Cotton event MON 88913 and compositions and methods for detection thereof

Invention Patent Descriptive Report for METHODS OF PRODUCTION OF A Glyphosate-Tolerant COTTON PLANT UNDERSTANDING EVENT MON 88913, OF DNA DETECTION, DETERMINATION OF COTTON PLANT ZYGOSITY AND WEED CONTROL.

[001] This patent application claims the benefit of provisional patent application n^O US 60 / 447,184, filed on February 12, 2003, the content of which is incorporated herein entirely by reference. Technical Field of the Invention [002] The present invention relates to the field of plant molecular biology. More specifically, the invention relates to a glyphosate-tolerant cotton event, MON 88913, and assays and methods for detecting the presence of the MON 88913 cotton event DNA in a plant sample, and its compositions.

Background to the Invention [003] Cotton is an important fiber crop in many areas of the world. Biotechnology methods have been applied to cotton to improve agronomic traits and product quality. The method for introducing transgenes into cotton plants was demonstrated in patent no.^O US 5,004,863. One of those important agronomic traits in cotton production is herbicide tolerance, particularly tolerance to glyphosate herbicide. This trait was introduced in cotton plants and is a successful product now used in cotton production. The current commercial Roundup Ready® cotton event (1445) provides excellent tolerance to glyphosate, the active ingredient in Roundup®, up to the quadrifoliate stage (Nida et al., J. Agric. Food Chem. 44: 1960-1966 (1996); Nida et al., J. Agric. Food Chem. 44: 1967-1974 (1996)). However, foliar application after the quadrifoliate stage should be limited due to

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2/42 to insufficient tolerance in male reproductive tissues under certain environmental conditions. This lack of tolerance in male breeders appears to be the result of insufficient expression of CP4 EPSPS in critical tissues, higher sensitivity of these tissues to glyphosate, and accumulation of high amounts of glyphosate in these resistant draining tissues (Pline et al., Weed Sci. 50: 438-447 (2002)). There is a need to obtain a cotton plant that is more highly tolerant to glyphosate than Roundup Ready ® 1445 cotton.

[004] It would be advantageous to be able to detect the presence of a specific event, to determine whether the offspring of a sexual cross contains a transgene of interest. In addition, it would be useful to obtain a method to detect a specific event to meet regulations that require approval before marketing or labeling of foods derived from recombinant plant cultures, for example. It is possible to detect the presence of a transgene by any well-known method of nucleic acid detection, such as polymerase chain reaction (PCR) or DNA hybridization, using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, 3 'transcription terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct, unless the sequence of the chromosomal genomic DNA adjacent to the inserted DNA (flanking genomic DNA) is known. Methods of detecting event-specific DNA for a glyphosate-tolerant cotton event, 1445, have been described (document no.^O US 20020120964, hereby incorporated by reference in its entirety).

[005] The present invention relates to a glyphosate-tolerant cotton event, MON 88913, compositions contained therein, and the

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3/42 method for the detection of the genomic / transgene insertion region in the MON 88913 cotton event and its progeny.

Summary of the Invention [006] The present invention relates to the transgenic cotton event designated MON 88913, which was deposited with the American Type Culture Collection (ATCC) under the N^O Access Code PTA-4854. Another aspect of the invention comprises progeny plants, or seeds, or regenerable parts of the MON 88913 cotton event plants and seeds. The invention also includes plant parts from the MON 88913 cotton event, which include, but are not limited to, pollen , egg, flowers, cocoons, fluff, buds, roots, and leaves. The invention relates to a cotton plant that has a glyphosate-tolerant phenotype and the unusual genetic compositions of MON 88913.

[007] One aspect of the invention provides DNA compositions and methods for detecting the presence of a genomic / transgene junction region of the MON 88913 cotton plant event. Isolated DNA molecules are provided that comprise at least one genomic / transgene junction DNA molecule, selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and their complements, where the junction molecule covers the site of insertion that comprises a heterologous DNA inserted into the cotton genome and the genomic DNA of the cotton cell that flanks the insertion site in the MON 88913 cotton event. A cotton seed and its plant material, which comprises these molecules, is an aspect of this invention.

[008] An unusual, isolated DNA molecule is supplied, which is a 5 'genomic / transgene region, SEQ ID NO: 3, or the complement of it, where this DNA molecule is unusual in the MON 88913 cotton event. cotton plant and the seed comprising SEQ ID NO: 3 in its genome is an aspect of this invention. According

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4/42 another aspect of the invention, an isolated DNA molecule is provided which is a 3 'genomic / transgene region, SEQ ID NO: 4, or the complement thereof, where this DNA molecule is unusual in the MON 88913 cotton event A cotton plant and the seed comprising SEQ ID NO: 4 in its genome is an aspect of this invention.

[009] According to another aspect of the invention, two DNA molecules are provided for use in a DNA amplification method, where the first DNA molecule comprises at least 11 or more contiguous polynucleotides from any part of the transgene region of the molecule of DNA of SEQ ID NO: 3, and a DNA molecule of similar length to any part of a region of SEQ ID NO: 3, flanking 5 'of cotton genomic DNA, where these DNA molecules, when used together, are useful as a set of primers in a DNA amplification method, which produces an amplicon. The amplicon produced using the DNA primer set in the DNA amplification method is diagnostic for the MON 88913 cotton event. Any amplicon produced from the MON 88913 DNA by DNA primers that are homologous or complementary to any part of SEQ ID NO: 3 is an aspect of the invention.

[0010] According to another aspect of the invention, two DNA molecules are provided for use in a DNA amplification method, where the first DNA molecule comprises at least 11 or more contiguous polynucleotides from any part of the transgene region of the molecule of DNA of SEQ ID NO: 4, and a DNA molecule of similar length from any part of a region of SEQ ID NO: 4, flanking 3 'of cotton genomic DNA, where these DNA molecules, when used together, are useful as a set of primers in a DNA amplification method, which

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5/42 produces an amplicon. The amplicon produced using the DNA primer set in the DNA amplification method is diagnostic for the MON 88913 cotton event. Any amplicon produced from the MON 88913 DNA by DNA primers that are homologous or complementary to any part of SEQ ID NO: 4 is an aspect of the invention.

[0011] In accordance with another aspect of the invention, methods are provided to detect the presence of DNA that specifically corresponds to the DNA of the DNA event 88913, in a sample. Such methods include: (a) placing the sample comprising DNA in contact with a set of DNA primers that, when used in a nucleic acid amplification reaction with the genomic DNA from the MON 88913 cotton event, produces an amplicon which is diagnosis for the MON 88913 cotton event; (b) carry out a nucleic acid amplification reaction, thereby producing amplicon; and (c) detecting the amplicon.

[0012] In accordance with another aspect of the invention, methods are provided to detect the presence of DNA that specifically corresponds to the DNA of the DNA event 88913, in a sample. Such methods include: (a) placing the sample comprising DNA in contact with a DNA probe comprising SEQ ID NO: 1 or SEQ ID NO: 2, which hybridize under stringent hybridization conditions with the genomic DNA of the MON cotton event 88913 and do not hybridize under stringent hybridization conditions with a cotton plant DNA that serves as a control; subject the sample and probe to stringent hybridization conditions; and (c) detecting probe hybridization to the 88913 cotton event DNA.

[0013] In accordance with another aspect of the invention, methods are provided to produce a cotton plant that tolerates the application of glyphosate, which comprise the steps of: (a) sexually crossing

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6/42 a first event of MON 88913 parental cotton, which comprises the expression strips of the present invention, which gives tolerance to the application of glyphosate, and a second parental cotton plant which lacks tolerance to glyphosate, thus producing a plurality of progeny plants; and (b) select the progeny plant that tolerates the application of glyphosate. Such methods may optionally comprise the additional step of backcrossing the progeny plant to the second parent cotton plant and selecting for the glyphosate tolerant progeny, to produce a true generation cotton variety that tolerates the application of glyphosate.

