BR122014009188B1 - DNA MOLECULES CONFERINING HERBICIDE TOLERANCE ISOLATED FROM COTTON COTTON FABRIC - Google Patents

Abstract

Patent specification: "Method for identifying an elite event, plants, cells, tissues and seeds comprising the same, kits for applying said methods, process for producing transgenic cotton plants, as well as DNA molecules". The invention pertains to transgenic cotton plants, plant material and seeds, characterized by housing a specific transformation event, particularly by the presence of a gene encoding a herbicide tolerant protein, at a specific location in the cotton genome. The cotton plants of the invention combine the herbicide tolerant phenotype with optimal agronomic performance.

Description

Patent Descriptive Report for "DNA Molecules CONFERING HERBICIDE TOLERANCE ISOLATED FROM PLANT COTTON FABRIC".

Divided from PI 0211726-6, filed July 19, 2002.

Field of the Invention This invention relates to transgenic cotton plants, plant material and seeds, characterized by housing a specific transformation event, particularly by the presence of a gene encoding a herbicide tolerant protein in a specific location in the cotton genome. The cotton plants of the invention combine the herbicide tolerant phenotype with agronomic performance, genetic stability and adaptability to different genetic fields equivalent to untransformed cotton lineage in the absence of weed pressure.

All documents cited herein are hereby incorporated by reference.

BACKGROUND OF THE INVENTION The phenotypic expression of a transgene in a plant is equally determined by the structure of the gene itself and its location in the plant genome. At the same time the presence of the transgene at different locations in the genome will influence the overall plant phenotype in different ways. The agronomically or industrially successful introduction of a commercially interesting trait into a plant by genetic manipulation can be a long procedure dependent on different factors. Current transformation and regeneration of genetically transformed plants are only the first in a series of selection steps, which include extensive genetic characterization, reproduction, and evaluation of field experiments.

[005] Cotton fiber is the single most important textile product in the world. About 80 million acres of cotton are harvested annually across the globe. Cotton is the fifth largest crop in the United States in terms of acres of production area, with over 15 million acres planted in 2000. Primary cotton weed species are Ipomoea sp. (Morning glory), Amaranthus spp. (anserine), Cyperus spp. (juniper), Xanthium spp. (thistle) and Sorgbum spp. (johnsongrass). Prior to the introduction of broadleaf herbicides that could be used in cotton fields, growers employed controlled postemergence applications of non-selective herbicides being careful not to contact growing crop plants. As this requires a difference in height between weeds and planting, this is not always possible. Especially for small cotton, this practice is time consuming and potentially harms the harvest.

The bar gene (Thompson et al., 1987, EMBO J 6: 2519-2523; Deblock et al., 1987, EMBO J. 6: 2513-2518) is a gene encoding the enzyme phosphinothricin acetyl transferase (PAT), the which, when expressed in a plant, confers resistance to the herbicidal compounds phosphinothricin (also called glufosinate) or bialaphos (see also for example US patents 5,646,024 and 5,561,236) and salts and optical isomers thereof. Phosphinothricin controls broadleaf weeds including morning glory and has a wide application opening.

Successful cotton genetic transformation has been achieved by a number of methods including Agrobacterium infection of cotton seedlings (Firoozabady et al., 1987, Plant Molecular Biology 10: 105-116; Umbeck et al. 1987, Bio / Technology). 5: 263-266 and WO 00/71733, US 5,004,863, and US 5,159,135), as well as direct gene transfer by microprojectile bombardment of meristematic cotton tissues (Finer and Mc Mullen, 1990, Plant Cell Reports , 5: 586-589; McCabe and Martinell, 1993, Bio / Technology 11: 596-598, WO92 / 15675, EPO 531 506). Increased transformation efficiency for Agrobacterium transformation has been reported using the methods described by Hansen et al. (1994, Proc. Nat. Acad. Sci. 91: 7603-7607), Veluthambi et al. (1989, Journal of Bacteriology 171: 3696 - 3703) and WO 00/71733.

Different methods for regeneration of cotton plants have also been described (WO 89/05344, US 5,244,802, US 5,583,036, WO 89/12102, WO 98/15622, and WO 97/12512).

However, previous documents fail to teach or suggest the present invention.

SUMMARY OF THE INVENTION The present invention relates to a transgenic cotton plant, or seed, cell or tissue thereof, comprising, stably integrated into its genome, an expression cassette which comprises a herbicide tolerance gene comprising the bar gene coding sequence (as described in Example 1.1 herein) which is herbicide tolerant and, in the absence of weed pressure, has an agronomic performance that is substantially equivalent to non-transgenic isoline. Under weed pressure and appropriate Liberty® treatment, the plant will have a higher agronomic phenotype compared to the non-transgenic plant.

In one embodiment of the invention, the cotton plant or seed, cells or tissues thereof, comprises the pGSV71 expression cassette (as described in Example 1.1, Table 1 annexes). In the preferred embodiment of the invention the cotton plant or seed, cells or tissues thereof comprise elite event EE-GH1. In another embodiment of the invention, the transgenic cotton plant or seed, cells or tissues thereof comprises: ] EE-GH1 event in your genome; or EE-GH1 event on the condition that the bar gene employed in the event is replaced with a nucleic acid sequence that hybridizes to the complement of the bar gene under stringent conditions.

More specifically, the present invention relates to a transgenic cotton plant, seed, cells or tissues thereof, the genomic DNA of which is characterized by the fact that when analyzed in a PCR identification protocol as described Here, employing two primers directed to the 5 'or 3' flanking region of EE-GH1 and foreign DNA, respectively, produce a fragment that is specific for EE-GH1. Preferably the primers are directed against the 5 'flanking region in SEQ ID NO: 1 and foreign DNA respectively; more preferably, the primers comprise the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and produce a DNA fragment of between 250 and 290 bp, preferably about 269 bp.

Reference seed comprising the elite event of the invention was deposited with the ATCC under accession number PTA-3343. Thus, a preferred embodiment of the invention is the seed comprising the elite event EE-GH1 deposited as ATTC accession number PTA-3343, which will grow into a glufosinate resistant cotton plant. ATCC bunker seed number PTA-3343, which is a seed batch consisting of about 50% non-transgenic seeds and 50% transgenic hemizigote transgenic seeds, comprising the elite event of the invention, which will develop -it is in glufosinate tolerant plants. The seed may be sown and growing plants may be treated with PPT or Liberty® as described herein to obtain 100% glufosinate tolerant plants comprising the elite event of the invention. The invention also relates to cells, tissues, progeny, and progeny of a plant comprising the elite event of the invention developed from the seed deposited at the ATCC having accession number PTA-3343. The invention also relates to plants obtainable by propagation and / or reproduction with a cotton plant comprising the elite event of the invention developed from seed deposited at the ATCC having accession number PTA-3343.

