFsp I. DNA integration also resulted in a two base pair deletion at the insertion locus of the corn genome e 6). In addition, one nucleotide difference (T to C) at the position of 5212 bp was observed in the non-translated 3' UTR region of the DNA insert (Seq ID No: 29). However, none of these changes seem to be critical to aad-l expression or create any new ORFs (>= 200 codons) across the junctions in the insert of DAS9.

Example 3.9. Confirmation of Corn Genomic Sequences To confirm the insertion site of event DAS9 transgene in the corn genome, PCR amplification was carried out with ent pairs of primers (Figure 4). Genomic DNA from event DAS9 and other transgenic or non-transgenic corn lines was used as a template.

Two aad-l specific primers, AI5End01 and AI5End02, and two primers ed according to the 5' end border sequence, 01 and lF5End02, were used to amplify DNA fragments spanning the aad-l gene to 5' end border sequence. Similarly, to amplify a DNA fragment ng the aad-\ to 3' end border sequence, lF3End05 primer derived from the 3' end border sequence and aad-l specific ABEndOl primer were used. DNA nts with expected sizes were amplified only from the genomic DNA of AAD-1 corn event DAS9, with each primer pair consisting of one primer located on the flanking border of AAD-1 corn event DAS- 40278-9 and one aad-l specific primer. The control DNA samples did not yield PCR products with the same primer pairs indicating that the cloned 5' and 3' end border sequences are indeed the upstream and downstream sequence of the ed aad- 1 gene construct, respectively. It is noted that a faint band with size of about 8 kb was observed in all the corn samples including AAD-1 corn event DAS9, AAD-1 corn event DAS-40474 and non enic corn line XHH13 when the primer pair of lF5End01 and AI5End01 were used for PCR amplification. An observed faint band (on a prepared gel) could be a result of nonspecific amplification in corn genome with this pair of primers.

To further confirm the DNA insertion in the corn genome, two primers located at the ' end border sequence, lF5End03 and 04, and two primers located at the 3' end border sequence, 03 and lF3End04, were used to amplify DNA fragments spanning the ion locus. PCR amplification with either the primer pair of lF5End03/lF3End03 or the primer pair of lF5End04/lF3End04 resulted in a fragment with expected size of approximately 8 kb from the c DNA of AAD-1 corn event DAS9. In contrast, no PCR ts resulted from the genomic DNA of AAD-1 corn event DAS7 or the non-transgenic corn line XHH13. Given that AAD-1 corn event DAS9 and event DAS7 were generated by transformation of Hill, followed by backcrossing the original transgenic events with the corn line XHH13, the majority of genome in each of these two events is theoretically from the corn line XHH13. It is very likely that only the flanking border sequences close to the aad-l transgene are carried over from the original genomic DNA and preserved during the AAD-1 event introgression process, while other regions of genome sequences might have been replaced by the genome sequences of XHH13. ore, it is not surprising that no fragments were amplified from the genomic DNA of AAD-1 corn event DAS7 and XHH13 with either the primer pair of !F5End03/lF3End03 or the primer pair of !F5End04/lF3End04.

Approximately 3.1 and 3.3 kb fragments were amplified with the primer pair of lF5End03/ lF3End03 and lF5End04/lF3End04 respectively in the genomic DNA of the corn lines Hill and B73 but not in the corn line A l 88. The results indicate that the border sequences originated from the genome of the corn line B73 . onal cloning of corn genomic DNA from B73/H P was performed to ensure validity of the flanking border sequences. The PCR amplified fragments were sequenced in order to prove the insert DNA region integrated into the specific location of B73/H II genomic DNA.

Primers were designed based on the sequence obtained. Primer set Amp IF/Amp 5R was used to amplify a 2 1 bp fragment spanning the ' to 3'junctions from native B73/H P genome without insert DNA. ce is revealed that there was a two base pair deletion from the native B73 genome in the ene insertion locus. Analysis of the DNA ces from the cloned native B73 genomic fragment identified one ORF (>= 200 codons) located downstream of the 3'- ation junction region. onally, there are no other ORFs across the original locus where the AAD-1 corn event DAS9 integrated. BLAST search also confirmed that both 5' end and 3' end border sequences from the event DAS40278-9 are located side by side on the same corn BAC clone.

Given the uniqueness of the egration junction of the AAD-1 corn event DAS- 40278-9, two pairs of specific PCR primers, 1F5EndT 1F/l F5EndT 1R and Corn278-F/Corn278- R, were designed to amplify this insert-to-plant genome junction. As predicted, the d DNA nt was only generated in the genomic DNA of the AAD-1 corn event DAS9 but not any other transgenic or non-transgenic corn lines. Therefore, those two primer pairs can be used as AAD-1 corn event DAS9 event-specific identifiers. e 4 . Genomic Characterization via Flanking SSR Markers of DAS9 To characterize and describe the genomic insertion site, marker sequences located in proximity to the insert were determined. A panel of polymorphic SSR markers were used to identify and map the transgene location. Event pDAS 1740-278 is located on chromosome 2 at approximately 20 cM between SSR markers UMC1265 and MMCOl 11 at approximately 20 cM on the 2008 DAS corn linkage map. Table 6 summarizes the primer information for these two makers found to be in close proximity to transgene pDAS1740-278.

Table 6. Primer names, dye labels, locus positions, d and reverse primer sequences, and icant notes for flanking makers associated with event pDAS 78.

Primer e mm Forward Primer Reverse Primer e Seq ID No: 30: 5' - Seq ID No: 3 1: 5'- TCGCC TGTGTTCTTGATT TACCCTACCAAT - GGGTGAGACAT - Left flanking umc1265 NED 2 20 2.02 3' 3' marker Seq ID No: 32: 5' - Seq ID No: 33: 5'- TACTGGGG AATCTATGT Right ATTAGAGCAGAAG GTGAACAGCAGC - flanking mmc01 11 FAM 2 20 2.03 - 3' 3' marker Example 4.1. gDNA Isolation gDNA was extracted from leaf punches using the DNEasy 96 Plant Test Kit (Qiagen, Valencia, California). Modifications were made to the protocol to accommodate for automation.

Isolated gDNA was quantified using the PicoGreen® dye from Molecular Probes, Inc. (Eugene, OR). The concentration of gDNA was diluted to 5 ng/mΐ for all samples using sterile deionized water.

Example 4.2. Screening of gDNA with Markers The diluted gDNA was genotyped with a subset of simple sequence repeats (SSR) markers. SSR s were synthesized by Applied Biosystems r City, California) with forward primers labeled with either 6-FAM, HEX/VIC, or NED (blue, green and yellow, respectively) fluorescent tags. The markers were divided into groups or panels based upon their fluorescent tag and amplicon size to facilitate post-PCR multiplexing and analysis.

PCR was carried out in 384-well assay plates with each reaction containing 5 ng of genomic DNA, 1.25X PCR buffer (Qiagen, Valencia, California), 0.20 mM of each forward and reverse primer, 1.25 mM MgCl2, 0.015 mM of each dNTP, and 0.3 units of rt Taq DNA polymerase (Qiagen, Valencia, California). Amplification was performed in a p PCR System 9700 with a al head module (Applied Biosystems, Foster City, rnia). The amplification program was as follows: (1) initial activation of Taq at 95°C for 12 minutes; (2) 30 sec at 94°C; (3) 30 sec at 55°C; (4) 30 sec at 72°C; (5) repeat steps 2-4 for 40 cycles; and (6) 30 min final extension at 72°C. The PCR products for each SSR marker panel were multiplexed together by adding 2 mΐ of each PCR product from the same plant to sterile deionized water for a total volume of 60 mΐ . Of the multiplexed PCR products, 0.5 ul were stamped into 384-well loading plates containing 5 mΐ of loading buffer comprised of a 1:100 ratio of GeneScan 500 base pair LIZ size standard and ABI HiDi Formamide (Applied Biosystems, Foster City, California).

The samples were then loaded onto an ABI Prism 3730x1 DNA Analyzer (Applied Biosystems, Foster City, California) for capillary electrophoresis using the manufacturer's recommendations with a total run time of 36 minutes. Marker data was collected by the ABI Prism 3730x1 ted Sequencer Data Collection software Version 4.0 and extracted via GeneMapper 4.0 software (Applied Biosystems) for allele characterization and fragment size labeling.

Example 4.3 SSR Marker Results The primer data for the flanking markers which were identified in the closest proximity to the transgene are listed in Table 6 . The two closest ated markers, 5 and MMCOl 11, are located imately 20 cM away from the transgene insert on chromosome 2 .

Example 5. Characterization of aad- 1 Protein in Event DAS9 The biochemical properties of the recombinant aad-l protein derived from the enic maize event DAS9 were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, d with Coomassie blue and glycoprotein detection s), western blot, immunodiagnostic test strip , matrix assisted laser desorption/ionization time-of-flight mass ometry (MALDI-TOF MS) and protein sequencing analysis by tandem MS were used to characterize the mical ties of the protein.

Example 5.1. Immunodiagnostic Strip Assay The presence of the aad - 1 protein in the leaf tissue of DAS9 was confirmed using commercially prepared immunodiagnostic test strips from American Bionostica. The strips were able to discriminate between transgenic and nontransgenic plants by testing crude leaf extracts (data not shown). The non-transgenic extracts (XHH13) did not contain detectable amounts of immunoreactive protein. This result was also confirmed by western blot analysis.

To test for the expression of the aad- 1 protein, an immunodiagnostic strip analysis was performed. Four leaf punches were collected from each plant for XHH13 (control plant) and event DAS9 by pinching the tissue between the snap-cap lids of individually labeled 1.5- mL microfuge tubes. Upon receipt in the lab, 0.5 mL of aad-\ extraction buffer (American tica, Swedesboro, NJ) was added to each tube, and the tissue was homogenized using a able pestle followed by shaking the sample for ~10 seconds. After homogenization, the test strip was placed in the tube and allowed to develop for ~ 5 minutes. The presence or absence of the aad-l protein in the plant extract was confirmed based on the appearance (or lack of appearance) of a test line on the immunodiagnostic strip. Once the expression of the aad-l n was confirmed for the transgenic event, the maize stalk tissue was harvested and lyophilized and stored at approximately -80°C until use Example 5.2. Purification of the aad- 1 Protein from Com Immuno-purified, derived aad-l protein ular weight: -33 kDa) or crude s extracts from corn stalk tissue were prepared. All leaf and stalk tissues were harvested and transported to the laboratory as follows: The leaves were cut from the plant with scissors and placed in cloth bags and stored at approximately -20 °C for future use. Separately, the stalks were cut off just above the soil line, placed in cloth bags and immediately frozen at approximately -80 °C for ~6 hours. The stalks were then placed in a lyophilizer for 5 days to remove water. Once the tissues were completely dried they were ground to a fine powder with dry ice and stored at approximately -80 °C until needed.

The maize-derived aad- 1 protein was extracted from lyophilized stalk tissue in a phosphate- based buffer (see Table 7 for buffer components) by weighing out ~30 grams of lized tissue into a chilled 1000 mL glass blender and adding 500 mL of extraction . The tissue was blended on high for 60 seconds and the soluble proteins were harvested by centrifuging the sample for 20 s at 30,000 xg. The pellet was re-extracted as described, and the supematants were combined and ed through a 0.45 m filter. The filtered supematants were loaded at approximately +4 °C onto an anti-aad - 1 immunoaffinity column that was conjugated with a monoclonal antibody ed by Strategic Biosolution Inc.