[0014] In accordance with another aspect of the invention, a method is provided to determine the zygosity of the MON 88913 cotton event progeny, comprising: (a) placing the sample comprising the cotton DNA in contact with a set of primers , comprising SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, which, when used in a nucleic acid amplification reaction with the MON 88913 cotton event genomic DNA, produces a first amplicon that is diagnostic for the MON 88913 cotton event; and (b) carry out a nucleic acid amplification reaction, thereby producing amplicon; and (c) detecting the amplicon first; and (d) placing the sample comprising the cotton DNA in contact with said set of primers, which, when used in a nucleic acid amplification reaction with the genomic DNA of cotton plants, produces a second amplicon which comprises the native genomic DNA of cotton, homologous to the cotton genomic region of a transgene insert identified as the MON 88913 cotton event; and (e) carry out a nucleic acid amplification reaction, thereby producing the second amplicon; and (f) detecting the second amplicon; and (g) comparing the first and second amplicons in a sample, where

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7/42 the presence of both amplicons indicates that the sample is heterozygous for insertion of the transgene.

[0015] A method for determining zygosity comprises placing a cotton DNA sample in contact with primers and probes comprising SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; use a Taqman® terminal PCR condition; and detect the products of the amplicons.

[0016] A method for controlling weeds in a MON 88913 cotton event field or crop comprises the step of applying an effective herbicide amount of glyphosate containing herbicide to the MON 88913 cotton.

[0017] The foregoing aspects and other aspects of the invention will become more evident from the following detailed description and the accompanying drawings.

Brief Description of the Drawings [0018] Figure 1 is the plasmid map of pMON51915.

[0019] Figure 2 is the genomic organization of insertion in the MON 88913 cotton event.

[0020] Figure 3 is the 5 'junction sequence of MON 88913 DNA (SEQ ID NO: 1) and the 3' junction sequence of MON 88913 DNA (SEQ ID NO: 2).

[0021] Figure 4 is the 5 'genomic / transgene DNA region of

MON 88913 (SEQ ID NO: 3).

[0022] Figure 5 is the 3 'genomic / transgene DNA region of

MON 88913 (SEQ ID NO: 4).

Detailed Description of Preferred Modalities [0023] The present invention relates to a glyphosate-tolerant cotton event, MON 88913, compositions contained therein, and the method for detecting the genomic / transgene insertion region in the MON 88913 cotton event and in your progeny. Definitions and

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The following methods are provided to further define the present invention and to guide those skilled in the art as to the practice of the present invention. Unless otherwise noted, terms and abbreviations must be understood according to conventional usage by those skilled in the relevant techniques. Definitions of common terms in molecular biology can also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5^Theedition, Springer-Verlag; New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for the DNA bases listed in 37 CFR § 1,822 is used.

[0024] As used herein, the term cotton means Gossypium hirsutum and includes all varieties of plants that can be created with the MON 88913 cotton event. The plant of the present invention is a cotton plant, more specifically the MON 88913 cotton plant. .

[0025] As used herein, the term comprise means includes, but without limitations.

[0026] As used herein, the term culture refers to plants or parts of cultivated plants, such as they are grown in a field, plot of land, row, greenhouse, land or container.

[0027] Glyphosate refers to N-phosphonomethylglycine and its salts. Nphosphonomethylglycine is a well-known herbicide that has activity over a wide spectrum of plant species. Glyphosate is the active ingredient in Roundup® (Monsanto Co.), a safe herbicide that has a desirably short half-life in the environment. Glyphosate is the active ingredient in the herbicide Roundup® (Monsanto Co.). Treatments with the glyphosate herbicide refer to treatments with the herbicide Roundup®, Roundup Ultra®, Roundup Pro®, or any other herbicidal formulation that contains glyphosate. Examples of glyphosate formulations on the market include, without limitation,

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9/42 those marketed by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® WEATHERMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP herbicides^® BIOACTIVE, ROUNDUP^® BIOFORCE, RODEO^®, POLARIS^®, SPARK^®, and ACCORD^®, all of which contain glyphosate as their isopropyl ammonium salt; those marketed by Monsanto Company as the ROUNDUP ® DRY and RIVAL® herbicides, which contain glyphosate as their ammonium salt; the one marketed by Monsanto Company as ROUNDUP ® GEOFORCE, which contains glyphosate as its sodium salt; and the one marketed by Syngenta Crop Protection as a TOUCHDOWN® herbicide, which contains glyphosate as its trimethyl sulfonium salt. When applied to a plant surface, glyphosate moves systemically through the plant. Glyphosate is phytotoxic due to its inhibition of the shikimic acid pathway, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the enzyme 5-enolpyruvyl-3-phosphochiquime synthase (EPSPS), found in plants. Tolerance to glyphosate can be achieved by the expression of bacterial variants of EPSPS and plant variants of EPSPS that have lower affinity for glyphosate, and therefore retain their catalytic activity in the presence of glyphosate (US patents 5,633,435, 5,094,945, 4,535 .060 and

6.040.497).

[0028] A transgenic event is produced by transforming a plant cell with heterologous DNA, such as, for example, a nucleic acid construct (pMON51915, Figure 1) that includes a transgene of interest; regeneration of a population of plants resulting from the insertion of the transgene in the genome of the plant cell, and selection of a specific plant characterized by insertion in a specific location of the genome. The term event refers to the original transforming plant and the progeny of the transformant. that include the

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10/42

Heterologous DNA. The term event also includes the progeny produced by a sexual heterocruzado between the event and another plant in which the progeny includes the heterologous DNA. Even after repeated backcrosses to a recurrent parent, the inserted DNA and the genomic DNA flanking the transformed parent event are present in the progeny of the crossing at the same chromosomal site. The term event also refers to the DNA of the original transformant that comprises the inserted DNA, and the flanking genomic sequence immediately adjacent to the inserted DNA, which would be expected to be transferred to a progeny that receives the inserted DNA that includes the transgene of interest as a result of a sexual crossing of a parental lineage that includes the inserted DNA (for example, the original transformant and the progeny resulting from self-pollination) and a parental line that does not contain the inserted DNA.

[0029] The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to the structure of chromatin (such as heterochromatin) or the proximity of transcriptional regulatory elements (such as enhancers) near the site of integration (Weising, et al., Ann. Rev. Genet. 22: 421-477, 1988). For this reason, it is often necessary to screen a large number of events to identify an event characterized by optimal expression of a introduced gene of interest. For example, it has been observed in plants and other organisms that there can be a wide variation in the levels of expression of a transgene introduced between events. There may also be differences in spatial or temporal patterns of expression, such as differences in the relative expression of a transgene in various plant tissues, which may not correspond to the expected patterns of transcriptional regulatory elements present in the construction of the introduced gene. For this reason, it is common to produce

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11/42 hundreds of thousands of different events and screen events for a single event that has the desired levels and patterns of transgene expression for commercial purposes. An event that has the desired levels or patterns of transgene expression is useful for the transgene's introgression into other genetic antecedents by sexual crossing, using conventional breeding methods. The progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in numerous varieties that are well adapted to growing conditions and local market demands.

[0030] A glyphosate-tolerant cotton plant can be created first by sexually crossing a first parent cotton plant, consisting of a cotton plant developed from the cell of the transgenic cotton plant derived from the transformation with the vegetable expression tapes contained in pMON51915 and which tolerates the application of the glyphosate herbicide, with a second parent cotton plant that lacks tolerance to the glyphosate herbicide, thus producing a plurality of plants of the first progeny; and then selecting a first progeny plant that is tolerant to the herbicide glyphosate; and effecting the self-pollination of the first progeny plant, thereby producing a plurality of second progeny plants; and then select a plant tolerant to the herbicide glyphosate from the second progeny plants. These steps may also include backcrossing the first glyphosate tolerant plant or the second glyphosate tolerant plant to the second parent cotton plant or third parent cotton plant, thereby producing a cotton plant that tolerates application of the glyphosate herbicide. In the present invention, the cotton plant

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Transgenic 12/42 is also defined as MON88913 cotton event and can be referred to here as MON 88913.