The invention also relates to plants, seeds, cells or tissues comprising a foreign DNA sequence, preferably a herbicide tolerance gene as described herein, integrated into chromosomal DNA in a region comprising the DNA sequence. of the plant of SEQ ID NO: 1 and / or SEQ ID NO: 4, more particularly comprising the DNA sequence of SEQ ID NO: 5, or a sequence that hybridizes under stringent conditions to a sequence that is complementary to a sequence comprising the plant DNA sequence of SEQ ID NO: 1, SEQ ID NO: 4 and / or SEQ ID NO: 5.

The invention also provides a process for producing a transgenic cell of a cotton plant which comprises inserting a recombinant DNA molecule into a region of the chromosomal DNA of a cotton cell, tissue or callus comprising the sequence of Plant DNA of SEQ ID NO: 1 and / or SEQ ID NO: 4, or comprising a sequence that hybridizes under stringent conditions to a sequence that is complementary to a sequence comprising the plant DNA sequence of SEQ ID NO: 1 and / or SEQ ID NO: 4.

The invention also relates to a method for identifying a transgenic plant, or cells or tissues thereof, comprising the elite EE-GH1 event whose method is based on identifying the presence of characterizing or sequencing DNA sequences. amino acids encoded by such DNA sequences in transgenic plants, cells or tissues. According to a preferred embodiment of the invention, such characterization DNA sequences are sequences of 15 bp, preferably 20 bp, more preferably 30 bp or more which comprise the insertion site of the event, that is also a part of foreign DNA. and a part of the cotton genome (or the 5 'or 3' flanking region) adjacent to it, allowing for specific identification of the elite event.

According to another aspect of the invention, DNA sequences are described comprising the event insertion site and sufficient polynucleotide length of both cotton genomic DNA and foreign (transgene) DNA in order to be useful as primer. or probe for detecting EE-GH1. Preferably, such sequences comprise at least 9 nucleotides of cotton genomic DNA and a similar number of EE-GH1 foreign DNA (transgene) nucleotides with these on either side of the insertion site respectively. More preferably, such DNA sequences comprise at least 9 nucleotides of cotton genomic DNA and a similar number of foreign DNA nucleotides contiguous with the insertion site in SEQ ID NO: 1 or SEQ ID NO: 4.

According to another preferred aspect of the invention, the method for identifying a transgenic plant or cells or tissues thereof comprising the elite event EE-GH1 comprises amplifying a sequence of a nucleic acid present in biological samples. using a polymerase chain reaction with at least two primers, one of which recognizes plant DNA in the 5 'or 3' flanking region of EE-GH1, the other that recognizes a sequence in the Foreign DNA. Preferably, the genomic DNA is analyzed using primers that recognize a sequence in the EE-GH1 5 'flanking region of the plant, more preferably in the plant DNA sequence in SEQ ID NO: 1, and a sequence in foreign DNA. respectively. Especially preferable, genomic DNA is analyzed according to the PCR identification protocol described herein whereby the initiator recognizing a sequence in the 5 'flanking region comprises the nucleotide sequence of SEQ ID NO: 2. Particularly, the primer recognizing a sequence in the 5 'flanking region comprises the nucleotide sequence of SEQ ID NO: 2 and the primer recognizing a sequence in the foreign DNA comprises the nucleotide sequence of SEQ ID NO: 3, so that the amplified fragment is a fragment is preferably between 250 and 290 bp, more preferably between 260 and 270 bp, most preferably about 269 bp.

Accordingly, the present invention relates to the transgenic plant, cells or tissues thereof which can be identified according to the identification methods for EE-GH1 described above.

[0023] The present invention relates to methods for identifying the EE-GH1 elite event in biological samples, the methods of which are based on primers or probes that specifically recognize the EE-GH1 5 'and / or 3' flanking sequence. . In a preferred embodiment of the invention these methods are based on primers or probes which recognize a sequence in SEQ ID NO: 1 and / or SEQ ID NO: 4, more particularly primers or probes comprising the sequence of SEQ ID NO: 2. with another embodiment of the invention such methods are based on primers or probes that recognize an EE-GH1 characterization sequence, which is a DNA sequence comprising the insertion site of the event, including sufficient 5 'or 3' flanking DNA nucleotides. and foreign DNA adjacent to it to be diagnostic for the event. More particularly, such methods are based on sequences comprising sufficient nucleotides of the 5 'or 3' flanking DNA and the foreign DNA adjacent thereto of SEQ ID NO: 1 or SEQ ID NO: 4. Such methods are known in the art and include, but are not limited to, the method described by Lyamichev et al. (1999, Nature Biotechn 17: 292-296).

The present invention also relates to the EE-GH1 specific flanking sequences described herein, which may be employed to develop EE-GH1 specific identification methods in biological samples. More particularly, the invention relates to EE-GH1 5 'and / or 3' flanking regions which may be employed for the development of specific primers and probes as well as to specific primers and probes developed from the 5 'and / or 3' flanking sequences of EE-GH1 and the insertion site adjacent to them. The invention also relates to identification methods for the presence of EE-GH1 in biological samples based on the use of such specific primers or probes.

The invention therefore also relates to an elite EE-GH1 event identification kit in biological samples, the kit comprising at least one primer or probe that specifically recognizes the EE 5 'or 3' flanking region. -GH1.

Preferably the kit of the invention comprises, in addition to a primer that specifically recognizes the EE-GH1 5 'or 3' flanking region, a second primer that specifically recognizes a sequence in the foreign DNA of EE-GH1, for use. in a PCR identification protocol. Preferably, the kit of the invention comprises two (or more) specific primers, one of which recognizes a sequence in the EE-GH1 5 'flanking region, more preferably a sequence in the plant DNA region of SEQ ID NO: 1, and another that recognizes a sequence in foreign DNA. Especially preferable, the primer recognizing the plant DNA sequence in the 5 'flanking region comprises the nucleotide sequence of SEQ ID NO: 2. Particularly, the primer recognizing the plant DNA sequence in the 5' flanking region comprises the sequence nucleotide sequence of SEQ ID NO: 2 and the primer recognizing foreign DNA comprises the nucleotide sequence of SEQ ID NO: 1 described herein.