(MAb 473F1 85.1 ; Protein A purified; Lot # : 609.03C4; 6.5 mg/mL (-35.2 mg total)) (Windham, ME); Conjugated to CNBr-activated Sepharose 4B (GE Healthcare, Piscataway, NJ). The non-bound proteins were collected and the column was washed extensively with pre- chilled 20 mM ammonium bicarbonate , pH 8.0. The bound proteins were eluted with 3.5 M NaSCN, (Sigma, St. Louis, MO), 50 mM Tris (Sigma, St. Louis, MO) pH 8.0 buffer. Seven -mL-fractions were collected and fraction numbers 2 7 were ed overnight at imately +4°C against 10 mM Tris, pH 8.0 buffer. The fractions were examined by SDS-PAGE and western blot and the remaining samples were stored at imately +4°C until used for subsequent analyses.

Table 7. The commercially available reference nces used in this study are listed in the following table: The protein that bound to the immunoaffinity column was examined by SDSPAGE and the results showed that the eluted fractions contained the aad-\ protein at an approximate molecular weight of 33 kDa. In addition, a western blot was also performed and was positive for the aad-l protein. The maize-derived aad-\ protein was isolated from ~30 g of lyophilized stalk material.

Example 5.3. SDS-PAGE and Western Blot Lyophilized tissue from event 278-9 and XHH13 stalk (-100 mg) were weighed out in 2-mL microfuge tubes and extracted with ~ 1 mL of PBST , St. Louis, MO) containing 10% plant protease tor cocktail (Sigma, St. Louis, MO). The extraction was facilitated by adding 4 small ball bearings and Geno-Grinding the sample for 1 minute. After grinding, the samples were centrifuged for 5 s at 20,000 g and the supernatants were mixed 4:1 with 5x Laemmli sample buffer (2% SDS, 50 mM Tris pH 6.8, 0.2 mg/mL bromophenol blue, 50% (w/w) glycerol containing 10% freshly added 2-mercaptoethanol) and heated for 5 minutes at . After a brief centrifugation, 45 m of the supernatant was loaded directly onto a BioRad Criterion SDS-PAGE gel (Bio-Rad, es, CA) fitted in a Criterion Cell gel module. A positive reference standard of microbe-derived aad- 1was resuspended at 1 mg/mL in PBST pH 7.4 and further diluted with PBST. The sample was then mixed with Bio-Rad Laemmli buffer with 5% 2-mercaptoethanol and processed as described earlier. The electrophoresis was conducted with lycine/SDS buffer (Bio-Rad, Hercules, CA) at es of 150 - 200 V until the dye front approached the end of the gel. After separation, the gel was cut in half and one half was stained with Pierce GelCode Blue protein stain and the other half was electro-blotted to a nitrocellulose membrane (Bio-Rad, Hercules, CA) with a Mini blot electrophoretic transfer cell (Bio-Rad, Hercules, CA) for 60 minutes under a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Tris/glycine buffer from Bio-Rad. For immunodetection, the membrane was probed with an aad-l specific polyclonal rabbit antibody (Strategic Biosolution Inc., Newark, DE, Protein A purified rabbit polyclonal antibody Lot # : DAS F l 197-15 1, 1.6 . A conjugate of goat anti-rabbit IgG (H+L) and alkaline phosphatase (Pierce Chemical, Rockford, IL) was used as the secondary antibody. ast BCIP/NBT substrate was used for development and visualization of the immunoreactive protein bands. The membrane was washed ively with water to stop the reaction and a record of the results was captured with a digital scanner (Hewlett Packard, Palo Alto, CA) In the rescens-producedaad-l the major protein band, as visualized on Coomassie stained SDS-PAGE gels, was approximately 33 kDa. As expected, the corresponding maizederived aad-l protein (event DAS9) was identical in size to the microbe-expressed proteins. Predictably, the plant purified fractions contained a minor amount of nonimmunoreactive impurities in addition to the aad-l protein. The co-purified ns were likely retained on the column by weak interactions with the column matrix or leaching of the monoclonal antibody off of the column under the harsh elution ions. Other researchers have also reported the non-specific adsorption of peptides and amino acids on cyanogen-bromide activated Sepharose 4B immunoadsorbents (Kennedy and Barnes, 1983; Holroyde et al., 1976; Podlaski and Stern, 2008).

The protein showed a positive signal of the expected size by polyclonal antibody western blot analysis. This was also observed in the DAS9 enic maize stalk extract. In the aad-l western blot analysis, no immunoreactive proteins were observed in the control XHH13 extract and no ate size proteins (aggregates or degradation products) were seen in the transgenic s.

Example 5.4. Detection of Post-translational Glycosylation The immunoaffinity chromatography-purified, maize-derived aad-l protein (Fraction #3) was mixed 4:1 with 5x Laemmli buffer. The microbe-derived aad-l, n trypsin inhibitor, bovine serum albumin and horseradish peroxidase were diluted with Milli-Q water to the approximate concentration of the plant-derived aad-l and mixed with Bio-Rad Laemmli buffer. The proteins were then heated at ~95°C for 5 minutes and centrifuged at 20000xg for 2 minutes to obtain a clarified supernatant. The ing supernatants were applied directly to a Bio-RadCriterion Gel and ophoresed with XT MES running buffer (Bio-Rad, Hercules, CA) essentially as bed above except that the electrophoresis was run at 170 V for ~60 minutes. After ophoresis, the gel was cut in half and one half was stained with GelCode Blue stain for total protein according to the manufacturers' protocol. After the staining was complete, the gel was scanned with a Molecular Dynamics ometer to obtain a permanent visual record of the gel. The other half of the gel was d with a GelCode Glycoprotein Staining Kit (Pierce Chemical, Rockford, IL) according to the manufacturers' protocol to visualize glycoproteins. The glycoproteins (with a detection limit as low as 0.625 ng per band) were visualized as a bands on a light pink background. After the glycoprotein staining was complete, the gel was scanned with a Hewlett Packard digital scanner to obtain a permanent visual record of the gel. After the image of the glycosylation staining was captured, the gel was stained with GelCode Blue to verify the ce of the nonglycosylated proteins. The results showed that both the maize- and microbe-derived aad-l proteins had no detectable covalently linked carbohydrates. This result was also med by peptide mass fingerprinting.

Example 5.5. Mass Spectrometry Peptide Mass Fingerprinting and Sequencing of Maize- and Pseudomonas- Derived aad- 1 Mass ometry analysis of the monas- and maize-derived aad-l was conducted. The aad-l protein derived from transgenic corn stalk (event DAS9) was subjected to ution ion by trypsin followed by MALDI-TOF MS and /MS.

The masses of the detected peptides were compared to those deduced based on potential protease cleavage sites in the sequence of maize-derived aad-l protein. The theoretical ge was generated in silico using Protein Analysis Worksheet (PAWS) freeware from Proteometrics LLC. The aad-l protein, once denatured, is readily digested by proteases and will generate numerous peptide peaks.

In the trypsin digest of the transgenic-maize-derived aad-l protein (event DAS 9), the detected peptide fragments covered nearly the entire n sequence lacking only one small tryptic fragment at the C-terminal end of the protein, F248 to R253 and one short (2 amino acids) peptide nt. This analysis confirmed the maize-derived protein amino acid sequence matched that of the microbe-derived aad-l protein. Results of these analyses indicate that the amino acid sequence of the derived aad-l protein was equivalent to the P. fluorescens-expressed n.

Example 5.5.1. Tryptic Peptide Fragment Sequencing In addition to the peptide mass fingerprinting, the amino acid residues at the N- and C- termini of the maize-derived aad-l protein (immunoaffinity purified from maize event DAS- 40278-9) were sequenced and compared to the sequence of the microbe-derived protein. The protein sequences were obtained, by tandem mass spectrometry, for the first 11 residues of the microbe- and derived proteins (Table 8). The amino acid sequences for both proteins were A ' H A A L S P L S Q R ' ' (SEQ ID NO:30) showing the N-terminal methionine had been removed by an aminopeptidase (Table 8). The inal aad- 1protein sequence was expected to be M' A H A A L S P L S Q R '2. (SEQIDNO:31) These results suggest that during or after translation in maize and P.fluorescens, the N-terminal methionine is cleaved by a methionine eptidase (MAP). MAPs cleave methionyl residues rapidly when the second residue on the n is small, such as Gly, Ala, Ser, Cys, Thr, Pro, and Val (Walsh, 2006). In addition to the methionine being removed, a small portion of the N-terminal peptide of the aad- 1 protein was shown to have been acetylated after the inal methionine was cleaved (Table 8). This result is encountered frequently with eukaryotic (plant) expressed proteins since approximately 80-90% of the N-terminal residues are ed (Polevoda and Sherman, 2003).

Also, it has been shown that proteins with serine and alanine at the N-termini are the most frequently acetylated (Polevoda and Sherman, 2002). The two cotranslational processes, cleavage of inal methionine residue and N-terminal ation, are by far the most common modifications and occur on the vast majority (-85%) of eukaryotic proteins oda and Sherman, 2002). However, examples demonstrating biological significance associated with N-terminal acetylation are rare (Polevoda and Sherman, 2000).

Table 8. Summary of N-terminal Sequence Data of AAD-1 Maize- and Microbe-Derived Proteins Source Expected N-terminal ce1 P.fluorescens NT A H A A L S P L S Q ( SEQ I D NO : 3 i Maize Event DAS9 M A H A A L S P L S Q R 2 Relative Source Detected N-terminal ce2 Abundance P.fluorescens A H A A L S P L S Q R 100% Maize Event DAS9 A H A A L S P L S Q R 12 31% Maize Event DAS9 H A A L S P L S Q R 12 3% ( SEQ I D NO : 3 0 : Maize Event DAS9 50% ( SEQ I D NO : 3 2 Maize Event DAS9 A A L S P L S Q R 12 6% ( SEQ I D NO : 3 3 Maize Event DAS9 A L S P L S Q R 12 12% (SEQ ID NO:34) Expected N-terminal sequence of the first 1 amino acid residues of P'. fluoresceins- and maizederived AAD- 1.

Detected N-terminal sequences of P.fluorescens- and maize-derived AAD- 1.

The tandem MS data for the N-terminal peptides revealed a mixture of AHAALSPLSQR (acetylated) and N-^ceiy/-AHAALSPLSQR (acetylated). "Ragged N -terminal ends" were also detected (peptides corresponding to amino acid sequences HAALSPLSQR, AALSPLSQR, and ALSPLSQR). The relative abundance, an te of relative peptide fragment quantity, was made based on the ponding LC peak areas measured at 214 run.

Notes: Numbers in superscript (Rx) te amino acid residue numbers in the sequence.

Amino acid residue abbreviations: alanine H : histidine leucine M : methionine proline Q : glutamine arginine S : serine In addition to N-acetylation, there was also slight N-terminal truncation that appeared during purification of the maize-derived aad-l protein (Table 8). These "ragged-ends" resulted in the loss of amino acids A2, H and A4 (in varying forms and amounts) from the maizederived protein. This truncation is thought to have occurred during the cation of the aad- 1 protein as the western blot probe of the crude leaf ts contained a single crisp band at the same MW as the microbe-derived aad-l protein. The extraction buffer for the western blotted s contained an excess of a protease inhibitor cocktail which contains a e of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic, and metalloproteases, and aminopeptidases.