[0031] It should also be understood that two different transgenic plants can also be mated to produce offspring that contain two exogenously added segregating genes. Self-pollination of the appropriate progeny can produce plants that are homozygous for both added exogenous genes. Backcrossing to a parental plant and heterocrossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods, which are commonly used for different traits and cultures, can be found in one of several references, for example, in Fehr, Breeding Methods for Cultivar Development, editor Wicox, J., American Society of Agronomy , Madison, WI (1987).

[0032] A probe is an isolated nucleic acid to which a detectable marker or conventional reporter molecule, such as a radioactive isotope, a ligand, a chemiluminescent agent, or an enzyme, is attached. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of the MON 88913 genomic DNA, either from a MON 88913 plant or from a plant that includes MON 88913 DNA. The probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of that target DNA sequence .

[0033] DNA primers are nucleic polyacid acids that are annealed to a complementary target DNA strand by nucleic acid hybridization, to form a hybrid between the primer

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13/42 and the target DNA strand. then extended along the strand of target DNA by a polymerase, such as a DNA polymerase. A pair of DNA primers or a set of DNA primers of the present invention refer to at least two molecules of DNA primers useful for amplifying a target DNA sequence, for example, by the polymerase chain reaction ( PCR) or other conventional nucleic acid amplification methods.

[0034] Probes and primers are generally 11 or more polynucleotides in length, often 18 polynucleotides or more, 24 polynucleotides or more, or 30 polynucleotides or more. Such probes and primers are selected to be of sufficient length to hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity to the target sequence, although probes that differ from the target sequence and retain the ability to hybridize to the target sequences can be designed by methods conventional.

[0035] Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2^The edition, volumes 1-3, editors Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter, Sambrook et al., 1989); Current Protocols in Molecular Biology, editors Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, Ausubel et al., 1992); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990. The pairs of DNA primers for PCR can be derived from a known sequence, for example, using computer programs

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14/42 intended for this purpose, such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

[0036] The primers and probes based on flanking genomic DNA and transgenic insertion sequences described in this case to confirm (and, if necessary, correct) the DNA sequences described by conventional methods, for example, by isolating the genomic DNA from MON 88913, once again cloning the genomic / transgene regions and sequencing such DNA molecules.

[0037] The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA molecule. Any conventional method of hybridizing or amplifying nucleic acids can be used to identify the presence of DNA from a transgenic event in a sample. Nucleic polyacid molecules and fragments are capable of hybridizing specifically to other nucleic acid molecules under certain circumstances. As used herein, two molecules of nucleic polyacids are said to be capable of hybridizing specifically to one another if the two molecules are capable of forming a double-stranded antiparallel nucleic acid structure. One nucleic acid molecule is said to be the complement of another nucleic acid molecule if they are completely complementary. As used herein, molecules are said to show complete complementarity when each 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 each other with sufficient stability to allow them to remain ringed together under conventional conditions of at least low severity. Similarly, molecules are said to be complementary if

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15/42 they can hybridize to each other with sufficient stability to allow them to remain ringed together under conventional conditions of high severity. Conventional severity conditions are described by Sambrook et al., 1989, and Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Distances from complete complementarity are therefore permissible, provided that such distances do not completely impede the ability of the molecules to form a double-stranded structure. For a nucleic acid molecule to serve as a primer or probe it needs only to be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the specific concentrations of solvents and salts employed.

[0038] As used herein, a substantially homologous DNA sequence is the sequence of a DNA molecule that hybridizes specific to the complement of a target DNA molecule with which it is being compared under conditions of high severity. The appropriate stringency conditions that promote DNA hybridization, such as 6.0 sodium chloride / sodium citrate (SSC) at about 45^OC, and then a wash of 2.0 x SSC at 50 ^OC, are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the concentration of salts in the washing step can be selected from a low severity of about 2.0 x SSC at 50^OC to a high severity of about 0.2 x SSC at 50 ^OC. In addition, the temperature in the washing step can be increased from low severity conditions to room temperature, about 22 ^OC, until severe conditions at about 65 ^OC. The temperature and also the salt can be varied, or the temperature or concentration of the salt can be kept constant,

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16/42 while the other variable is changed. In a preferred embodiment, a nucleic polyacid of the present invention must hybridize specifically to one or more of the nucleic acid molecules listed in SEQ ID NO: 3 or 4, or their complements or fragments of either under moderately severe conditions, such as example at 2.0 x SSC and about 65^OC. In a particularly preferred embodiment, a nucleic acid of the present invention must hybridize specifically to one or more of the nucleic acid molecules listed in SEQ ID NO: 3 or 4 or complements or fragments of any of them under conditions of high severity. In one aspect of the present invention, a preferred marker nucleic acid molecule of the present invention has the nucleic acid sequence set forth in SEQ ID NO: 1 or 2 or its complements or fragments thereof. In another aspect of the present invention, a preferred marker nucleic acid molecule of the present invention shares a substantial part of its sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or its complement or fragments of any of them, where the sequence identity is between 80% and 100% or 90% and 100%. In another aspect of the present invention, a preferred marker nucleic acid molecule of the present invention shares between 95% and 100% of the sequence identity set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or its complement or fragments of any one from them. SEQ ID NO: 1 or SEQ ID NO: 2 can be used as markers in plant breeding methods to identify the progeny of crosses similar to the methods described for the analysis of DNA markers with repetition of simple sequences, in DNA markers: Protocols , applications, and overviews, 1997, 173-185, editors Cregan etal., Wiley-Liss, NY, hereby incorporated by reference

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17/42 in its entirety. Hybridization of the probe to the target DNA molecule can be detected by any of the methods known to those skilled in the art, and they may include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

[0039] As for the amplification of a target nucleic acid sequence (such as by PCR), using a pair of specific amplification primers, severe conditions are conditions that allow the pair of primers to hybridize only to the target sequence of nucleic acids to which a primer that has the corresponding wild-type sequence (or its complement) would bind, and preferably, to produce a unique amplification product, amplicon, in a DNA thermal amplification reaction.

[0040] The specific term for (a target sequence) indicates that a probe or primer hybridizes under severe hybridization conditions only to the target sequence in a sample comprising the target sequence.

[0041] As used herein, the term amplified DNA or amplicon refers to the product of the nucleic acid amplification method directed at a target nucleic polyacid molecule that is part of a nucleic polyacid model. For example, to determine whether a cotton plant resulting from a sexual crossing contains the genomic DNA from the MON 88913 cotton event plant of the present invention, the DNA that is extracted from a tissue sample from the cotton plant can be subjected to a nucleic polyacid amplification method, using a primer pair that includes a primer derived from the DNA sequence in the MON 88913 plant genome, adjacent to the insertion site of the inserted heterologous DNA (transgenic DNA) and a second primer derived from the inserted heterologous DNA, to produce

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18/42 an amplicon that is diagnostic for the presence of the MON 88913 event DNA. The diagnostic amplicon has a length and a DNA sequence that are also diagnostic for the event. The amplicon may have a length in the range between the combined length of the primer pairs plus a pair of nucleotide bases, preferably plus about fifty nucleotide base pairs (pbs), more preferably, plus about two hundred and fifty pairs of nucleotide bases, and even more preferably, about four hundred and fifty pairs of nucleotide bases or more. Alternatively, a pair of primers can be derived from the genomic sequence on both sides of the inserted heterologous DNA, in order to produce an amplicon that includes the entire polynucleotide sequence of the insert (such as, for example, an advance primer isolated from the genomic part of SEQ ID NO: 3 and a reverse primer isolated from the genomic part of SEQ ID NO: 4, which amplifies a DNA molecule comprising the two expression strands of the DNA fragment pMON51915 that was inserted into the MON 88913 genome, with the insert comprising about 8,512 bp of the insert, Figure 2). A member of a pair of primers derived from the plant's genomic sequence can be located at a distance from the inserted DNA sequence, and this distance can be in the range between a pair of nucleotide bases and about twenty thousand base pairs of nucleotides. The use of the term amplicon specifically excludes primer dimers that can be formed in the DNA thermal amplification reaction.

