MXPA01004419A - Glufosinate tolerant rice - Google Patents

Abstract

This invention pertains to rice plants, plant material and seeds characterized by harboring a specific transformation event particularly by the presence of the bar gene under control of a CaMV 35S promoter, at a specific location in the rice genome. The rice plants of the invention combine glufosinate tolerance with optimal overall agronomic performance, genetic stability and adaptability to different genetic backgrounds.

Description

RESISTANCE OF RICE TO GLUFOS1NATE FIELD OF THE INVENTION This invention relates to rice plants, plant material and seeds characterized by preserving a specific transformation event particularly by the presence of a bar gene under the control of a 35S CaMV promoter. At a specific site in the rice genome. The rice plants of the invention combine the tolerance to glufosinate with an optimum agronomic development in general, genetic stability and adaptability to different genetic environments. All citations documented here are incorporated as references.

BACKGROUND OF THE INVENTION The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time, the presence of the transgene in different places of the genome will influence the general phenotype of the plant. The agronomically or industrially successful introduction of a commercially interesting trait into a plant by genetic manipulation can be a slow procedure dependent on several factors. The 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 in field trials. Rice production is commonly threatened by several weeds. Some of these can be highly competitive and in cases of severe infestation can result in yield losses of such magnitude that make the harvest economically unattractive. For the cultivation of mechanized rice planted directly, typical of moderate production, both cultivation practices (for example crop rotation, irrigation management) and herbicides are necessary to control weeds (Hill et al., 1994). The bar gene (Thompson et al, 1978, EMBO J. 6: 2519-2523, Deblock et al., 1987, EMBO J. 6: 2513-2518) is a gene that codes for the enzyme phosphinothricin acetyltranfraside (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 . Other genes encoding PAT have been described (see for example: Wohlleben et al., 1988, Gene 70: 25-37, EP 275,957, US 5,276,268, US 5,637, 489, US 5,273,894). The transformation of monocotyledonous plants by electroporation of intact tissue capable of forming compact embryogenic calli or compact embryogenic calli obtained from said tissue is described in the U.S. patent. 5,641, 664. Here, the transformation of compact embryogenic calluses of rice by electroporation of a bar gene and the regeneration of transgenic rice plants is described. Transgenic rice plants containing the gus gene have been described either with the bar gene, or with the hyg gene that confers hygromycin resistance, obtained by the transformation of immature rice embryo cells by bombardment with DNA coated with particles of gold (Christou et al, 1991: Biotechnology 9: 957). The transformation of the rice with the bar gene by electroporation of cells in added suspension is described in the patent of E.U.A. 5,679,558. However, the foregoing documents do not teach or suggest what is claimed in the present invention.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to transgenic rice plants, cells, tissue or seeds, resistant to glufosinate, which are characterized by one or more of the following characteristics: a) the genomic DNA of the plant, cell, tissue or seed is capable of of giving one or more, such as at least two, advantageously at least three, preferably at least four, for example at least five, more preferably six restriction fragments or pairs of restriction fragments selected from the group of: i) an EcoRI fragment with a length between about 1159 and about 1700 bp, preferably about 1327 bp; ii) a pair of BamHI fragments of which one has a length between about 805 and about 1093 bp, preferably about 805 bp and the other has a length between about 1700 and about 2140 bp, preferably about 2.0 kpb; Ii) a pair of EcoRV fragments in which one has a length between about 2838 and about 4507 bp, preferably about 3.8 kbp and the other has a length of more than about 5077 bp, preferably around 12 kbp; iv) a Hindlll fragment with a length between about 5077 and about 11,497 bp, preferably about 5.3 bp; v) a pair of Ncol fragments both having lengths between about 2838 and about 4507 bp, preferably one of about 3.1 kbp and one of about 4.1 kbp; vi) a Nsil fragment with a length between about 4749 and about 11497 bp, preferably about 5.1 kbp; wherein each of the restriction fragments is capable of hybridizing under standard severe conditions, with the fragment of about 1327 bp obtained by digestion with EcoRI of the plasmid having the nucleotide sequence of SEQ ID No 8; and / or, b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify a DNA fragment of between 290 and 350 bp, preferably around 313 bp, using a polymerase chain reaction with two initiators having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively (or including a DNA fragment of about 290 to about 350 bp, preferably about 313 bp amplified using a chain reaction of the polymerase with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively). The present invention relates to plants, cells, tissue or seeds of transgenic rice, with resistance to glufosinate, which is characterized in that the genomic DNA of the plant, cell, tissue or seed is capable of giving at least one, advantageously at least two or more, for example at least three, preferably at least four, for example at least five, more preferably six of the restriction fragments or pairs of restriction fragments selected from the group described above comprising the restriction fragments or pairs of restriction fragments described under i), ii), ii), iv), v) and vi) above, where the selection may include any combination of i), ii), iii), iv), v) and vi) described above.