The C-terminal sequence of the maize- and microbe-derived aad-l proteins were determined as described above and compared to the ed amino acid sequences (Table 9). The results indicated the ed sequences were identical to the expected sequences, and both the maize- and microbe-derived aad-l ns were identical and unaltered at the C- terminus.

Table 9. Summary of C-terminal Sequence Data of AAD-1 Maize- and Microbe-Derived Proteins Source ed C-terminal Sequence1 P.fluorescens 2 8 7 T T V G G V R P A R2 9 6 Maize Event DAS9 2 8 7 T T V G G V R P 2 9 6 ( SEQ I D NO : 3 5 Source Detected C-terminal Sequence P.fluorescens 2 8 7 T T V G G V R P R2 9 6 Maize Event DAS9 2 8 7 v G G V R P A R2 6 ( SEQ I D NO : 3 5 : Expected C-terminal sequence of the last 10 amino acid residues of P.fluorescens- and maizederived AAD-1.

Detected C-terminal sequences of P.fluorescens- and maize-derived AAD-1 .

Notes: Numbers in superscript ( x) indicate amino acid residue numbers in the sequence.

Amino acid e abbreviations: A : e G : glycine P : proline R : ne T: ine V: valine Example 6. Field Expression. Nutrient Composition Analysis and Agronomic Characteristics of a Hybrid Maize Line Containing Event DAS9 The purpose of this study was to determine the levels of AAD-1 protein found in corn tissues. In addition, compositional analysis was performed on corn forage and grain to investigate the lency between the isogenic non-transformed corn line and the transgenic corn line DAS9 (unsprayed, sprayed with 2,4-D, d with quizalofop, and sprayed with 2,4-D and quizalofop). Agronomic characteristics of the isogenic non-transformed corn line were also compared to the DAS9 corn. The Field expression, composition, and mic trials were conducted at six test sites located within the major corn-producing regions of the U.S and Canada. These sites represent regions of diverse agronomic practices and environmental conditions. The trials were located in Iowa, Illinois (2 sites), Indiana, ka and Ontario, Canada.

All site mean values for the control, unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D and AAD-1 + both entry samples were within literature ranges for corn. A limited number of significant differences between unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D or AAD- 1 + both corn and the control were observed, but the differences were not considered to be biologically meaningful because they were small and the results were within ranges found for commercial corn. Plots of the composition results do not indicate any biologically-meaningful treatment-related compositional differences among unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D or AAD-1 + both corn and the control corn line. In conclusion, unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D and AAD-1 + both corn ition results m equivalence of AAD-1 (Event DAS 40278-9) corn to conventional corn lines.

Example 6.1. Corn Lines Tested Hybrid seed ning the DAS9 event and control plants which are conventional hybrid seed of the same genetic background as the test substance line, but do not contain the DAS9 event, are listed in Table 10.

Table 10.

The corn plants described above were grown at ons within the major corn growing regions of the U.S. and Canada. The six field testing facilities, Richland, IA; Carlyle, IL; Wyoming, IL; Rockville, IN; York, NE; and Branchton, Ontario, Canada (referred to as IA, ILl, IL2, IN, NE and ON) represent regions of e agronomic practices and environmental conditions for corn.

The test and control corn seed was planted at a seeding rate of approximately 24 seeds per row with seed spacing within each row of approximately 10 inches (25 cm). At each site, 4 replicate plots of each treatment were established, with each plot consisting of 2-25 ft rows.

Plots were arranged in a randomized complete block (RCB) design, with a unique ization at each site. Each com plot was bordered by 2 rows of a ansgenic maize hybrid of similar maturity. The entire trial site was surrounded by a minimum of 12 rows (or 30 ft) of a non- transgenic maize hybrid of similar relative maturity.

Appropriate insect, weed, and disease control practices were applied to e an agronomically acceptable crop. The monthly maximum and minimum temperatures along with rainfall and tion were average for the site. These ranges are typically encountered in corn production.

Example 6.2. Herbicide Applications Herbicide treatments were applied with a spray volume of approximately 20 gallons per acre (187 L/ha). These applications were designed to replicate maximum label rate commercial practices. Table 11 lists the herbicides that were used.

Table 11. ae = acid equivalent. ai = active ient. 2,4-D (Weedar 64) was d as 3 broadcast over-the-top applications to Test Entries 4 and 5 (seasonal total of 3 lb ae/A). Individual applications were at ergence and approximately V4 and V8 -V8.5 stages. Individual target application rates were 1.0 lb ae/A for Weedar 64, or 1120 g ae/ha. Actual application rates ranged from 1096 - 1231 g ae/A.

Quizalofop (Assure II) was applied as a single broadcast over-the-top application to Test Entries 3 and 5. Application timing was at approximately V6 growth stage. The target application rate was 0.0825 lb ai/A for Assure II, or 92 g ai/ha. Actual application rates ranged from 90.8 - 103 g ai/ha.

Example 6.3. Agronomic Data Collection and Results Agronomic characteristics were recorded for all test s within Blocks 2, 3, and 4 at each location. Table 12 lists the following characteristics that were measured.

Table 12. 1r I/ lua i n 1i h . I ' c i i i ii ol a i Early Population V I and V4 Number of plants emerged per plot. ng Vigor V4 Visual estimate of average vigor of emerged plants per plot Plant Vigor/Injury Approximately 1-2 Injury from ide applications. weeks after applications Time to Silking imately 50% The number of accumulated heat units from the time of Silking planting until approximately 50% of the plants have emerged silks.

Time to Pollen Approximately 50% The number of accumulated heat units from the time of Shed Pollen shed planting until approximately 50% of the plants are ng pollen Pollen Viability Approximately 50% Evaluation of pollen color and shape over time Plant Height Approximately R6 Height to the tip of the tassel Ear Height Approximately R6 Height to the base of the primary ear Stalk g Approximately R6 Visual estimate of percent of plants in the plot with stalks broken below the primary ear Root Lodging Approximately R6 Visual estimate of percent of plants in the plot leaning approximately 30° or more in the first -1/2 meter above the soil surface Final Population Approximately R6 The number of plants remaining per plot Days to Maturity Approximately R6 The number of accumulated heat units from the time of planting until approximately 50% of the plants have reached physiological maturity.

Stay Green imately R6 Overall plant health Disease Incidence Approximately R6 Visual estimate of foliar disease incidence Insect Damage Approximately R6 Visual estimate of insect damage Note: Heat Unit = ((MAX temp + MIN temp) / 2) - 50°F An analysis of the agronomic data collected from the control, aad- 1 unsprayed, aad- 1 + 2,4-D, aad-l + quizalofop, and aad- l + both entries was conducted. For the across-site analysis, no statistically significant differences were observed for early population (VI and V4), vigor, final population, crop injury, time to silking, time to pollen shed, stalk lodging, root lodging, disease incidence, insect damage, days to maturity, plant height, and pollen viability (shape and color) values in the across location summary is (Table 13). For stay green and ear , significant paired t-tests were observed between the control and the aad-l + quizalofop entries, but were not accompanied by significant overall treatment effects or False Discovery Rates (FDR) adjusted es (Table 13).

Table 13. Summary Analysis of Agronomic Characteristics Results Across Locations for the DAS9 aad-1 Corn (Sprayed and Unsprayed) and Control 49.2 50.8 46.4 48.1 51.9 Pollen Shape . (0486) 0 (0618,0819) (0.409, (0.739, (0.409, “mums (4’) 0.819 0.889 0.819 74.4 74.7 73.6 73.9 75.0 P011?“ Shari? (0.724) (0.809, 0.924) (0.470, (0.629, (0.629, 60 m1nutes(A)) 0.819 0.819 0.819 82.6 82.6 82.6 82.6 82.5 Pollen Shape 0 816 (100,1.00) (100,100) (100,100) (0.337, 120 minutes (%) 0.819) Overall d Sprayed Sprayed 2,4D Both p (P-value, (P-value, C51 9 52.5 48.9 50.3 Pollen Color rmnutes (A). (0524) 0 (0.850, 0960) (0.306, (0.573, , 0.819 0819 0.819 .3 75.9 74.2 74.2 75.9 Pollen Color. 60 rmnutes (A). 0 (0332) (0612,0819) (0.315, (0.315, (0.612, 0.819 0.819 0.819 Pollen Color 84.0 84.0 84.0 84.0 84.0 120 minutes % .11 5.22 5.00 5.00 5.00 Stalk Lodging (%) ) (0356,0819) (0.356, (0.356, (0.356, 0.819) 0.819) 0.819) 0.44 0.17 0.72 0.17 0.11 Root Lodging (%) ) (0.457, 0.819) (0.457, (0.457, (0.373, 0.819) 0.819) 0.819) 4.67 4.28 3.92 4.17 4.11 Stay Green‘ (0.260) , 0,819) (0.034“: (0.144, (0.106, 0.819 0.819) 0.819) 6.42 6.22 6.17 6.17 6.17 Disease Incidence] (0.741) (0.383, 0,819) (0.265, (0.265, (0.265, 0.819) 0.819) 0.819) 7.67 7.78 7.78 7.72 7.56 Insect Damagek ) (0.500, 0.819) (0.500, (0.736, (0.500, 0.819) 0.889) 0.819) - 2411 2413 2415 2416 2417 Days to Mailturflty (0.487) (0.558, 0.819) (0.302, (0.185, (0.104, (heat unlts) 0.819) 0.819) 0.819) 294 290 290 291 291 Plant Height (cm) (0.676) (0.206, 0.819) (0.209, (0.350, (0.286, 0.819 0.819 0.819 Overall Sprayed Sprayed Sprayed Overall treatment effect estimated using an F-test.

Comparison of the d and unsprayed treatments to the control using a t-test.

P-values adjusted using a False Discovery Rate (FDR) procedure.

Visual estimate on 1-9 scale; 9 = tall plants with large robust leaves. 0-100% scale; with 0 = no injury and 100 = dead plant.

The number of heat units that have accumulated from the time of ng. 0-100% scale; with % pollen grains with collapsed walls. 0- 100% scale; with % pollen grains with intense yellow color.

Visual te on 1-9 scale with 1 no visible green tissue.

Visual estimate on 1-9 scale with 1 being poor disease resistance.

Visual estimate on 1-9 scale with 1 being poor insect resistance.

NA = statistical analysis not performed since no variability across replicates or treatment.

Statistical difference indicated by e <0.05 . e 6.4. Sample Collection Samples for expression and composition analysis were collected as listed in Table 14.

Table 14.

Approx S l per l-n Gro h Sample Control T M ni es Block Tissue Stage" Si/e I . Ί II I 2-5 1 Leaf V2-4 3 leaves 3 (expression) Leaf V9 3 leaves 3 Pollen R l 1 plant 3 Rootb R l 1 plant 3 Leaf R l 1 leaf 3 Forage R4 2 plants Whole Plant R6 2 plants 3 Grain R6-Maturity 1 ear 3 2 - 4 Forage R4 3 plants (composition) Grain R6-Maturity 5 ears Approximate growth stage.

The pollen, root, and leaf s collected at R l ted from the same plant.

Two plants d, combined and sub-sampled for expression, or 3 plants for composition.

Example 6.5. Determination of aad-l Protein in Corn s Samples of corn were analyzed for the amount of aad-l protein. Soluble extractable aad- 1 n is quantified using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Beacon Analytical , Inc. (Portland, ME).