According to another embodiment, the invention relates to an EE-GH1 elite event identification kit in biological samples, which kit comprises at least one specific primer or probe having a sequence that corresponds (or is complementary to) a) a sequence that hybridizes under stringent conditions to a characterization or specific region of EE-GH1. Preferably the probe sequence corresponds to a specific region comprising part of the foreign DNA and part of the 5 'or 3' flanking region (plant DNA) of contiguous EE-GH1 therewith. More preferably the specific probe comprises (or is complementary to) a sequence that hybridizes under stringent conditions to a sequence of 15 to 30 nucleotides traversing the insertion site in the 5 'or 3' region of foreign DNA in SEQ ID NO: 1 or SEQ Especially preferable is that the specific probe comprises (or is complementary to) a sequence that hybridizes under stringent conditions to a sequence of 9 nucleotides of plant DNA and a similar number of foreign DNA nucleotides contiguous with the insertion site, in the sequences of SEQ ID NO: 1 or SEQ ID NO: 4. According to a particular embodiment, such a primer comprises a sequence that is identical to a sequence of 9 plant DNA nucleotides and a similar number of nucleotides of the DNA. Foreign DNA contiguous with the insertion site in the sequences of SEQ ID NO: 1 or SEQ ID NO: 4.

The methods and kits encompassed by the present invention may be employed for different purposes such as, but not limited to the following: identifying EE-GH1 in plants, plant material or in products such as, but not limited to food, or products. feed (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention may be employed to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; Additionally or alternatively, the methods or kits of the present invention may be employed to determine the quality (i.e. percentage of pure material) of plant material comprising EE-GH1. The present invention also relates to a method for locating plants comprising elite event EE-GH1 in their bud at introduction in different crops.

It should be understood that particular embodiments of the invention are described by the dependent claims cited herein. Brief Description of the Figure The following detailed description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figure, incorporated herein by reference, in which: Fig. 1. PCR analysis of other EE-GH1 events and elite events using the EE-GH1 FCR identification protocol.

Gel loading sequence: lane 1, molecular weight marker (100 bp ladder), lane 2, DNA sample from a cotton plant comprising EE-GH1 transgenic event, lane 3, DNA samples from a cotton plant comprising another transgenic event, lane 4, wild-type cotton DNA, lane 5, wild-type + 1 copy of pGSV71-BamHI (positive control) digest, lane 6, negative control (no standard), lane 8, molecular weight marker (100 bp ladder).

Detailed Description of Preferred Embodiments The term "gene" as used herein refers to any DNA sequence comprising various operably linked DNA fragments such as a promoter region, a 5 '(5'UTR) untranslated region. ), an encoding region (which may or may not encode for a protein), and an untranslated 3 'region (3'UTR) comprising a polyadenylation site. Typically in plant cells, the 5'UTR, the coding region and the 3'UTR are transcribed into an RNÂ from which, in the case of a protein coding gene, the coding region is translated into a protein. A gene may include additional DNA fragments such as, for example, introns. As used herein, a genetic site is the position of a given gene in the genome of a plant.

The term "chimeric" when referring to a DNA sequence or gene is used to refer to the fact that the DNA sequence or gene comprises at least two functionally relevant DNA fragments (such as promoter, 5'UTR, coding region, 3'UTR, intron) which are not naturally associated with each other and / or originate, for example, from different sources. "Alien" referring to a DNA or gene sequence with respect to a plant species is employed to indicate that the DNA or gene sequence is not naturally found in that plant species, or is not naturally found in that genetic site in that species. of plant. The term "foreign DNA" will be used herein to refer to a DNA sequence as incorporated into the genome of a plant as a result of transformation. "Transformation DNA" as used herein refers to a recombinant DNA molecule employed for transformation. Transformation DNA generally comprises at least one "gene of interest" (for example a chimeric gene) which is capable of conferring one or more specific characteristics to the transformed plant. The term "recombinant DNA molecule" is used to exemplify and thus may include an isolated nucleic acid molecule which may be DNA and which may be obtained by recombinant or other procedures.

As used herein the term "transgene" refers to a gene of interest (or all foreign DNA) as incorporated into the genome of a plant. A "transgenic plant" refers to a plant comprising at least one transgene in the genome of all of its cells.

The foreign DNA present in the plants of the present invention will preferably comprise a herbicide tolerance gene, more specifically a 35S-bar gene as the gene of interest.

A "herbicide tolerance" gene as used herein refers to a gene that makes the plant tolerant to an herbicide. An example of a herbicide tolerance gene is a gene comprising a sequence encoding the enzyme phosphinothricin acetyl transferase that detoxifies phosphinothricin under the control of a constitutive promoter. More specifically, in the elite event of the present invention the herbicide tolerance gene comprises the coding sequence of the Streptomyces hygroscopicus bialaphos (bar) resistance gene (Thompson et al. (1987) EMBO J 6: 2519-2523) under the control of Cauliflower Mosaic Virus 35S promoter (Odell et al. (1985), Nature 313: 810-812), also referred to as "35S-baf here. Expression of the 35S-aro gene confers tolerance to the phosphinothricin herbicidal compounds or bialaphos or glufosinate, or more generally glutamine synthase inhibitors, or optical salts or isomers thereof which will generally be referred to as "glufosinate tolerance" herein.

By hybridization under "stringent conditions" is meant conventional hybridization conditions as described by Sam-brook et al. (1989) (Molecular Cloning: A Lab Mouse rat Manual, Second Edition, Cold Spring Harbor Lab Rat Press , NY) which for example may comprise the following steps: 1) immobilization of plant genomic DNA fragments on a filter, 2) prehybridization of the filter for 1 to 2 hours at 42 ° C in 50% formamide, 5 X SSPE , 2 X Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68 ° C in 6 X SSC, 2 X Denhardt's reagent and 0.1% SDS, 3) addition of the labeled hybridization probe , 4) incubating for 16 to 24 hours, 5) washing the filter for 20 minutes at room temperature in 1X SSC, 0.1% SDS, 6) washing the filter three times for 20 minutes each at 68 ° C at 0 ° C, 2 X SSC, 0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at -70 ° C with an intensification analysis.

Incorporation of a recombinant DNA molecule into the plant genome typically results from transformation of a cell, tissue or callus (or other genetic manipulation). The particular embedding site is either random or at a predetermined location (if a targeted integration process is employed).

DNA introduced into the plant genome as a result of transforming a plant cell or tissue with a recombinant DNA or "transforming DNA" is hereinafter referred to as "exogenous DNA" comprising one or more "transgenes". Thus, exogenous DNA can equally comprise recombinant as well as newly introduced, rearranged DNA from the plant. However the term "plant DNA" in the context of the present invention will refer to plant DNA which is generally found at the same genetic locus in the wild type plant.