The present invention relates to transgenic rice plants, cells, tissues or seeds, with resistance to glufosinate, which is preferably characterized by both characteristics described under a) and b) above. The invention also relates to a deposit of seeds in ATCC under the number ATCC 203352, a plant which is generated from this seed, and cells or tissues of a plant generated from this seed. The invention further relates to plants obtained by propagation of, and / or reproduction from, a rice plant that is generated from the seed deposited in ATCC under the number ATCC 203352. The invention furthermore relates to plants, seeds, cells or tissues (e.g., rice plants, seeds, cells or tissues) comprising the flanking regions discussed herein with the bar-35S gene (as discussed herein) among them, or plants, seeds, cells or tissues (e.g., plants) of rice, seeds, cells or tissues) comprising a nucleotide sequence with at least 65%, for example, at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, by example at least 95% or even 97% or 100% similar to the sequence described herein, such as the sequence of the construction of the flanking region of the bar-35S gene of the flanking region or the insertion region. The invention further relates to a process for growing rice plants of the invention as described above, more particularly a method which comprises the application of a herbicide with glufosinate as an active ingredient to rice plant crops. It is believed that the rice plants of the invention, when grown according to the process described above, which comprises the application of a herbicide with glufosinate as the active ingredient, exhibit an improvement in growth compared to unprocessed rice of the same cultivate (US 5,739,082). Thus, the invention may comprise a method for improving the yield or growth of rice plants. The invention also provides a process for the recroduction of rice which comprises crossing with the rice plants of the invention. The invention further provides a method for producing a transgenic cell of a rice plant or a plant obtained therefrom, which consists in inserting a recombinant DNA molecule into a part of the chromosomal DNA of a rice cell characterized by the sequence of SEQ ID No. 9 and, optionally, regenerating a rice plant from the transformed rice cell. The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, with methods that consist of establishing one or more of the following characteristics of the genomic DNA of the transgenic plant, or of its cells or tissues: a) the genomic DNA of the plant, cell, tissue or seed is capable of giving a yield of at least three, preferably of at least four, for example of at least 5, more preferably six of the restriction fragments or pairs of fragments of restriction selected from the group of: i) an EcoRI fragment with a length between 1 159 and 1700 bp, preferably about 1327 bp; ii) a pair of BamHI fragments in which one has a length between 805 and 1093 bp, preferably about 805 bp and the other has a length between 1700 and 2140 bp, preferably about 2.0 kbp; iii) a pair of EcoRV fragments of which one has a length between 2838 and 4507 bp, preferably about 3.8 kbp and the other has a length of more than 5077 bp, preferably about 12 kbp; V) a Hindlll fragment with a length between 5077 and 1497 bp, preferably about 5.3 kbp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp, preferably one of about 3.1 kbp and one of about 4.1 kbp; vi) a Nsil fragment with a length between 4749 and 1497 bp, preferably about 5.1 kbp; wherein each of the restriction fragments is capable of hybridizing under standard severe conditions, with the 1327 bp fragment obtained by digestion with EcoRI of the plasmid having the nucleotide sequence of SEQ ID NO: 8; and / or, b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify a DNA fragment of between 290 and 350 bp, preferably around 313 bp, using a polymerase chain reaction with two initiators that have the SEC nucleotide sequence ID No. 4 and SEQ ID No. 5, respectively. The invention further relates to a kit for identifying the transgenic plants comprising the selected event of the present invention, said kit comprising PCR probes that recognize the external DNA and the 3 'or 5' flanking sequence of GAT-OS2, preferably that have the nucleotide sequence SEQ ID No. 4 and SEQ ID No. 5, respectively, for use in the PCR identification protocol.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, which is given by way of example, but is not intended to limit the invention to specific embodiments described, can be understood in conjunction with the appended figures, incorporated herein by reference, in which: Figure 1. Restriction map obtained after digestion of the GAT-OS2 genomic DNA. Sequence loaded gel analyzed by Southern blot: line 1, Lambda DNA digested with PstI, line 2, DNA GAT-OS2 digested with EcoRI, lane 3, DNA GAT-OS2 digested with BamHI, lane 4, DNA GAT-OS2 digested with EcoRV line 5 DNA GAT-OS2 digested with HindIII, line 6, DNA GAT-OS2 digested with Ncol, line 7, DNA GAT-OS2 digested with NsiI, line 8, DNA non-transgenic rice line 9, plasmid DNA Control digested with EcoRI. Figure 2. PCR analysis of different lines using the GAT-OS2 identification protocol by PCR. Sequence loaded on the gel: line 1, molecular weight marker (leader 100 bp), lines 2 to 11, DNA samples from rice plants comprising different transgenic events, line 12, wild type DNA M202, line 13, DNA of the Bengal wild type, line 14, negative control (water), line 15, molecular weight marker (leader 100 bp).

DETAILED DESCRIPTION OF THE INVENTION The term "gene", as used herein, refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter and a 5 'untranslated region (5' UTR), which together form the promoter region , a coding region (which may or may not code for a protein), and a 3 'untranslated 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 RNA which, in the case of a gene encoding a protein, is translated into the protein. A gene can include additional DNA fragments such as, for example, introns. As used here, a genetic locus is the position of a given gene in the genome of a plant. The term "chimeric", when referring to a gene or a DNA sequence, is used to refer to the fact that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5 'UTR, coding region, 3 'UTR, intron) and that are not naturally associated with one another and that originate, for example, from different sources. "External" refers to a gene or a DNA sequence with respect to a plant species that is used to indicate that the gene or DNA sequence is not naturally found in plant species. As used herein, the term "transgene" refers to a recombinant DNA molecule incorporated in the genome of a plant. The term "recombinant DNA molecule" is used to illustrate and therefore may include an isolated nucleic acid molecule which may be DNA and which may be obtained through recombination or other methods. This recombinant DNA molecule usually comprises at least one copy or at least one "gene of interest" (eg, a chimeric gene) which is capable of conferring one or more specific characteristics to the transformed plant. A "transgenic plant" refers to a plant that comprises a transgene in the genome of all its cells.

The incorporation of a recombinant DNA molecule into the plant genome typically results in the transformation of a cell or tissue (or other genetic manipulation). The particular site of incorporation may be either random or a predetermined site (if a directed integration procedure is used). The transgene 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 transgene has been inserted is also referred to as "insertion site" or "white site". Insertion of the transgene into the plant genome can be associated with a deletion of plant DNA, referred to as "directed site deletion". A "flanking region" or "flanking sequence" as used herein refers to a sequence of at least 20 bp, preferably at least 50 bp, and greater than 5,000 bp of the plant genome which is located either immediately to the extreme 5 'and contiguous with or immediately towards the 3' end and contiguous with the transgene. The transformation procedures leading to the random integration of the transgene will result in a transformant with different flanking regions, which are characterized and unique to each transformant. When the transgene is introduced into a plant through a traditional cross, it will generally not change its insertion site in the plant genome, or its flanking regions. An "insertion region" as used herein refers to the region corresponding to the region encompassed by the insertion site (and possible deletion of target site), towards the 5 'end and towards the 3' end of the flanking regions of a transgene in the plant genome (not transformed). The expression of the transgene is used to indicate that the gene (s) of interest included in the transgene is expressed in a manner that confers on the plant one or more phenotypic traits (eg resistance to herbicides) that were intended to be conferred by the introduction of the recombinant DNA molecule - the transforming DNA - used during the transformation (based on the structure and function of the gene (s) of interest in part or complete). An event is defined as a genetic (artificial) locus which, as a result of genetic manipulation, carries a transgene comprising at least one copy of a gene of interest. The typical allelic state of a TS event the presence or absence of the transgene. An event is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic set of a plant. At the molecular level, an event is characterized by the restriction map (for example as determined by Southern blot) and / or by the flanking sequences towards the 5 'end and / or towards the 3' end of the transgene, and / or the Molecular configuration of the transgene. Usually the transformation of a plant with a transforming DNA comprises at least one gene of interest that leads to a multitude of events, each of which is unique. A select event, as used here, is an event that is selected from a group of events obtained by transformation by the same transforming DNA, based on the expression and stability of the transgene and its compatibility with optimal agronomic characteristics of the plant that understand Thus the criterion for the selection of the selected event is one or more, preferably two or more, advantageously all of the following: a) that the presence of the transgene does not compromise other desirable characteristics of the plant, such as those related to its agronomic development or value commercial; b) that the event is characterized by a well-defined molecular configuration which is stably inherited and for which appropriate diagnostic tools for identity control can be developed; c) that the gene (s) of interest in the transgene show a phenotypic expression that is correct, appropriate and stable spatially and temporally, under both heterozygous (or hemizygous) and homozygous conditions of the event, at a commercially acceptable level in a medium range of conditions environmental conditions in which the plants that carry the event are probably exposed to normal agronomic use; It is preferred that the transgene be associated with a position in the plant genome that allows introgression to the desired commercial genetic base. The status of an event as a selected event is confirmed by the introduction of the selected event in different relevant genetic environments and observing compliance with one, two or all of the criteria a), b) and c) above.