Samples of corn tissues were isolated from the test plants and prepared for expression analysis by coarse grinding, lyophilizing and fine-grinding (if necessary) with a Geno/Grinder (Certiprep, Metuchen, New ). No additional preparation was required for . The aad-l protein was extracted from corn s with a phosphate buffered saline solution containing the detergent Tween-20 (PBST) containing 0.5% Bovine Serum Albumin (BSA). For pollen, the n was extracted with a 0.5% PBST/BSA buffer containing 1 mg/mL of sodium ascorbate and 2% protease inhibitor cocktail. The plant tissue and pollen extracts were centrifuged; the aqueous supernatant was collected, diluted with appropriate buffer if necessary, and analyzed using an aad-l ELISA kit in a ch format. The kit used the following steps.

An aliquot of the diluted sample and a biotinylated anti-aaii-1 monoclonal antibody are incubated in the wells of a microtiter plate coated with an immobilized anti-aad-l monoclonal antibody. These dies bind with aad-l protein in the wells and form a "sandwich" with aad-l protein bound between soluble and the immobilized antibody. The d samples and conjugate are then removed from the plate by washing with PBST. An excess amount of streptavidin-enzyme (alkaline phosphatase) conjugate is added to the wells for incubation. At the end of the incubation period, the unbound reagents were removed from the plate by washing.

Subsequent addition of an enzyme substrate generated a colored product. Since the aad-l was bound in the antibody sandwich, the level of color development was d to the concentration of aad-l in the sample (i.e., lower residue concentrations result in lower color development).

The absorbance at 405 nm was measured using a Molecular Devices V-max or Spectra Max 190 plate reader. A calibration curve was generated and the aad- 1 concentration in unknown samples was calculated from the polynomial regression equation using Soft-MAX Pro™ software which was compatible with the plate reader. Samples were analyzed in duplicate wells with the average concentration of the duplicate wells being reported.

A summary of the aad-l n trations (averaged across sites) in the s corn matrices is shown in Table 15. aad-\ average protein concentration ranged from 2.87 ng/mg dry weight in R l stage root to 127 ng/mg in pollen. Expression results for the unsprayed and sprayed plots were similar. The aad-l protein was not detected in any control samples, with the exception of one control root sample from the Indiana site.

Table 15. Summary of Mean tration Levels of aad-1 Protein Measured in the aad-1 Unsprayed, aad-1 + Quizalofop, aad-1 + 2,4-D and aad-1 + Quizalofop and 2,4-D in Maize Tissues Corn AAD-1 ng/mg Tissue Dry Weight Tissue ent Mean Std. Dev. Range V2-V4 Leaf AAD-1 Unsprayed 13.4 8.00 1.98-29.9 AAD-1 + Quizalofop 13.3 6.89 4.75-24.5 AAD-1 + 2,4-D 14.2 7.16 4.98-26.7 AAD-1 + Quizalofop and 2,4-D 12.3 7.09 2.5 V9 Leaf AAD-1 yed 5.96 2.50 2.67-10.9 AAD-1 + Quizalofop 5.38 1.84 2.52-9.15 AAD-1 + 2,4-D 6.37 2.41 3.03-10.9 AAD-1 + Quizalofop and 2,4-D 6.52 2.38 3.1 1-11.1 R l Leaf AAD-1 Unsprayed 5.57 1.66 3.47-9.34 AAD-1 + Quizalofop 5.70 1.63 2.70-7.78 AAD-1 + 2,4-D 5.99 1.90 2.40-9.42 AAD-1 + Quizalofop and 2,4-D 6.06 2.27 1.55-10.2 Pollen AAD-1 Unsprayed 127 36.2 56.3-210 AAD-1 + Quizalofop 108 29.9 52.2-146 AAD-1 + 2,4-D 113 30.2 37.5-137 AAD-1 + ofop and 2,4-D 112 32.6 45.4-162 R l Root AAD-1 Unsprayed 2.92 1.87 0.42-6.10 AAD-1 + Quizalofop 3.09 1.80 0.56-6.06 AAD-1 + 2,4-D 3.92 2.03 0.91-7.62 AAD-1 + Quizalofop and 2,4-D 2.87 1.23 1.09-5.56 R4 Forage AAD-1 Unsprayed 6.87 2.79 2.37-12.1 AAD-1 + Quizalofop 7.16 2.84 3.05-11.6 AAD-1 + 2,4-D 7.32 2.46 2.36-10.6 AAD-1 + Quizalofop and 2,4-D 6.84 2.31 2.25-10.3 Whole plant AAD-1 Unsprayed 4.53 2.55 .88 AAD-1 + Quizalofop 4.61 2.22 0.75-8.77 AAD-1 + 2,4-D 5.16 2.53 0.83-10.2 AAD-1 + Quizalofop and 2,4-D 4.55 1.77 1.30-8.21 Grain AAD-1 Unsprayed 5.00 1.53 2.66-8.36 AAD-1 + Quizalofop 4.63 1.51 1.07-6.84 AAD-1 + 2,4-D 4.98 1.78 2.94-9.10 AAD-1 + Quizalofop and 2,4-D 4.61 1.62 1.81-7.49 a ND = value less than the method Limit Of Detection (LOD).

Values in parentheses are between the method LOD and Limit Of Quantitation (LOQ) .

Example 6.6. Compositional Analysis Samples of corn forage and grain were analyzed at for nutrient content with a variety of tests. The analyses performed for forage included ash, total fat, re, protein, carbohydrate, crude fiber, acid detergent fiber, neutral detergent fiber, calcium and phosphorus. The analyses performed for grain included proximates (ash, total fat, re, protein, ydrate, crude fiber, acid detergent , l detergent fiber (NDF), minerals, amino acids, fatty acids, vitamins, secondary lites and anti-nutrients. The results of the nutritional analysis for corn forage and grain were compared with values reported in literature ( see; Watson, 1982 (4); Watson, 1984 (5); ILSI Crop Composition Database, 2006 (6); OECD Consensus Document on Compositional erations for maize, 2002 (7); and Codex Alimentarius Commission 2001 (8))· Example 6.6.1. Proximate, Fiber and Mineral Analysis of Forage An analysis of the protein, fat, ash, moisture, carbohydrate, ADF, NDF, calcium and phosphorus in corn forage samples from the control, unsprayed aad-l, aad-l + quizalofop, aad-l + 2,4-D and aad-l +both entries was performed. A summary of the results across all locations is shown in Table 16. For the across-site and individual-site analysis, all proximate, fiber and mineral mean values were within literature . No statistical differences were observed in the across-site is between the control and transgenic entries for moisture, ADF, NDF, calcium and phosphorus. For protein and ash, significant paired t-tests were observed for the unsprayed AAD-1 (protein), the aad-l + quizalofop (protein), and aad-l +both (ash), but were not anied by significant l treatment effects or FDR adjusted p-values. For fat, both a significant paired t-test and adjusted p-value was observed for aad-l + quizalofop compared with the control, but a significant overall treatment effect was not observed. For carbohydrates, a tically significant overall ent effect, paired t-test and FDR adjusted p-value was observed between the aad-l + quizalofop and the control. Also for carbohydrates, a significant paired t-test for the unsprayed aad-l entry was observed, but without a significant FDR adjusted p-value. These differences are not biologically meaningful since all across-site results for these analytes were within the ed literature ranges for corn, and differences from the control were small (<23 %).

Table 16. Summar of the Proximate. Fiber and Mineral Anal sis of Corn Forage Minerals (% dry weight) Calcium 0.212 0.203 0.210 0.215 0.231 0.071-0.6 (0.321) (0.532, (0.930, (0.815, (0. 150, 0.708) 0.970) 0.91 1) 0.296) Phosphorus 0.094- 0.197 0.189 0.202 0.203 0.200 (0. 163) (0. 198, (0.427, (0.288, (0.608, 0.55 0.354) 0.615) 0.450) 0.762) a Combined range.

Overall treatment effect estimated using an F-test.

Comparison of the transgenic treatments to the control using t-tests. d P-values ed using a False Discovery Rate (FDR) procedure.

Statistical difference indicated by P-Value <0.05.

Example 6.6.2. Proximate and Fiber Analysis of Grain A summary of the results for proximates (protein, fat, ash, moisture, cholesterol and carbohydrates) and fiber (ADF, NDF and total dietary fiber) in corn grain across all locations is shown in Table 17. All results for proximates and fiber were within literature ranges, and no significant differences in the across-site analysis were observed between the l and transgenic entries for fat, ash, NDF and total dietary fiber. For moisture, a significant overall ent effect was observed, but not accompanied by significant paired t-tests or FDR ed p-values. For ADF, a significant paired t-test was observed for aad-l +both, but no significant overall treatment effect or FDR adjusted p-value was seen. For both protein and carbohydrates, significant pair-tests, ed p-values and l treatment effects were found for the yed aad-l, aad-l + ofop, and aad-l +both. Since these differences were small (< 12%) and all values were within literature ranges, the differences are not ered biologically meaningful.

Table 17. Summary of the Proximate and Fiber Analysis of Corn Grain from All Sites.

Overall Sprayed Sprayed Sprayed Treatment Unsprayed Quizalofop 2,4-D Both Proximate Literature Effect (P-value, (P-value, (P-value, (P-value, (% dry ) Values (Pr>F) Control Adi. ) Adj. P) Adi. P) Adj. P) Protein 9.97 10.9 11.1 10.5 10.9 6-17.3 (0.003 ) (0.002 , (0.0004 , (0.061, (0.002 , 0.016 ) 0.013 ) 0.161) 0.015 ) Fat 4.26 4.19 4.16 4.26 4.22 1.2-18.8 (0.369) (0.238, (0.095, (0.955, (0.427, 0.397) 0.215) 0.977) 0.615) Ash 1.45 1.55 1.52 1.45 1.51 0.62-6.28 ) (0. 178, (0.364, (0.982, (0.397, 0.330) 0.557) 0.985) 0.587) Moisture 25.1 25.5 24.4 24.5 24.5 6.1-40.5 (0.038 ) (0.406, (0.056, (0. 117, (0. 114, 0.594) 0 .152) 0.254) 0.250) Cholesterol < LOQ < LOQ < LOQ < LOQ < LOQ NR NA Carbohydrate 84.3 83.3 83.2 83.8 83.4 63.3-89.8 (0.005 ) (0.002 , (0.001 , (0.074, (0.003 , 0.015 ) 0.013 ) 0.185) 0.019 ) Fiber (% dry ) Acid Detergent 4.23 3.94 3.99 3.89 3.82 Fiber (ADF) 1.3 (0.247) (0. 130, (0. 197, (0.078, (0.035 , 0.269) 0.354) 0.193) 0 .106) Neutral Detergent 10.6 10.3 9.89 9.90 10.3 Fiber (NDF) 5.59-22.6 (0.442) (0.455, (0. 120, (0. 121, (0.552, 0.638) 0.254) 0.254) 0.708) Total y 13.4 12.8 12.9 13.1 12.9 Fiber 8.3-35.3 (0.579) (0. 164, (0. 195, (0.487, (0.215, 0.3 13) 0.353) 0.679) 0.370) a Combined range.

Overall treatment effect estimated using an F-test.

Comparison of the transgenic treatments to the control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure.

