Cotton event MON 88913 and compositions and methods for detection thereof

This application claims the benefit of US Provisional Application No. 601447184, filed on February 12, 2003, the complete content of which is incorporated by reference in this documentation.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology of plants. More specifically, the invention relates to a glyphosate tolerance event in cotton, MON 88913, and to assays and methods for detecting the presence of DNA from the cotton event MON 88913 in a plant sample and in compositions comprising it.

BACKGROUND OF THE INVENTION

Cotton is an important fiber crop in many areas of the world. Biotechnology methods have been applied to cotton to improve agronomic characteristics and product quality. The method of introducing transgenes into cotton plants is demonstrated in US Patent No. 5,004,863. One such important agronomic character in cotton production is herbicide tolerance, in particu lar, glyphosate herbicide tolerance. This character has been introduced in cotton plants and is a successful product now used in cotton production. The Roundup Ready® cotton event (1445) provides excellent tolerance to glyphosate, the active ingredient in Roundup®, up to the four-leaf stage (Nida et al., J. Agrie. Food Chem. 44: 1960-1966, 1996; Nida al., J. Agrie. Food

Chem. 44: 1967-1974, 1996). However, foliar application beyond the four-leaf stage should be limited due to insufficient tolerance in male reproductive tissues under certain environmental conditions. This lack of tolerance in reproductive tissues seems to be the result of insufficient expression of CP4 EPSPS in critical tissues, the increased sensitivity of these tissues to glyphosate and the accumulation of large amounts of glyphosate in these strong reserve tissues (Pline et al., Weed Sci. 50: 438447, 2002). There is a need for cotton plants with greater glyphosate tolerance than Roundup Ready® 1445 cotton.

It would be advantageous to be able to detect the presence of a particular event, in order to determine if the progeny of a sexual cross contain a transgene of interest. In addition, a method to detect a particular event would be useful to meet the regulations required by the market to approve and mark foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene through any well known method of nucleic acid detection, such as nucleic acid amplification or nucleic acid hybridization techniques using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, and so on. 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 DNA adjacent to the inserted DNA ("cyclosing DNA") is known. Methods of detection of event-specific DNA have been described in a glyphosate tolerance event in cotton 1445 (US Patent No. 20020120964, fully incorporated herein by reference).

The present invention relates to a glyphosate tolerance event in cotton MON 88913, with compositions containing it and with a method for detecting the region of transgenic / genomic insertion in the cotton event MON 88913, and the progeny thereof.

SUMMARY OF THE INVENTION

The present invention relates to the transgenic cotton event called MON 88913, which has seeds deposited in the American Culture Type Collection (ATCC) with Accession No. PTA-4854. Another aspect of the invention comprises the progeny plants, or the seeds, or the regenerable parts of the plants and the seeds of the MON 88913 cotton event. The invention also includes parts of MON 88913 cotton event plants, which include, without limitation, pollen, ovules, flowers, flakes, threads, buds, roots and leaves. The invention relates to a cotton plant that has a glyphosate tolerance phenotype, and to the new genetic compositions of MON 88913.

One aspect of the invention provides DNA compositions and methods for detecting the presence of a transgenic / genomic binding region in a cotton plant with the MON 88913 event. Isolated DNA molecules are provided comprising at least one transgenic / genomic binding DNA molecule that is selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2, and complements thereof, where the molecule Binding encompasses the insertion site comprising a heterologous DNA inserted into the cotton genome and the genomic DNA of the cotton cell surrounding the insertion site in the MON 88913 cotton event.

A cottonseed, and plant material thereof comprising these molecules, constitutes an aspect of this invention.

A new DNA molecule is provided which is a 5 'transgenic / genomic binding region of SEQ ID No. 3, or the complement thereof, where this DNA molecule is novel because it has the MON 88913 cotton event. Cotton plants and seeds that comprise SEQ ID No. 3 in their genome are an aspect of this invention. In accordance with another aspect of the invention, an isolated DNA molecule is provided which is a 3 'transgenic / genomic region of SEQ ID No. 4, or the complement thereof, where this DNA molecule is novel because it possesses the MON 88913 cotton event. Cotton plants and seeds that comprise SEQ ID No. 4 in their genome are an aspect of this invention.

According to another aspect of the invention, two DNA molecules are provided for use in a DNA amplification method, wherein the first DNA molecule comprises at least 11 or more contiguous polynucleotides of any portion of the transgenic region of the molecule. DNA of SEQ ID No. 3, and a DNA molecule of similar length from any portion of a surrounding 5 'region of cotton genomic DNA of SEQ ID No. 3, where these DNA molecules, when used together, They are useful as a set of DNA primers in a DNA amplification method that allows an amplicon to be obtained. The amplicon produced using the set of DNA primers in the DNA amplification method is diagnostic for the MON 88913 cotton event.

Any amplicon produced from MON 88913 DNA by DNA primers that are homologous or complementary to any portion of the

SEQ ID No. 3 constitutes an aspect of the invention.

In accordance with another aspect of the invention, two DNA molecules are provided for use in a DNA amplification method, wherein the first DNA molecule comprises at least 11 or more contiguous polynucleotides of any portion of the transgenic region of the molecule. DNA of SEQ ID No. 4, and a DNA molecule of similar length from any portion of a surrounding 3 'region of cotton genomic DNA of SEQ ID No. 4, where these DNA molecules are useful as a set of DNA primers in a DNA amplification method. The amplicon produced using the DNA primer set in the DNA amplification method is diagnostic for the MON 88913 cotton event. The amplicons produced from MON 88913 DNA by DNA primers that are homologous or complementary to any portion of SEQ ID No. 4 is an aspect of the invention.

In accordance with another aspect of the invention, methods are provided to detect the presence of DNA that corresponds specifically to DNA from the MON 88913 cotton event in a sample. Such methods comprise: (a) placing the sample comprising DNA in contact with a set of DNA primers that, when used in a nucleic acid amplification reaction with genomic DNA of the MON 88913 cotton event, produce an amplicon that is diagnostic for the MON 89913 cotton event; (b) perform a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detect the amplicon.

In accordance with another aspect of the invention, methods are provided to detect the presence of DNA that corresponds specifically to the DNA of the MON 88913 cotton event in a sample. Such methods comprise: (a) placing the sample comprising DNA in contact with a DNA probe comprising SEQ ID No. 1 or SEQ ID No. 2 that hybridizes under strict hybridization conditions with the genomic DNA of the event of MON 88913 cotton and does not hybridize under strict hybridization conditions with the DNA of a control cotton plant; (b) subject the sample and the probe to strict hybridization conditions; and (c) detect hybridization of the probe with the DNA of the MON 88913 cotton event.

In accordance with another aspect of the invention, methods are provided for producing a plant that tolerates the application of glyphosate, comprising the steps of: (a) sexually crossing a first parent of a MON 88913 cotton event, comprising the expression cassettes of the present invention that confer tolerance to glyphosate application, and a second cotton progenitor plant that lacks glyphosate tolerance, thereby producing a plurality of progeny plants; and (b) select a progeny plant that tolerates the application of glyphosate. Such methods may optionally comprise the additional step of backtracking the progeny plant with the second cotton progenitor plant and selecting the progeny that exhibits tolerance to glyphosate, to produce a viable cotton variety that tolerates the application of glyphosate.

In accordance with another aspect of the invention, a method is provided for determining the cygosity of the progeny of the MON 88913 cotton event, comprising: (a) placing the sample comprising 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 genomic DNA of the MON 88913 cotton event, allows to obtain a first amplicon that is diagnostic for the MON 88913 cotton event; (b) perform a nucleic acid amplification reaction, thus producing the first amplicon; (c) detect the first amplicon; (d) placing the sample comprising cotton DNA in contact with said set of primers, which, when used in a nucleic acid amplification reaction with genomic DNA from cotton plants, produces a second amplicon comprising the native genomic DNA of cotton homologous to the cotton genomic region of a transgenic insert identified as the MON 88913 cotton event; (e) perform a nucleic acid amplification reaction, thereby producing the second amplicon, (f) detecting the second amplicon; and (g) compare the first and second amplicons in a sample, where the presence of both amplicons indicates that the sample is heterozygous for transgenic insertion.

A method is provided for determining the zygosity which comprises putting a sample of cotton DNA 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; using the Taqman® terminal PCR reaction; and detect the amplicon produced.

A method is provided for controlling weeds in a crop or a field of plants with the MON 88913 cotton event, which comprises the step of applying an effective amount as a glyphosate herbicide, which contains the herbicide, to the MON 88913 cotton field.

The previous section and other aspects of the invention will become more apparent from the following detailed description and the attached figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1, Map of plasmid pMON51915.

Figure 2. Genomic organization of the insert in the MON 88913 cotton event.

Figure 3. Sequence of 5 'MON 88913 binding DNA (SEQ ID No. 1) and 3' binding DNA sequence (SEQ ID No. 2).

Figure 4. 5 'transgenic / genomic DNA region of MON 88913 (SEQ ID

No. 3).

Figure 5. 3 'transgenic / genomic DNA region of MON 88913 (SEQ ID

NOT).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a glyphosate tolerance event in cotton MON 88913, with compositions containing it and with a method for detecting the region of transgenic / genomic insertion in the cotton event MON 88913, and the progeny thereof. The following definitions and methods are provided to better define the present invention and guide those skilled in the art in the practice of the present invention. Unless otherwise indicated, the terms should be interpreted according to the conventional use given by those trained in the relevant technique. Definitions of common molecular biology terms can also be found in Rieger et al., Glossary of Genetics: Classical and

Molecular, 5^to edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,

Oxford University Press: New York, 1994. The nomenclature is used for the DNA bases indicated under 37 CFR § 1822.

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

As used in this documentation, the term "understand" means "that includes without limitations."

As used herein, the term "crop" refers to cultivated plants or parts of plants, such as those grown in a field, a plot, a row, a greenhouse, a pontoon or a container.

Glyphosate refers to N-phosphonomethylglycine and its salts. Nphosphonomethylglycine is a well-known herbicide that has activity on a broad 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. Treatments with a glyphosate herbicide refer to treatments with Roundup®, Roundup Ultra®, Roundup Pro® herbicides or any other herbicidal formulation containing glyphosate. Examples of commercial glyphosate formulations include, without restrictions, those marketed by Monsanto Company such as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® WEATHERMAX, ROUNDUP® CT, herbicides.

ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD®, all of which contain glyphosate in the form of a sopropylammonium salt; those marketed by Monsanto Company such as ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate in the form of an ammonium salt; those marketed by Monsanto Company as ROUNDUP® GEOFORCE, which contains glyphosate in the form of a sodium salt; and those marketed by Syngenta Crop Protection as the herbicide TOUCHDOWN®, which contains glyphosate in the form of a trimethylsulfonium salt. When applied to the surface of a plant, glyphosate travels systemically through the plant. Glyphosate is phytotoxic because it inhibits the pathway of shichemical acid, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the enzyme 5-enolpiruvil-3-phosphoshiquimate synthetase (EPSPS) present in plants. Glyphosate tolerance can be obtained by expressing variants of EPSPS that have a lower affinity for glyphosate and, therefore, retain their catalytic activity in the presence of glyphosate (US Pat. Nos. 5,633,435, 5,094,945, 4,535 .060 and 6,040,497).

A transgenic "event" occurs through the transformation of plant cells with a heterologous DNA, for example, a nucleic acid construct (pMON51915, Figure 1) that includes a transgene of interest, the regeneration of the resulting plant population of the insertion of the transgene in the genome of the plant, and the selection of a particular plant characterized by an insertion in a particular site of the genome. The term "event" refers to the original transformant and the progeny of the transformant that includes the heterologous DNA. The term "event" also refers to the progeny produced by a sexual cross between the event and another whose progeny includes heterologous DNA. Even after repeated backcrossing with a recurring parent, the heterologous DNA and the surrounding genomic DNA of the transformed parent event are present in the same chromosomal location of the crossing progeny. The term "event" also refers to the DNA of the original transformant comprising the inserted DNA and the surrounding genomic sequence immediately adjacent to the inserted DNA, from which it would be expected to be transferred to the progeny receiving the inserted DNA, which comprises the transgene of interest, as a result of a sexual crossing of a parent line that included the inserted DNA (for example, the original transformant and the progeny resulting from the autocross) and a progenitor line that did not contain the inserted DNA.

It is known that the expression of exogenous genes in plants is affected by their position in the chromosomes, probably due to chromatin structures (for example, heterochromatin) or to the proximity of transcriptional regulatory elements (eg, enhancers) near from the integration site (Weising et al., Ann. Rev. Genet 22: 421-477, 1988). For this reason, it is usually necessary to analyze a large number of events in order to identify an event characterized by the optimal expression of a gene of interest introduced. For example, it has been observed in plants and other organisms that there may be a great variation in the expression levels of a gene introduced between events. There may also be differences in spatial or temporal expression patterns, for example, differences in the relative expression of a transgene in various plant tissues, which may respond to expected patterns due to the transcriptional regulatory elements present in the Genetic construction introduced. For this reason, it is common to produce between hundreds and thousands of different events, and analyze these events in search of a single event that has the desired transgenic expression levels and patterns for commercial purposes. An event that has the desired levels or patterns of transgenic expression is useful for introducing the transgene into other genetic backgrounds by sexual exocruza, using conventional culture methods. The progeny of these crosses maintains the characteristics of transgenic expression of the original transformant. This strategy is used to ensure reliable genetic expression in a number of varieties that are well adapted to local growing conditions and market demands.

A cotton plant with glyphosate tolerance can be produced by first sexually crossing a first cotton progenitor plant, which consists of a cotton plant grown from a transgenic cotton plant cell derived from a transformation with the contained vegetable expression cassettes in pMON51915, and which tolerates the application of the glyphosate herbicide, with a second cotton progenitor plant that lacks tolerance to the glyphosate herbicide, thus producing a plurality of plants of the first progeny; and then selecting a plant of the first progeny that presents tolerance to the application of the herbicide glyphosate; and self-cross the plant of the first progeny, thus producing a plurality of plants of the second progeny; and then select among the plants of the second progeny those plants that have tolerance to the herbicide glyphosate. These steps may also include the backcross of the plant of the first progeny with tolerance to glyphosate or the plant of the second progeny with tolerance to glyphosate, with the second cotton progenitor plant or with a third cotton progenitor plant, thus producing a plant of cotton that tolerates the application of glyphosate herbicide. In the present invention, the transgenic cotton plant is also defined as the MON 88913 cotton event, and can be known as MON 88913 in the present documentation.

It should also be understood that two different transgenic plants can be crossed to produce offspring containing two exogenous independent segregating transgenes. Self-crossing of the appropriate progeny can produce plants that are homozygous for both exogenous genes added. The backcross is also contemplated with a parent plant and the exocruza with a non-transgenic plant, such as vegetative propagation. Descriptions of other culture methods that are commonly used for different characters can be found in any of numerous references, for example, Fehr, in Breeding Methods for Cultivar Development, Wilcox J. editor, American Society of Agronomy, Madison Wl (1987).

A "probe" is an isolated nucleic acid that binds to a conventional detectable or reporting brand molecule, for example, a radioactive isotope, a ligand, a chemiluminescent agent or an enzyme. Said probe is complementary to the chain of a desired nucleic acid, in the case of the present invention, with a genomic DNA chain of MON 88913, either from a MON 88913 plant or from a sample that includes MON 88913 DNA. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides or other probe materials that specifically bind to a white DNA sequence and that can be used to detect the presence of the white DNA sequence.

DNA primers are isolated polynucleic acids that are aligned with a complementary strand of white DNA by nucleic acid hybridization, to form a hybrid between the primer and the white DNA strand, then extend along the white strand of DNA. by means of a polymerase, for example, DNA polymerase. Pairs or sets of primers can be used for amplification of a nucleic acid sequence, for example, by polymerase chain reaction (PCR) or by other conventional methods of nucleic acid amplification.

The probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more or 30 polynucleotides or more. Said probes and polynucleotides specifically hybridize with a white sequence under strict hybridization conditions. Preferably, the probes and primers according to the present invention have a complete sequence identity with the white sequence, although probes that differ from the white sequence and retain the ability to hybridize with white sequences under conditions of high severity by conventional methods can be designed .

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2^to edition, volumes 1 to 3, Sambrook et al., Coid Sping Harbor Laboratory Press, Coid Sping Harbor,

1989, 1989 (hereinafter, "Sambrook et al., 1989"); Current Protocols ¡n Molecular Biology, edited by Ausubel et al., Greene Publishing and WileyInterscience, New York, 1992 (with periodic updates) (hereafter, “Ausuibel et al., 199”); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. Pairs of DNA primers for PCR can be derived from a known sequence, for example, using computer programs for this purpose, such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

Primers and probes based on the surrounding genomic DNA and the inserted sequences described herein can be used to confirm (and, if necessary, correct) the sequences described by conventional methods, for example, by isolating MON genomic DNA. 88913, again cloning the transgenic / genomic regions and sequencing said DNA molecules.

The nucleic acids that constitute the probes and the primers of the present invention hybridize under strict conditions with a white DNA sequence. Any conventional method of nucleic acid amplification or hybridization can be used to identify the presence of DNA from a transgenic event in a sample. The polynucleic acid molecules, or fragments thereof, are capable of hybridizing specifically with other nucleic acid molecules under certain circumstances. As used herein, it is said that two nucleic acid molecules are capable of hybridizing specifically to each other if the two molecules are capable of forming a nucleic acid structure of two antiparallel chains. It is said that a nucleic acid molecule is the "complement" of another nucleic acid molecule if it shows complete complementarity. As used herein, two molecules are said to show "complete complementarity" when each nucleotide of one of the molecules is complementary to one nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize with each other with sufficient stability to allow them to remain aligned with one another under conventional "low severity" conditions. Similarly, the molecules are said to be "complementary" if they can hybridize with each other with sufficient stability to allow them to remain aligned with one another under conventional conditions of "high severity." Common conditions of severity are described in Sambrook et al., 1989, and in Haynes et al., Nucleic Acid Hybridization, A Practic Approach, IRL Press, Washington DC (1985). Therefore, deviations from complete complementarity are allowed, as long as such distances do not completely annul the ability of molecules to form a double chain structure. In order for a nucleic acid molecule to serve as a probe or primer, it is necessary that it only has a sufficiently complementary sequence to be able to form a double chain structure under the particular concentrations of solvent and salt employed.

As used herein, a substantially homologous DNA sequence is a nucleic acid sequence that specifically hybridizes with the complement of the nucleic acid sequence with which it is compared under conditions of high severity. Appropriate severity conditions that promote DNA hybridization, for example, sodium chloride / sodium citrate (SSC) 6.0X at approximately 45 ° C, followed by washing of 2.0X SSC at 50 ° C, are known. by those trained in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected between a low severity of about SSC 2.0X at 50 ° C and a high severity of about SSC 2.0X at 50 ° C. In addition, the temperature in the washing step can be increased from low severity conditions at room temperature, approximately 22 ° C, to high severity conditions at approximately 65 ° C. The temperature and concentration of salts can be varied or the temperature or concentration of salts can be kept constant while the other variable is altered. In a preferred embodiment, a nucleic acid of the present invention will specifically hybridize with one or more of the nucleic acid molecules indicated in SEQ ID No. 3 or 4, supplements thereof or any fragment thereof, under conditions of moderate severity, for example, at about SSC 2.0X and about 65 ° C. In a particularly preferred embodiment, a nucleic acid of the present invention will specifically hybridize with one or more of the nucleic acid molecules indicated in SEQ ID No. 3 or 4, supplements thereof or any fragment thereof, under conditions of great severity. In one aspect of the present invention, a preferred marker nucleic acid molecule of the present invention comprises the nucleic acid sequence indicated in SEQ ID No. 1 or 2, supplements thereof or fragments thereof. In another aspect of the present invention, a preferred marker nucleic acid molecule of the present invention shares a substantial portion of its sequence identity with the nucleic acid sequence indicated in SEQ ID No. 1 or SEQ ID No. 2. , complements thereof or fragments of any of them, where the sequence identity comprises between 80% and 100%, or between 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% sequence identity with the sequence indicated in SEQ ID No. 1 or SEQ ID No. 2, complements thereof or fragments of any of them. SEQ ID No. 1 or SEQ ID No. 2 can be used as markers in plant cultivation methods to identify the progeny of genetic crossings, where the methods are similar to those described for the analysis of repetitions of simple sequences of DNA in DNA markers: Protocols, applications, and overviews: (1997) 173-185, Cregan, etal., Editors, Wiley-Liss NY; which is fully incorporated in this documentation by reference. Hybridization of the probe with the desired DNA molecule can be detected by any of the many methods known to those skilled in the art, which may include, without limitation, fluorescent tags, radioactive tags, antibody-based tags and chemiluminescent tags.