Exogenous DNA can be characterized by the location and configuration at the site of incorporation of the recombinant DNA molecule into the plant genome. The site in the plant genome where a recombinant DNA has been inserted is also referred to as the "insertion site" or "target site". Insertion of recombinant DNA into the plant genome may be associated with a plant DNA deletion, referred to as a "target site deletion". The "flanking region" or "flanking sequence" as used herein refers to a sequence of at least 20 bp, preferably at least 50 bp, and up to 5000 bp of the plant genome that is located or immediately upstream of e contiguous with or immediately downstream of and contiguous with exogenous DNA. Transformation procedures inducing random integration of exogenous DNA will result in transformants with different flanking regions, which are characteristic and unique to each transformant. When recombinant DNA present in a plant is introduced into a different plant through traditional crossbreeding, its insertion site in the plant genome or its flanking regions will generally not be changed (apart from occasional changes due to mutations or crossover and trans -posons). An "insertion region" as used herein refers to the region corresponding to the region of at least 40 bp, preferably at least 100 bp, and up to more than 10,000 bp, encompassed by the sequence comprising the upstream flanking region and / or downstream of an exogenous DNA in the (non-transformed) plant genome (and including the insertion site and possible deletion of the target site). Taking into account minor differences due to mutations within a species, an insertion region will retain at least 85%, preferably 90%, more preferably 95%, and most preferably 100% sequence identity with the sequence comprising the flanking regions. upstream and downstream of exogenous DNA in a given plant of that species.

The expression of a gene of interest refers to the fact that the gene confers on the plant one or more phenotypic characteristics (eg herbicide tolerance) that were intended to be conferred by the introduction of the recombinant DNA molecule. - Transformation DNA - used during transformation (based on the structure and function of part or all of the gene (s) of interest).

An "event" is defined as a (artificial) genetic locus that, as a result of genetic construct, carries an exogenous DNA comprising at least one copy of the gene (s) of interest (also referred to as an event). of transformation). An event is phenotypically characterized by the expression of transgenes. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, an event is characterized by the restriction map (e.g., as determined by Southern blotting) and / or flanking sequences upstream and / or downstream of exogenous DNA, and / or the molecular configuration of exogenous DNA comprising the transgenes. Usually when transforming a plant cell, tissue or callus with a transforming DNA, a multitude of events are generated, each of which is unique.

An "elite event" as used herein is an event that is selected from the group of events obtained by transformation with the same transformation DNA or by plant breeding obtained by such transformation, based on phenotypic expression and stability. transgenes and in the absence of negative impact on the agronomic characteristics of the plant comprising it (ie selected transformation event). Thus, the criteria for elite event selection are one or more, preferably two or more, advantageously all of the following: that the presence of exogenous DNA in the plant does not comprise other desired plant characteristics, such as those relating to agronomic performance or business values;

[0046] that the event is characterized by a well-defined molecular configuration that is stably inherited and for which appropriate diagnostic tools for identity control can be developed;

That the gene (s) of interest show stable and phenotypic appropriate spatial and temporal expression in ho-mozigota condition of the event, at a commercially acceptable level over a range of environmental conditions in which plants carrying the event are likely to be exposed in normal agronomic use.

It is preferred that exogenous DNA be associated with a position in the plant genome that allows introgression into the desired commercial genetic bases.

[0049] The state of an event as an elite event is confirmed by introgressing the elite event on different relevant genetic bases and observing compliance with one, two or all of the criteria, for example a), b) and c) above .

An "elite event" thus refers to a genetic locus comprising an exogenous DNA that meets the criteria described above. A plant, plant material or progeny such as seeds may comprise one or more elite events in its genome. Thus, when referring to a plant, cell or seed tissue comprising the elite EE-GH1 event in its genome, a plant, cell or plant tissue is intended to comprise the exogenous DNA described herein (comprising the 35S-ibar gene) integrated into its genome at the integration site described herein.

Tools for identifying an elite event or the plant or plant material comprising an elite event, or products comprising plant material comprising the elite event are based on the specific genomic characteristics of the elite event, such as, a restriction map of the genomic region comprising exogenous DNA, molecular markers or sequence of exogenous DNA flanking region (s).

Once one or both of the flanking regions of exogenous DNA has been sequenced, primers and probes can be developed that specifically recognize this nucleic acid sequence (s) (DNA or RNA) from a sample by a molecular biological technique. For example, a PCR method may be developed to identify the elite event in biological samples (such as plant samples, plant material or products comprising plant material). Such a PCR is based on at least two specific "primers", one recognizing a sequence within the 5 'or 3' flanking region of the elite event, and the other recognizing a sequence within the exogenous DNA. Primers preferably have a sequence within 15 and 35 nu-cleotids which under optimized PCR conditions "specifically recognize" a sequence within the 5 'or 3' flanking region of the elite event and the exogenous DNA of the elite event, respectively. so that a specific fragment ("integrating fragment") is amplified from a nucleic acid sample comprising the elite event. This means that only the targeted integration fragment, and no other sequence (of that size) in the plant genome or exogenous DNA, is amplified under optimized PCR conditions. Preferably, the integrating fragment is between 50 and 500 nucleotides, most preferably between 100 and 350 nucleotides. Preferably, the specific primers have a sequence that is between 80 and 100% identical to a sequence within the 5 'or 3' flanking region of the elite event and the exogenous DNA of the elite event, respectively, as long as the bad ones. Comparisons also allow specific identification of the elite event with these primers under optimized PCR conditions. The range of permissible mismatches, however, can easily be determined experimentally and is known to a person skilled in the art. Since the primer sequence and genome-recognized sequence are unique to the elite event, the integration fragment amplification will occur only in biological samples comprising (the nucleic acid of) the elite event. Preferably, when performing a PCR to identify the presence of EE-GH1 in known samples, a control is included from a group of primers with which a fragment within a "housekeeping gene" of the event plant species. can be amplified. Routine genes are genes that are expressed in most cell types and are related to basic metabolic activities common to all cells. Preferably, the amplified fragment of the routine gene is a fragment that is larger than the amplified integrating fragment. Depending on the samples to be analyzed, other controls may be included.

Standard PCR protocols are described in the art, as in "PCR Applications Manual" (Roche Molecular Biochemicals, 2nd Edition, 1999). Optical conditions for PCR, including the sequence of specific primers, are specified in a "PCR identification protocol" for each elite event. It is however understood that several parameters in the PCR identification protocol may need to be adjusted for specific laboratory conditions, and may be slightly modified to obtain similar results. For example, use of a different method for DNA preparation may require adjustment of, for example, the amount of primers, polymerase and annealing conditions employed. Similarly, the selection of other primers may dictate other ideal conditions for the identification protocol. These adjustments however will be apparent to the person skilled in the art, and are therefore detailed in standard PCR application manuals such as the above.

Alternatively, specific primers may be employed to amplify an integrating fragment that may be employed as a "specific probe" to identify EE-GH1 in biological samples. Contacting the nucleic acid of a biological sample with the probe under conditions that allow the probe to hybridize to its corresponding fragment in the nucleic acid results in the formation of a nucleic acid / probe hybrid. Formation of this hybrid can be detected (eg nucleic acid labeling or probe), so formation of this hybrid indicates the presence of EE-GH1. Such identification methods based on hybridization to a specific probe (either in a solid phase vehicle or in solution) have been described in the art. The specific probe is preferably a sequence which, under optimized conditions, hybridizes specifically to a region comprising part of the 5 'or 3' flanking region of the elite event and also comprising part of the exogenous DNA contiguous thereto (hereinafter also referred to as as a "specific region" of the event). Depending on the method employed, such a specific probe may comprise a sequence of from 15 to 30 bp or from 50 to 500 bp, preferably from 100 to 350 bp, which hybridize under stringent nucleotide sequence (or complement to sequence) conditions. such sequence) from a specific region.