[0042] Nucleic polyacid amplification can be performed by any of the various nucleic polyacid amplification methods known in these techniques, including polymerase chain reaction (PCR). Amplification methods are known in these techniques and are described, among others, in US Patent Nos. 4,683,195 and 4,683,202, and in PCR Protocols: A Guide to Methods and

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19/42

Applications, editors Inis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb (kilobase) of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl Acad. Sci. USA 91: 5695-5699, 1994). These methods, as well as other methods known in these DNA amplification techniques, can be used in the practice of the present invention. The sequence of insertion of heterologous DNA or the genomic DNA sequence flanking MON 88913 can be verified (and corrected, if necessary) by amplifying such DNA molecules from MON 88913 seeds or plants developed from the seed deposited with the ATCC with n^O of accession PTA-4854, using primers derived from the sequences provided herein, and then by standard DNA sequencing of the PCR amplicon or its cloned DNA fragments. DNA detection kits, which are based on DNA amplification methods, contain DNA primers that specifically amplify a diagnostic amplicon. The kit can provide a detection method based on agarose gel, Taqman® terminal, or any of a number of methods for detecting the diagnostic amplicon known in these techniques. A kit containing DNA primers homologous or complementary to any part of SEQ ID NO: 3 or SEQ ID NO: 4 is an object of the invention. [0043] The amplicon produced by these methods can be detected by a plurality of techniques. One of these methods is Genetic Bit Analysis (Nikiforov et al., Nucleic Acids Res. 22: 4167-4175, 1994), where a DNA oligonucleotide is drawn that overlaps the adjacent flanking genomic DNA sequence and also the sequence of DNA inserted. The oligonucleotide is immobilized in wells of a microtiter plate. After PCR of the region of interest (using a primer in the inserted sequence and one in the adjacent flanking genomic sequence), a PCR product with

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20/42 a single strand can be hybridized to the immobilized oligonucleotide and serves as a model for a single base extension reaction, using DNA polymerase and specific labeled dideoxynucleotide triphosphates (ddNTPs) for the next expected base. The reading can be fluorescent or ELISA-based. A signal indicates the presence of the genomic / transgenic sequence due to the successful amplification, hybridization and extension of a single base.

[0044] Another method is the Pyrosequencing technique described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is drawn that overlaps the genomic DNA and the junction of the insertion DNA. The oligonucleotide is hybridized to the single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a 5 'DNA polymerase, ATP, sulfurylase, luciferase, apyrase, 5' -adenosine phosphosulfate and luciferin. Deoxynucleotide triphosphates (dNTPs) are added individually and the incorporation results in a light signal that is measured. A light signal indicates the presence of the genomic / transgenic sequence due to the successful amplification, hybridization or extension of a single base.

[0045] Polarization under Fluorescence, described by Chen et al.

(Genome Res. 9: 492-498, 1999), is a method that can be used to detect the amplicon of the present invention. Using this method, an oligonucleotide is drawn that overlaps the genomic flanking and the joining of the inserted DNA. The oligonucleotide is hybridized to the 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 fluorescence-labeled ddNTP. The extension of a single base results in the incorporation of ddNTP. Incorporation can be measured

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21/42 as a change in polarization using a fluorimeter. A change in polarization indicates the presence of the genomic / transgenic sequence due to the successful amplification, hybridization or extension of a single base.

[0046] Taqman® (PE Applied Biosystems, Foster City, CA) is described as a method for detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, the FRET oligonucleotide probe is designed, which overlaps the genomic flanking and the junction of the inserted DNA. The FRET probe and the PCR primers (one primer in the inserted DNA and one in the sequence of the flanking genomic DNA) undergo a cycle in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the release cleavage of the fluorescent cluster out of the switch portion on the FRET probe. A fluorescent signal indicates the presence of the genomic / transgenic sequence due to successful amplification and hybridization.

[0047] Molecular beacons have been described (molecules that emit light when certain chemical reactions occur) Molecular beacons for use in sequence detection, as described by Tyangi et al. (Nature Biotech. 14: 303-308, 1996). Briefly, a FRET oligonucleotide probe is designed that overlaps the genomic flanking and the joining of the inserted DNA. The unique structure of the FRET probe results in the inclusion of a secondary structure that keeps the fluorescent and switch groups in close proximity. The FRET probe and the PCR primers (one primer in the inserted DNA and one in the sequence of the flanking genomic DNA) undergo a cycle in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in

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22/42 removal of the secondary structure of the probe and in the spatial separation of the fluorescent and switch groups. A fluorescent signal results. A fluorescent signal indicates the presence of the sequence of the flanking / transgenic insertion sequence due to successful amplification and hybridization.

[0048] DNA detection kits can be developed using the compositions described here and methods well known in DNA detection techniques. The kits are useful for identifying the DNA of the MON 88913 cotton event in a sample, and can be applied to methods of growing cotton plants that contain the MON 88913 DNA. The kits contain DNA sequences that are useful as primers or probes and that are homologous or complementary to any part of SEQ ID NO: 3 or SEQ ID NO: 4, or to DNA sequences homologous or complementary to the DNA contained in any of the transgenic genetic elements of pMON51915 that were inserted into MON 88913 DNA (Figure 2). These DNA sequences can be used in DNA amplification (PCR) methods or as probes in nucleic polyacid hybridization methods, i.e., Southern analysis, Northern analysis. The transgenic genetic elements contained in the MON 88913 DNA (Figure 2) include a first expression cassette comprising the scrofulary mosaic promoter, constructed as a chimeric promoter element with the Arabidopsis alpha-1 elongation factor (At. Ef1a) (FMV35S / Ef1a, US Patent No. 6,462,258, SEQ ID NO: 28, hereby incorporated by reference in its entirety), operationally linked to the leader and translational intron of the Arabidopsis alpha factor 1 stretching (Genebank accession number X16430, as described in Axelos et al., Mol. Gen. Genet. 219: 106-112, 1989), operably linked to Arabidopsis EPSPS chloroplast transit peptide (TS-At.EPSPS: CTP2, Klee et al., Mol. Gen. Genet.

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210: 47-442, 1987), operationally linked to 5-enol-pyruvylchiquimato3-phosphate synthase (EPSPS) from the CP4 strain of Agrobacterium sp. tolerant to glyphosate (aroA: CP4, patent no.^O 5,633,435), operably linked to the 3 'termination region of pea ribulose 1,5-bisphosphate carboxylase E9 (T-Ps.RbcS2: E29, Coruzzi et al., EMBO J. 3: 1671-1679, 1984), and a second expression cassette comprising the CaMV35SAct8 promoter, including the first intron of the Act8 gene (SEQ ID NO: 29, US Patent No. 6,462,258) operatively connected to a transit peptide of EPSPS chloroplasts of Arabidopsis (TS.At.EPSPS: CTP2), operationally connected to a 5-enol-pyruvylchiquimate-3-phosphate synthase (EPSPS) of the CP4 strain of Agrobacterium sp. tolerant to glyphosate (aroA: CP4, patent no.^O 5,633,435, hereby incorporated by reference in its entirety), operably linked to the 3 'termination region of pea ribulose 1,5-bisphosphate carboxylase E9.