Regarding the amplification of a white nucleic acid sequence (eg, by PCR) using a pair of particular amplification primers, "severe conditions" are conditions that allow the pair of primers to hybridize only with the white acid sequence. nucleic with which a primer having the corresponding wild-type sequence (or its complement) is attached, preferably to produce a single amplification product, the amplicon, in a DNA thermal amplification reaction.

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

As used herein, an amplified DNA or amplicon refers to the product of nucleic acid amplification of a desired nucleic acid sequence that is part of a template nucleic acid. For example, to determine if the cotton plant obtained through a sexual cross contains in its genome transgenic DNA of the cotton plant with the event MON 88913 of the present invention, the DNA extracted from a sample of cotton plant tissue may be subjected to a method of amplifying polynucleic acids using a pair of primers that includes a primer derived from the DNA sequence in the genome of the MON 88913 plant, adjacent to the insertion site of the inserted heterologous DNA (transgenic DNA); and a second primer derived from the inserted heterologous DNA, to produce an amplicon that is diagnostic for the presence of DNA from the MON 88913 event. The diagnostic amplicon has a length and a sequence that are also diagnostic for the event. The amplicon may vary in length, from the combined length of the primer pairs, plus a pair of nucleotide bases, more preferably about fifty pairs of nucleotide bases (bp), more preferably even about two hundred and fifty pairs of nucleotide bases and even more preferably approximately four hundred and fifty pairs of nucleotide bases or more. Alternatively, a pair of primers can be obtained from the surrounding genomic sequence on both sides of the inserted DNA, so as to produce an amplicon that includes the complete inserted nucleotide sequence (eg, a direct primer isolated from the genomic portion of SEQ ID No. 3 and a reverse primer isolated from the genomic portion of SEQ ID No. 4, which amplify a DNA molecule comprising the two expression cassettes of the DNA fragment pMON51915 that was inserted into the genome of MON 88913, where the insert comprises approximately 8512 bp of the insert, Figure 2). A member of the pair of primers derived from the genomic sequence of the plant may be located at a certain distance from the inserted DNA sequence, where this distance may vary between a pair of nucleotide bases and approximately twenty thousand pairs of nucleotide bases. The use of the term amplicon specifically excludes the pairs of primers that may be formed in the DNA thermal amplification reaction.

Polynucleic acid amplification can be achieved using any nucleic acid amplification method known in the art, including polymerase chain reaction (PCR). A variety of amplification methods are known in the art, and are described, among others, in US Pat. Nos. 4,683,195 and 4,683,202, and in PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Nati. Acad. Sci. USA 9T.

5695-5699, 1994). These methods, as well as other methods known in the art of DNA amplification, can be used in the practice of the present invention. The sequence of the inserted heterologous DNA or the surrounding sequence of MON 88913 can be verified (and corrected, as necessary) by amplifying said DNA molecules from MON 88913 seeds or plants grown from the seeds deposited in the ATCC with the number of PTA-4854 access, using primers derived from the sequences provided herein, followed by conventional sequencing of the DNA from the PCR amplicon, or cloned DNA fragments thereof. The sets of DNA detection elements that are based on DNA amplification methods contain DNA primers that specifically amplify a diagnostic amplicon. The set of elements may comprise a detection method based on agarose gel, Taqman®, or any method for detecting the amplicon that is known in the art. A set of elements containing DNA primers that are homologous or complementary to any portion of SEQ ID No. 3 or SEQ ID No. 4 constitutes an object of the invention.

The amplicon produced by these methods can be detected using a plurality of techniques. One such method is the analysis of genetic fragments (Nikiforov, et al. Nucleic Acid Res. 22: 4167-4175, 1994), where a DNA oligonucleotide is designed that overlaps with the adjacent surrounding genomic DNA sequence and the sequence Transgenic DNA inserted. The oligonucleotide is immobilized in the cavities of a plate for mlcrotulation. After PCR of the region of interest (using a primer of the inserted sequence and one of the adjacent surrounding genomic sequence), a single chain PCR product can be hybridized with the immobilized oligonucleotide and used as a template for an extension reaction of single base using a labeled DNA polymerase and dideoxynucleotide triphosphate (ddNTP) specific for the next expected base. The reading can be based on fluorescence or ELISA. A signal indicates the presence of the transgenic / genomic sequence due to the successful single base amplification, hybridization and extension.

Another method is the pyrosequencing technique, as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is designed that overlaps with the adjacent genomic DNA and the DNA inserted into the junction. The oligonucleotide is hybridized with the single chain PCR product of the region of interest (a primer in the inserted sequence and one in the surrounding genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5 'phosphosulfate and luciferin. DNTP is added individually, and incorporation results in a light signal that is measured. A light signal indicates the presence of the transgenic / genomic sequence due to the successful amplification, hybridization and extension of a single or multiple bases.

Fluorescence polarization, as 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 designed that overlaps with the surrounding genomic DNA and the DNA inserted into the junction. The oligonucleotide is hybridized with a single chain PCR product of the region of interest (a primer in the inserted DNA and one in the surrounding genomic DNA sequence) and incubated in the presence of fluorescently labeled DNA polymerase and ddNTP. A single base extension results in the incorporation of ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgenic / genomic sequence due to the amplification, hybridization and extension of a single successful base.

Taqman® (PE Applied Biosystems, Foster City, CA) is described as a method of detecting and quantifying the presence of a DNA sequence, and will be used according to the instructions provided by the manufacturer. Briefly, a FRET probe oligonucleotide is designed that overlaps with the surrounding genomic DNA and the DNA inserted into the junction. The FRET probe and the PCR primers (a primer in the inserted DNA and one in the surrounding genomic sequence) are cyclized in the presence of a thermostable polymerase and dNTP. Hybridization of the FRET probe results in the cutting and release of the fluorescent portion of the neutralization portion in the FRET probe. A fluorescent signal indicates the presence of the transgenic / genomic sequence due to successful amplification and hybridization.

Molecular markers have been described for use in sequence detection, as described by Tyangi, et al. (Nature Biotech. 14: 303-308, 1996). Briefly, a FRET oligonucleotide is designed that overlaps with the surrounding genomic DNA and the DNA inserted into the junction. The unique structure of the FRET probe is characterized by a secondary structure that keeps the fluorescent and neutralization portions close to each other. FRET probe and primer PCR primer (a primer in the inserted DNA and one in the surrounding genomic sequence) are cycled in the presence of a thermostable polymerase and dNTP. After successful PCR amplification, hybridization of the FRET probe with the white sequence results in the removal of the secondary structure of the probe and the spatial separation of the fluorescent and neutralization portions. A fluorescent signal is obtained. A fluorescent signal indicates the presence of the inserted transgene / surrounding sequence due to successful amplification and hybridization.

Sets of elements for DNA detection can be developed using the compositions described herein and methods well known in the art of DNA detection. Element sets are useful for identifying the DNA of the MON 88913 cotton event in a sample, and can be applied to methods for growing cotton plants that contain the MON 88913 DNA. Element sets contain DNA sequences that are useful as primers or probes, which are homologous or complementary to any portion of SEQ ID No. 3 or SEQ ID No. 4, or with homologous or complementary DNA sequences of DNA. contained in the transgenic genetic elements of pMON51915 that have been inserted into the MON 88913 DNA (Figure 2). These DNA sequences can be used in DNA amplification methods (PCR) or as probes in polynucleic acid hybridization methods, that is, Southern analysis or Northern analysis. The transgenic elements contained in the MON 88913 DNA (Figure 2) include a first expression cassette comprising the mosaic virus promoter of the scrofularia, constructed as a chimeric promoter element with the Arabidopsis elongation factor 1-alpha promoter ( At.Efla) (FMV35S / Efla, US Patent No. 6,462,258, SEQ ID No. 28, fully incorporated herein by reference), operatively linked to the guideline sequence and to the intron of Arabidopsis elongation factor 1-alpha (Genbank accession number X16430, described in Axelos et al ,, Mol. Gen. Genet. 219: 106-112, 1989), operatively linked to Arabidopsis EPSPS chloroplast transit peptide (TS-At.EPSPS: CTP2, Klee et al., Mol. Gen. Genet. 210: 47-442, 1987), operatively linked to a 5-enol-pyruvilshiquirnato-3 -phosphate synthetase (EPSPS) with glyphosate tolerance of the strain of Agrobacierium sp. CP4 (aroA: CP4, US Patent No. 5,633,435), operatively attached to the 3 'termination region of pea 1,5-bisphosphate carboxylase E9 (T-Ps.RbcS2.E9, Coruzzi, at al., EMBO J. 3: 1671-1679, 1484), and a second expression cassette comprising the CaMV35S-Act8 promoter, which includes the first intron of the Act8 gene (SEQ ID No. 29, US Patent No. 6,462,258), attached operatively to the Arabidopsis EPSPS chloroplast transit peptide (TS-At.EPSPS: CTP2), operatively linked to a 5enol-pyruvilshiquimato-3-phosphate synthetase (EPSPS) with glyphosate tolerance of the Agrobacierium sp. CP4 (aroA: CP4, US Patent No. 5,633,435, fully incorporated herein by reference), operably linked to the 3 'termination region of pea 1,5-bisphosphate carboxylase E9 ribulose .

The following examples are included to demonstrate examples of some preferred embodiments of the invention. Those skilled in the art should appreciate that the procedures described in the examples below represent approaches that the inventors found to work well in the practice of the invention and, therefore, may be considered to be examples of preferred modes for their practice.

However, those skilled in the art should, in the light of the present description, appreciate that several changes can be made to the specific embodiments described and still obtain a similar or similar result, without departing from the spirit and scope of the invention.