Statistical difference indicated by e <0.05. f NR = not reported.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Example 6.6.3 Mineral Analysis of Grain An is of corn grain samples for the minerals calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium, and zinc was performed. A summary of the results across all locations is shown in Table 18. All results were within the reported literature ranges. For the across-site analysis, no significant differences were observed for calcium, copper, iron, and potassium. Mean results for um, iodine, um and sodium were below the limit of quantitation of the method. For magnesium and phosphorus, significant paired t-tests were observed for the unsprayed aad-l and the aad-l + ofop entries, but were not accompanied by significant overall ent effects or FDR adjusted p-values. For ese and molybdenum, a significant paired t-test was observed for the unsprayed aad- 1, but a significant FDR adjusted p-value and overall ent effect was not found. For the aad-l + both entry, a significant paired t-test was observed for zinc, but a significant FDR adjusted e or overall treatment effect was not t. Additionally, these differences from the l were small (< 13%), and all values were within literature ranges, when available.

Table 18. Summary of the Mineral Analysis of Corn Grain from All Sites.

Overall Sprayed Sprayed Sprayed Treatment Unsprayed Quizalofop 2,4-D Both Minerals Literature Effect (P-value, (P-value, (P-value, (P-value, (mg/lOOg dry wt.) Values (Pr>F) Control Adj. ) Adj. P) Adi. P) Adj. P) Calcium 4.05 4.21 4.12 4.04 4.06 1.27-100 (0.493) (0. 146, (0.505, (0.944, (0.898, 0.289) 0.687) 0.977) 0.957) um 0.006- < LOQ < LOQ < LOQ < LOQ < LOQ 0.016 Copper 0.073- 0.144 0.151 0.146 0.141 0.149 (0.963) (0.655, (0.890, (0.817, (0.749, 1.85 0.782) 0.957) 0.91 1) 0.863) Iodine < LOQ < LOQ < LOQ < LOQ < LOQ 7.3-81 NA Iron 2.49 2.60 2.56 2.51 2.59 0.1-10 (0.333) (0.086, (0.3 10, (0.801, (0. 145, 0.206) 0.482) 0.91 1) 0.289) Magnesium 59.4- 122 129 128 126 127 (0.072) 1000 (0.010 , (0.017 , (0. 145, , 0.051) 0.066) 0.289) 0 .177) Manganese 0.525 0.551 0.524 0.526 0.532 0.07-5.4 (0.099) (0.025 , (0.884, (0.942, (0.505, 0.082) 0.957) 0.977) 0.687) Molybdenum 261 229 236 244 234 NR (0. 143) (0.020 , , (0.206, (0.046, 0.072) 0 .173) 0.362) 0 .132) Phosphorus 289 303 300 299 298 147-750 (0. 102) (0.012 , (0.035 , (0.055, (0.085, 0.057) 0 .106) 0.150) 0.206) Potassium 362 368 359 364 357 181-720 (0.453) (0.330, (0.655, (0.722, (0.454, 0.5 10) 0.782) 0.839) 0.638) Selenium < LOQ < LOQ < LOQ < LOQ < LOQ 0.001-0.1 NA Sodium < LOQ < LOQ < LOQ < LOQ < LOQ 0-150 NA Zinc 2.26 2.32 2.34 2.29 2.37 0.65-3.72 (0. 166) (0. 183, (0. 108, (0.627, f, 0.336) 0.238) 0.768) 0.085) a Combined range.

Overall treatment effect ted using an F-test.

Comparison of the transgenic treatments to the control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Statistical difference indicated by P-Value <0.05. e 6.6.4 Amino Acid Analysis of Grain Corn samples were analyzed for amino acid content in the control, unsprayed aad-1, aad- 1 + quizalofop, aad-\ + 2,4-D and aad-l +both corn, and a summary of the results over all locations and by dual field site are shown in Table 19. Levels of all amino acids were within the reported literature ranges, and no significant differences in the -site analysis were observed for arginine, lysine, and ne. Significant differences were observed for several of the amino acids in the across-site analysis. In these ces, the amino acid content of the control was lower than the aad-l transgenic lines, which may be related to the overall lower protein content in the control grain compared with the aad-l lines. For the unsprayed aad- 1 entry, significant overall treatment effects along with icant paired t-tests and FDR adjusted p-values were found for all amino acids except ne, glycine, lysine, tryptophan and ne. For the aad-l + quizalofop entry, significant overall treatment effects along with significant paired t-tests and FDR adjusted p-values were found for all amino acids except arginine, cysteine, glycine, lysine, tryptophan and tyrosine. For the aad-l + 2,4-D entry, significant overall treatment effects along with significant paired t-tests (with significant FDR adjusted p-values) were found for all amino acids except arginine, ic acid, glycine, histidine, lysine, tyrosine and valine. For the aad-l +both entry, significant overall treatment effects along with significant paired t-tests and FDR adjusted p-values were found for all amino acids except arginine, glycine, lysine, serine, tryptophan and tyrosine. Although there were many differences observed for amino acids, the differences were small (< 15%), not observed across all sites, and all mean values were within reported ture ranges.

Table 1 . Summary of the Amino Acid Analysis of Corn Grain from All Sites.

Overall Sprayed Sprayed Sprayed Treatment Unsprayed ofop 2,4-D Both Amino Acids Literature Effect (P-value, ue, (P-value, (P-value, (% dry weight) Values (Pr>F) Control Adj. P) Adj. P) Adi. P) Adj. P) Alanine 0.44-1.39 0.806 0.901 0.900 0.863 0.894 (0.002 ) (0.0005 , (0.0005 , (0.021 , (o.oor, 0.013 ) 0.013 ) 0.074) 0.013 ) Arginine 0.12-0.64 0.486 0.499 0.505 0.487 0.484 (0.371) (0.286, (0. 139, (0.929, (0.897, 0.450) 0.283) 0.970) 0.957) Aspartic Acid 0.34-1.21 0.712 0.768 0.764 0.743 0.762 (0.010 ) (0.002 , (0.003 , (0.060, (0.004 , 0.015 ) 0.021 ) 0.160) 0.027 ) Cysteine 0.08-0.51 0.213 0.225 0.223 0.223 0.226 (0.033 ) (0.009 , (0.020 , (0.018 , (0.005 , 0.050 ) 0.072) 0.067) 0.028 ) Glutamic Acid 0.97-3.54 1.97 2.22 2.21 2.12 2.20 (0.001 ) (0.0003 , (0.0004 , (0.017 , (0.001 , 0.013 ) 0.013 ) 0.067) 0.013 ) Glycine 0.18-0.54 0.383 0.397 0.398 0.390 0.397 (0.052) (0.018 , (0.013 , (0.217, (0.016 , 0.067) 0.059) 0.371) 0.066) Histidine 0.14-0.43 0.283 0.303 0.302 0.295 0.302 (0.005 ) (0.001 , (0.002 , (0.036, (0.002 , 0.013 ) 0.014 ) 0.109) 0.014 ) Isoleucine 0.18-0.71 0.386 0.427 0.427 0.410 0.431 (0.003 ) (0.001 , (0.001 , (0.044 , (0.001 , 0.014 ) 0.014 ) 0.127) 0.013 ) e 0.64-2.49 1.35 1.54 1.54 1.47 1.53 (0.001 ) (0.0003 , (0.0003 , (0.013 , (0.001 , 0.013 ) 0.013 ) 0.059) 0.013 ) Lysine 0.05-0.56 0.310 0.315 0.316 0.309 0.316 (0.21 1) (0.210, (0. 128, (0.879, (0. 102, 0.367) 0.265) 0.956) 0.226) Methionine 0.10-0.47 0.195 0.209 0.209 0.205 0.208 (0.003 ) (0.001 , (0.001 , (0.014 , (o.oor, 0.013 ) 0.013 ) 0.061) 0.014 ) Phenylalanine .93 0.551 0.617 0.619 0.592 0.615 (0.002 ) (0.001 , (0.001 , (0.023 , (0.001 , 0.013 ) 0.013 ) 0.077) 0.013 ) Proline .63 0.910 1.01 1.01 0.975 0.997 (0.002 ) 4 , (0.001 , (0.012 , (o.oor, 0.013 ) 0.013 ) 0.059) 0.014 ) Serine 0.24-0.91 0.498 0.550 0.550 0.529 0.536 (0.009 ) (0.002 , (0.001 , (0.042 , (0.015 , 0.014 ) 0.014 ) 0.122) 0.061) l Sprayed Sprayed Sprayed Treatment Unsprayed Quizalofop 2,4-D Both Amino Acids Literature Effect (P-value, (P-value, (P-value, (P-value, (% dry weight) Values (Pr>F) Control Adj. P) Adj. P) Adi. P) Adj. P) Threonine 0.22-0.67 0.364 0.394 0.394 0.384 0.390 (0.005 ) (o.oor, (o.oor, (0.023 , (0.003 , 0.014 ) 0.013 ) 0.077) 0.020 ) Tryptophan 0.03-0.22 0.052 0.055 0.056 0.056 0.056 (0.088) (0.067, (0.025 , (0.014 , (0.029 , 0 .173) 0.082) 0.060) 0.092) Tyrosine 0.10-0.79 0.336 0.355 0.375 0.339 0.314 (0.390) (0.535, , (0.907, (0.500, 0.708) 0.370) 0.964) 0.687) Valine .86 0.495 0.537 0.538 0.519 0.538 (0.005 ) (0.002 , (0.002 , (0.054, (o.oor, 0.014 ) 0.014 ) 0.148) 0.014 ) Combined range.

Overall ent effect estimated using an F-test.

Comparison of the enic ents to the l using t-tests.

P-values ed using a False Discovery Rate (FDR) procedure.

Statistical difference indicated by P-Value <0.05.

Example 6.6.5. Fatty Acid Analysis of Grain An analysis of corn grain s for fatty acids was performed. A summary of the results across all locations is shown in Table 20. All results for the control, unsprayed aad-l, aad-l + quizalofop, aad-l + 2,4-D and aad-l +both corn grain samples analyzed for these fatty acids were within the published literature ranges. Results for caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), myristoleic (14:1), pentadecanoic (15:0), pentadecenoic (15:1), heptadecanoic (17:0), heptadecenoic (17:1), gamma linolenic (18:3), dienoic , eicosatrienoic (20:3), and arachidonic (20:4) were below the method Limit of Quantitation (LOQ). In the across-site analysis, no significant differences were observed for 16:0 palmitic, 16:1 pamitoleic, 18:0 stearic, 18:2 linoleic, 18:3 linolenic, and 20:0 arachidic. For 18:1 oleic and : 1 eicosenoic, significant paired t-tests were observed for the unsprayed aad-l (18:1) and the aad-l + 2,4-D (18:1 and 20:1) entries, but were not accompanied by significant overall treatment s or FDR adjusted p-values. For 22:0 behenic, a significant overall treatment effect and significant paired t-tests for aad-l + 2,4-D and aad-l + both were found, but significant FDR adjusted p-values were not present.

Table 20. Summa ofthe Fatt Acid Anal sis of Corn Grain from All Sites.