[0049] The following examples are included to demonstrate examples of certain preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the examples that follow represent approaches that the inventors have found to work well in the practice of the invention, and therefore, can be considered to be examples of preferred modes for their practice. However, those skilled in the art should appreciate that, in the light of this specification, many changes can be made in the specific modalities described and still obtain a similar or similar result without departing from the spirit and scope of the invention. Examples

Example 1 [0050] The MON 88913 transgenic cotton event was generated by a cotton cell transformation, mediated by Agrobacterium, with a DNA fragment derived from pMON51915 (Figure 1). The construction of the transformation of the plant, pMON51915, was mated in

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Agrobacterium using a triparental mating procedure (Ditta et al., Proc. Natl. Acad. Sci. 77: 7347-7351, 1980). The transformation of the cotton cell with transgenes can be carried out using the methods described, for example, in patents no.^the US 5,004,863, US 5,159,135, and US 5,518,908, incorporated herein by reference in their entirety. The processing of cotton is carried out essentially as described in document n^O WO / 0036911, or as described in patent no.^O US 5,846,797, hereby incorporated by reference in their entirety. A modification of these methods may include, but is not limited to, the following example. The Coker 130 seed is superficially sterilized and germinated in the dark. Hypocotyl explants are cut from the germinated seedlings to lengths of about 1 to 1.5 cm. The ABI strain of Agrobacterium tumefaciens, transformed to contain pMON51915, is grown in Luria broth without antibiotics for 16 ha 28^OC, and then diluted to approximately 2 x 10⁸ bacteria per milliliter (mL). The hypocotyl explant is immersed in the Agrobacterium inoculum for 2 to 5 min, and then co-cultured for about 45 h in MS + 1.9 mg / L KNO3 + m 3% glucose (TRM), 30 explants per plate, 24^OC, in the dark. The explants are transferred to TRM containing 150 mg / mL of cefotaxime and 300 MS of glyphosate during four culture periods, each period for approximately six weeks. Embryogenic calluses are secreted from the primary explant at the end of 3^O or 4^O culture period and placed on the same medium. Embryogenic calluses are subcultured once by briefly suspending liquid TRM + 3% glucose, and then pouring the suspension onto plates with TRM + 150 mg / mL cefotaxime + glyphosate 300 μM. The somatic embryos are collected 3 to 8 weeks after the liquid subculture, and then cultured in Stewart & Hsu medium with 0.5% glucose. The plants derived from somatic embryos are matured to 4 to 7 cm (3 to 6 leaves) in boxes

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Magenta with Steart & Hsu medium modified with 40 mM NO3 / 10 mM NH4 + 2% sucrose. These plants are then transplanted to soil in 10 cm (4 inch) pots, 100% humidity, 16 hours of light per day, for 4 to 6 days, and then 50% humidity for 5 to 10 days.

[0051] The DNA fragment of pMON51915 contains two transgenes expression cassettes inserted in the MON 88913 genome (figure 2) that collectively confer tolerance to glyphosate to MON 88913 and its progeny.

[0052] The MON 88913 plant and the seed have regenerable parts.

The regenerable parts of the seed include, but are not limited to, the embryo, the cotyledon, and the sprout or root meristem. The regenerable parts of the plant include, but are not limited to, the leaves, petiole, hypocotyl, branch sections, and apical or root meristems. The invention also includes plant parts from the MON 88913 cotton event, which include, but are not limited to, pollen, egg, flowers, cocoons, fluff, buds, roots and leaves. The invention also includes extractable MON 88913 seed components, which include, but are not limited to, protein, bran, flour, husks, oil and short fiber fluff.

Example 2 [0053] The glyphosate-tolerant cotton event, MON88913, was selected from many transgenic cotton events for vegetative and reproductive glyphosate lesions. The successful production of a transgenic event with commercial quality currently requires producing a large number of events. In the present invention, MON 88913 was one event out of approximately 1,000 R0 events that had been transformed with many different DNA constructs that included pMON51915. The MON 88913 event was selected from the many events by a series of molecular analyzes and screening of

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26/42 glyphosate tolerance.

[0054] The events were screened in a greenhouse glyphosate tolerance test, with the plants screened for vegetative and reproductive tolerance. Fifteen to twenty-five R1 seeds from each event were planted in 15-compartment trays with Metro-Mix growth medium, which contains a combination of peat, vermiculite, nutrients, wetting agents, and processed tree fern and ash. Additional fertilizers included in the medium were Osmocote 14-14-14, Osmocote Plus 15-9-12, and MicroMax micronutrients. All plants were grown in a greenhouse. The average daytime temperature during the growing season was 32 degrees Celsius (^OC), while the average night temperature was 24 ^OC. The photoperiod was adjusted to 16 hours of light and eight hours of darkness, with maximum light intensity. The average relative humidity during the growth cycle was 45 percent. The plants were then sprinkled in the stages of 4 and 8 leaves, sequentially, with 3.50 liters (L) per hectare (ha) (48 ounces per acre) of Roundup Ultra® (herbicide containing glyphosate). Seven days after the application of glyphosate in the 4-leaf stage, the plants were classified according to the vegetative lesion, and the segregation of the glyphosate-tolerant phenotype was collected. These data were used to confirm that the transgenic insertion of the event was behaving as a single dominant gene, depending on Mendelian genetic models. The events with good vegetative tolerance were subsequently transplanted in a 25 cm (10 in) pot with the same Metro-Mix growth medium described above, and grown until maturity. The plants were treated with Pix Plus (BASF, Research Triangle Park, NC) as needed, to regulate the plant height. Three months after planting, the plants were mapped for cocoon retention in the first fruiting positions of the

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27/42 five first fruiting branches. The maximum retention value for this plant map is 5 (five cocoons retained). This provided a quick indirect measure of the plant's fertility. With this screening in the greenhouse, the average retention for the current commercial event (cotton RR 1445) is less than 1.0. Events with an average cocoon retention value greater than or equal to three were collected and advanced for additional event selection. Events that have cocoon retention values ​​greater than or equal to two have value as new selections of glyphosate-tolerant plants.

[0055] The events that met the criteria for vegetative and reproductive tolerance to Roundup® Ultra were analyzed for the number of copies through the Southern blot analysis. Single copy events, which showed good tolerance in initial greenhouse experiments, were further characterized in (1) additional greenhouse tolerance tests with higher glyphosate ratios, (2) replicated field experiments, and (3) additional molecular screens . The greenhouse tolerance tests were conducted using homozygous plants. All experiments contained the 1445 Roundup Ready cotton event^® currently commercialized (commercial standard) for comparison. The seeds were planted in trays with 15 compartments and treated with 4.7 L / ha (64 ounces / acre) of Roundup Ultra® in the 4-leaf stage and 7.0 L / ha (96 ounces / acre) in the 8-stage sheets. The plants were then transplanted into 25 cm (10 inch) pots and four plants were mapped in the mid-season in the first fruiting positions of the first five fruiting branches. End-of-season data were also collected at all events and included cotton weight per seed, number of cocoons, cocoon size, and cocoon retention.

[0056] Field tests were used to select the event

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28/42 that had better growth rates, fruit retention, and production. Field tests were arranged in a randomly divided batch layout with three replications and three treatments. The events were planted in two-row, 9.1-meter (30-foot) lots. The treatments consisted of 4.7 L / ha (64 ounces / acre or 1.5 pounds of acid equivalent per acre) of Roundup Ultra® in the stages of 4, 6, 10 and 14 nodes, and 7.0 L / ha ( 96 ounces / acre or 2.25 pounds of acid / acre equivalent) of Roundup Ultra® in the 4, 6, 10 and 14 node stages. A mid-season plant map was completed with ten plants per lot. Cocoon retention data was collected for the first and second positions of the first five fruiting nodes, which provides a range of cocoon retention values ​​from 0 to 10. A plant with a cocoon retention value equal to or greater than that 3 on the scale of 0 to 10 has value as a new selection of glyphosate tolerant plant.