EXAMPLES

Example 1

The MON 88913 cotton transgenic event was generated by Agrobacterium-mediated transformation of cotton cells with a DNA fragment derived from pMON51915 (Figure 1). The plant transformation construct, pMON51915, was introduced into Agrobacterium using a triparental conjugation procedure (Ditta et al., Proc. Nati. Acad. Sci. 77: 7347-7351, 1980). The transformation of cotton cells with transgenes can be carried out using the methods described, for example, in US Patent No. 5,004,863, US Patent No. 5,159,135 and US Patent No. 5,518,908, fully incorporated in this documentation by reference. The transformation of cotton is performed essentially as described in WO 1003691, or as described in US Patent No. 5,846,791, fully incorporated herein by reference. A modification of these methods may include, without limitation, the following example. Coker 130 seed surfaces are sterilized and germinated in the dark. Hypocotyl explants are trimmed from germinated shoots to lengths of approximately 1-1.5 centimeters. The ABI strain of Agrobacterium tumefaciens, transformed to contain pMON51915, is grown in Luria broth without antibiotics for 16 hours at 28 ° C, then diluted to approximately 2 x 10⁸ bacteria / milliliters (mi). The hypocotyl explants are immersed in the inoculum of Agrobacterium for 2-5 minutes, then co-cultured for approximately 45 hours in MS⁺ KNO3 1.9 mg / l + 3% glucose (TRM), 30 explants per plate, at 24 ° C in the dark. The explants are transferred to a TRM medium containing 150 mg / l of cefotaxime and 300 μato glyphosate for four periods of culture, where each period is extended for approximately six weeks. Embryogenic cells are secreted from primary explants in 3^er and 4th cultivation period, and they are placed in the same medium. Embryogenic cells are subcultured once, submerging them briefly in TRM liquid medium + 3% glucose, followed by placing the suspension on plates with TRM + cefotaxime 150 mg / l + glyphosate 300 μΜ. Somatic embryos are harvested 3-8 weeks after liquid subculture, then grown in Stewart and Hsu medium with 0.5 glucose. Seedlings derived from somatic embryos are allowed to mature until they reach approximately 4-7 cm (3-6 leaves) in Magenta boxes with Stewart medium and Hsu modified with 40 mM / NH NO3₄ 10 mM + sucrose

two. These plants are then transplanted to planting soil, in pots of 4, with 100% humidity and 16 hours of light per day, for 4-6 days, followed by 50% humidity for 5-10 days.

The DNA fragment of pMON51915 contains two transgenic expression cassettes inserted into the genome of MON 88913 (Figure 2), which collectively confer glyphosate tolerance to MON 88913 and the progeny thereof.

MON 88913 plants and seeds have regenerable parts. Regenerable parts of the seeds include, without limitation, the embryo, cotyledon and roots or root meristems. The regenerable parts of the plant include, without limitation, the leaves, the petiole, the hypocotyl, stem sections and the apical and root meristems. The invention also includes parts of MON 88913 cotton event plants, which include, without limitation, pollen, ovules, flowers, flakes, threads, buds, roots and leaves. The invention also includes the removable components of MON 88913, which include, without limitation, proteins, foods, flours, shells, oils and lnters.

EXAMPLE 2

The glyphosate tolerance event in cotton MON 88913 was selected from several transgenic events of vegetative and reproductive tolerance to lesions with glyphosate in cotton. The successful production of a commercial quality transgenic event currently requires the production of a large number of transgenic events. In the present invention, MON 88913 was an event among approximately 1000 R0 events that had been transformed with several different DNA constructs that included pMON51915. The MON 88913 event was selected among the different events through a series of molecular analyzes and glyphosate tolerance searches.

Events were analyzed in a greenhouse glyphosate tolerance test, where vegetative and reproductive tolerance was evaluated in plants. Between fifteen and twenty-five Ri seeds of each event were sown in 15 cell trays with Metro-Mix 350 culture medium, which contains a combination of humus, vermiculite, nutrients, wetting agents and processed bark 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 day temperature during the growing season was 32 degrees Celsius (° C), while the average night temperature was 24 ° C. The photoperiod was set at 16 hours of light and eight hours of darkness, with maximum lighting intensity. The average relative humidity during the crop cycle was 45 percent. The plants were then sprayed in the 4 and 6 leaf stages, consecutively with 48 oz / a (oz = ounces, a = acre) of Roundup Ultra® (herbicide containing glyphosate). Seven days after the application of glyphosate in the 4-leaf stage, the vegetative damage was determined in the plants and the segregated glyphosate tolerance phenotypes were collected. These data were used to confirm that the transgenic event insert was performing as a single dominant gene, according to Mendelian genetic models. Events with good vegetative tolerance were subsequently transplanted into 10-inch pots with the same Metro-Mix 350 culture medium described previously, and grown until maturity. The plants were treated with Pix Plus (BASF, Research Triangle Park, NC), as necessary, to regulate the height of the plant. Three months after planting, the retention of flakes in the first fruiting position in the first five branches with fruits was determined in the plants. The maximum retention value for this determination in plants is 5 (five flakes retained). This provided a rapid indirect measurement of plant fertility. With this greenhouse test, the average retention for the current commercial event (RR 1445 cotton) is less than 1.0. Events with an average value of retention of flakes greater than or equal to three were harvested and subjected to another procedure for selecting events: events with retention values ​​of flakes greater than or equal to two have value as new plant selections with tolerance to glyphosate

In the events that met the criteria of vegetative and reproductive tolerance to Roundup® Ultra, the quantity of copies was analyzed by means of a Southern blot analysis. Events with single copies that showed good tolerance in the initial greenhouse experiments were further characterized by 1) additional greenhouse tolerance tests with higher glyphosate application rates, 2) field test replicas and 3) more molecular analysis Greenhouse tolerance tests were performed using homozygous plants. All experiments contained the current Roundup Ready® 1445 cotton commercial event (commercial reference) by way of comparison. The seeds were sown in 15 cell trays and treated with 64 oz / y of Roundup Ultra® in the 4-leaf stage and 96 oz / a in the 8-leaf stage. The plants were transplanted into 10-inch pots and the first fruiting positions of the first five branches with fruits were determined in four plants during the middle of the season. At the end of the season, data were also collected from all events, which included the weight of the cottonseed, the number of flakes, the size of the flakes and the retention of the flakes.

Field tests were used to select the events with the best growth rates, fruit retention and yield. Field tests were arranged in a randomized division design, with three replications and three treatments. The events were sown in two plots with two rows of 30 feet. The treatments consisted of 64 oz / a (1.5 kb ae / a) of Roundup Ultra® without spraying, in the stages of 4, 6, 10 and 14 nodes, and 96 oz / a (2.25 Ib of ae / a) Roundup Ultra® in stages 4, 6, 10 and 14 nodes. In the middle of the season, the evaluation of the plants on ten plants per plot was completed. The flake retention data were collected for the first and second fruiting position of the first five nodes with fruits, which resulted in a flake retention value on a scale of 0-10. A plant with a flake retention value equal to or greater than 3 on a scale of 0-10 has value as a new plant selected on the basis of glyphosate tolerance.

Field tests (10 sites) where the performance of cotton yarn (pounds / acre, Ib / a) of MON 88913 and cotton RR 1445 was compared showed that MON 88913 provided substantial protection against glyphosate (pounds of acid / acre equivalent , Ib de ea / a), affecting performance (Table 1). The yield is a measure of the retention of the flakes, and the modified cotton plants with glyphosate tolerance that retain a substantial amount of flakes in the first and second fruiting position will maintain an average yield superior to that of the cotton plants that do not show tolerance to glyphosate. An effective dose of a herbicide containing glyphosate to control weeds in a field with MON 88913 plants comprises approximately 4 oz / y, and may exceed 128 oz / y, depending on the amount of weed to be controlled and the state of development of weeds. Glyphosate can be mixed with other herbicides to improve herbicidal activity against certain weed species.

Table 1. Comparison of the yield of cotton yarn MON 88913 and 1445 after treatment with glyphosate.

Performance (Ib / a) in 10 sites

Glyphosate

Treatment

1445

MON 88913

EXAMPLE 3

Ib de ae / a

2421,84

2551,58

1.5 Ib of ae / a

1044,19

2587,61

2.5 Ib of ae / a

831,47

2412,3

Genomic DNA was isolated from all POR reactions and Southern blot analysis using a CTAB procedure (Rogers et al., Plant Mol. Biol. 5: 69-76, 1985) or the set of elements for Dneasy ™ 96 plants ( Cat. No., Qiagen Inc., Valencia, CA), according to the manufacturer's instructions. Foliar tissue was collected from plants in the 2-4 leaf stage. The smallest true leaves of each plant were collected and immediately frozen in dry ice. The DNA was extracted using, for example, the following method. The tissue was ground using plastic spheres with liquid nitrogen. Five ml of extraction buffer solution was added to 0.75 grams (g) of tissue and incubated at 55 ° C for 45 minutes. The CTAB extraction buffer solution consisted of 100 mM Tris, pH 8.0, 1.4 M NaCI, 20 mM EDTA, 29% CTAB with the addition of 5 μΙ (microliters) of beta-mercaptoethanol, 5 μΙ of RNase and PVPP one%. Then, the samples were extracted with an equal volume of chloroform (5 ml) and centrifuged at 3700 rpm for 15 minutes 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 3700 rpm for 15 minutes, the precipitates were washed with 70% ethanol, air dried and suspended again in 250 µΙ of water.

Genomic cotton DNA was obtained adjacent to the transgen insertion site of the MON 88913 event using TAIL-PCR (Liu et al., Plant Journal 8: 457-463, 1995). Genomic DNA extension was performed using the GenomeWalker element set (CloneTech Laboratories, Palo Alto, CA), according to the manufacturer's protocol. Briefly, the DNA (approximately 5 pg) was digested, isolated using the CTAB protocol described previously, with various restriction endonucleases (EcoRV, Seal), at 37 ° C overnight, in a total volume of 100 μΙ. Restriction endonucleases with QIAquick PCR purification columns (Cat # 28104, Qiagen, Inc.) were removed. The binding of the adapter molecules was performed in accordance with what is 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.