Thus, a "select event" refers to a genetic locus that comprises a transgene, which responds to the criteria described above. A plant, plant material or progeny such as seeds can comprise the selected event in this genome. The "diagnostic tools" developed to identify a selected event or plant or plant material that comprises a selected event, are based on the specific genomic characteristics of the selected event, such as, a specific restriction map of the genomic region comprises the transgene and / or the sequence of the flanking region (s) of a transgene. A "restriction map", as used herein, refers to a group of Southern blot patterns obtained after the disruption of plant genomic DNA with a particular restriction enzyme, or group of restriction enzymes and hybridization with a probe that shares similarity of sequence with the transgene (under specific conditions). Due to the restriction sites (endogenous) present in a plant genome before the incorporation of the transgene, the insertion of a transgene will alter the specific restriction map of that genome. Thus, a particular transformant or progeny derived therefrom can be identified by one or more specific restriction patterns. The conditions for determining the restriction map of an event are displayed in a restriction map identification protocol. Alternatively, plants or plant material comprising a selected event can be identified by assays according to the PCR identification protocol. This is a PCR that uses primers which specifically recognize the selected event. Essentially, a group of primers is developed which recognize a) a sequence with the 3 'or 5' flanking sequence of the selected event and b) a sequence within the external DNA whose primers amplify a fragment (integration fragment) preferably between 100 and 350 nucleotides. Preferably, a control of a group of primers is included which amplify a fragment within a maintenance gene of the plant species (preferably a fragment which is larger than the amplified integration fragment). The optimal conditions for PCR, including the sequence of the specific primers, are specified in the PCR identification protocol. The term "similarity", for example, with respect to a nucleotide sequence, is intended to indicate a quantifiable amount of homology between two sequences. The sequence similarity percentage can be calculated as (Nmf - Nrt7) * 100 / Nre where Ndif is the total number of non-identical residues in the two sequences when they are aligned and where Nre ^ is the number of residues in one of the sequences. Thus, the AGTCAGTC DNA sequences will have a sequence similarity of 75% with the sequence of AATCAATC (Nre = 8, Ndtf = 2). The invention comprises the nucleic acid molecules and sequences having at least 65%, for example, at least 70%, such as at least 75%, or at least 80% or advantageously at least 85%, for example at least 90% , such as at least 95% or even 97% or 7 100% similarity to the sequences described herein, as well as plants, cells, tissues, seeds, and progeny thereof (e.g., rice plants, cells, tissues, seeds and progeny thereof) comprising said acid molecules nucleic. Alternatively or additionally, the "similarity" with respect to the sequence refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences where the alignment of the two sequences can be determined according to the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983 PNAS USA 80: 726) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a penalty for a space of 4, and a computer-aided analysis and the interpretation of the data sequence including the alignment that can be carried out using suitably Intelligenetics ™ series programs (Intelligenetics Inc. CA). Sequences that are "essentially similar" have a sequence or identity similarity of at least about 75%, advantageously of about 80%, such as at least about 85%, preferably at least about 90%, especially about 95%, such as at least 97%, and especially about 100%. It is clear that when it is said that the RNA sequences are essentially similar or similar, or that they have a degree of sequence identity with the DNA sequences, the thymidine (T) in the DNA sequence is considered equal to the uracil (U) in the RNA sequence.