[0057] Field tests (10 sites), comparing the production of cotton fluff in kg / ha (pound of acid / acre equivalent) of MON 88913 and cotton RR 1445, indicated that MON 88913 provided substantial protection against the effects of glyphosate on production (Table 1). Production is a measure of cocoon retention, cotton plants produced by genetic engineering in terms of tolerance to glyphosate, which retain a substantial number of cocoons in the first and second fruiting positions, must maintain a production advantage over cotton plants that are not so tolerant of glyphosate. An effective dose of a glyphosate-containing herbicide to control weeds in a MON 88913 field comprises about 0.29 L / ha (4 ounces / acre) and can exceed 9.3 L / ha (128 ounces / acre), depending on the species of weed to be controlled and the stage of weed development. Glyphosate can be mixed with other herbicides to enhance herbicidal activity

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Table 1

Comparison of MON 88913 and 1445 Lanugo Production After Glyphosate Treatment

<td></td><td colspan="3">Production in kg / ha (lb / acre) from 10 sites</td>

<td>Glyphosate treatment</td><td>0 kg ea^*/ ha (0 pound ea / acre)</td><td>1.68 kg ea * / ha (1.5 pound ea / acre)</td><td>2.52 kg ea * / ha (2.25 pound ea / acre)</td>

<td> 1445</td><td> 2.714,41 (2.421,84)</td><td> 1.170,33 (1.044,19)</td><td> 932,08 (831,47)</td>

<td>MON 88913</td><td> 2.860,32 (2.551,58)</td><td> 2.900,71 (2.587,61)</td><td> 2.704,19 (2.412,3)</td>

* ea = acid equivalent

Example 3 [0058] Genomic DNA from cotton for all PCR reactions and Southern blot analyzes was isolated using a CTAB procedure (Rogers et al., Plant Mol. Biol. 5: 69-76, 1985) or Dneasy® 96 Plant Kit (catalog number 69181, Qiagen, Inc., Valencia, CA), following the manufacturers' instructions. Leaf tissue was collected from plants in the 2- to 4-leaf stage. The smallest true leaves were collected from each plant and immediately frozen on dry ice. DNA was extracted using, for example, the following method. The fabric was ground using plastic beads with liquid nitrogen. Five milliliters of extraction buffer was added to 0.75 g of tissue and incubated at 55^OC for 45 min. The CTAB extraction buffer consisted of 100 mM Tris pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2% CTAB with the addition of 5 μL of beta-mercaptoethanol, 5 μL of RNase and 1% of PVPP. The samples were then extracted with an equal volume of chloroform (5 mL) and then centrifuged at 3,700 rpm for 15 min at room temperature. The aqueous phase was transferred to a new tube and the DNA was precipitated with an equal volume of isopropanol. After centrifuging at 3,700 rpm for 15 min, the pellets were washed with 70% ethanol, air-dried, and resuspended in 250 μL of water.

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30/42 [0059] The genomic DNA of the cotton adjacent to the transgenic insertion was obtained for the MON 88913 event, using TAIL-PCR (Liu et al., Plant Journal 8: 457-463, 1995). Genomic DNA extension was carried out using the GenomeWalker kit (CloneTech Laboratories, Palo Alto, CA), following the manufacturer's protocol. Briefly, the DNA (~ 5 pg), isolated using the CTAB protocol described above, was digested with several restriction endonucleases (EcoRV, Sca1) at 37^OC overnight, in a total volume of 100 gL. Restriction endonucleases were removed with QIAquick PCR purification columns (n^O No. 28104, Qiagen, Inc.). The attachment of adapter molecules was as described in the manufacturer's protocol. The DNA was amplified using the FMV-1 primer (SEQ ID NO: 5) with the API primer (CloneTech Laboratories) for the primary reaction and the nested FMV-2 primer (SEQ ID NO: 6) with the AP2 primer (CloneTech Laboratories ) for the secondary reaction.

[0060] The genomic / transgenic DNA 3 'of MON 88913 was isolated using inverse PCR. The total genomic DNA (~ 10 gg) was digested with three restriction enzymes: BclI, NcoI and HindIII. QIAquick PCR Purification columns were used to purify DNA after digesting overnight at 37 ° C^OC. The DNA was eluted from the columns with 50 gL of water and then diluted to 1 mL. The diluted eluate (85 gL) was combined with 10 gL of buffer (10X) and 5 gL T4 ligase to recirculate the fragments. After an overnight incubation at 16^OC, the ligase was inactivated by heat at 70 ^OC. The samples were amplified by PCR with a series of nested primers. Primer combinations for PCR included: primer pair 8099-E9-1 / E9-2 (SEQ ID NO: 7 / SEQ ID NO: 8) for samples with BclI and NcoI and primer pair 8099-E9 -1 / Act8 rev (SEQ ID NO: 7 / SEQ ID NO: 9) for the sample with HindIII; the 8099-E9-2 / E9-1 primer pair (SEQ ID NO: 10 / SEQ ID NO: 11) for samples with BclI and NcoI; the pair of

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31/42 primers 8099-E9-2 / Act8 (SEQ ID NO: 10 / SEQ ID NO: 12) for the sample with HindIII; the 8099-E9-3 / E9-1 primer pair (SEQ ID NO: 13 / SEQ ID NO: 11) for samples with BclI and NcoI and the 8099-E9-3 / Act8 primer pair (SEQ ID NO: 13 / SEQ ID NO: 12) for the sample with HindIII. Conditions for PCR included: Primary PCR = 7 cycles of 94^OC for 2 seconds, 72 ^OC for 10 min; 37 cycles of 94^OC for 2 seconds, 67 ^OC for 10 min; 1 cycle of 67^OC for 10 min; Secondary and tertiary PCR = 5 cycles of 94^OC for 2 seconds, 72 ^OC for 10 min; 24 cycles of 94^OC for 2 seconds, 67 ^OC for 10 min; 1 cycle of 67^OC for 10 min.

[0061] Alternatively, DNA amplification by PCR of the 3 'end of the MON 88913 event can be performed with conditions that include: 7 cycles of 94 ^OC for 25 seconds, 72 ^OC for 3 min; 37 cycles of 94^OC for 25 seconds, 67 ^OC for 3 min; 1 cycle of 67^OC for 7 min. All subsequent amplifications were performed with the following conditions: 7 cycles of 94^OC for 2 seconds, 72 ^OC for 4 min; 37 cycles of 94^OC for 2 seconds, 67 ^OC for 4 min; 1 cycle of 67^OC for 7 min. All amplicons are visualized in 0.8% agarose gels stained with ethidium bromide. DNA is prepared for sequencing by purifying the PCR samples directly with the QIAquick PCR Purification kit (n^O catalog 28104, Qiagen, Inc.) or by extracting the appropriate gel fragment, and using the QIAquick Gel Extraction kit (n^O catalog 28704, Qiagen, Inc.).

[0062] A series of DNA primers were designed to sequence the insertion of the transgene and the flanking genomic regions of MON 88913. DNA primers were designed that allowed the amplification of the entire transgene and the flanking genomic regions into five overlapping fragments. Unique primers were designed to allow amplification of each EPSPS-CTP2 / aroA-CP4 / RbcS2: E9 region separately.

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For all fragments used in the sequencing, the amplifications were performed in triplicate. The combinations of DNA primer pairs, used as sequencing primers for the 5 'transgene / genomic region (SEQ ID NO: 14 and SEQ ID NO: 15), 3' transgene / genomic region (SEQ ID NO: 16 and SEQ ID NO: 17) and insertion genetic elements (SEQ ID NO: 18 and SEQ ID NO: 11; SEQ ID NO: 19 and SEQ ID NO: 15; SEQ ID NO: 20 and SEQ ID NO: 11). Total genomic DNA was used for all PCR reactions. All amplicons are visualized in 0.8% agarose gels stained with ethidium bromide. DNA is prepared for sequencing by purifying the PCR samples directly with the QIAquick PCR Purification kit, or extracting the appropriate fragment from the gel, and using the QIAquick Gel Extraction kit. The DNA sequence was produced using DNA sequence analysis equipment (ABI Prism® 377, PE Biosystems, Foster City, CA) and DNASTAR sequence analysis software (DNASTAR, Inc., Madison, WI).

[0063] The DNA fragments of the flanking regions of the MON 88913 transgene / genomic insert were subcloned using a TOPO TA Cloning® kit (Invitrogen). The DNA sequence of the transgene / genomic region 5 'is illustrated in Figure 4 and the DNA sequence of the transgene / genomic region 3' is illustrated in Figure 5. In the DNA sequence illustrated in Figures 4 and 5, the sequence of insertion of the transgene is in italics.