Preferably, the specific probe will comprise a sequence of from about 15 to about 100 identical (or complementary) contiguous nucleotides for a specific region of the elite event. Most preferably such a probe comprises at least 9 nucleotides identical to (or complementary to the 3 'or 5' flanking region and a similar number of nucleotides in the adjacent exogenous DNA. More preferably, such a probe comprises at least 9 identical nucleotides). (or complementary to the 3 'or 5' flanking region and a similar number of nucleotides in the exogenous DNA adjacent to it of SEQ ID NO: 1 or SEQ ID NO: 4.

A "restriction map" as used herein refers to a group of Southern blot patterns obtained after dividing the plant genomic DNA (and / or the exogenous DNA comprised therein) with a particular restriction enzyme, or group. of restriction and hybridization enzymes with a probe sharing sequence similarity with exogenous DNA under standard stringency conditions. Standard stringency conditions as employed herein refer to the hybridization conditions described herein or to conventional hybridization conditions as described by Sambrook et al (1989) (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY ) which for example may comprise the following steps: 1) immobilization of plant genomic DNA fragments onto a filter, 2) prehybridization of the filter for 1 to 2 hours at 42 ° C in 50% formamide, 5 X SSPE, 2 X Denhardt reagent and 0.1% SDS, or for 1 to 2 hours at 68 ° C in 6 X SSC, 2 X Denhardt reagent and 0.1% SDS, 3) addition of the hybridization probe that was labeled, 4) incubation for 16 to 24 hours, 5) wash the filter for 20 minutes at room temperature in 1 X SSC, 0.1% SDS, 6) wash the filter three times for 20 minutes each at 68 ° C 0.2 x SSC, 0.1% SDS, and 7) filter exposure for 24 to 48 hours to film and X-ray at -70 ° C with an intensification analysis.

Due to the (endogenous) restriction sites present in a plant genome prior to incorporation of exogenous DNA, insertion of an exogenous DNA will alter that genome-specific restriction map. Thus, a particular transformant or progeny thereof can be identified by one or more specific restriction patterns.

Alternatively, plants or plant material comprising an elite event may be identified by testing according to a PCR identification protocol. This is a PCR that specifically recognizes the elite event. Essentially, a group of PCR primers is developed which recognizes a) a sequence within the 3 'or 5' flanking sequence of the elite event and b) a sequence within the outer DNA, which primers amplify a fragment (fragment of preferably between 100 and 300 nucleotides. Preferably, a control is included from a group of primers which amplify a fragment within a routine plant species gene (preferably a fragment which is larger than the amplified integrating fragment). Optimal PCR conditions, including the sequence of specific primers, are specified in a PCR identification protocol.

Other methods for identifying plants, plant material or products comprising plant material comprising elite event EE-GH1 are also considered. These methods include all methods based on detection of the outer DNA sequence and elite event flanking sequence (s) with a specific probe. More particularly, chip-based technologies such as those described by Hacia et al. 1996 (Nat Genet 14 (4): 441-447) and Shoemaker et al. 1996 (Nat Genet 14 (4): 450-456) are contemplated. These methods allow segregation of target molecules as high density series by use of fixed probe series or by labeling genes with oligonucleotides, after which they can be displayed by hybridization. Alternatively, third wave detection methods, such as those described by Lyamichev et al. (1999, above) are contemplated.

Identification of the protein (s) encoded by the elite event outer DNA may be done by classical protein detection methods described in the art, such as those based on the protein's electromagnetic or chromatographic properties or detection by specific monoclonal antibodies (as described in "Guide to protein purification, Murray P. Deutscher editor).

A kit as used herein refers to a group of reagents for the purpose of carrying out the method of the invention, more particularly the identification of the EE-GH1 elite event in biological samples. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers as described above. Optionally, the kit may also comprise any other reagent described herein in the PCR identification protocol.

Alternatively, according to another embodiment of this invention, the kit may comprise a specific probe as described above that specifically hybridizes to nucleic acid from biological samples to identify the presence of EE-GH1 in it. Optionally, the kit may also comprise any other reagent (such as but not limited to hybridization buffer, label) for identifying EE-GH1 in biological samples using the specific probe.

The kit of the invention may be used, and components thereof may be specifically adjusted, for quality control purposes (eg seed lot purity), elite event detection in plant material or material comprising or derived from. of plant material, such as but not limited to food or food products.

The present invention relates to the development of an elite cotton event, EE-GH1, for plants comprising this event, offspring obtained from these plants and for plant cells, or plant material derived from from this event. Plants comprising the elite event EE-GH1 were obtained by transformation with pGSV71 as described in example 1.

Cotton plants or plant material comprising EE-GH1 may be identified according to the PCR identification protocol described for EE-GH1 in Example 4 herein. Briefly, cotton genome DNA is PCR amplified using a primer that specifically recognizes a sequence within the 5 'or 3' flanking sequence of EE-GH1, particularly the primer with the sequence of SEQ ID NO: 2, and a primer. which recognizes a sequence in the outer DNA, particularly the primer with the sequence of SEQ ID NO: 3. Endogenous cotton DNA primers are used as controls. If the plant material produces a fragment between 250 and 290 bp, preferably about 269 bp, the cotton plant is determined to host the elite EE-GH1 event.

Plants housing EE-GH1 are characterized by their glufosinate tolerance, which in the context of the present invention includes that plants are tolerant to the Liberty® herbicide. Liberty® tolerance can be tested in different ways. The leaf painting method as described herein is most useful when it is desired to identify both resistant and sensitive plants, but does not need to kill the sensitive units. Alternatively, tolerance can be tested by spraying Liberty®. Vaporization treatments should be done between leaf stages V3 and V4 for best results. Tolerant plants are characterized by the fact that vaporization of plants with at least 200 grams of active ingredient / hectare (gai / ha), preferably 400 gai / ha, and possibly up to 1600 gai / ha (4X normal field rate) , doesn't kill the plants. A broadcasting application should be applied at a rate of 793.72 g - 963.87 g (28-34 ounces) of Liberty®. This is best for applying to a volume of 75.71 I (20 gallons) of water per acre using a flat fan type nozzle while being careful not to direct spray applications directly into the plant vortex to avoid burning of surfactants in the leaves. The herbicide effect should appear within 48 hours and be clearly visible within 5-7 days.

Plants housing EE-GH1 may also be characterized by the presence in their cells of phosphinothricin acetyl transferase as determined by a PAT assay (De Block et al., 1987, supra).