3 'transgenic / genomic DNA from MON 88913 was isolated using reverse PCR. Total genomic DNA (approximately 10 μg) was digested with three restriction enzymes: Bell, Ncol and Hindlll. QIAquick PCR purification columns were used to purify the DNA after digesting it overnight at 37 ° C. DNA from the columns was eluted with 50 μΙ of water, and then diluted to 1 ml. The eluted portion was combined after dilution (85 μΙ) with μΙ of buffer (10x) and 5 μΙ of T4 ligase to obtain circular fragments. After an overnight incubation at 16 ° C, the ligase was quenched with heat at 70 ° C. The samples were amplified by PCR with a series of nested primers. Combinations of primers for PCR included: primary pair 8099-E9-1 / E9-2 (SEQ ID No. 7 / SEQ ID No. 8) for samples with Bell and Ncol, and primer pair 8099-E9 -1 / Act8 rev (SEQ ID No. 7 / SEQ ID No. 9) for the sample with Hindlll; primer pair 8099-E9-2 / E9-1 (SEQ ID No. 10 / SEQ ID No. 11) for samples with Bell and Ncol; primer pair 8099-E9-2 / Act8 (SEQ ID No. 10 / SEQ ID No. 12) for the Hindlll sample; primer pair 8099-E9-3 / E9-1 (SEQ ID No. 13 / SEQ ID No. 11) for samples with Bell and Ncol; and 8099-E9-3 / Act8 (SEQ ID No. 13 / SEQ ID No. 12) for the sample with Hindlll. The PCR conditions included: Primary PCR = 7 cycles of 94 ° C for 2 seconds, 72 ° C for 10 minutes; 37 cycles of 94 ° C for 2 seconds, 67 ° C for 10 minutes; 1 cycle of 67 ° C for 10 minutes; Secondary and tertiary PCR = 5 cycles of 94 ° C for 2 seconds, 72 ° C for 10 minutes; 24 cycles of 94 ° C for 2 seconds, 67 ° C for 10 minutes; 1 cycle of 67 ° C for 10 minutes.

Alternatively, DNA amplification can be performed by PCR at the 3 'end of the MON 88913 event with conditions including: 7 cycles of 94 ° C for 25 seconds, 72 ° C for 3 minutes; 37 cycles of 94 ° C for 25 seconds, 67 ° C for 3 minutes; 1 cycle of 67 ° C for 7 minutes. All subsequent amplifications were performed under the following conditions: 7 cycles of 94 ° C for 2 seconds, 72 ° C for 4 minutes; 37 cycles of 94 ° C for 2 seconds, 67 ° C for 4 minutes; 1 cycle of 67 ° C for 7 minutes. All amplicons were visualized on 0.8% agarose gels colored with ethidium bromide. DNA was prepared for sequencing by purifying the PCR samples directly with the set of elements for QIAquick PCR purification (Cat. No. 29104, Qlagen Inc.) or extracting the appropriate gel fragment and using the set of gel extraction elements QIAquick (Cat. No. 428704, Qiagen Inc.).

A series of DNA primers was designed to sequence the transgenic insert and adjacent surrounding genomic regions of MON 88913. DNA primers were designed to allow amplification of the transgene and the entire surrounding regions using five overlapping fragments. Unique primers were designed to allow amplification of each EPSPS-CTP2 / aroA-CP4 / RbcS2: E9 region separately. For all fragments used in sequencing, triplicate amplifications were performed. Combinations of pairs of DNA primers were used as sequencing primers for the 5 'transgenic / genomic region (SEQ ID No. 14 and SEQ ID No. 15), the 3' transgenic / genomic region (SEQ ID No. 16 and SEQ ID No. 17) and the genetic elements inserted (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 were visualized in 0.8% agarose gels colored with ethidium bromide. DNA was prepared for sequencing by purifying the PCR samples directly with the set of elements for QIAquick PCR purification or by extracting the appropriate gel fragment and using the set of QIAquick gel extraction elements. The DNA sequence was obtained using a set of elements for DNA sequence analysis (ABI Prism ™ 377, PE Biosystems, Foster City, CA) and DNASTAR sequence analysis software (DNASTAR Inc., Madison, Wl).

DNA fragments from the surrounding regions of the transgenic / genomic insert were subcloned into MON 88913 using a set of TOPO TA® cloning elements (Invitrogen). The DNA sequence of the 5 'transgenic / genomic region is detailed in Figure 4, while the DNA sequence of the 3' transgenic / genomic region is detailed in Figure 5. In the DNA sequence illustrated in the Figures 4 and 5, the sequence of the transgenic insert in italics is indicated.

EXAMPLE 4

Event DNA primers are used to produce a diagnostic amplicon for the MON 88913 cotton event genome. Diagnostic amplicons for the MON 88913 genome comprise at least one binding sequence, SEQ ID No. 1 or SEQ ID No. two. Event primers that produced a diagnostic amplicon for MON 88913 were used, where primer pairs included, without limitation 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 3 'sequence of the amplicon, in the protocol detailed in Table 2. In addition to these primer pairs, any pair of primers, homologues or complementary to SEQ ID No. 3 or SEQ ID No. 4, which produces a diagnostic amplicon for the genome of MON 88913 in a DNA amplification reaction, It is an aspect of the present invention. Any single primer molecule composed of DNA polynucleotides 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. Any single primer molecule composed of DNA polynucleotides comprising less than 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. An example of the amplification conditions for this analysis is illustrated in Table 2 and Table 3; however, modifications for these methods that use homologous or complementary DNA primers with SEQ ID No. 3 or SEQ ID No. 4, or DNA sequences of the genetic elements contained in the transgenic insert of MON 88913 that produce a diagnostic amplicon for MON 88913, they will be within the knowledge of those trained in the art. A diagnostic amplicon comprises a homologous or complementary DNA molecule with at least one transgenic / genomic DNA (SEQ ID No. 1 or SEQ ID No. 2), or a substantial portion thereof.

An analysis for a sample of plant tissue with the MON 88913 event should include a positive tissue control of the MON 88913 event, a negative control of a cotton plant that does not contain the MON 88913 event, and a negative control that does not contain genomic DNA of cotton. Those skilled in the art may select additional primer sequences between SEQ ID No. 3 and SEQ ID No. 4, for use in DNA amplification methods, and the conditions selected for the production of an amplicon using these methods may be different from those which are detailed in Table 2 and Table 3, but can still result in a diagnostic amplicon for the MON 88913 event. The use of these primer DNA sequences with modifications of the methods of

<img file="AR096584A2_D0001.tif" />

Tables 2 and 3 are within the scope of the invention. The amplicon produced by at least one primer DNA sequence derived from SEQ ID No. 3 or SEQ ID No. 4, which is diagnostic for MON 88913, is an aspect of the invention.

The sets of DNA detection elements containing 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 , are an aspect of the invention. The amplicon produced by at least one primer sequence derived from any of the genetic elements of pMON31915, which is diagnostic for MON 88913, is an aspect of the invention. A plant or a cottonseed whose genome produces an amplicon comprising SEQ ID No. 1 or SEQ ID No. 2, when evaluated in a DNA amplification method, will be an aspect of the present invention. The presence of the MON 89913 amplicon can be determined using a Stratagene-Robodycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercicler Gradient thermal cycler, as indicated in Table 3, or using methods and apparatus known to those skilled in the art.

Table 2. PCR procedure and reaction mixture conditions for the identification of the transgenic / genomic binding region of

MON 88913 5 *.

Reactive Step

Nuclease free water

Reaction damper

Quantity Comments

It is added up to a final volume of 10μΐ

2.0 μΙ Final concentration

10x (with MgCb)

0.4 μΙ Solution of dATP, dCTP, ^H10 mM dGTPydTTP

Event primer (SEQ0.4 μΙ (ID No. 14) (suspended again in 1x TE buffer or nuclease-free water ^ up to a concentration of 10 μΜ)

Event primer (SEQ 0.4 μΙ

ID No. 15) (suspended again in 1x TE buffer or nuclease-free water up to ^ 10 μΜ concentration)

1x buffer, 1.5 mM final concentration of MgCl2 Final concentration of 200 μΜ for each dNTP

Final concentration 0.2 μΜ

Final concentration 0.2 μΜ

DNase-free RNase (500 ng / μΙ)

0.1 μΙ ng / reaction

1.0 μΙ (REDTaq DNA polymerase is recommended to change (1 unit / μΙ) pipettes before the next step) unit / reaction

DNA extracted (template). 'Samples to be analyzed: - individual sheets - grouped sheets (maximum of 50 sheets / set)' Negative control 'Negative control * 10-200 ng of genomic DNA I * 200 ng of genomic DNA * 50 ng of DNA

<img file="AR096584A2_D0002.tif" />

* Positive control * Cotton genomic positive control (other than MON 88913) * without template DNA * 50 ng MON 88913 genomic DNA

Mix gently, add 1-2 drops of mineral oil in each reaction

Table 3. Suggested PCR parameters for the different thermal cyclers.

The PCR is performed on a Stratagene Roboclcler, MJ device

Engine, Perkin-Elmer 9700, or in an Eppendorf Mastercicler Gradient thermal cycler, using the following cycling parameters. The MJ Engine or Eppendorf Mastercicler Gradient thermal cycler should be operated in calculated mode. The Perkin-Elmer thermal cycler is operated

9700 with the ramp speed set to maximum.

Cycle No. Parameters; Stratagene Robocycler

94 ° C 3 minutes

94 ° C 1 minute

60 ° C 1 minute

72 ° C 1 minute and 30 seconds

I 72 ° C 10 minutes

Cycle No. Parameters; MJ Engine or Perkin-Elmer 9700

94 ° C 3 minutes

94 ° C 10 seconds

60 ° C 30 seconds 72 ° C 1 minute

Cycle No.