Example 4 [0064] Pairs of DNA event primers are used to produce a diagnostic amplicon for the MON 88913 event genome. The diagnostic amplicons for the MON 88913 genome comprise at least one junction sequence, SEQ ID NO: 1 or SEQ ID NO: 2. The event primer pairs, which will produce a diagnostic amplicon for MON 88913, where the primer pairs

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33/42 include, but are not limited to, SEQ ID NO: 14 and SEQ ID NO: 15 for the 5 'sequence of the amplicon, and SEQ ID NO: 16 and SEQ ID NO: 17 for the amplicon 3', when used in the protocol outlined in Table 2. In addition to these primer pairs, any pair of primers, homologous or complementary to SEQ ID NO: 3 or SEQ ID NO: 4, which in a DNA amplification reaction produces a diagnostic amplicon for the MON 88913 genome , is an aspect of the present invention. Any single isolated DNA primer polynucleotide molecule, comprising at least 11 contiguous nucleotides of SEQ ID NO: 4, or its complement, which is useful in a DNA amplification method to produce a diagnostic amplicon for MON 88913, is an aspect of the invention. Any single isolated DNA primer polynucleotide molecule, comprising at least 11 contiguous nucleotides of SEQ ID NO: 3, or its complement, which is useful in a DNA amplification method to produce a diagnostic amplicon for MON 88913, is an aspect of the invention. An example of the amplification conditions for this analysis is illustrated in Table 2 and Table 3, however, any modification of these methods using DNA primers homologous or complementary to SEQ ID NO: 3 or SEQ ID NO: 4, or DNA sequences of the genetic elements contained in the insertion of the MON 88913 transgene, which produce a diagnostic amplicon for MON 88913, is within the knowledge of those skilled in the art. A diagnostic amplicon comprises a DNA molecule homologous or complementary to at least one transgene / genomic junction DNA sequence (SEQ ID NO: 1 or SEQ ID NO: 2) or a substantial part of it.

[0065] An analysis of the plant tissue sample from the MON event

88913 must include a positive tissue control for the MON 88913 event, a negative control of a cotton plant that is not the 88913 event, and a negative control that does not contain any DNA

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34/42 cotton genomics. Additional primer sequences can be selected from SEQ ID NO: 3 and SEQ ID NO: 4 by those skilled in the art of DNA amplification methods, and the conditions selected for the production of an amplicon by the methods indicated in Table 2 and T Table 3 may differ, but result in a diagnostic amplicon for the MON 88913 event. The use of these DNA primer sequences with modifications in the methods of Tables 2 and 3 are within the scope of the invention. The amplicon produced by at least one sequence of the DNA primer, derived from SEQ ID NO: 3 or SEQ ID NO: 4, which is diagnostic for MON 88913, is an aspect of the invention.

[0066] DNA detection kits, which contain at least one DNA primer derived from SEQ ID NO: 3 or SEQ ID NO: 4, which, when used in a DNA amplification method, produces a diagnostic amplicon for MON 88913, is an aspect of the invention. The amplicon produced by at least one primer derived from any of the genetic elements of pMON51915, which is diagnostic for MON 88913, is an aspect of the invention. A cotton plant or seed, where its genome will produce an amplicon comprising SEQ ID NO: 1 or SEQ ID NO: 2, when tested in a DNA amplification method, is an aspect of the present invention. The MON 88913 amplicon assay can be performed using a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient Thermocycler, as indicated in Table 3, or by methods and apparatus known to those skilled in the art.

Table 2

PCR Procedure and Reaction Mixing Conditions for the Identification of the Junction Region 5 'Transgene / Genomics Insertion of MON 88913

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<td>Stage</td><td>Reagent</td><td>Amount</td><td>comments</td>

<td> 1</td><td>nuclease-free water</td><td>add up to a final volume of 20 pL</td><td> -</td>

<td> 2</td><td>10X reaction buffer (with MgCl2)</td><td>2.0 pL</td><td>1X final concentration of buffer, final concentration of 1.5 mM MgCl2</td>

<td> 3</td><td>10 mM dATP, dCTP, dGTP and dTTP solution</td><td>0.4 pL</td><td>final concentration 200 pM of each dNTP</td>

<td> 4</td><td>event initiator (SEQ ID NO: 14) (resuspended in 1X TE buffer or nuclease-free water to a concentration of 10 pM)</td><td>0.4 pL</td><td>final concentration 0.2 pM</td>

<td> 5</td><td>event initiator (SEQ ID NO: 15) (resuspended in 1X TE buffer or nuclease-free water to a concentration of 10 pM)</td><td>0.4 pL</td><td>final concentration 0.2 pM</td>

<td> 6</td><td>RNase, free of DNase (500 ng / pL)</td><td>0.1 pL</td><td>50 ng / reaction</td>

<td> 7</td><td>REDTaq DNA polymerase (1 unit / pL)</td><td>1.0 pL (recommended to change pipettes before the next step)</td><td>1 unit / reaction</td>

<td> 8</td><td>Extracted DNA (model): - Samples to be analyzed * individual sheets * sheets collected (maximum 50 sheets / selection) - Negative control - Negative control - Positive control</td><td>- 10-200 ng of genomic DNA - 20 ng of DNA genomic - 50 ng of genomic cotton DNA (not MON 88913) - no model DNA - 50 ng of MON genomic DNA 88913</td><td></td>

<td> 9</td><td>Mix gently and add 1-2 drops of mineral oil on top of each reaction.</td><td></td><td></td>

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Table 3

Suggested PCR Parameters for Different Thermal Recycling Devices [0067] Proceed with the amplification of DNA in a

Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient Thermocycler, using the parameters of the following cycles. The MJ Engine or Eppendorf Mastercycler Gradient Thermocycler must be operated in the calculated mode. The Perkin-Elmer 9700 Thermocycler is operated with the ascension speed set at maximum.

<td>N of Cycles</td><td>Adjustments: Stratagene Robocycler</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 1 minute 60 ^OC, 1 minute 72 ^OC, 1 minute and 30 seconds</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

<td>N of Cycles</td><td>Settings: MJ Engine or Perkin-Elmer 9700</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 10 seconds 60 ^OC, 30 seconds 72 ^OC, 1 minute</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

<td>N of Cycles</td><td>Settings: Eppendorf Matercycler Gradient</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 15 seconds 60 ^OC, 15 seconds 72 ^OC, 1 minute and 30 seconds</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

Example 5 [0068] The genomic DNA of MON 88913 and the genomic DNA of

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37/42 control cotton (~ 15 μg each) is digested with various restriction enzymes (140 U) in a total volume of 150 gL, including 15 μL of the corresponding manufacturer's buffer (NEB, Beverely, MA). Restriction endonucleases, such as BgII, BamHI, NcoI, HindIII, and BcII, are used in the Southern analysis of MON 88913. Digestions with endonucleases are carried out at the appropriate temperature for at least 6 hours. After incubating, the DNA is precipitated with 3 M sodium acetate and 2.5 volumes of ethanol. Subsequently, the DNA is washed with 70% ethanol, dried, and resuspended in 40 gL TBE. The loading buffer (0.2x) is added to the samples and then the electrophoresis is conducted in agarose gels (0.8%) for 16-18 h at 30 volts. The gels are stained with ethidium bromide, and then treated with a depurinization solution (0.125 N HCl) for 10 min, and with a denaturing solution (0.5 M sodium hydroxide, 1.5 M sodium chloride) for 30 minutes. min, and finally, with a neutralizing solution (0.5 M Trizma base, 1.5 M sodium chloride) for 30 min. The DNA is transferred to a Hybond-M membrane (Amersham Pharmacia Biotech, Buckinghamshire, England), using a Turboblotter (Schleicher and Schuell, Dassel, Germany) for 4-6 hours and then fixed to the membrane using UV light. [0069] The membranes are pre-hybridized with 20 ml of solution

DIG Easy Hyb (Roche Molecular Biochemicals, Indianapolis, IN; n^O catalog 1603558) for 2-4 hours at 45 ^OC. Radioactive DNA probes (³²P dCTP) homologous or complementary to SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 3 or SEQ ID NO: 4, or a portion thereof, are prepared using a Radprime DNA Labeling kit (Invitrogen, Carlsbad, CA; catalog number 18428-011). Unincorporated nucleotides are removed using Sephadex G-50 columns (Invitrogen). The prehybridization solution is replaced by 10 mL of preheated DIG Easy Hyb solution, containing the denatured probe until

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38/42 a final concentration of 1 million counts per mL. The spots are hybridized at 45^OC for 16-18 h.