The present invention relates to the development of a selected event in rice, GAT-OS2, and plants, plant cells or plant material derived from this event. Plants comprising the selected GAT-OS2 event were obtained through transformation with a 1501 bp Pvul-Hindlll fragment of plasmid PB5 / 35Sbar (SEQ ID No 8) as described in example 1. The recombinant DNA molecule used for the generation of this select event comprises a DNA sequence encoding the enzyme phosphinothricin acetyltransferase and the 35S promoter of cauliflower mosaic virus, wherein the sequence encoding phosphinothricin acetyltransferase is under the control of the 35S promoter of the Cauliflower mosaic virus (called "bar-35S gene"). The 35S promoter has a "constitutive" expression pattern in rice (Battraw et al, 1990, Plant Mol Biol 15: 527-538), which means that it is significantly expressed in most plant cell types, for the most part part of the plant life cycle. Expression of the bar-35S gene in rice plants confers resistance to the herbicidal compound phosphinothricin or bialaphos or glufosinate or more generally, to inhibitors of glutamine synthetase, or salt or optical isomers thereof. The plants or plant material comprising GAT-OS2 can be identified according to the restriction map identification protocol described herein in Example 3b) (1). Briefly, rice genomic DNA is digested with a selection (preferably at least one such as at least two, advantageously at least three, or at least four, or at least five such as three to six) of the following restriction enzymes: EcoRI, BamHI, EcoRV, HindIII, Ncol, Nsil, is then transferred to nylon membranes and hybridized with the EcoRI fragment of about 1327 bp of plasmid PB5 / 35Sbar. Then it is determined for each restriction enzyme used where the following fragments can be identified: -EcoRI: a fragment of between about 1 159 and about 1700 bp, preferably about 1327 bp -BamHI: a fragment of between about 1700 and about 2140 bp, preferably about 2.0 kbp and a fragment between about 805 and about 1093 bp, preferably about 805 bp -EcoRV: a fragment of more than about 5077 bp, preferably around of 12 kbp and a fragment of between about 2838 and about 4507 bp, preferably about 3.8 kbp -Hindll: a fragment of between about 5077 and about 11497 bp, preferably about 5.3 kbp -Ncol: two fragments of between about 2838 and about 4507 bp, preferably one of about 4.1 kbp and one of about 3.1 kbp -Nsil: a fragment of between around 4749 and around 1497 bp, preferably about 5.1 kbp The lengths of the DNA fragments are determined by comparison with the group of DNA fragments of known length, particularly the PstI fragments of the lambda phage DNA. If the plant material after digestion with at least one of such or at least two, advantageously at least three, preferably at least four, especially with at least five, more preferably with all the restriction enzymes, gives DNA fragments with the same length as those described above, it is determined that the rice plant contains the selected event GAT-OS2. Plants or plant material comprising GAT-OS2 can also be identified according to the PCR identification protocol described herein in Example 3b) (2). Briefly rice genomic DNA is amplified by PCR using an primer which specifically recognizes a GAT-OS2 flanking sequence, particularly the primer with the sequence of SEQ ID No 5, and an initiator which recognizes a sequence in the transgene, particularly the primer with the sequence of SEQ ID No 4. The endogenous rice primers are used as controls. If the plant material gives a fragment of between about 290 and about 350 bp, preferably around 313 bp, it is determined that the rice plant contains the selected event GAT-OS2. Plants containing GAT-OS2 are also characterized by their resistance to glufosinate, which in the context of the present invention includes plants that are resistant to the Liberty ™ herbicide. The resistance to Liberty ™ is defined by the criterion that the sprinkling of the plants in the 3 or 4 leaf stage (3V to 4V) with at least 200 grams of the active ingredient / hectare (gi a / ha), preferably 400 gai / ha, and possibly more than 1600 gai / ha, does not kill the plants. Plants containing GAT-OS2 can therefore be characterized by the presence in their cells of phosphinothricin acetyltransferase as determined by the PAT assay (De Block et al, 1987, supra). Plants containing GAT-OS2 can, for example, be obtained from seeds deposited in ATCC under the number ATCC 203352. Said plants can furthermore be propagated and / or used in conventional cross schemes to produce more transformed plants with the same characteristics or to introduce the selected event of the invention within other cultivars of the same plant species. The seeds obtained from these plants contain the select event stably incorporated in their genomes. The rice plants of this invention can be grown in a conventional manner. The presence of the transgene ensures that they are resistant to glufosinate. Therefore, weed in the fields where said rice plants grow can be controlled by the application of herbicides comprising glufosinate as an active ingredient (such as Liberty ™). Plants that contain GAT-OS2 are also characterized by having agronomic characteristics that are comparable with the following rice varieties commercially available in the United States: Priscilla, Cypress, Bengal, Cocadrie, Jefferson, Madison, M202, M201, M103, Drew, Kaybonnet, Lagrue. The relevant agronomic characteristics are: height of the plant, strength / stiffness of the straw, resistance to storage, morphology of the leaf (length, width, and angle of the bulrush leaf), time of maturity, configuration of the foil, fertility of the panicle, complete closure of the seed shell, grain size and shape, and grain yield and yield. It has been observed that the presence of the transgene in this region of the genome of the rice plant, more particularly at this site in the rice plant genome, confers phenotypic and molecular characteristics that are particularly interesting to this event. More specifically, the presence of a transgene at this particular site in the genome results in the expression of the phenotypically stable transgene without significantly compromising any aspect of the desired agronomic development of the plant. Thus, the insertion region, which corresponds to SEQ ID No. 9, more particularly the GAT-OS2 insertion site here, is particularly suited for the introduction of a gene (s) of interest, such as a resistance gene. to herbicides, more specifically a gene encoding phosphinothricin acetyltransferase under the control of a 35S promoter, particularly the Pvul-HindIII fragment of plasmid pB5 / 35Sbar. A recombinant DNA molecule can be inserted specifically into this 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, any FLP recombinase from Saccharomyces cervisiae (US patent 5,527,695), CRE recombinase from Escherichia phage P1. coli (published PCT application WO 9109957, the pSRI recombinase of Saccharomyces rouxii (Araki et al., 1985, J. Mol Biol 182: 191-203), or the lambda phage recombination system such as that described in the US patent 4,673,640 DNA can be inserted into the plant genome, such as the rice genome by techniques including electroporation methods, bombardment with DNA coated with gold particles or biolistic methods, or methods mediated by agrobacterium or polyethylene glycol, and the like. As used herein, "comprising" is interpreted as the specification of the presence of established, integral, step or component features as they relate, but which do not prevent the presence or addition of one or more features, intakes, steps or components, or groups of the same. Thus, for example, a nucleic acid or protein comprising a nucleotide or amino acid sequence can comprise more nucleotides or amino acids than those currently cited, ie it can be embedded in a larger nucleic acid or a protein. A chimeric gene comprises a DNA sequence which is functionally or structurally defined, and which may comprise additional DNA sequences, etc. The following examples describe the development and characteristics of the rice plants that contain the selected event GAT-OS2.

Unless stated otherwise, all recombinant DNA techniques are carried out in accordance with standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and volume 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. The materials and standard methods for molecular work in plants are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy published by BIOS Scientific Publications Ltd (United Kingdom) and Blackwell Scientific Publications, United Kingdom. In the description and examples, reference is made to the following sequences: SEQ ID No. 1: sequence comprising the 5 'flanking region of GAT-OS2 SEQ ID No. 2: sequence comprising the 3' flanking region of GAT-OS2 SEQ ID No 3: sequence comprising the insertion site of GAT-OS2 SEQ ID No 4: OSA03: primer for the PCR identification protocol SEQ ID No 5: OSA04: GAT-OS2 specific primer for the PCR identification protocol. SEQ ID No 6: OSA01 endogenous rice initiator SEQ ID No 7: OSA02 endogenous rice initiator SEQ ID No 8: plasmid pB5 / 35Sbar SEQ ID No 9: insertion region EXAMPLES EXAMPLE 1 Transformation of rice with a gene that codes for herbicide resistance a) Construction of the chimeric DNA comprising the bar gene under the control of a 35S promoter (pB5 / 35Sbar) A plasmid pB5 / 35Sbar was constructed following the standard procedures. The sequence of plasmid pB5 / 35Sbar is given in SEQ ID No. 8. Digestion with Pvul-Hindlll gave a 1501 bp fragment which comprises the following genetic elements: The 1501 bp Pvul-Hindlll fragment was purified by extraction of this fragment after electrophoresis. b) Rice transformation The Bengal variety is a medium grain of rice grown in the rice research station of the Louisiana experiment station in agriculture. The variety was officially released in 1992. The pedigree includes MARS and M201 (Linscombe, S.D. et al., 1993, Crop Science: 33: 645-646). The transformation of the rice plants with the 1501 bp Pvul-Hindlll fragment of PB5 / 35Sbar was carried out using direct DNA transfer. The selection was made on phosphinothricin (PPT) at all stages except seedling regeneration, which was done in the absence of PPT to accelerate growth. This resulted in a group of primary transformants (plants of the To generation).

EXAMPLE 2 Development of events a) Development of transgenic homozygous lines The various T0 homozygous seedlings were changed from tissue culture, and transferred to greenhouse soil, and allowed to flower and yield seeds. Seedlings were evaluated for fertility, fertility and glufosinate-ammonium resistance. 19 plants were selected for further analysis. The T-t seeds produced by these were collected from these plants and planted in the soil. The T-i plants were sprayed with Liberty ™ herbicide at 800 grams of active ingredient per hectare (g.a.i./ha, the recommended dose for farmers is 400 g.a.i./ha). The events that survived the application of the herbicide and that were segregated 3: 1 for herbicide resistance were selected for further evaluation. The resistant plants were evaluated for their damage (burn of the tip of the leaf). T2 seeds were harvested from panicles of all resistant plants of selected events. These were planted in a row and the T2 plants were sprayed with Liberty ™ herbicide (1600 g.a.i./ha) to evaluate the segregation of the herbicide resistance. Those rows that had 100% of survivors and that corresponded to the lines that were homozygous for the transgene were selected. These were evaluated again for herbicide damage and for phenotypic traits. Additional selection of events was made based on the uniformity of the phenotype within the rows of panicles (for the desired characteristics). b) Characterization of transgenic events - selection of GAT-OS2 The transgenic events were also characterized by Southern blot patterns, phenotypic and agronomic development in general, and yield. Where appropriate, these characteristics were determined under field conditions.