Overall Fatty Acids Treatment (% total fatty Literature Effect aCIdSY Valuesb (Pr>F)° Control 8:0Ca lic <LO <LO <LO <LO <LO -------- --------0687 14:0 Myristic < LOQ ND—0.3 \A 14:1 oleic < LOQ NR \A :0 Pentadecanoic < LO < LO < LO < LO < LO :1 Pentadecenoic < LO < LO < LO < LO < LO 16:0 Palmitic 7—20.7 (0.559) 16: 1 Palmitoleic ND—1.0 (0.552) 17:0 He tadecanoic < LO < LO < LO < LO < LO 17:1 He tadecenoic < LO < LO < LO < LO < LO 18:0 Stearic ND-3.4 ) 18:1 Oleic 17.4 - 46 ) 18:2 Linoleic 47.5 483 48.4 48.0 48.5 34.0-70 (0.474) (0.189, (0.144, (0.453, (0.119, 0.345) 0.289) 0.638) 0.254) 18:3 Gamma < LOQ < LOQ < LOQ < LOQ < LOQ Linolenic NR NA Overall Sprayed Sprayed d Fatty Acids ent Unsprayed Quizalofop 2,4-D Both (% total fatty Literature Effect (P-value, (P-value, ue, (P-value, acids) Values' 3 (Pr>F) Control Adi. P) Adj. P) Adj. P) Adj. P) 18:3 Linolenic 1.04 1.05 1.06 1.04 1.06 ND-2.25 ) (0.537, (0.202, (0.842, (0.266, 0.708) 0.357) 0.932) 0.428) :0 Arachidic 0.400 0.386 0.393 0.390 0.390 0.1-2 (0.379) (0.061, (0.341, (0. 153, (0. 175, 0 .161) 0.525) 0.297) 0.328) :1 Eicosenoic 0.232 0.226 0.230 0.223 0.227 0.17-1.92 (0. 107) (0.089, (0.497, (0.013 , (0. 121, 0.210) 0.687) 0.059) 0.254) :2 Eicosadienoic < LOQ < LOQ < LOQ < LOQ < LOQ ND-0.53 NA :3 trienoic < LOQ < LOQ < LOQ < LOQ < LOQ 0.275 NA :4 Arachidonic < LOQ < LOQ < LOQ < LOQ < LOQ 0.465 NA 22:0 Behenic 0.136 0.088 0.076 0.086 0.108 ND-0.5 (0.044s ) (0.093, (0.887, (0.011s (0.023g, 0.213) 0.957) 0.054) 0.077) Results converted from units of % dry weight to % fatty acids. ed range.

Overall treatment effect estimated using an F-test. d Comparison of the transgenic treatments to the l using t-tests.

P-values adjusted using a False Discovery Rate (FDR) procedure.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Statistical difference indicated by P-Value <0.05.

Example 6.6.6. Vitamin Analysis of Grain The levels of vitamin A , Bl, B2, B5, B6, B12, C, D , E , niacin, and folic acid in corn grain samples from the control, unsprayed aad-l, aad-l + quizalofop, aad-l + 2,4-D and aad-l + both corn entries were determined. A summary of the results across all locations is shown in Table 2 1. Vitamins B12, D and E were not quantifiable by the ical methods used. All mean s reported for vitamins were similar to reported literature values, when ble.

Results for the vitamins without reported literature ranges (vitamins B5 and C) were similar to control values obtained (< 22% difference from control). For the across-site analysis, no statistical differences were observed, with the exception of vitamins B 1, C and niacin.

Significant paired t-tests for Vitamins B 1 were observed between the control and unsprayed aad- 1, aad-l + quizalofop, and aad-l + both, but were not accompanied by significant overall treatment effects or FDR adjusted p-values. For n C, a significant overall treatment effect was observed along with significant paired t-tests and FDR adjusted p-values for aad-l + quizalofop and aad-l + 2,4-D. Similarly for niacin, a significant overall treatment effect was observed along with significant paired s and FDR adjusted p-values for aad-l + quizalofop and aad-l +both. A significant paired t-test for the aad-l + 2,4-D was also found for niacin for the aad-l + 2,4-D entry, but was not anied by a significant overall treatment effect or FDR adjusted p-value. Since the differences were not observed across sites and values were within literature ranges (when available), the differences are not ered biologically meaningful.

Table 2 1. Summary of Vitamin Analysis of Corn Grain from All Sites.

Overall Sprayed d Sprayed Vitamins Treatment Unsprayed Quizalofop 2,4-D Both (mg/kg dry Literature Effect (P-value, (P-value, (P-value, (P-value, weight) Values (Pr>F) Control Adi. ) Adj. P) Adi. P) Adj. P) Beta Carotene 0.19 - 1.80 1.85 1.80 1.82 1.87 (Vitamin A) (0.649) (0.372, (0.967, (0.770, (0.221, 46.8 0.566) 0.983) 0.883) 0.376) Vitamin B 1 3.47 3.63 3.67 3.54 3.64 (Thiamin) 1.3 - 40 (0.068) (0.041s, (0.013s, (0.375, (0.032 , 0 .121) 0.059) 0.567) 0 .100) Vitamin B2 2.15 2.05 2.08 1.99 2.07 (Riboflavin) 0.25 - 5.6 (0.803) (0.443, , (0.227, (0.543, 0.63 1) 0.756) 0.383) 0.708) n B5 5.28 5.17 5.09 5.29 5.10 (Pantothenic acid) NR (0.820) (0.623, , (0.968, (0.424, 0.766) 0.582) 0.983) 0.615) Vitamin B6 3.68 - 6.52 6.57 6.66 6.66 7.08 (Pyridoxine) (0.43 1) (0.859, , (0.652, (0.088, 11.3 0.938) 0.782) 0.782) 0.210) Vitamin B 12 < LOQ < LOQ < LOQ < LOQ < LOQ NR NA Vitamin C 22.4 21.2 17.5 18.0 20.4 NR (0.018 ) , (0.005 , (0.004s, (0.068, 0.429) 0.028 ) 0.026 ) 0 .173) Vitamin D < LOQ < LOQ < LOQ < LOQ < LOQ NR NA Vitamin E (alpha < LOQ < LOQ < LOQ < LOQ < LOQ erol) 1.5 - 68.7 (0.558) Niacin (Nicotinic 26.1 24.2 22.9 23.7 22.9 it. B3) 9.3 - 70 (0.013 ) (0.050, (0.002e, (0.018 , (0.002 , 0 .140) 0.017 ) 0.067) 0.016 ) Folic Acid 0.594 0.588 0.574 0.592 0.597 0.15 - 683 (0.88 1) (0.779, (0.403, (0.93 1, (0.916, 0.890) 0.592) 0.970) 0.970) a Combined range.

Overall treatment effect ted using an F-test.

Comparison of the transgenic treatments to the control using s. d P-values adjusted using a False Discovery Rate (FDR) procedure.

Statistical difference indicated by P-Value <0.05.

NR = not reported.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Example 6.6.7. Anti-Nutrient and Secondary Metabolite is of Grain The secondary metabolite (coumaric acid, ferulic acid, furfural and inositol) and antinutrient c acid, raffinose, and trypsin tor) levels in corn grain samples from the control, unsprayed aad-l, aad-l + quizalofop, aad-l +2,4-D and aad-l +both corn entries were determined. A summary of the results across all locations is shown in Table 22 and 23. For the across-site analysis, all values were within literature ranges. No significant differences between the aad-l entries and the control entry results were observed in the across-site analysis for inositol and trypsin inhibitor. Results for furfural and raffinose were below the method's limit of quantitation. Significant paired t-tests were observed for coumaric acid (unsprayed aad-l, aad-l + 2,4-D and aad-l +both), and ferulic acid (aad-l + quizalofop and aad-l +both). These differences were not accompanied by significant overall treatment effects or FDR adjusted pvalues and were similar to the control (< 10% difference). A significant overall treatment effect, paired t-test, and FDR adjusted p-value was found for phytic acid (unsprayed aad-l). Since all results were within literature ranges and similar to the control (<1 1% ence), these differences are not considered to be ically meaningful.

Table 22. Summary of Secondary lite Analysis of Corn Grain from All Sites. l Sprayed Sprayed Sprayed ary Treatment Unsprayed ofop 2,4-D Both lite Literature Effect (P-value, (P-value, (P-value, ue, (% dry weight) Values (Pr>F) Control Adj. ) Ad). P) Adi. P) Adi. P) Coumaric Acid 0.021 0.020 0.020 0.019 0.020 0.003- (0. 119) 0.058 (0.038 , (0.090, (0.022 , (0.029 , 0 .113) 0.21 1) 0.074) 0.091) Ferulic Acid 0.02- 0.208 0.199 0.196 0.200 0.197 (0.077) (0.05 1, (0.010 , (0.080, 0.389 (0.019 , 0 .141) 0.05 1) 0.196) 0.069) Furfural 0.0003- < LOQ < LOQ < LOQ < LOQ < LOQ 0.0006 Inositol 0.0089- 0.218 0.224 0.218 0.213 0.211 (0.734) 0.377 (0.548, (0.973, (0.612, (0.526, 0.708) 0.984) 0.763) 0.708) a Combined range.

Overall treatment effect estimated using an F-test.

Comparison of the transgenic treatments to the control using t-tests.

P-values adjusted using a False ery Rate (FDR) procedure.

Statistical difference indicated by P-Value <0.05.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Table 23. Summary of Anti-Nutrient Analysis of Corn Grain from All Sites. a Combined range. l treatment effect estimated using an F-test.

Comparison of the transgenic treatments to the control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure.

Statistical difference indicated by P-Value <0.05. f NA= statistical analysis was not performed since a majority of the data was < LOQ.

Example 7 - Additional agronomic trials Agronomic characteristics of corn line 40278 ed to a near-isoline corn line were evaluated across diverse environments. ents included 4 genetically distinct hybrids and their appropriate near-isoline l hybrids tested across a total of 2 1 locations.

The four test hybrids were medium to late maturity hybrids g from 99 to 113 day ve maturity. Experiment A tested event DAS9 in the genetic background Inbred C x BC3S1 conversion. This hybrid has a relative maturity of 109 days and was tested at 16 locations (Table 24). Experiment B tested the hybrid background Inbred E x BC3S1 conversion, a 113 day relative maturity hybrid. This hybrid was tested at 14 locations, using a slightly different set of locations than Experiment A (Table 24). Experiments C and D tested hybrid ounds BC2S1 conversion x Inbred D and BC2S1 conversion x Inbred F, respectively.

Both of these hybrids have a 99 day relative maturity and were tested at the same 10 locations.

Table 24. Locations of agronomic trials {TC "Table 11. Locations of Experiment 2 agronomic trials" \f \ 1 "1" } Patteville, WI X X X own, WI X X For each trial, a randomized complete block design was used with two replications per location and two row plots. Row length was 20 feet and each row was seeded at 34 seeds per row. Standard regional mic practices were used in the management of the trials.

Data were collected and analyzed for eight agronomic characteristics; plant height, ear height, stalk lodging, root lodging, final population, grain moisture, test weight, and yield. The parameters plant height and ear height provide information about the appearance of the hybrids.

The agronomic characteristics of percent stalk g and root lodging determine the harvestability of a hybrid. Final population count measures seed y and seasonal growing conditions that affect yield. Percent grain moisture at t defines the maturity of the hybrid, and yield (bushels/acre adjusted for moisture) and test weight (weight in pounds of a bushel of corn adjusted to 15.5% moisture) be the reproductive capability of the hybrid.

Analysis of variance was conducted across the field sites using a linear model. Entry and location were included in the model as fixed effects. Mixed models including location and location by entry as random effects were explored, but location by entry ned only a small portion of variance and its variance component was often not icantly different from zero.

For stock and root g a logarithmic transformation was used to stabilize the variance, however means and ranges are reported on the original scale. Significant differences were declared at the 95% ence level. The significance of an overall treatment effect was estimated using a t-test.