[0070] The stains are washed with a low severity solution (5X SSC, 0.1X SDS) at 45 ^OC then repeatedly washed with a high severity solution (0.1X SSC, 0.1% SDS) at 65 ° C. The spots are exposed to phosphorous screening (Amersham Biosciences, Piscataway, NJ) for&gt; 2 h and the exposure is read using a Data Storm 860 machine (Amersham Biosciences).

Example 6 [0071] The methods used to identify the heterozygous cotton progeny of the homozygous that contains the MON 88913 event are described in a zygosity assay, whose examples of conditions are described in Table 4 and Table 5. The DNA primers used in the zygosity test are the triggers SQ1099 (SEQ IS NO: 21), SQ1100 (SEQ ID NO: 22), SQ1353 (SEQ ID NO: 23), 6FAM® labeled primer (SEQ ID NO: 24) , and VIC® labeled primer (SEQ ID NO: 25); 6FAM and VIC are fluorescent dye products from Applied Biosystems (Foster City, CA) attached to the DNA primer.

[0072] SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, when used in these reaction methods, produce one DNA amplicon for non-transgenic cotton, two DNA amplicons for heterozygous cotton that contains the MON event 88913, and a DNA amplicon for homozygous MON 88913 cotton that is distinct from any other non-MON 88913 cotton. The controls for this analysis should include a positive control of homozygous and heterozygous cotton containing MON 88913 DNA, a negative control of non-transgenic cotton, and a negative control that does not contain any model DNA. This assay is optimized for use with a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient Thermocycler. Other methods and apparatus known to

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39/42 skilled in the art, which produce amplicons that identify the zygony of the progeny of crosses made with MON 88913 cotton plants, are within the knowledge of those skilled in the art. Table 4

Zygosity Test Reaction Solutions

<td>Stage</td><td>Reagent</td><td>Amount</td><td>comments</td>

<td> 1</td><td>nuclease-free water</td><td>add up to a final volume of 10 μL</td><td> -</td>

<td> 2</td><td>2X Universal Master Mix (Applied Biosystems, n^Ocatalog 4304437)</td><td>5 μL</td><td>1X final concentration</td>

<td> 3</td><td>SQ1099 initiators, SQ1100, SQ1353 (replaced in suspension in water free from nucleases to a concentration of 20 μM)</td><td>0.5 μL</td><td>final concentration 0.25 μM</td>

<td> 4</td><td>6FAM® initiator (replaced in suspension in water free from nucleases to a concentration of 10 μM)</td><td>0.2 μL</td><td>final concentration 0.4 μM</td>

<td> 5</td><td>VIC® initiator (replaced in suspension in water free from nucleases to a concentration of 10 μM)</td><td>0.2 μL</td><td>final concentration 0.15 μM</td>

Continuation...

<td>Stage</td><td>Reagent</td><td>Amount</td><td>comments</td>

<td> 6</td><td>REDTaq DNA polymerase (1 unit ^ L)</td><td>1.0 μL (recommended to change pipettes before the next step)</td><td>1 unit / reaction</td>

<td> 7</td><td>Extracted DNA (model): - Samples to be analyzed (sheets individual) - Negative control</td><td>3.0 μL - 4-80 ng of genomic DNA - 4 ng of genomic cotton DNA</td><td></td>

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<td></td><td>- Negative control - Positive control - Positive control</td><td>non-transgenic - no DNA model (solution in which the DNA was resuspended) - 4 ng of the known genomic DNA of heterozygous cotton MON 88913 - 4 ng of the DNA of homozygous cotton MON 88913</td><td></td>

<td> 8</td><td>Mix gently and add 1-2 drops of mineral oil on top of each reaction.</td><td></td><td></td>

Table 5

Conditions of the Thermocycler of the Zygosity Assay [0073] Proceed with the amplification of the DNA in a

Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient Thermocycler, using the parameters of the following cycles. When PCR is operated on the Eppendorf Mastercycler Gradient Thermocycler or MJ Engine, the Thermocycler must be operated in the calculated mode. When PCR is operated on the Perkin-Elmer 9700, the Thermocycler must be operated with the ascension speed set at maximum.

<td>N of Cycles</td><td>Adjustments: Stratagene Robocycler</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 1 minute 60 ^OC, 1 minute 72 ^OC, 1 minute and 30 seconds</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

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<td>N of Cycles</td><td>Settings: MJ Engine or Perkin-Elmer 9700</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 30 seconds 60 ^OC, 30 seconds 72 ^OC, 1 minute and 30 seconds</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

<td>N of Cycles</td><td>Settings: Eppendorf Matercycler Gradient</td>

<td> 1</td><td> 94 ^OC, 3 minutes</td>

<td> 38</td><td> 94 ^OC, 15 seconds 60 ^OC, 15 seconds 72 ^OC, 1 minute and 30 seconds</td>

<td> 1</td><td> 72 ^OC, 10 minutes</td>

Example 7 [0074] Analysis of the genomic DNA samples from cotton was conducted using a Taqman® termination method. The production of diagnostic amplicons for the MON 88913 genomic DNA was done using a set of A primers that included the event primers: SEQ ID NO: 21, SEQ ID NO: 22, and the 6-FAM probe SEQ ID NO: 24 ; and a set of B primers that included the event primers: SEQ ID NO: 26, SEQ ID NO: 27, and probe 6-FAM SEQ ID NO: 28. The method uses a 96-well or 384-well format and a GeneAmp 9700 PCR System from Applied Biosystems, or the MJ Research PT-225 DNA Machine. The DNA extracted from the cotton tissue samples, as previously described, should be in the range between 5 and 10 ng per PCR reaction. Each reaction contains a final volume of 10 gL, consisting of 0.5 gL of equal concentration of the event initiators (20 gM), 5.0 gL of 2X Universal Master Mix, 0.2 gL of the 6-FAM probe (10 gM ), 3 gL of the DNA sample (5-10 ng) and water up to 10 gL. The parameters of the thermal cycles are 1 cycle of 50^OC for 2 minutes, 1 cycle of 95 ^OC for 10 min; 10 cycles of 95^OC for 15 seconds, 64 ^OC for 1 min; and

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42/42 later, -1 ^OC / cycle, 30 cycles of 95 ^OC for 15 seconds; 54^OC for 1 minute, then keep at 10 ^OC. Amplicon production was determined by a microplate reader, such as TECAN Safire (Durham, NC), using the conditions described by the manufacturer. The data analysis program (TaqPro®) was used to score the production of the marked amplicon. Other equipment and methods of analysis known in these DNA detection techniques can be used to detect the amplicons of the present invention.

[0075] A deposit from Monsanto Technology LLC, the MON 88913 cotton seed, described above and set out in the claims, was made in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 Universitiy Boulevard, Manassas, Va 20110. The access number is PTA-4854. The deposit will be kept in the depository for a period of 30 years, or 5 years after the last application, or for the effective validity of the patent, whichever is the longest, and will be replaced as needed during that period.

[0076] Having illustrated and described the principles of the present invention, it should be evident to those skilled in the art that the invention can be modified in the arrangement and detail without running away from such principles. All modifications that are within the spirit and scope of the attached claims are claimed.

[0077] All publications and published patent documents cited in this specification are hereby incorporated by reference to the same degree as if each publication or individual patent application had been specifically and individually indicated as being incorporated by reference.