72 ° C 10 minutes

Parameters: Eppendorf Mastercycler Gradient

94 ° C 3 minutes

94 ° C 15 seconds

60 ° C 15 seconds

72 ° C 1 minute and 30 seconds

72 ° C 10 minutes

EXAMPLE 5

MON 88913 genomic DNA and control cotton genomic DNA (approximately 15 pg each) are digested with several restriction enzymes (140 U) in a total volume of 150 μΙ, including 15 μΙ of the corresponding supplier's buffer solution (NEB, Beverely , MA). Restriction endonucleases, for example, Bgll, Bamlll, Ncol, Hindlll and Bell, are used in the Southern analysis of MON 88913. Endonuclease digestions are performed at the appropriate temperature for at least 6 hours. After incubation, the DNA is precipitated with 3M sodium acetate and 2.5 volumes of ethanol. Subsequently, the DNA is washed with 70% ethanol, dried and suspended again in 40 μΙ of TBE. Charge buffer (0.2x) is added to the samples and an electrophoresis is performed on agarose gels (0.8%) for 16-18 hours at 30 volts. The gels are colored with ethidium bromide, then treated with a depurination solution (0.125 N HCI) for 10 minutes, with a denaturation solution (0.5M sodium hydroxide, 1.5M sodium chloride) for 30 minutes. minutes, and finally with a neutralization solution (0.5 M Trizrna base, 1.5 M sodium chloride) for 30 minutes. The DNA is transferred to a Hybond-N membrane (Amersham Pharmacia Biotech, Buckinghamshire, England) using a Turboblotter device (Scleicher and Schuell, Daasel, Germany) for 4-6 hours, and then fixed to the membrane using a UV light .

The membranes are pre-hybridized with 20 ml of Easy Hyb DIG solution (Roche Molecular Biochdmicals, Indianapolis, IN, Cat. No. 1603558) for 2-4 hours at 45 ° C. Radioactive DNA probes are prepared (³²P dCTP) homologous or complementary to SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, or portions thereof, using the set of elements to mark Radprime DNA (Invitrogen, Carlsbad, CA; Cat. No. 15428-011). Unincorporated nucleotides are removed using Sephadex G50 columns (Invitrogen). The prehybridization solution is replaced with 10 ml of a previously hybridized Easy Hyb DIG solution, containing the denatured probe, to a final concentration of 1 million counts per ml. The transferred ones are hybridized at 45 ° C for 16-18 hours.

The transferred ones are washed with a low severity solution (5x SSC, 0.1x SDS) at 45 ° C and then washed repeatedly with a higher severity solution (0.1x SSC, 0.1% SDS) at 65 ° C. Transfers to a phosphor screen (Amersharn Bloeciences, Piscataway, NJ) are exposed for&gt; 2 hours, and the exposed plate is read using a Data Storm 860 (Amersharn Biosciences).

EXAMPLE 6

The methods used to separate the heterozygous progeny from the homozygous progeny containing the MON 88913 event in a cygosity assay are described for which examples of conditions are described in Table 4 and Table 5. The DNA primers used in the assay of zygosity are primer SQ1099 (SEQ ID No. 21), SQ1100 (SEQ ID No. 22), SQ1353

<img file="AR096584A2_D0003.tif" />

(SEQ ID No. 23), the primer labeled 6-FAM ™ (SEQ ID No. 24, and the primer labeled VIC ™ (SEQ ID No. 23), 6-FAM and VIC are fluorescent dyes produced by Applied Biosystems (Foster City, CA), which bind to the DNA primer.

When SEQ ID N ° 21, SEQ ID N ° 22 and SEQ ID N ° 23 are used in these reaction methods, they produce a DNA amplicon for non-transgenic cotton, two DNA amplicons for the heterozygous cotton that contains the MON event 88913, and a DNA amplicon for homozygous cotton MON 88913 that is different from any other cotton other than MON 88913. Controls in these analyzes should include a positive control of homozygous and heterozygous cotton containing MON 88913 event DNA, a negative non-transgenic cotton control and a negative control that does not contain template DNA. This test has been optimized to use a Strategene Robocicler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercicler Gradient thermal cycler. Those skilled in the art know other methods and apparatus for producing amplicons that identify the cygosity of the progeny of the crosses made with MON 88913 cotton plants.

Table 4. Reaction solution of the cygosity test.

Reagent Step Quantity Comments

It adds up to a

Nuclease-free water final volume of 10μΙ μΙ

2x Universal Master Mix (Applied Biosystems cat. # 4304437) Primers SQ1099,0,5pl

1x final concentration

Concentration

SQ1100, SQ1353 final 0.25 μΜ (again suspended in nuclease-free water to a concentration of μΜ)

6-FAM ™ 0.2 μΙ primer (suspended again in nuclease-free water to a concentration of

Final concentration 0.4 μΜ μΜ)

VIC ™ primer (suspended again in nuclease-free water up to a concentration of 10 μΜ)

0.2 μΙ

Final concentration 0.15 μΜ

1.0 μΙ (se REDTaqire DNA polymerase exchange recommendation

<td rowspan="2">(1 unit / μΙ) DNA extracted (template):</td><td colspan="2">pipettes before the unit / reaction</td>

<td>next step)</td><td>It is diluted in</td>

<td>* Samples to analyze:</td><td>* 3.0 μΙ</td><td>Water</td>

<td>(individual sheets)</td><td>* 4-80 ng genomic</td><td>DNA</td>

<td>'Negative control</td><td>* 4 ng of genomic cotton</td><td>DNA from no</td>

Transgenic 'Negative control' Positive control 'without template DNA (solution where DNA was suspended again) * 4 ng of known genomic DNA from heterozygous cotton

<img file="AR096584A2_D0004.tif" />

event MON 88913 * &lt;

'Positive control * 4 ng of known genomic cDNA of homozygous cotton from event MON 88913 of

Mix gently, add 1-2 drops of mineral oil in each reaction

Table 5. Conditions for thermocyclers in the cygosity test.

It proceeds with the amplification of DNA in a Stratagene device

Robocicler, MJ Engine, Perkin-Elmer 9700, or in an Eppendorf Mastercicler Gradient thermal cycler, using the following cycling parameters. When using an Eppendorf Mastercicler Gradient thermal cycler or an MJ Engine device it should be operated in calculated mode. When using a Perkin-Elmer 9700 thermal cycler, the ramp speed set to maximum should be used.

Cycle No. Parameters: Stratagene Robocycler

94 ° C 3 minutes

94 ° C 1 minute

Cycle No.

60 ° C 1 minute

72 ° C 1 minute and 30 seconds

72 ° C 10 minutes

Parameters: MJ Engine or Perkin-Elmer 9700

94 ° C 3 minutes

94 ° C 30 seconds

60 ° C 30 seconds

72 ° C 1 minute and 30 seconds

72 ° C 10 minutes

Parameters: Eppendorf Mastercycler Gradient

94 ° C 3 minutes

Cycle No. 1

94 ° C 15 seconds

60 ° C 15 seconds

72 ° C 1 minute and 30 seconds

72 ° C 10 minutes

EXAMPLE 7

Analysis of genomic DNA samples was performed using a terminal method of Taqman®. Diagnostic amplicons were produced for genomic DNA from MON 88913 using a set of primers A, which included the primers for event SEQ ID No. 21, SEQ ID No. 22, and the 6-FAM SEQ ID No. 24 probe; and a set of primers B, which included the primers for the event SEQ ID No. 26 and SEQ ID No. 27, and the 6-FAM SEQ ID No. 28 probe. The method uses a format of 96 cavities or 394 cavities, and an Applied Biosystems GeneAmp PCR 9700 system or an M1 Research DNA Engine FT225 device. The DNA extracted from the cotton tissue samples, as previously described, should be in the range of 5 to 10 ng per PCR reaction. Each reaction contains a final volume 100 μΙ consisting of 0.5 μΙ of an equal concentration of each of the primers for the event (20 μΜ), 5.0 μΙ of 2x universal master mix, 0.2 μΙ of the probe 6-FAM (10 μΜ), 3 μΙ of the DNA sample (5-10 ng) and water to complete 10 μΙ. The thermal cycling parameters are: 1 cycle at 50 ° C for 2 minutes, 1 cycle at 95 ° C for 10 minutes, 10 cycles at 95 ° C for 15 seconds, 64 ° C for 1 minute, then -1 ° C / cycle, 30 cycles of 95 ° C for 15 seconds, 54 ° C for 1 minute, then kept at 10 ° C. Amplicon production was determined using a microplate reader, for example, TECAN Safire (Durham, NC), using the conditions described by the manufacturer. A data analysis program (TaqPro ™) was used to evaluate the production of the labeled amplicon. Other equipment and other methods of analysis known in the art of DNA detection can be used to detect the amplicons of the present invention.

A deposit was made in the name of Monsanto Technology LLC, which consisted of MON 88913 cotton seeds previously described and detailed in the claims, under the conditions of the Budapest Treaty, in the American Culture Type Collection (ATCC), 10801 University Boulevard , Manassas, Go. 20110. The ATCC access number is PTA-4854. The deposit in the institution would be kept for a period of 30 years or 5 years after the last request, or during the effective duration of the patent, whatever the most extended period, and will be replaced during that period, as necessary .

Having illustrated and described the principles of the present invention, it should be apparent to those skilled in the art that the invention can be modified in its arrangement and details, without departing from said principles. All modifications within the spirit and scope of the appended claims are claimed.

All published publications and patent documents cited in this specification are incorporated herein by reference, to the same extent that it was indicated that each individual patent publication or application was incorporated specifically and individually as a reference.