Southern blot analysis The presence of the transgene was checked by standard analysis of Southern blot using enzymatic digestion of rice genomic DNA with EcoRV and hybridization with the 1327 bp EcoRI fragment of pB5 / 35Sbar. The relative intensity of the band provided an indication of whether the plants were homozygous or hemizygous for the transgenic locus. We found two events that had simple inserts. This was confirmed by the fact that the segregation pattern of the transgene can be explained by Mendelian inheritance from a single locus.

General plant phenotype and agronomic development The Ti and T2 plants were evaluated for a number of phenotypic traits including plant height, straw strength / stiffness, storage tendency, leaf morphology (too thin or incorrect angle of bulrush leaf), late maturity, foil configuration, panicle infertility or incomplete fertility, incomplete closure of the seed shell (which could lead to an increase in disease susceptibility) grain size and shape, and production of the grain and yield. The lines were evaluated to be similar (or better) in the agronomic characteristics that were exhibited compared to the untransformed Bengal cultivar and the following rice varieties: Priscilla, Cypress, Cocadrie, Jefferson, Madison, M202, M201, M103, Drew, Kaybonnet, Lagrue. In some cases, plants within a panicle row were segregated by somaclonal variation for one or more of the above-mentioned traits. Unless this resulted in the introduction of a commercially interesting phenotypic trait, these plants were discarded.

Field trials for yield evaluation T2 seeds were harvested as a group from the selected homozygous populations and compared to a standard Bengal variety. The seeds were planted as rows of panicles in isolated blocks representing each event. The transgenic batches were sprayed with 1600 g.a.i./ha of Liberty ™ herbicide or not sprayed ("unsprayed" batches). Batches with nontransgenic standard varieties were not sprayed with Liberty ™. Standard herbicide treatment to control local weeds was applied to all lots. The transgenic events were tested for field development in different places including Louisíana and Puerto Rico (winter nursery). Statistical analyzes of the agronomic parameters and range statistics of plant morphology and other non-parametric data were completed to identify the best commercial candidate to compete with the progenitor variety, Bengal and the following rice varieties: Priscilla, Cypress, Cocadrie, Jefferson, Madison, M202, M201, M103, Drew, Kaybonnet, Lagrue. The GAT-OS2 event was the one that showed the greatest utility to produce a range of reproduction lines.

EXAMPLE 3 Characterization of the GAT-OS2 event a) Deep molecular genetic analysis of the locus Once the GAT-OS2 event was identified as the event in which the expression of the transgene as well as other general agronomic developments was optimal, the transgene locus was analyzed in detail at the molecular level. This included detailed analysis of Southern blot and sequencing of the flanking regions of the transgene. (1) Southern blot analysis using multiple restriction enzymes Leaf tissue was harvested from transgenic and control plants.

Genomic DNA was isolated from the leaf tissue according to Dellaporta et al. (1983, Plant Molecular Biology Reporter, 1, vol.3, p.19-21). The concentration of DNA for each preparation was determined by measuring the optical density in a spectrophotometer at a wavelength of 260 nm. 10 μg of genomic DNA were digested with restriction enzyme in a final reaction volume of 40 μl, applying the conditions proposed by the manufacturer. The digestion time and / or amount of restriction enzymes was adjusted to ensure complete digestion of the genomic DNA samples without non-specific degradation. After digestion, 4 μl of loading dye was added to the digested DNA samples, and loaded onto a 1% agarose gel. The following control DNAs were also loaded into the gel: A negative control with genomic DNA prepared from non-transgenic rice plant. This negative control is used to confirm the absence of background hybridization. A control positive DNA: with a single heterozygous copy of integration of the transgene within the genome of Oryza sativa, 10 μg of genomic DNA having the same number of molecular equivalents as ± 19 picograms of the Pvul-Hindlll fragment of 1501 bp of pB5 DNA / 35Sbar (Oryza sativa diploid genomic size: 0.8x109 bp). The amount representing a plasmid copy per genome is added to 1 μg of digested non-transgenic Oryza sativa DNA. This reconstitution sample is used to show that the hybridization is carried out under conditions that allow the hybridization of the probe with the target sequences. Lambda phage DNA (strain Clind 1 ts 857 Sam 7, Life Technologies) digested with Pstl was included as the standard size. After electrophoresis, DNA samples (digested rice genomic DNA, controls and DNA of standard size) were transferred to nylon membranes by capillary blotting for 12 to 16 hours. The DNA templates used for the preparation of the probe were prepared by restriction digestion of plasmid PB / 35Sbar with EcoRI. This released a 1327 bp DNA fragment that covers a relevant part of the transforming DNA (Pvul-Hindlll fragment of 1501 bp). After purification, the DNA fragment was labeled according to standard procedures and used to hybridize the membrane. Hybridization was carried out under standard severe conditions: the labeled probe was denatured by heat for 5 to 10 minutes in a water bath of 95 ° C to 100 ° C and chilled on ice for 5 to 10 minutes and added to the hybridization solution (SSC 6 X (SSC20 X is NaCl 3.0 M, citrate Na 0.3 M, pH 7.0), Denhardt's 5 X (Denhardt's 100 X = Ficol 2%, Polyvinylpyrrolidone 2%, Bovine serum albumin 2%) , SDS 0.5% and 20 μg / ml denatured DNA vehicle (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) Hybridization was carried out overnight at 65 ° C. were washed three times for 20 to 40 minutes at 65 ° C, with the washing solution (SSC 2 X, SDS 0.1%) Restriction patterns obtained after digestion of genomic DNA GAT-0S2 with different restriction enzymes were presented in Figure 1 and summarized in Table 1.