Plants housing EE-GH1 can, for example, be obtained from seeds deposited to the ATCC under accession number PTA-3343, which contains 50% nuclei that are hemizygous for the elite event. Such plants may also be propagated to introduce the elite event of the invention into other crops of the same plant species. The selected seeds obtained from these plants contain the elite event stably incorporated into their genome. The invention also relates to plants derived from the ATCC accession number PTA-334, comprising EE-GH1. The term "derived from" here indicates that plants are related, that is, they are both progeny (direct or two or more generations) of the same transformant by mating.

Plants housing EE-GH1 are also characterized by having agronomic characteristics that are comparable to commercially available cotton varieties in the United States in the absence of weed pressure and the use of Liberty® for weed control. It has been observed that the presence of an outer DNA in the insertion region of the cotton plant genome described herein confers particularly interesting phenotypic and molecular characteristics to plants comprising this event. More specifically, the presence of the outer DNA in this particular region in the genome of these plants results in plants showing stable phenotypic expression of the gene of interest without significantly compromising any aspect of the desired plant agronomic performance. Thus, the insertion region corresponding to a sequence comprising the plant DNA of SEQ ID NO: 1 and / or SEQ ID NO: 4, more particularly a sequence corresponding to SEQ 10 NO: 5, more particularly the insertion site. of EE-GH1 herein is shown to be particularly adapted for the introduction of a gene (s) of interest. More particularly, the EE-GH1 insertion region (corresponding to a DNA sequence of at least 40 bp in the cotton genome within SEQ ID NO: 5), or a sequence of at least 40 bp that hybridizes under stringent conditions to the genome. complement of the sequence of SEQ ID NO: 5, is particularly suited for the introduction of exterior DNA comprising a herbicide tolerance gene, guarantee expression of each of these genes in the plant without compromising agronomic performance.

A recombinant DNA molecule can be specifically inserted into an insertion region by targeted insertion methods. Such methods are well known to those skilled in the art and comprise, for example, homologous recombination using a recombinase such as, but not limited to Saccharomyces cerevisiae FLP recombinase (published PCTP application WO 99/25821), phage CRE recombinase Escherichia coli P1 (published PCT application WO 99/25840), Saccharomyces rouxii pSR1 recombinase (Araki et al. 1985, J Mol Biol 182: 191-203), the Mu phage Gin / gix system (Maeser et al. Kahlmann, 1991, Mol Gen Genetics 230: 170-176) or the lambda phage recombination system (as described in US Patent 4,673,640).

As used herein, "sequence identity" to nucleotide sequences (DNA or RNA) refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shortest of the two sequences. Alignment of the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983, Proc Nat Acad Sci USA 80: 726) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Interpretation and computer-assisted analysis of sequence data, including sequence alignment as described above, can, for example, be conveniently performed using programs from the Wisconsin Package (from Genetics Computer Group, Inc). Sequences are indicated as "essentially similar" when such sequences have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least about 85%, most particularly about 90%, especially about 95%, more especially about 100%, very especially are identical. Of course, when RNA sequences are said to be essentially similar or have a certain degree of sequence identity to DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. "Complementary to" as used herein refers to the complementarity between nucleotides A and T (U), and G and C in nucleotide sequences.

As used herein, a "biological sample" is a sample of a plant, plant material or products comprising plant material. The term "plant" is intended to cover cotton (such as but not limited to Gossypium hirsutum) plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken from or derived from any plant. similar, including without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts. "Plant material" as used herein refers to material that is obtained or derived from a plant. Products comprising plant material refer to food, food or other products that are produced using plant material or may be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are preferably tested for the presence of EE-GH1 specific nucleic acids, implying the presence of nucleic acids in the samples. Thus the methods referred to herein for identifying elite event EE-GH1 in biological samples, preferably refer to the identification in biological samples of nucleic acids comprising the elite event.

As used herein "comprising" is to be construed as specifying the presence of the specified characteristics, integers, steps or components as referred to, but does not exclude the presence or addition of one or more characteristics, integers, steps or components. , or groups of these. Thus, for example, a nucleic acid or protein comprising a nucleotide or amino acid sequence may comprise more nucleotides or amino acids than the actual units mentioned, that is, be embedded in a larger protein or nucleic acid. A chimeric gene comprising a DNA sequence that is functionally or structurally defined may comprise additional DNA sequences, etc.

[0073] The following examples describe the development and characteristics of cotton plants housing the EE-GH1 elite event as well as the development of tools for elite event identification EE-GH1 in biological samples.

Unless otherwise stated, all recombinant DNA techniques are performed according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and Volumes 1 and 2 by Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Cray published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

In the description and examples, reference is made to the following sequences: SEQ ID NO: 1: sequence comprising the 5 'flanking region SEQ ID NO: 2: primer GHI06 SEQ ID NO: 3: primer GHI05 SEQ ID NO : 4: sequence comprising the 3 'flanking region SEQ ID NO: 5: insertion region SEQ ID NO: 6: plasmid pGSV71 SEQ ID NO: 7: plasmid pRVA44 SEQ ID NO: 8 primer MDB327 SEQ ID NO: 9 : primer MLD015 SEQ ID NO: 10: primer MLDÜ16 SEQ ID NO: 11: primer MDB612 SEQ ID NO: 12: primer MDB053 SEQ ID NO: 13: primer MDB356 SEQ ID NO: 14: primer DPA017 SEQ ID NO: 15: primer MLD019 SEQ ID NO: 16: sequence comprising target site suppression SEQ ID NO: 17: primer GHI01 SEQ ID NO: 18: primer GHI02 Examples Example 1: Cotton transformation with a herbicide tolerance gene Construction of chimeric DNA comprising the gene bar under the control of a constitutive promoter.

A plasmid pGSV71 was constructed following standard procedures. The sequence of the genetic elements of plasmid pGSV71 is given in Table 1 (SEQ ID NO: 6): Table 1. Nucleotide positions of the genetic elements in PGSV71 Continued ,,, Transformation of Gossvoium hirsutum Coker312 plant cotton tissue was transformed with pGSV71 using Agrobacterium transformation (US 5,986,181) and regenerated on plants in appropriate vehicles.

Small seedlings initiated in selective regeneration vehicles were transferred to new vehicle for germination (the whole vehicle is hormone free). The plants were then transferred to growth chambers or greenhouses.

[0079] Selection was performed on phosphinothricin (PPT) at all stages except seedling regeneration, which was performed in the absence of PPT to accelerate growth. This resulted in a group of primary transformants (TO generation plants).

Example 2: Event Development 2.1. Development of lines bearing the characteristic of the event [0080] TO buds were transferred to greenhouse soil and plants were evaluated for glufosinate tolerance and presence of PAT enzyme with a PAT ELISA (Steffens Biotechnische Analysen GmbH, Ebringen, Germany) ).