<td></td><td>SEQUENCE LIST</td>

<td> &lt;110&gt;</td><td>Cerny, Eric Duong, Can Hart, Jesse Huber Scott Krieb, Rachel</td>

<td> &lt;120&gt;</td><td>Listello, Jennifer Martens, Amy Sammons, Bernard COTTON EVENT MON 88913 AND COMPOSITIONS AND METHODS FOR YOUR</td>

DETECTION

<td> &lt;130&gt;</td><td>11899.0239.00PC00 (MOBT: 239P)</td>

<td> &lt;150&gt;</td><td> 60/447.184</td>

<td> &lt;151&gt;</td><td> 2003-02-12</td>

<td> &lt;160&gt;</td><td> 28</td>

<td> &lt;170&gt;</td><td>Patent version 3.2</td>

<td> &lt;210&gt;</td><td> 1</td>

<td> &lt;211&gt;</td><td> 20</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>Chimeric DNA from cotton genomic DNA and transgenic DNA</td>

inserted &lt;400&gt; 1 attcaatgta gtcaaacact &lt;210&gt; 2 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Chimeric DNA of cotton genomic DNA and transgenic DNA inserted &lt;400&gt; 2 ttgaatatat attacaaagc 20 &lt;210&gt; 3 &lt;211&gt; 2880 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; chimeric DNA of cotton genomic DNA and transgene insert DNA &lt;400&gt; 3 gcttggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgcccttt 60 tttactacga tgttaagtcc tattttacac agtttcttta agacagattt gaccgctcct 120 acgatacttg gagaaacgtt ggtcgaatgt ctcttagaat acaacaacac gatgatcaaa 180 gcagtagcac ctctgtagtg attaacgaac aagcgttgtc tttttctatc accaaaacat 240 tggaaaacat ggagaggaaa agagtagaat tttggaaaga aaataatctt ggtatgagag 300 agtgagattg agcaaaaaat tttgaagagg tcttagcctt ttatatgcgt tcaaagtgga 360 ggaattttgg aaatatccat gtataatgag acaaaatctg catttaaaat ggcatttcgc 420 gtcgcctgcg tcgtgcgagt gcgccccaac cctgacgggt ttggacttac accctcatac 480

<img file="AR096584A2_D0005.tif" />

<td>acgcgaggca</td><td>ggattccaag</td><td>tttagtcatt</td><td>caatcactct</td><td>taaagtgagc</td><td>ttcaagctta</td><td> 540</td>

<td>gacattacaa</td><td>attaaattaa</td><td>ataatataag</td><td>ataattgcgc</td><td>sooooo</td><td>aacatttttt</td><td> 600</td>

<td>ttgtgatcct</td><td>gaacgtaatc</td><td>aacgagggta</td><td>tgatggttat</td><td>gattcacgga</td><td>aagagcgaga</td><td> 660</td>

<td>gaagagaacc</td><td>gtcgctcgaa</td><td>gaggatgatg</td><td>attcatccta</td><td>ttcatgcacg</td><td>actgtccaac</td><td> 720</td>

<td>tccccaccca</td><td>atcaaattcc</td><td>aaattatgac</td><td>atgagaagaa</td><td>catcatccca</td><td>cgtggtctgt</td><td> 780</td>

<td>gcttcacgcc</td><td>accatgtccc</td><td>acgtgggctc</td><td>cattttggtg</td><td>gggcccttcc</td><td>ccaccgccca</td><td> 840</td>

<td>agctgatccc</td><td>gggttggcca</td><td>tccctacttt</td><td>taattatcag</td><td>agccacctcc</td><td>ccaatctgca</td><td> 900</td>

<td>aaacgacgga</td><td>aatggaaaac</td><td>tataattttc</td><td>ttttttttca</td><td>acgtacttat</td><td>aaaatatttt</td><td> 960</td>

<td>tcaaaaaagt</td><td>atgaataaaa</td><td>ttgtgatatt</td><td>gcttggccta</td><td>agaggccaat</td><td>cttttgcaaa</td><td> 1020</td>

<td>tctcgaagtc</td><td>gggaggcaca</td><td>ataaaaactt</td><td>ggaaagtttt</td><td>ttcaagtgtc</td><td>tgctttataa</td><td> 1080</td>

<td>aattattgaa</td><td>atgcatgtat</td><td>tcgtacttgc</td><td>cttatttatc</td><td>gacaatttaa</td><td>acattattat</td><td> 1140</td>

<td>ttcatgaaaa</td><td>tgtccttcca</td><td>ccgatttcaa</td><td>tgacaaaacc</td><td>aataattact</td><td>actttttatt</td><td> 1200</td>

<td>ttcaattatg</td><td>tcacggttca</td><td>catgtttatt</td><td>agggtttagg</td><td>ttgaggttaa</td><td>aactttcgac</td><td> 1260</td>

<td>tctctattcg</td><td>taacgcttaa</td><td>agatgtaggg</td><td>tttaggttga</td><td>ggttaaaaca</td><td>atcatgtaat</td><td> 1320</td>

<td>gtaaggatac</td><td>ctgaaaagct</td><td>gtcattagtg</td><td>taagtgttta</td><td>ttactagggt</td><td>tgtttaaatt</td><td> 1380</td>

<td>catgttgatg</td><td>tcaagcttgg</td><td>ataacccatt</td><td>ttactaaaaa</td><td>aataaatgaa</td><td>gtcccaaagg</td><td> 1440</td>

<td>gcattgggca</td><td>tcctatcaaa</td><td>gatgggaaat</td><td>tttttcaaaa</td><td>ttttaaccta</td><td>aaaaagaggt</td><td> 1500</td>

<td>ggaaagtctt</td><td>agtccaaata</td><td>atcagccaca</td><td>tcagaatttg</td><td>attcgtttct</td><td>ttcaagcaaa</td><td> 1560</td>

<td>ttatacctat</td><td>tggctgcaat</td><td>atctttaagt</td><td>ggaatggtcg</td><td>gccaaacttt</td><td>tccatatcag</td><td> 1620</td>

<td>cttgattcat</td><td>ctctaaactt</td><td>gattattctt</td><td>ttttattaat</td><td>attaaattcc</td><td>acaacttgaa</td><td> 1680</td>

<td>ctttaatttt</td><td>tttaattaat</td><td>sooooooooo</td><td>gtcacctttt</td><td>caagctgaaa</td><td>aagaaaaaga</td><td> 1740</td>

<td>aaccttaatt</td><td>attatcacta</td><td>gtattaaatt</td><td>tcaaaacttg</td><td>atttgtccta</td><td>aatttgaaaa</td><td> 1800</td>

<td>ggggtctcct</td><td>tcaattcata</td><td>tatgtagtca</td><td>tgaagattat</td><td>aacttagctg</td><td>aaaatggcct</td><td> 1860</td>

<td>ccattatttg</td><td>gcttattcaa</td><td>tcaaaagttt</td><td>acaaaactag</td><td>tgcaaattta</td><td>atatgataat</td><td> 1920</td>

<td>gtctacaaga</td><td>accaaatacg</td><td>aattgagtaa</td><td>atttttttgg</td><td>ctaaaataaa</td><td>ttacgaattg</td><td> 1980</td>

<img file="AR096584A2_D0006.tif" />

<td>atgaattatc</td><td>attttaaaaa</td><td>gttcttttta</td><td>accatttctt</td><td>ttactgaatt</td><td>aaaaaaaggt</td><td> 2040</td>

<td>tttattaatc</td><td>atatatatta</td><td>caaattaccc</td><td>attaagtagc</td><td>caaattacaa</td><td>attttaattc</td><td> 2100</td>

<td>aatgtagtca</td><td>aacactgata</td><td>gtttaaacat</td><td>gactctctta</td><td>aggtagccaa</td><td>agcccgggct</td><td> 2160</td>

<td>taattaaggc</td><td>gcgccggcca</td><td>agtcggccgc</td><td>ggccgcgtta</td><td>tcaagcttct</td><td>gcaggtcctg</td><td> 2220</td>

<td>ctcgagtgga</td><td>agctaattct</td><td>cagtccaaag</td><td>cctcaacaag</td><td>gtcagggtac</td><td>agagtctcca</td><td> 2280</td>

<td>aaccattagc</td><td>caaaagctac</td><td>aggagatcaa</td><td>tgaagaatct</td><td>tcaatcaaag</td><td>taaactactg</td><td> 2340</td>

<td>ttccagcaca</td><td>tgcatcatgg</td><td>tcagtaagtt</td><td>tcagaaaaag</td><td>acatccaccg</td><td>aagacttaaa</td><td> 2400</td>

<td>gttagtgggc</td><td>atctttgaaa</td><td>gtaatcttgt</td><td>caacatcgag</td><td>cagctggctt</td><td>gtggggacca</td><td> 2460</td>

<td>gacaaaaaag</td><td>gaatggtgca</td><td>gaattgttag</td><td>gcgcacctac</td><td>caaaagcatc</td><td>tttgccttta</td><td> 2520</td>

<td>ttgcaaagat</td><td>aaagcagatt</td><td>cctctagtac</td><td>aagtggggaa</td><td>caaaataacg</td><td>tggaaaagag</td><td> 2580</td>

<td>ctgtcctgac</td><td>agcccactca</td><td>ctaatgcgta</td><td>tgacgaacgc</td><td>agtgacgacc</td><td>acaaaagaat</td><td> 2640</td>

<td>tagcttgagc</td><td>tcaggattta</td><td>gcagcattcc</td><td>agattgggtt</td><td>caatcaacaa</td><td>ggtacgagcc</td><td> 2700</td>

<td>atatcacttt</td><td>attcaaattg</td><td>gtatcgccaa</td><td>aaccaagaag</td><td>gaactcccat</td><td>cctcaaaggt</td><td> 2760</td>

<td>ttgtaaggaa</td><td>gaattcgata</td><td>tcaagcttga</td><td>tatcggaagt</td><td>ttctctcttg</td><td>agggaggttg</td><td> 2820</td>

<td>ctcgtggaat</td><td>gggacacata</td><td>tggttgttat</td><td>aataaaccat</td><td>ttccattgtc</td><td>atgagatttt</td><td> 2880</td>

&lt;210&gt; 4 &lt;211&gt; 1675 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Chimeric DNA of cotton genomic DNA and inserted transgenic DNA &lt;400&gt; 4 tgaccgaagt taatatgagg agtaaaacac ttgtagttgt accattatgc ttattcacta 60 ggcaacaaat atattttcag acctagaaaa gctgcaaatg ttactgaata caagtat 120 caagtat

<img file="AR096584A2_D0007.tif" />

<td>ctcttgtgtt</td><td>ttagacattt</td><td>atgaactttc</td><td>ctttatgtaa</td><td>ttttccagaa</td><td>tccttgtcag</td><td> 180</td><td>I |</td>

<td>attctaatca</td><td>ttgctttata</td><td>attatagtta</td><td>tactcatgga</td><td>tttgtagttg</td><td>agtatgaaaa</td><td> 240</td><td>t</td>

<td>tattttttaa</td><td>tgcattttat</td><td>gacttgccaa</td><td>ttgattgaca</td><td>acatgcatca</td><td>atcgacctgc</td><td> 300</td><td>j i ? I</td>