TABLE 1 Restriction map of GAT-OS2 (*) The lengths of these fragments are those predicted from the restriction map of pB5 / 35S of the Pvul-Hindlli fragment of 1501 bp. (2) Identification of the flanking sequences The sequences of the transgenic flanking regions inserted in the GAT-OS2 event was determined using the thermal asymmetric entanglement PCR (TAIL-) method as described in Liu et al. (1995, The Plant Journal 8 (3): 457-463). This method uses three specific primers housed in successive reactions together with a short-range arbitrary degenerate primer (AD), so that the efficiencies in the relative amplification of the specific and non-specific products can be thermally controlled. The specific primers were selected to be coupled to the transgene boundary and were based on their coupling conditions. A small amount (5μl) of unpurified secondary and tertiary PCR products was analyzed on a 1% agarose gel. The tertiary PCR product was used for preparative amplification, purification and sequencing in an automated sequencer using the DieDeoxy equipment 1. TAIL-PCR from the Hindlll site The primers used were: where: N = A, C, T or g; C or g; W = A or T The fragment amplified using MDB556-MDB41 1 was approximately 400 bp of which 113 bp was sequenced (flank 5 ': SEQ ID No 1). The sequence between bp 1 and bp 92 comprised plant DNA, while the sequence between bp 93 and bp 1 13 corresponded to the pB5 / 35Sbar DNA. 2. TAIL-PCR of the Pvul site The primers used were: where: N = A, C, T. or g; S = C or g; W = A or T The fragment amplified using MDB285-MDB410 was approximately 1200 bp (3 'flank: SEQ ID No 2). The sequence between bp 1 and bp 604 corresponded to DNA, while bp 605 to bp 1279 comprised e! Plant DNA (3) Identification of the deletion in the blank site. The insertion site of the transgene was identified using the primers corresponding to the sequences within the flanking regions of the wild type Oryza sativa transgene variety Bengal as a template. The following initiators were used: Sequence (5 '- »3') Position in the position on the flank 5 '(SEQ ID flank 3 (SEQ No 1) ID No 2) YTP059 TCg.gAC.AAC.CgC.gAT.AgT.TCg 56? 76 OSA04 TCg.CAT.ATg.TAT.gTA.ACA.CgC 717? 697 This gives a fragment of 168 bp (SEQ ID No 3) in which bp 38 to 55 corresponds to the target deletion site.

Thus, the complete rice insertion region (SEQ ID No. 9) as sequenced comprises: 1 - . 1 -92: Flanking region 5 'pb 1 -92 of SEQ ID No 1 93-110: White site of deletion bp 38 to 55 of SEQ ID No 3 11 1-785: Flanking region 3 'bp 605 to 1279 of SEQ ID No 2 (4) Genetic analysis of the locus The genetic stability of the insert was verified by molecular and phenotypic analysis in the progeny of the plants after several generations. Southern blot analyzes of the glufosinate-resistant plants of the GAT-OS2 rice plants of the T0, and T2 generations were compared and found identical. This proves that the molecular configuration of the transgene in plants containing GAT-OS2 was stable. The GAT-OS2 event exhibited Mendelian segregation for the transgene as a unique genetic locus in at least three subsequent generations indicating that the insert is stable. Based on the previous results, GAT-OS2 was identified as a select event. b) Development of diagnostic tools for identity control The following protocols were developed to identify any rice plant material that comprises the selected event GAT-OS2. (1) Protocol for the identification of the restriction map of the selected event GAT-OS2 The rice plants containing the selected event GAT-OS2 can be identified by Southern blot using essentially the same procedure as that described in example 3 a) (1 ). Thus rice genomic DNA is 1) digested with at least three, preferably at least 4, particularly with at least 5, more particularly with all of the following restriction enzymes: EcoR1, BamHI, EcoRV, HindIII, Ncol, Nsil, 2), is transferred to nylon membranes and 3) hybridizes with the EcoRI fragment of 1327 bp of plasmid PB5 / 35Sbar. If, with respect to each of the restriction enzymes used, the DNA fragments are identified with the same length as those listed in Table 1, it is determined that the rice plant contains the selected event GAT-OS2. (2) Protocol for the identification of the selected event GAT-OS2 by polymerase chain reaction An assay was carried out, with all appropriate controls, to be carried out before attempting to select unknown parts. The presented protocol may require optimization for components that may differ between laboratories (preparation of template DNA, Taq DNA polymerase, quality of primers, dNTP's, thermal cycler, etc.). The amplification of endogenous sequences plays a key role in the protocol. One has to achieve PCR and thermocycler conditions that amplify the equimolar amounts of both endogenous and transgenic sequences in a known transgenic genomic DNA template. In cases where the target endogenous fragment is not amplified or when the target sequences are not amplified with the same intensity as the ethidium bromide stain, as judged by agarose gel electrophoresis, optimization conditions may be required for the PCR.

DNA template The DNA template is prepared according to Edwards et al. (Nucleic Acid Research, 19, p1349, 1991). When DNA prepared by other methods is used, a test should be done using different amounts of mold. Usually 50 ng of the genomic template DNA gives good results.

Assigned negative and positive controls The following positive and negative controls should be included in a PCR run: - Control of the master mix (negative DNA control). This is a PCR in which no DNA is added to the reaction. When the expected result, absence of PCR products, is observed, it indicates that the PCR cocktail was not contaminated with white DNA. - A positive DNA control (sample of genomic DNA that is known to contain the transgenic sequence). The successful amplification of this positive control demonstrates that the PCR was run under conditions that allow the amplification of the target sequences. - A control of wild DNA. This is a PCR in which the DNA template provided is a genomic DNA prepared from a non-transgenic plant. When the expected result, absence of amplification of the transgenic PCR product but amplification of the endogenous PCR product, is observed, it indicates that there was no background amplification of the transgene detectable in a sample of genomic DNA. The following primers are used, which specifically rearrange the transgene and a flanking sequence of GAT-OS2: OSA03: 5'-gAC.TCT.gTA.TgA. ACT.gTT.CgC-3 '(SEQ ID 4) (white: P35S) OSA04: 5'-TCg.CAT.ATg.TAT.gTA.ACA.CgC-3' (SEQ ID 5) (white: plant DNA) Initiators that target an endogenous sequence are always included in the PCR cocktail. These primers serve as internal control in unknown samples and in the positive control of DNA. A positive result with the pair of endogenous primers shows that there is enough DNA of adequate quality in the preparation of genomic DNA for a PCR product to be generated. The endogenous primers used are: OSA01: 5'gAT.Cag.TgC.Agg.CAA.TAC.Tgg-3 '(SEQ ID 6) (phospholipase gene D Acc. No. AB001919,3836- ^ 3856) OSA02: 5'-TTC.CTA.ACA.TgT.ggg.TgT.Cg-3 '(SEQ ID 7) (Phospholipase D Acc. No. AB001919,4291 ^ 4272 gene) Amplified fragments The expected amplified fragments in the PCR reaction are: For primer pair OSA01-OSA02: 457 bp (endogenous control) For primer pair OSA03-OSA04: 313 bp (select event GAT-OS2) PCR conditions The PCR mixture for 50μl of the reaction contains: 5 μl DNA template 5 μl of 10x amplification buffer (supplemented with Taq polymerase) 1 μlNTPIOmM 1 μlOSAOI (10 pmol / μl) 1 μlOSA02 (10 pmol / μl) 1 μl ! OSA03 (10 pmol / μl) 1 μlOSA04 (10 pmol / μl) 0.2 μl Taq DNA polymerase (5 units / μl) water up to 50 μl The perfusion of the thermocycle to be followed for optimal results is as follows: 4 min. at 95 ° 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 22 to 25 cycles followed by: 5 minutes at 72 ° C Analysis of the aqarose gel Between 10 and 20 μl of the PCR samples should be applied to a 1.5% agarose gel (Tris-borate buffer) with a suitable molecular weight marker (e.g., 100 bp PHARMACIA scale).