Results from these agronomic characterization trials can be found in Table 2 . No tically significant differences were found for any of the four 40278 hybrids compared to the isoline controls (at p<0.05) for the parameters of ear height, stalk lodging, root lodging, grain moisture, test weight, and yield. Final population count and plant height were statistically different in ments A and B, respectively, but similar differences were not seen in comparisons with the other 40278 hybrids tested. Some of the variation seen may be due to low levels of genetic variability remaining from the backcrossing of the DAS9 event into the elite inbred lines. The overall range of values for the measured parameters are all within the range of values obtained for traditional corn hybrids and would not lead to a conclusion of increased weediness. In summary, agronomic characterization data indicate that 40278 corn is ically equivalent to conventional corn.

Table 25. Analysis of agronomic characteristics! TC "Table 12. is of agronomic characteristics from Experiment 2 " \f D \ 1 "1" } Experiment A Experirner t B AAD-1 23.71 14.34 28.70 Grain Moisture (%) 0.9869 Control 23.72 13.39 31.10 AAD-1 56.96 50.90 59.50 Test Weight (lb/bushel) 0.2796 Control 56.67 52.00 60.10 AAD-1 200.08 102.32 258.36 Yield ls/acre) 0.2031 Control 205.41 95.35 259.03 Table 25. (cont.) is of agronomic teristics Experiment C Range Parameter unit Treat ment Mean in Ma AAD-1 95.92 94.00 96.00 Plant Height (inches) Control 90.92 90.00 90.00 AAD-1 47.75 41.00 50.00 Ear Height (inches) Control 43.75 37.00 46.00 AAD-1 6.74 0.00 27.47 Stalk Lodging (% Control 5.46 0.00 28.12 AAD-1 0.3512 0.00 7.58 Root Lodging (%) Control 0.3077 0.00 33.33 Final Population AAD-1 32.78 29.00 36.00 (plants/acre in 1OOP's) Control 31.68 24.00 35.00 AAD-1 19.09 13.33 25.90 Grain Moisture (%) Control 19.36 13.66 26.50 AAD-1 54.62 42.10 58.80 Test Weight (lb/bushel) Control 55.14 52.80 58.40 AAD-1 192.48 135.96 243.89 Yield (bushels/acre) Control 200.35 129.02 285.58 Experiment D Ra e Parameter (units) ent Mean Mi Max AAD-1 7.29 0.00 9.26 Stalk Lodging (% Control 4.17 0.00 39.06 Final Population AAD-1 29.93 27.P0 34.00 (plants/acre in 1OOP's) Control 31.86 29.00 35.00 AAD-1 18.74 19.40 24.40 Grain re (%) Control 19.32 13.35 25.70 AAD-1 56.59 54.80 58.30 Test Weight (lb/bushel) Control 55.50 52.70 57.40 AAD-1 203.55 196.51 240.17 Yield (bushels/acre) Control 199.82 118.56 264.11 Example 8 - Use of Corn Event 278-9 Insertion Site for Targeted Integration Consistent agronomic performance of the transgene of corn event DAS9 over several generations under field conditions suggests that these identified regions around the corn event DAS9 insertion site provide good genomic ons for the targeted integration of other enic genes of interest. Such targeted integration overcomes the problems with so- called "position effect," and the risk of creating a mutation in the genome upon integration of the transgene into the host. Further advantages of such targeted integration include, but are not d to, reducing the large number of transformation events that must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene sion without also exhibiting alities resulting from the inadvertent insertion of the transgene into an important locus in the host genome. Moreover, such targeted integration allows for stacking transgenes rendering the breeding of elite plant lines with both genes more efficient.

Using the disclosed teaching, a skilled person is able to target cleic acids of interest to the same insertion site on chromosome 2 as that in corn event DAS9 or to a site in close proximity to the insertion site in corn event 278-9. One such method is disclosed in International Patent Application No. WO2008/021207, herein incorporated by reference in its entirety.

Briefly, up to 20 Kb of the genomic sequence flanking 5' to the insertion site and up to Kb of the genomic sequence flanking 3' to the insertion site (portions of which are identified with reference to SEQ ID NO:29) are used to flank the gene or genes of st that are intended to be inserted into a genomic location on chromosome 2 via homologous recombination. The gene or genes of interest can be placed exactly as in the corn event DAS9 insertion site or can be placed anywhere within the 20 Kb regions around the corn event DAS9 insertion sites to confer consistent level of transgene expression without ental s on the plant. The DNA vectors containing the gene or genes of interest and ng sequences can be delivered into plant cells via one of the l methods known to those skilled in the art, including but not limited to Agrobacterium-mediated transformation. The insertion of the donor DNA vector into the corn event DAS9 target site can be further enhanced by one of the several s, ing but not limited to the co-expression or up-regulation of recombination enhancing genes or down-regulation of endogenous recombination ssion genes. Furthermore, it is known in the art that double-stranded cleavage of specific sequences in the genome can be used to increase homologous recombination frequency, therefore insertion into the corn event DAS9 insertion site and its flanking regions can be enhanced by expression of natural or designed sequence-specific endonucleases for cleaving these ces.

Thus, using the teaching provided herein, any heterologous nucleic acid can be inserted on corn chromosome 2 at a target site located between a 5' molecular marker sed in e 4 and a cular marker discussed in Example 4, preferably within SEQ ID NO:29, and/or regions thereof as discussed elsewhere herein.

Example 9 - Excision of the pat Gene Expression Cassette from Corn Event DAS9 The removal of a selectable marker gene expression cassette is advantageous for targeted ion into the genomic loci of corn event DAS9. The removal of the pat selectable marker from corn event DAS9 allows for the re-use of the pat selectable marker in targeted integration of polynucleic acids into chromosome 4 in subsequent generations of corn.

Using the disclosed teaching, a skilled person is able to excise polynucleic acids of interest from corn event DAS9. One such method is disclosed in Provisional US Patent ation No. 61/297,628, herein incorporated by reference in its entirety.

Briefly, sequence-specific endonucleases such as zinc finger nucleases are designed which recognize, bind and cleave specific DNA sequences that flank a gene expression cassette.

The zinc finger nucleases are delivered into the plant cell by crossing a parent plant which contains transgenic zinc finger nuclease sion cassettes to a second parent plant which contains corn event DAS9. The resulting progeny are grown to maturity and analyzed for the loss of the pat expression cassette via leaf painting with a ide which contains glufosinate. Progeny plants which are not resistant to the herbicide are confirmed molecularly and ed for self-fertilization. The excision and removal of the pat expression cassette is molecularly confirmed in the progeny obtained from the self-fertilization. Using the teaching provided herein, any heterologous nucleic acid can be excised from corn chromosome 2 at a target site located between a 5' lar marker and a 3'molecular marker as discussed in Example 4, ably within SEQ ID NO:29 or the indicated regions thereof.

Example 10 - Resistance to brittlesnap Brittlesnap refers to breakage of corn stalks by high winds following applications of growth regulator herbicides, usually during periods of fast growth. Mechanical "push"' tests, which use a bar to ally push the com to simulate damage due to high winds, were performed o hybrid corn containing event 278-9 and control planis not containing event DAS9. The treatments were completed at four different geographical locations and were replicated four times (there was an exception for one trial which was o ly replicated three times).

The plots consisted of eight rows: four rows of each of the two hybrids, with two rows contammg event DAS9 and two rows t the event. Each row was twenty feet in length. Co plants were grown to the V4 developmental stage, and a commercial herbicide containing 2.4-D (Weedar 64, Nufarm Inc., Burr Ridge, IL) was applied at rates of 120 g ae/ha, 2240 g ae/ha and 44 80 g ae/ha . Seven days after application of the herbicide, a ical push test was performed. The mechanical push test for esnap consisted of pulling a 4-foot bar down the two rows of corn to simulate wind damage. Height of the bar an speed of travel were set to provide a low level of stalk breakage (10% or less) with untreated plants to ensure a test severe enough to demonstrate a difference between treatments. The directionali ty of the brittlesnap treatment was applied against leaning com.

Two of the trial locations experienced high winds and thunderstorms 2-3 days after application of the 2,4-D herbicide. On two consecutive days, a thunderstorm commenced in Huxley IA. Wind speeds of 2 to 1 m s with high speeds of 33 m s were reported at the site of the field p ot. The wind direction was variable. On one day, a thunderstorm was ed i Lanesboro MN. Winds of high velocity were reported at the site of this field plot In addition, both storms produced rain. The combination of rain and wind attributed to the reported brittlesnap damage.

Assessments of the brittlesnap damage which resulted from the ical push test (and ent weather) were made by visually rating the percentage of injur/. Prior to the mechanical brittlesnap bar treatment plant stand counts were made for the hybrid corn containing event 278-9 and controls. Several days after the brittlesnap bar treatment the plot stand counts were ssed. The percentage of leaning and percentage of reduced stand within the plot was determined (Table 26). The data from the trials demonstrated that hybrid corn containing event DAS9 has less propensity for brittlesnap as compared to the null plants following an application of 2,4-D.

Table 26: DAS9 Co Bri esr ap Tolerance to V4 Application of 2,4-D Amine. Th percentage of brittiesnap was ated for hybrid corn plants containing event DAS-40278 1Thunderstorm and high winds occurred 2-3 days after application in two trials Treatments replicated four times in a randomized complete block design (one trial was only completed for three replications) Means corrected for occurrences in untreated (untreated means forced to zero) Example 11 - Protein Analysis of Grain Grain with increased total protein content was produced from hybrid corn containing event DAS9 as compared to control plants not containing the event. Two consecutive multisite field trails were conducted that included rayed and herbicide-treatments with three different ide ations. In 7 of the 8 statistical comparisons, the 278-9 event produced grain with significantly higher total protein content (Table 27). This data is corroborated by analyses of individual amino acids.

Table 27: Protein content of grain from multisite field trials Agronomic characteristics of hybrid corn containing event DAS9 compared to nearisoline corn were collected from multiple field trials across diverse geographic nments for a growing season. The data were collected and analyzed for agronomic characteristics as described in e 7. The results for hybrid corn lines containing event DAS9 as compared to null plants are listed in Table 28. Additionally, agronomic characteristics for the hybrid corn lines containing event DAS9 and null plants sprayed with the ides quizalofop (280 g ae/ha) at the V3 stage of pment and 2,4-D (2,240 g ae/ha) sprayed at the V6 stage of development are described in Table 29.

Table 28: yield, percent re, and final population results for hybrid corn containing event DAS- Table 29: yield, percent moisture, percentage stock lodging, percentage root lodging and total population for hybrid corn lines containing event DAS9 as compared to the near-isoline control.

Corn #l LSD (0.5) 6.9 0.358 0.98 1.65 0.7 Spray Trial Hybrid Corn #2 198.6 26.76 0.38 2.08 29.29 Containing DAS- 40278-9 Control Hybrid 172.3 23.76 1.5 39.16 28.86 Corn #2 LSD (0.5) 13.3 1.107 0.89 10.7 1.1 Non Spray Hybrid Corn #2 207.8 24.34 0.22 0.59 3 1 Containing DAS- 40278-9 Control Hybrid 206.2 24.88 0.35 0.12 30.94 Corn #2 LSD (0.5) 8.0 0.645 0.55 1.79 0.9 e 13 - Pre-plant and/or Pre-emergence Applications Preplant burndown ide applications are intended to kill weeds that have emerged over winter or early spring prior to planting a given crop. Typically these applications are applied in no-till or reduced tillage ment systems where physical removal of weeds is not completed prior to planting. A herbicide program, therefore, must control a very wide um of broadleaf and grass weeds present at the time of planting. Glyphosate, gramoxone, and glufosinate are examples of non-selective, non-residual herbicides widely used for preplant burndown herbicide applications.