Plants T1 to T3 were germinated in the greenhouse and tested for Liberty * tolerance at a rate of 2x (vaporizing 0.1656 mtJm2 (56 oz / ha)). Positive plants were tested for bar gene expression using the Pat assay as described by Deblock et al 1987 (EMBO J. 6: 2513-2518).

The presence of exogenous DNA and copy number was verified by Southern blot analysis. Total genomic DNA was isolated from 1 g of leaf tissue according to the CETAB method of Doyle et al. (1987, Phytochem. Bull. 19:11) and digested with restriction enzyme EcoRI.

Probes as follows were used for Southern analysis: "baf probe: 474 bp Kpnl-Bg11 digestion of plasmid pDE110 (WO 92/09696)" 35S probe ": 892 bp Ncol-Munl digestion pRVA44 (SEQ ID NO: 7) [0084] T2 plants have also been evaluated for general phenotypic characteristics compared to non-transgenic isogenic strains.In more recent generations, parasitic strains for which no penalty in phenotype or agronomic yield has been observed. observed for the presence of the transgene in hemizygote or homozygote condition, when compared to wild type were selected.

T4 material was germinated in the field and tested under field conditions for Liberty® tolerance at different times.

[0086] In more recent generations, plants have been compared to commercial varieties for field, fiber quality and plant mapping data. Agronomic characteristics such as plant height, node height, cotton cocoon retention, position, vigor, fiber size, fiber strength and lint yield were evaluated.

It was determined that an event performed equally or better than comparable controls and that for this event production was dependent on the base rather than the presence of the transgene. 2.2. Selecting an Elite Event This selection procedure produced an elite event that exhibited optimal expression of the bar 35S gene, ie tolerance to glufosinate ammonia, without penalizing the production of agronomic performance. This elite event was called EE-GH1. 2.3. EE-GH1 testing on cotton varieties with different genetic bases and at different locations [0089] The selected event was introduced on different commercial genetic bases including FM989, FM 832, FM958, and FM966 and results from field experiments from four sites. different were compared. Plants were sprayed at 1600 gai / ha using different treatments (1x3-5 leaf stage, 4x, 3-5 leaf stage, 1x + 1x, 3-5 leaf stage, 4x + 4x, 3-5 leaf stage). sheet, 0 as a control).

Moulting emergence and vigor assessment for the elite event was very good.

No visible damage as a result of herbicide application has never been observed after application regardless of rate or stage of development at the time of application. There were no harmful effects on plant morphology or habit of herbicide application.

In addition »the event had normal leaf» cotton and flower cocoon morphology »excellent fertility» and did not show abnormal insect disease or susceptibility on multiple genetic bases. During introgression on multiple genetic bases no aberrant problems or abnormalities were observed in four generations. 2.4. Genetic analysis of locus The genetic stability of the insertion for the EE-GH1 event was verified by phenotypic and molecular analysis in progeny plants over several generations.

Southern blot analyzes of T1, T2 and T3 generation plants were compared for EE-GH1 event. The obtained patterns were found to be identical in the different generations. This proves that the molecular configuration of exogenous DNA in EE-GH1 was stable. The EE-GH1 event exhibited Mendelian segregation for the transgene as a single genetic locus for at least three subsequent generations indicating that the insertion is stable.

Example 3: Characterization of the elite event EE-GH1. 3.1 In-Site Genetic and Molecular Analysis [0096] Once the EE-GH1 event was identified as an event in which transgene expression as well as total agronomic performance was optimal, the transgene locus was analyzed in detail over a molecular level. . This included the sequencing of the flanking regions of the transgene.

The sequence of the regions flanking the transgene inserted into the EE-GH1 event was determined using the TAIL-PCR protocol as described by Líu et al. (1995, Plant J. 8 (3); 457 - 463) Determination of the flanking region 5 'The primers used were: whereby N = A, C, T or g; S = C or g; W = A or T

The amplified fragment using MDB327-GHI05 was approximately 1200 bp which was sequenced (5'flanq: SEQ ID NO: 1). The sequence between base pairs 1 and base pairs 677 comprised the plant DNA at the same time. while the sequence between base pairs 678 and base pairs 850 corresponded to pGSV71 DNA. b) Determination of the 3 'flanking region The primers used were: By which: N = A, C, T or g; S = C or g; W = A or T

The fragment amplified using MDB612-DPA017 was approximately 400 bp, the complete sequence of which was determined (SEQ ID NO: 4). The sequence between nucleotide 1 and 179 corresponds to T-DNA, while the sequence between nucleotide 180 and 426 corresponds to plant DNA. c) Identification of target site deletion Using primers corresponding to sequences within the transgene flanking regions in wild type Gossypium hirsutum as a standard, the transgene insertion site was identified.

The following primers were used: Sequence (5 '-> 3') Position in Flanking Position 5 'flanking (SEQ ID 3' (SEQ ID NO: 1} NO: 4) GHI06 TTg.CAC.CAT.CTA.gCT. 815 ^ 795 --- CAC.TC (SEQ ID NO: 2) MLD019 CAA.gAT.gCg.AgC.AAC. -— 285 ^ 266 TAT.gT (SEQ ID NO: 15) [00103] This produced a fragment of 200 bp (SEQ ID NO: 16) wherein 85 to 122 bp corresponds to a target site deletion.

Thus, the insertion region (SEQ ID NO: 5) as sequenced comprises: 1-677: flanking region- 1 to 677 base pairs of SEQment 5 'ID NO: 1 678-714: deletion of the target site 85 to 122 bp of SEQ ID NO: 16 715-916: flanking region- 180 to 426 bp of SEQ ID NO: 4 ment 3 '3.2. Genetic analysis of locus The genetic stability of the insert was verified by phenotypic and molecular analysis in progeny plants over several generations.

Southern blot analyzes on glu-phosinate tolerant plants of T0-generation EE-GH1 cotton plants were compared and found to be identical. This proves that the molecular configuration of the transgene in plants containing EE-GH1 was stable.

The EE-GH1 event exhibited Mendelian segregation for the transgene as a single genetic locus for at least three subsequent generations indicating that the insertion is stable.

Based on the above results, EE-GH1 was identical to an elite event.

Example 4: Development of diagnostic tools for identity control.

[00109] An Elite EE-GH1 Event PCR Identification protocol has been developed to identify the presence of EE-GH1 in plants, plant material or biological samples.

Elite EE-GH1 Event Polymerase Chain Reaction Identification Protocol.