Validation of results Data from transgenic plant DNA samples with a single run of PCR and a single PCR cocktail should not be accepted unless 1) positive DNA controls show the expected PCR products (transgenic fragments) and endogenous), 2) the negative DNA control is negative for the PCR amplification (absence of fragments) and 3) the control of wild-type DNA shows the expected result (amplification of the endogenous fragment). The lines showing the visible quantities of the transgenic and endogenous PCR products of the expected sizes indicate that the corresponding plants from which the DNA genomic template was prepared, have inherited the selected event GAT-OS2. The lines whose quantities are not visible from the transgenic PCR products and which show visible amounts of the endogenous PCR product, indicate that the corresponding plant from which the genomic DNA template was prepared does not comprise the selected event. The lines that do not show visible amounts of endogenous and transgenic PCR products indicate that the quality and / or quantity of the genomic DNA did not allow PCR products to be generated. These plants can not be evaluated. The preparation of genomic DNA must be repeated and a new PCR must be carried out, with the appropriate controls, to be carried out.

Use of the PCR discriminator protocol to identify GAT-OS2 The rice leaf material of plants comprising different transgenic events (samples 1 to 10) was evaluated according to the protocol described above. Samples of wild type M202 and wild type Bengal were taken as negative controls. The results of the PCR analysis are illustrated in Figure 2. Samples 8 and 9 (which in fact contain the DNA of the plants derived from the same event) were recognized as comprising the selected event GAT-OS2. All the other lines evaluated do not include this selected event.

EXAMPLE 4 Introduction of GAT-OS2 within preferred cultivars The selected GAT-OS2 event is introduced by repeated crossing within the following cultivars: - Températe Japonicas de California (such as but not limited to M204, M202, M201, M103) - Tropical Japonicas de California (such as but not limited to L201) -L202 - Températe Japanese Japanese and Korean (such as but not limited to Koshihikari and Milyang) - Temprate Japanese Japanans (such as but not limited to Millin and Jarrah) - Températe Japonicas Mediterránea (such as but not limited to Ballila, Arborio) - Indica of China (such as but not limited to Guichao, Congui 314, Teqing) - Tropical Japonicas of the Southeastern United States., Large grain (such as but not limited to Drew, Cypress, Jefferson, Priscilla, Cocadrie) - Tropical Japonicas from the Southeast of the United States, medium grain (such as but not limited to Bengal, Mars, Arms, Mercury) - Tropical Japonicas from South America, large grain (such as but not limited to El Paso 144, IRGA 409) - Basmati and Jasmine types from the Far East (Kasmir, Kwao Dak Mali) - African javanica types (rice bulu) It was noted that the introduction of the select event within these cultivars do not significantly influence any of the desirable phenotypic or agronomic characteristics of these cultivars (there are no harmful effects by the linkage) while the expression of the transgene, as determined by the resistance to glufosinate, reached commercially acceptable levels. This confirms the status of the GAT-OS2 event as a select event. As used in the claims below, unless the stated clarification is made, the term "plant" is intended to encompass plant tissues, of any maturity stage, as well as any cells, tissues or organs taken from or derived from any of said plants, including without limitation, any seeds, leaves, stems, root flowers, particular cells, gametes, cell cultures, tissue cultures or protoplasts. The seeds comprising the selected event GAT-OS2 were deposited as GAT-OS2 in ATCC under the number: ATCC 203352.

Claims (42)

NOVELTY OF THE INVENTION CLAIMS

1. - A transgenic rice plant resistant to glufosinate, cell, tissue or seed, which is characterized because: a) the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least three restriction fragments or pairs of fragments of restriction, wherein said restriction fragments or pairs of restriction fragments are selected from the group of: i) an EcoRI fragment with a length between 1159 and 1700 bp; ii) a pair of BamHI fragments of which one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; iv) a Hindlll fragment with a length between 5077 and 11,497 bp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp; vi) a Nsil fragment with a length between 4749 and 11497 bp; wherein each of the restriction fragments is capable of hybridizing, under standard severe conditions, with the 1327 bp fragment that was obtained by digestion with EcoRI of the plasmid having the nucleotide sequence of SEQ ID No. 8; and / or b) a DNA fragment between 290 and 350 bp, preferably about 313 bp, which can be amplified with the genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

2. The plant, cell, tissue or seed according to claim 1, further characterized in that the DNA fragment between 290 and 350 bp, preferably around 313 bp, can be amplified from genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

3. The plant, cell, tissue or seed according to claim 1, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least three restriction fragments or pairs of restriction fragments, wherein said restriction fragments are selected from the group of: i) an EcoRI fragment with a length between 1159 and 1700 bp; ii) a pair of BamHI fragments of which one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; iv) a Hindlll fragment with a length between 5077 and 11,497 bp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp; vi) a Nsil fragment with a length between 4749 and 11497 bp; wherein each of the restriction fragments is capable of hybridizing, under standard severe conditions, with the 1327 bp fragment obtained by EcoRI digestion of the plasmid having the nucleotide sequence of SEQ ID No. 8.

4. - The plant, cell, tissue or seed according to claim 3, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least four restriction fragments or pairs of restriction fragments selected from said group.

5. The plant, cell, tissue or seed according to claim 4, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least five restriction fragments selected from said group.

6. The plant, cell, tissue or seed according to claim 5, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding the six restriction fragments or pairs of restriction fragments selected from said group.

7. The plant, cell, tissue or seed of any of claims 3 to 6, further characterized in that the DNA fragment between 290 and 350 bp, preferably around 313 bp, can be amplified from the genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

8.- A plant which is generated from a seed deposited in ATCC under the number ATCC 203352.

9. - A cell or tissue of the plant according to claim 8.

10.- A seed deposited in ATCC under number 203352.

11.- A transgenic rice plant with glufosinate resistance, according to claims 1 to 7, further characterized because it is obtained by propagation of, and / or reproduction with, a rice plant that is generated from the seed deposited in ATCC under number 203352.

12. A process for growing rice plants comprising growing plants according to any of claims 1 to 8.

13. The method according to claim 12, further characterized in that it comprises the application of a herbicide with glufosinate as an active ingredient to the cultivated rice plants.

14. A process for the reproduction of rice which comprises crossing with a plant according to any of claims 1 to 8.

15. A process for producing a transgenic cell of a rice plant which comprises inserting a molecule of recombinant DNA within a part of the chromosomal DNA of a rice cell characterized by the sequence of SEQ ID No. 9.

16.- A transgenic cell of a rice plant obtained by the method according to claim 15.

17. - A process for producing a transgenic rice plant which comprises inserting a recombinant DNA molecule into a part of the chromosomal DNA of a rice cell characterized by the sequence of SEQ ID No. 9, and the regeneration of a rice plant from the transformed rice cell.