Some weeds, however, are difficult to control at this time of the season due to one or more of the following: inherent insensitivity of the weed species or biotype to the herbicide, relatively large size of winter annual weeds, and cool weather ions limiting herbicide uptake and activity. l herbicide options are available to tank mix with these herbicides to increase spectrum and ty on weeds where the non-selective herbicides are weak. An example would be 2,4-D tank mix applications with glyphosate to assist in the control of Conyza canadensis (horseweed). Glyphosate can be used from 420 to 1680 g ae/ha, more typically 560 to 840 g ae/ha, for the preplant burndown control of most weeds t; however, 280 - 1120 g ae/ha of 2,4-D can be d to aid in control of many broadleaf weed species (e.g., horseweed). 2,4-D is an herbicide of choice because it is ive on a very wide range of broadleaf weeds, effective even at low temperatures, and extremely inexpensive. However, if the subsequent crop is a sensitive dicot crop, 2,4-D residues in the soil (although short-lived) can negatively impact the crop. Crops that contain an aad-1 gene are tolerant to 2,4-D and are not negatively impacted by 2,4-D residues in the soil. The increased ility and reduced cost of tankmix (or commercial premix) partners will improve weed control options and increase the robustness of burndown applications in important no-till and reduced tillage situations. aad-1 Corn Transgenic hybrid corn (pDAS 78) containing the aad-l gene which encodes the yalkanoate dioxygenase (AAD-1) protein was evaluated for tolerance to preemergence applications of 2,4-D in the field. Trials were ted at multiple locations in Mississippi, Indiana, and Minnesota using a randomized complete block design with three replications of two row plots, approximately 6 m in length, at each site. Herbicide-treated plots were paired with untreated plots to e accurate evaluation of emergence and early season growth. Herbicide treatments of 1120, 2240, and 4480 g ae/ha of 2,4-D amine were applied shortly after planting but before crop emergence (0-2 days after planting). Soil and precipitation information for these trials is contained in Table 30.

Table 30. Soil and precipitation ation for tions of pDAS1740-278 hybrid tolerance Approximately 16-21 days after planting and application of 2,4-D, injury averaged from 12 to 31% for the conventional control hybrid as rates increased from 1120 to 4480 g ae/ha of 2,4-D amine. Injury to hybrid corn containing pDAS 1740-278 ranged from 3 to 9% across the same rate range. The current proposed 2,4-D target application rates for transgenic hybrid corn (pDAS 78) containing the aad-\ gene are at or below 1,120 g ae/ha for 2,4-D. Results of field testing indicate that hybrid corn containing pDAS 1740-278 provided robust tolerance of 2,4-D herbicide treatments at rates more than two to four times the proposed target use rates with minimal damage (Table 31). aae = acid equivalent, ha = e d 0-2 days after planting, before crop emerg Evaluations taken 16-21 days after application.

Preemergence applications of 2,4-D amine are applied at rates of 1120, 2240, 4480 g ae/ha at 7 days, 15 days, or 30 days preplanting to hybrid corn containing the aad-1 gene and conventional control hybrids. The preemergence applications are applied using art recognized procedures to field plots which are located at geographically distinct locales. ide-treated plots are paired with untreated plots to provide accurate evaluation of emergence and early season growth. imately 16-21 days after planting and 2,4-D applications at 7, 15 or 30 days preplanting; injury of the conventional l hybrids and hybrid corn containing aad-1 are measured. Results of field testing indicate that hybrid corn containing aad-1 provides robust tolerance of rgence treatments of 2,4-D herbicide at 7, 15, or 30 days preplanting. aad-1 Cotton Transgenic cotton containing the aad-1 gene which encodes the aryloxyalkanoate dioxygenase (AAD-1) protein was evaluated for tolerance to preemergence applications of 2,4-D in the field. Trials consisted of three replications and were conducted at multiple locations in Mississippi, Georgia, Tennessee, and Arkansas. A randomized complete block design of a single row separated by a guard row, approximately 10 feet (20 feet for the sippi trial) in length was used. Seed was planted using 8 seed per foot, plants were then hand thinned to 3.5 plants/foot of row. Herbicide-treated plots were paired with untreated plots to provide accurate evaluation of emergence and early season growth. All plots received at least ½inch of rain or irrigation water within 24 hours of application. Herbicide treatments of 560, 1120, 2240, and 4480 g ae/ha of 2,4-D amine (WEEDAR 64, Nufarm, Burr Ridge, II) were applied shortly after planting but before crop emergence. Soil and precipitation information for these trials is contained in Table 32.

Table 32. Soil and reci n information for evaluations of aad-l containin cotton.

The preemergence ations of 2,4-D at 560, 1120, and 2240 gm ae/ha did not significantly affect plant stands of transgenic cotton containing the aad-l gene as compared to the untreated plot.

Approximately 7 to 8 days after planting and preemergence application of 2,4-D at concentrations of 560, 1120, 2240, and 4480 gm ae/ha, stand reductions of 3%, no reduction, 19%, and 34%, respectively, were reported for the aad-l containing cotton as compared to stand ion of the untreated control. At 11 to 14 days after planting and application of 2,4-D at concentrations of 560, 1120, 2240, and 4480 gm ae/ha, stand reductions of 5%, 2%, 14%, and %, tively, were reported for the aad-l ning cotton as compared to stand reduction of the untreated l.

Preemergence application of 2,4-D at concentrations of 560, 1120, 2240, and 4480 gm ae/ha did not cause ty. Chlorosis was observed at <5% visual rating, 7-8 days after application for the 2240 and 4480 gm ae/ha treatments. Chlorosis was not ed for the 560 and 1120 gm ae/ha treatments at 7 to 8 days after application. Moreover, chlorosis was not detected in any of the aad-l containing cotton, treated with concentrations of 560, 1120, 2240, and 4480 gm ae/ha, for the remainder of the season Preemergence applications of 2,4-D at concentrations of 560 and 1120 gm ae/ha resulted in minimal growth inhibition of less than 2% (which is statistically insignificant from the untreated control plot). These s were consistent throughout the trial: 7 to 8; 11 to 14; 27 to ; and, 52 to 57 days after application. Preemergence application of 2,4-D at a tration of 2240 gm ae/ha resulted in a growth inhibition of <10%. Preemergence application of 2,4-D at a concentration of 4480 gm ae/ha resulted in growth tion of 27%, 28%, 17% , and 8.3% when rated at 7 to 8, 11 to 14, 27 to 30, and 52 to 57 days after application.

Injury caused by preemergence application of 2,4-D at concentrations of 560 and 1120 gm ae/ha was not significantly different from the untreated l plot. The injury ratings for the aad-1 cotton plants treated with 2,4-D at a concentration of 2240 gm ae/ha ranged from 3% to 15 % over the course of the trial. The injury ratings for the aad-1 cotton plants treated with 2,4-D at a concentration of 4480 gm ae/ha ranged from 8% to 34% over the course of the trial. ences in plant height, fruiting pattern, and yield were not detected in aad-1 cotton which had been treated with preemergence application of 2,4-D at concentrations 560, 1120, 2240, and 4480 gm ae/ha.

Preemergence applications of 2,4-D amine are applied at rates of 560, 1120, 2240, 4480 g ae/ha at 7 days, 15 days or 30 days preplanting to cotton containing the aad-1 gene and control cotton. The preemergence applications are applied using art recognized procedures to field plots which are located at geographically distinct locales. Herbicide-treated plots are paired with untreated plots to provide accurate evaluation of emergence and early season . After planting and 2,4-D applications at 7, 15 or 30 days nting; injury of the cotton containing the aad-1 gene and control is measured. Results of field testing indicate that cotton containing the aad-1 gene provides tolerance of preemergence treatments of 2,4-D herbicide at 7, 1 , or 30 days preplanting.

This e discusses some of many options that are available. Those skilled in the art of weed l will note a variety of other applications including, but not limited to gramoxone + 2,4-D or glufosinate + 2,4-D by utilizing products described in federal herbicide labels (CPR, 2003) and uses described in Agriliance Crop tion Guide (2003), as es. Those skilled in the art will also recognize that the above example can be applied to any 2,4-D-sensitive (or other phenoxy auxin herbicide) crop that would be protected by the AAD-1 (v3) gene if stably transformed. 1001482000

Claims (19)

1. A method of controlling weeds, said method comprising planting seed in an area, and applying an aryloxyalkanoate herbicide to said area Within 30 days before planting seed in said area, said seed comprising a polynucleotide that encodes an AAD-l protein having at least 75% identity with the AAD-l protein encoded by nucleotides 4265-5152 of SEQ ID NOZ29.

2. The method of claim 1, wherein said herbicide is selected from the group consisting of 2,4—D; 234—DB; MCPA; MCPB; and an aryloxyphenoxypropionate.

3. The method of claim 1 or 2, wherein said method comprises applying a second herbicide to said area.

4. The method of claim 3, wherein said second ide is selected from the group 15 consisting of glufosinate, glyphosate, and dicamba.

5. The method of any one of claims 1 to 4, wherein said area is a field, and said herbicide is applied within 7 days, 11 days, 14 days, or 15 days before planting said seed in said field. 20

6. The method of any one of claims 1 to 5, wherein said method is used for controlling sate—resistant weeds in said area, wherein said seed is grown into a plant further comprising a glyphosate tolerance trait, and said method comprises applying an aryloxyalkanoate herbicide to at least a portion of said area. 25

7. The method of any one of claims 1, or 3 t0 6 wherein said ide is a phenoxy auxin.

8. The method of claim 6 wherein said herbicide is applied from a tank mix with glyphosate. 30

9. The method of claim 6 or 8 wherein at least one of said weeds is a glyphosate-resistant volunteer of a different species than said plant.

10. The method of any one of the preceding , wherein said weeds are in an area under cultivation, said area comprising a plurality of plants grown from said seeds, said method r 35 comprising applying an yalkanoate herbicide over the top of the plants. 1001482000

11. The method of claim 10, said method comprising applying said herbicide jointly or separately with a second herbicide.

12. The method of any one of the preceding , wherein said aryloxyalkanoate herbicide is a 2,4-D amine, and said 2,4—D amine is applied at a rate within a range selected from the group consisting of 560, 1120, 2240, and 4480 g ae/ha.

13. The method of claim 4, wherein said glyphosate is applied at a rate from 420 to 1680 g 10 ae/ha.

14. The method ofclaim 13, wherein said glyphosate is d at a rate from 560 to 840 g ae/ha.

15 15. The method of any one of claims 1 to l l or 13 to 14, n said aryloxyalkanoate herbicide is a 2,4—D amine, and said 2,4—D amine is applied at a rate from 280 — l 120 g ae/ha.

16. The method of any one of the preceding claims, wherein said seed is from a corn plant. 20

17. The method of any one of the preceding claims, n said polynucleotide comprises SEQ ID NOz29,

18. The method of any one of the preceding claims, wherein said seed comprises a genome comprising AAD—l event DAS—40278—9 as present in seed deposited with American Type 25 Culture Collection (ATCC) under Accession No. PTA—10244.

19. The method of claim 1, substantially as hereinbefore described.