[00110] A test run, with all appropriate controls, must be performed before attempting to evaluate unknowns. The protocol presented may require optimization for components that may differ between labels (preparation of standard DNA, Taq DNA polymerase, primer quality, dNTP's, term cyclizer, etc.), [00111] Endogenous sequence amplification plays a key role. in the protocol. It must achieve PCR and thermocycle the conditions that amplify the equimolar amounts of both the endogenous and transgenic sequence in a standard transgenic genomic DNA pattern. When the targeted endogenous fragment is not amplified or when the targeted sequences are not amplified with the same ethidium bromide staining intensities as evaluated by agarose gel electrophoresis, optimization of PCR conditions may be required. Standard DNA Standard DNA is prepared according to the CTAB method described by Doyle and Doyle (1987, Phytochem, Bull. 19: 11). When using DNA prepared with other methods, a test run using different amounts of standard must be performed. Usually 50 ng of genomic standard DNA produces the best results.

Designated Negative and Positive Controls The following negative and positive controls must be included in a PCR run: A Master Mix control (negative DNA control). This is a PCR where no DNA is added to the reaction. When the expected result, no PCR product, is observed, this indicates that the PCR cocktail was not contaminated with target DNA.

[00114] - A positive DNA control (genomic DNA sample known to contain transgenic sequences.) Successful amplification of this positive control demonstrates that PCR was determined under conditions that allow amplification of the target sequences.

[00115] - A wild type DNA control. This is a PCR where the standard DNA provided is genomic DNA prepared from a non-transgenic plant. When the expected result, no transgene PCR product amplification but endogenous PCR product amplification, is observed, this indicates that there is no detectable transgene base amplification in a genomic DNA sample. Primers The following primers, which specifically recognize the transgene and an EE-GH1 flanking sequence are used: Primers targeting an endogenous sequence are always included in the PCR cocktail. These primers serve as an internal control on unknown samples and positive DNA control. A positive result with the endogenous primer pair demonstrates that there is ample DNA of adequate quality in the preparation of genomic DNA for the PCR product to be generated. The endogenous primers used are: GH101: 5-AAC.CTA.ggC.TgCTgA.Agg.AgC-3 '(SEQ ID NO: 17) (Alcohol Dehydrogenase Gene Acc. NO: AF036569, 107Ü- »109G GHI02: 5' -CAA.CTC, CTC.CAg, TCA, TCT, CCg-3 '(SEQ ID NO: 18) (Alcohol dehydrogenase gene Acc. NO: AF036569, 1515-> 1495) Amplified Fragments The expected amplified fragments in PCR reaction are: For GHI01-GFII02 primer pair: 445 bp (endogenous control) For GHI05-GHI06 primer pair: 269 bp (EE-GH1 Elite Event) PCR conditions

The 50 µl reaction PCR mix contains: 5 µl standard DNA 5 µl 10x Amplification Buffer (supplied with Taq polymerase) 1 d of 10 mM GHIQ1 0.5x1 dNTPs (10 pmoles / xl ) 0.5 od GHI02 (10 pmoles / d) 1 od GHI05 (10 pmoles / d) 1 od GHI06 (10 pmoles / d) 0.2 od DMA Taq polymerase (5 units / d) water up to 50 od The thermocycling profile to be followed for optimal results is as follows: 4 min. at 95 ° C

Followed by: 1 min. at 95 ° C

1 min at 57 ° C 2 min. at 72 ° C for 5 cycles Followed by: 30 sec. at 92 ° C 30 sec. at 57 ° C 1 min. at 72 ° C For 25 cycles Followed by: 5 minutes at 72 ° C

Agarose Gel Analysis Between 10 and 20 d of the samples would be applied to a 1.5% agarose gel (Tris-borate buffer) with an appropriate molecular weight marker (eg 100 bp graded PHARMACIA ).

Validation of Results Data from transgenic plant DNA samples within a single PCR run and a simple PCR cocktail would not be acceptable except 1) positive DMA control shows the expected PCR products (transgenic and endogenous fragments). us), 2) negative DNA control is negative for PCR amplification (no fragment) and 3) wild type DNA control shows expected result (endogenous fragment amplification).

Bands showing visible amounts of the expected sizes of endogenous and transgenic PCR products indicate that the corresponding plant from which genomic standard DNA was prepared has inherited the elite event EE-GH1. Bands not showing visible amounts of the transgenic PCR product and showing visible amounts of the endogenous PCR product indicate that the corresponding plant from which genomic standard DNA was prepared does not understand the elite event. Bands not showing visible amounts of transgenic and endogenous PCR products indicate that the quality and / or quantity of genomic DNA did not allow a PCR product to be generated. These plants cannot be classified. Genomic DNA preparation would be repeated and a new PCR run, with the appropriate controls, must be performed.

Use of discriminant PCR protocol to identify EE-GH1 Plant cotton leaf material comprising different transgenic events (samples 1 to 4) was tested according to the protocol described above. Wild-type cotton samples were taken as negative controls.

The results of the PCR analysis are illustrated in Figure 1. Sample 1 is recognized as comprising the elite event EE-GH1. All other test strains do not understand this elite event.

Example 5: Ingression of EE-GH1 into Preferred Growers The elite event EE-GH1 is introduced by repeated crossbreeding in the following commercial cotton crops: FM5013, FM5015, FM5Q17, FM989, FM832, FM966 and FM958.

It is observed that the introgression of the elite event in these crops does not significantly influence any of the desirable agronomic or phenotypic characteristics of these crops (no binding resistance) while transgene expression as determined by glufosinate tolerance reaches levels. commercially acceptable. This confirms the state of the EE-GH1 event as an elite event.

As used in the claims below, unless otherwise clearly stated, the term "plant" is intended to encompass plant tissues at any stage of maturity as well as any cells, tissues, or organs taken from or derived from any such plant. plant including without limitation any seeds, leaves, stems, flowers, roots, single cells, gametes, cell culture, tissue cultures or protoplasts.

The reference seed comprising the elite event EE-GH1 was deposited as EE-GH1 in ATCC (10801 Uversity Blvd., Manassas, VA 20110-2209) on April 26, 2001, under ATCC accession number PTA-3343. .

As used in the claims below, unless otherwise clearly stated, the term "plant" is intended to encompass plant tissues at any stage of maturity as well as any cells, tissues, or organs taken from or derived from any such plant. plant including without limitation any seeds, leaves, stems, flowers, roots, single cells, gametes, culture cells, tissue cultures or protoplasts.

The above description of the invention is intended to illustrate and not to limit. Various changes or modifications to the described embodiments may occur by those skilled in the art. These may be made without departing from the spirit and scope of the invention.

CLAIM

Claims (1)

1. DNA molecule, characterized in that it comprises a contiguous sequence of nucleotides 1 to 677 of SEQ ID NO: 1, nucleotides 221 to 2540 of SEQ ID NO: 6, and nucleotides 180 to 426 of SEQ ID NO: 4, and said DNA molecule can be obtained from cotton seeds that were deposited in the ATCC under accession number PTA-3343 or their progeny.