18. A transgenic rice plant obtained by the method according to claim 17.

19. A method for identifying a transgenic plant, or cells or tissues thereof, comprising the selected event GAT-OS2, whose The method comprises establishing one or more of the following characteristics: a) the genomic DNA of the plant, cell, tissue or seed is capable of yielding at least three of the restriction fragments or pairs of restriction fragments selected from the group of: i) An EcoRI fragment with a length between 1159 and 1700 bp; ii) a pair of BamHI fragments where one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; iv) a Hindlll fragment with a length between 5077 and 11,497 bp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp and one of about 4.1 kbp; vi) a Nsil fragment with a length between 4749 and 11497 bp; wherein each of the restriction fragments is capable of hybridizing, under severe standard conditions, with the 1327 bp fragment that was obtained by EcoRI digestion of the plasmid having the nucleotide sequence of SEQ ID No. 8; and / or, b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify some DNA fragment between 290 and 350 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

20. The method according to claim 19, further characterized in that it comprises establishing whether the genomic DNA of the transgenic plant or its cells or tissues are capable of yielding the said 6 restriction fragments or pairs of restriction fragments.

21. The method according to claim 19, further characterized in that it comprises establishing whether the genomic DNA of the transgenic plant or its cells or tissues can be used to amplify a DNA fragment of about 313 bp using a cadei reaction. of the polymerase with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

22.- A team to identify a transgenic plant, its cells or tissues that comprise a selected event MS-B2, said equipment comprises at least two PCR probes, one of which recognizes a sequence within the external GAT-OS2 DNA, the other which recognizes a sequence within the 3 'or 5' flanking region of GAT-OS2.

23. The equipment according to claim 22, further characterized in that said equipment comprises the PCR probes having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

24.- A transgenic rice plant resistant to glufosinate, cell, tissue or seed, which is characterized because: a) the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least three restriction fragments or pairs of restriction fragments, wherein said restriction fragments or pairs of restriction fragments are selected from the group of: i) an EcoRI fragment with a length between 1159 and 1700 bp; ii) a pair of BamHI fragments of which one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; V) a Híndlll fragment with a length between 5077 and 11,497 bp; v) a pair of NCO fragments? both with lengths between 2838 and 4507 bp; vi) a Nsil fragment with a length between 4749 and 11497 bp; wherein each of the restriction fragments is capable of hybridizing, under standard severe conditions, with the 1327 bp fragment that was obtained by digestion of the GAT-OS2 genomic DNA (ATCC number 203352) with EcoRI; and / or b) a DNA fragment between 290 and 350 bp, preferably about 313 bp, which can be amplified with the genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

25. - The plant, cell, tissue or seed according to claim 24, further characterized in that the DNA fragment between 290 and 350 bp, preferably around 313 bp, can be amplified from the genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

26.- The transgenic rice plant resistant to glufosinate, cell, tissue or seed, which is characterized because: a) the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least three restriction fragments or pairs of restriction fragments, wherein said restriction fragments or pairs of restriction fragments are selected from the group of: i) an EcoRI fragment with a length between 1159 and 1700 bp; ii) a pair of BamHI fragments of which one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; iv) a Hindlll fragment with a length between 5077 and 1497 bp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp; vi) a Nsil fragment with a length between 4749 and 11497 bp; wherein each of the restriction fragments is capable of hybridizing, under standard severe conditions, with the 1327 bp fragment that was obtained by digestion of the GAT-OS2 genomic DNA (number ATCC 203352) with EcoRI.

27. - The plant, cell, tissue or seed according to claim 26, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least four restriction fragments or pairs of restriction fragments selected from said group.

28. The plant, cell, tissue or seed according to claim 27, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least five restriction fragments or pairs of selected restriction fragments. of said group.

29. The plant, cell, tissue or seed according to claim 28, further characterized in that the genomic DNA of said plant, cell, tissue or seed is capable of yielding at least six restriction fragments or pairs of selected restriction fragments. of said group.

30. The plant, cell, tissue or seed according to any of claims 26 to 29, further characterized in that the DNA fragment between 290 and 350 bp, preferably around 313 bp, can be amplified from the genomic DNA of said plant, cell, tissue or seed using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.

31.- A plant which is generated from a seed deposited in ATCC under the number ATCC 203352.

32. - A cell or tissue of the plant according to claim 31.

33.- A seed deposited in ATCC under number 203352.

34.- A transgenic rice plant with resistance to glufosinate, in accordance with claims 24 to 30 , characterized further because it is obtained by propagation of, and / or reproduction with, a rice plant that is generated from the seed deposited in ATCC under number 203352.

35.- A process for cultivating rice plants which comprises generating the plants according to any of claims 24 to 31.

36. The method according to claim 35, further characterized in that it comprises the application of a herbicide with glufosinate as an active ingredient to the cultivated rice plants. 37.- A method for reproducing rice which comprises a cross with a plant according to any of claims 24 to 31. 38.- A method for identifying a transgenic plant, or cells or tissues thereof, comprising the event select GAT-OS2, whose method comprises establishing one or more of the following characteristics: a) the genomic DNA of the plant, cell, tissue or seed is capable of yielding at least three of the restriction fragments or pairs of restriction fragments selected from the group of: i) An EcoRI fragment with a length between 1 159 and 1700 bp; ii) a pair of BamHI fragments where one has a length between 805 and 1093 bp and the other has a length between 1700 and 2140 bp; iii) a pair of EcoRV fragments where one has a length between 2838 and 4507 bp and the other has a length of more than 5077 bp; iv) a Hindlll fragment with a length between 5077 and 1497 bp; v) a pair of Ncol fragments both with lengths between 2838 and 4507 bp and one of about 4.1 kbp; vi) a Nsil fragment with a length between 4749 and 1497 bp; wherein each of the restriction fragments is capable of hybridizing, under severe standard conditions, with the 1327 bp fragment that was obtained by digestion of the GAT-OS2 genomic DNA (number ATCC 203352) with EcoRI; and / or, b) the genomic DNA of the plant, cell, tissue or seed can be used to amplify some DNA fragment between 290 and 350 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively. 39.- The method according to claim 38, further characterized in that it comprises establishing whether the genomic DNA of the transgenic plant, or cells or tissues are capable of yielding all six of said restriction fragments or pairs of restriction fragments. The method according to claim 38, further characterized in that it comprises establishing whether the genomic DNA of the transgenic plant, or its cells or tissues, can be used to amplify a DNA fragment of about 313 bp using a chain reaction of the polymerase with two primers having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively. 41.- A team to identify a transgenic plant, its cells or tissues comprising the selected event MS-B2, said equipment comprises at least two PCR probes, one of which recognizes a sequence within the external DNA of GAT-OS2, the other which recognizes a sequence within the 3 'or 5' flanking region of GAT-OS2. The equipment according to claim 41, further characterized in that said equipment comprises the PCR probes having the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 5, respectively.