<td>agccactcga</td><td>gtggaggcct</td><td>catctaagcc</td><td>cccatttgga</td><td>cgtgaatgta</td><td>gacacgtcga</td><td> 360</td><td>I i F</td>

<td>aataaagatt</td><td>tccgaattag</td><td>aataatttgt</td><td>ttattgcttt</td><td>cgcctataaa</td><td>tacgacggat</td><td> 420</td><td>í F 1.</td>

<td>cgtaatttgt</td><td>cgttttatca</td><td>aaatgtactt</td><td>tcattttata</td><td>ataacgctgc</td><td>ggacatctac</td><td> 480</td><td>í í</td>

<td>atttttgaat</td><td>tgaaaaaaaa</td><td>ttggtaatta</td><td>ctctttcttt</td><td>ttctccatat</td><td>tgaccatcat</td><td> 540</td><td>F</td>

<td>actcattgct</td><td>gatccatgta</td><td>gatttcccgg</td><td>acatgaagcc</td><td>atttacaatt</td><td>gaatatatat</td><td> 600</td><td> 1</td>

<td>tacaaagcta</td><td>tttgcttata</td><td>acatatgcga</td><td>aaaattttgt</td><td>actataatca</td><td>ggggtaaatt</td><td> 660</td><td> 1</td>

<td>taggaggggg</td><td>cttgtaggtc</td><td>tcgcttctct</td><td>sooooooooo</td><td>aattttctat</td><td>ttagttattt</td><td> 720</td><td></td>

<td>aaaattttaa</td><td>aagtaaaata</td><td>taaaaatttc</td><td>atttaatcct</td><td>ttaaaaatta</td><td>taaagatata</td><td> 780</td><td></td>

<td>gactattaaa</td><td>atgatgaaat</td><td>tacaatttta</td><td>ttatcataaa</td><td>aattataatt</td><td>taatttcgac</td><td> 840</td><td></td>

<td>ccctaacaaa</td><td>attttctgat</td><td>tttgccccta</td><td>actgtaatat</td><td>ttgtataaaa</td><td>acattttctt</td><td> 900</td><td> 1 1</td>

<td>tttgcattta</td><td>atgatttctt</td><td>taattcagtc</td><td>caagaaagaa</td><td>atttattaat</td><td>tgcatatgcg</td><td> 960</td><td>r</td>

<td>aaagttagtc</td><td>cttgcctagt</td><td>gatattaaag</td><td>gaaagaaaca</td><td>soooooat</td><td>aaattaattt</td><td> 1020</td><td></td>

<td>ttaaagcaaa</td><td>tagtaaaaat</td><td>aaggaaaaac</td><td>tttctacgat</td><td>agtctataat</td><td>tcaaaaaaag</td><td> 1080</td><td></td>

<td>aaataataat</td><td>ctttaaccat</td><td>tgaattttaa</td><td>aataacatca</td><td>gaataatcta</td><td>tttatttaat</td><td> 1140</td><td></td>

<td>ttaataaata</td><td>ataataacat</td><td>atatattaat</td><td>attaaaattt</td><td>ttattgagct</td><td>tagtgtcaca</td><td> 1200</td><td></td>

<td>aatcaataaa</td><td>aaatttctta</td><td>caaaataaat</td><td>tatattattt</td><td>tgagggtgtt</td><td>ttattatttt</td><td> 1260</td><td></td>

<td>atatatttta</td><td>tacagacata</td><td>tagaaatata</td><td>aatacacata</td><td>ataaaatttg</td><td>aatccaaatt</td><td> 1320</td><td></td>

<td>tttaattttt</td><td>aacatttata</td><td>atttactatt</td><td>caaccaaaat</td><td>tttatttatt</td><td>atttatatca</td><td> 1380</td><td></td>

<td>aatttttata</td><td>aatatattta</td><td>tcagataatg</td><td>cgattttttt</td><td>tacctatata</td><td>tagatgacat</td><td> 1440</td><td></td>

<td>aatctacttt</td><td>aaattaagtc</td><td>ctaaaaataa</td><td>tatatcatac</td><td>caaaaaaatt</td><td>cttaaaatga</td><td> 1500</td><td></td>

<td>atctgataat</td><td>acttaacccc</td><td>ttttataaaa</td><td>caatcttaac</td><td>cccttatata</td><td>ttttaatatt</td><td> 1560</td><td></td>

aatatcatta taaatataaa tctattgagc atatgtttta aaccaagtaa tgttgagtgc 1620 ggtagtaaaa ctcattacac attttaagta gaacgtagtt cgaaccttgg agaag 1675 &lt;210&gt; 5

<td> &lt;211&gt;</td><td> 29</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>scrofular mosaic virus</td>

<td> &lt;400&gt;</td><td> 5</td>

ccactaactt taagtcttcg gtggatgtc 29

<td> &lt;210&gt;</td><td> 6</td>

<td> &lt;211&gt;</td><td> 28</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>scrofular mosaic virus</td>

<td> &lt;400&gt;</td><td> 6</td>

ctgaaactta ctgaccatga tgcatgtg 28 &lt;210&gt; 7

<td> &lt;211&gt;</td><td> 26</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Pisum sativum</td>

<td> &lt;400&gt;</td><td> 7</td>

catttggacg tgaatgtaca cacgtc 26 &lt;210&gt; 8

<td> &lt;211&gt;</td><td> 21</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Pisum sativum</td>

<td> &lt;400&gt;</td><td> 8</td>

gttgtcgaaa ccgatgatac g

<td> &lt;210&gt;</td><td> 9</td>

<td> &lt;211&gt;</td><td> 27</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Arabidopsis thaliana</td>

<td> &lt;400&gt;</td><td> 9</td>

tctgcaatca aaaacataaa gatctga 27 &lt;210&gt; 10

<td> &lt;211&gt;</td><td> 30</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Pisum sativum</td>

<td> &lt;400&gt;</td><td> 10</td>

cctataaata cgacggatcg taatttgtcg 30

<td> &lt;210&gt;</td><td> 11</td>

<td> &lt;211&gt;</td><td> 21</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Pisum sativum</td>

<td> &lt;400&gt;</td><td> 11</td>

accgagate cgaacgaaag c 21 &lt;210&gt; 12

<td> &lt;211&gt;</td><td> 25</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Arabidopsis thaliana</td>

<td> &lt;400&gt;</td><td> 12</td>

aatcgtaatc gagatccaac acaag 25 &lt;210&gt; 13 i

ί t

<td> &lt;211&gt;</td><td>³¹ í</td>

<td> &lt;212&gt;</td><td>DNA f</td>

<td> &lt;213&gt;</td><td>Pisum sativum J</td>

<td> &lt;400&gt;</td><td>13 f</td>

ccatattgac catcatactc attgctgatc c 31 &lt;210&gt; 14

<td> &lt;211&gt;</td><td>²⁷ í</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Agrobacterium turnefaciens I</td>

<td> &lt;400&gt;</td><td> 14</td>

ttttactacg atgttaagtc ctatttt 27 &lt;210&gt; 15

<td> &lt;211&gt;</td><td> 21</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Agrobacterium turnéfaciens</td>

<td> &lt;400&gt;</td><td> 15</td>

gaccagagga cttacgagca g 21 &lt;210&gt; 16

<td> &lt;211&gt;</td><td> 24 ₅</td>

<td> &lt;212&gt;</td><td>T DNA</td>

<td> &lt;213&gt;</td><td>Agrobacterium tumefaciens {</td>

<td> &lt;400&gt;</td><td> 16 ’</td>

gattccgaat tcaagcttca tggg 24 &lt;210&gt; 17 &lt;211&gt; 27

<img file="AR096584A2_D0008.tif" />

&lt;212&gt; DNA &lt;213&gt; Agrobacterium tumefacíens &lt;400&gt; 17 ctaagatcga actctccgac actaagg &lt;210&gt; 18 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Arabidopsis thaliana &lt;400&gt; 18 gtctactgtt tttattgatt caatatttga ttg &lt;210&gt; 19 &lt;210 &lt;192 212&gt; DNA &lt;213&gt; Agrobacterium swelling &lt;400&gt; 19 gcagctgtcg ctacccacct cg &lt;210&gt; 20 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; Arabidopsis thaliana &lt;400&gt; 20 ggttttctcg atcaagattc agatc &lt;210&gt; 21 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Gossypium hirsutum &lt;400&gt; 21 aattacccat taagtagcca aattacaaa 29

<td> &lt;210&gt;</td><td> 22</td>

<td> &lt;211&gt;</td><td> 24</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>scrofular mosaic virus</td>

<td> &lt;400&gt;</td><td> 22</td>

gggctttggc taccttaaga gagt 24 &lt;210&gt; 23

<td> &lt;211&gt;</td><td> 27</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Gossypium hirsutum</td>

<td> &lt;400&gt;</td><td> 23</td>

cgcatatgtt ataagcaaat agctttg 27 &lt;210&gt; 24

<td> &lt;211&gt;</td><td> 17</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>PCR labeled DNA probe</td>

<td> &lt;400&gt;</td><td> 24</td>

caatgtagtc aaacact 17 &lt;210&gt; 25 &lt;211&gt; 20

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>PCR labeled DNA probe</td>

<td> &lt;400&gt;</td><td> 25</td>

agtgtacata tagggaatat 20 &lt;210&gt; 26

<td> &lt;211&gt;</td><td> 24</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>PCR DNA primer</td>

<td> &lt;400&gt;</td><td> 26</td>

ggacatgaag ccatttacaa ttga 24 &lt;210&gt; 27

<td> &lt;211&gt;</td><td> 23</td>

<td> &lt;212&gt;</td><td>DNA</td>

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>PCR DNA primer</td>

<td> &lt;400&gt;</td><td> 27</td>

cctacaagcc ccctcctaaa ttt 23 &lt;210&gt; 28 &lt;211&gt; 23 &lt;212&gt; DNA

<img file="AR096584A2_D0009.tif" />

<td> &lt;213&gt;</td><td>Artificial Sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>PCR DNA probe</td>

<td> &lt;400&gt;</td><td> 28</td>

agctatttgc ttataacata tgc

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