HK1051295B

HK1051295B - Hybrid winter oilseed rape and methods for producing same

Info

Publication number: HK1051295B
Application number: HK03103700.2A
Authority: HK
Inventors: De Both Greta; De Beuckeleer Marc
Original assignee: Bayer Cropscience Nv
Priority date: 1999-12-08
Filing date: 2000-12-06
Publication date: 2005-12-09

Description

Hybrid winter oilseed rape and method of producing the same

Technical Field

The present invention relates to winter oilseed rape (WOSR) plants, and more particularly to a pair of winter oilseed rape plants particularly suitable for producing hybrid seed. More specifically, one plant is characterized by male sterility because of the presence of a male sterility gene in its genome, while the other plant is characterized by carrying a fertility restorer gene which can prevent the activity of the male sterility gene. The pair of WOSR plants of the invention combine the ability to form hybrid seeds with optimal overall agronomic performance, genetic stability and adaptability to different genetic backgrounds.

All documents cited herein are incorporated herein by reference.

Background

The phenotypic expression of a transgene in a plant is determined by the structure of the gene itself and its location in the plant genome. While transgenes are present at different locations in the genome, the overall phenotype of the plant can be affected in different ways. The successful introduction of a commercially interesting trait agriculturally or industrially into a plant by genetic manipulation is a lengthy process influenced by different factors. The actual transformation and regeneration of genetically transformed plants is only the first step in a series of selection steps, including extensive genetic identification, breeding and evaluation in field trials.

Oilseed rape (OSR) (Brassica napus, AACC, 2n ═ 38) is a natural hybrid product from interspecific crosses of Oilseed rape (Brassica Oleracea), CC, 2n ═ 18) and turnip (Brassica, AA, 2n ═ 20). Winter oilseed rape is sown in the last 10 days of august and in the first 10 days of september and harvested in july of the next year, which requires a period of temperate climate for vernalization. Faster growing spring rape was sown at the end of three and early april and harvested in august to september. The major types of OSR currently grown are the low erucic acid and high erucic acid varieties. The double low (00) variety contains low (typically less than 1%) levels of erucic acid (which is found to be difficult for humans to digest) and low levels of glucosinolates (which make the pomace by-product difficult to digest by animals). The current uses of the "00" breed include oil for human consumption and high protein feed for feeding animals. Industrial applications include pharmaceuticals and hydraulic oil supply materials. High Erucic Acid Rape (HEAR) varieties are grown specifically for their erucic acid content-typically 50-60% oil in oil. The primary end use of the HEAR is to produce erucamide, which is the "slip agent" used in the production of polyethylene. A small portion is used to produce behenyl alcohol, which is added to the waxy crude mineral oil to improve the fluidity of the latter.

Oilseed rape plants are amphoteric, typically 60-70% being self-pollinating. As a basis for selection, hybrid generation and introduction of genetic variation often rely on adaptation to naturally occurring phenomena such as self-incompatibility and cytoplasmic male sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides have not been widely used in OSR breeding because of their limited availability and high cost, respectively.

Transgenic methods for producing male or female sterile plants have been developed, which provide interesting alternatives to conventional techniques.

EP 0,344,029 describes a system for obtaining nuclear male sterility in which plants are transformed with a male sterility gene comprising DNA encoding a bacillus rnase, for example under the control of the tapetum-specific promoter PTA29, which gene, when incorporated into a plant, selectively destroys tapetum cells. Transformation of tobacco and oilseed rape plants with this chimeric gene leads to plants in which pollen formation is completely hindered (Mariani et al, 1990, Nature 347: 737-one 741).

To restore fertility to progeny of male sterile plants, systems have been developed in which male sterile plants are crossed with transgenic plants carrying a fertility restorer gene, which when expressed suppresses or prevents the activity of the male sterile gene (US 5,689,041; US 5,792,929). The fertility restorer gene is placed under the control of a promoter which directs its expression at least in cells expressing the male sterility gene. Mariani et al (1992, Nature 357: 384-387) showed that the expression of the protein by pTA 29: sterility encoded by the barnase gene of bacillus may be determined by the presence of chimeric pTA 29: barstar gene recovery.

De Block and De Brouwer (1993, Planta 189: 218-225) describe the cytochemical and histochemical analysis of anther development of Brassica napus plants which comprise only the chimeric pTA 29: a bacillus rnase gene or a gene comprising pTA 29: bacillus rnase gene and pTA 29: a barnase inhibitor gene of bacillus.

Successful transformation of Brassica species has been achieved by several methods including Agrobacterium infection (described, for example, in EP 0,116,718 and EP 0,270,882), particle bombardment (described, for example, in Chen et al, 1994, theory and applied genetics (the or. appl. Genet.) 88: 187-.

The foregoing documents, however, do not teach or suggest the present invention.

Summary of The Invention

The present invention relates to a pair of WOSR plants particularly suitable for producing hybrid seed. More specifically, the present invention relates to a first transgenic WOSR plant or seed, cell or tissue thereof comprising an expression cassette comprising a male-sterility gene integrated into its genome and a second transgenic WOSR plant or seed, cell or tissue thereof comprising an expression cassette comprising a fertility-restorer gene integrated into its genome, and hybrid seed obtained by crossing the first and second plants comprising a male-sterility gene and/or a fertility-restorer gene integrated into their genomes.

In one embodiment of the invention, the first WOSR plant or seed, cell or tissue thereof comprises a pTHW107 expression cassette. In a preferred embodiment of the invention, the first WOSR plant or seed, cell or tissue thereof comprises event MS-BN 1. In another embodiment of the invention, the second WOSR plant or seed, cell or tissue thereof comprises a pTHW1118 expression cassette. In a preferred embodiment of the invention, the WOSR plant or seed, cell or tissue thereof comprises event RF-BN 1. In a particularly preferred embodiment of the invention, the first WOSR plant comprises event MS-BN1 and the second WOSR plant comprises event RF-BN1, and the hybrid seeds obtained therefrom comprise event MS-BN1 and RF-BN1 or only RF-BN 1.

The present invention relates to transgenic WOSR seeds or plants that can grow from such seeds, whose genomic DNA has one or both of the following characteristics:

a) the genomic DNA may produce at least two, preferably at least three, more preferably at least four, most preferably five sets of restriction fragments selected from the following sets of restriction fragments:

i) a set comprising two EcoRI fragments, one of which is between 2140 and 2450bp in length, preferably about 2266bp, and one of which is greater than 14kbp in length;

ii) a set comprising two EcoRV fragments, one of which is between 1159 and 1700bp in length, preferably about 1.4kbp, and the other of which is greater than 14kbp in length;

iii) a set of two Hpal fragments, one of length between 1986 and 2140bp, preferably of length about 1990bp, and one of length between 2140 and 2450bp, preferably about 2229 bp;

iv) a set of three AflIII fragments, one of length between 514 and 805bp, preferably of length about 522bp, and one of length between 2140 and 2450bp, preferably about 2250bp, and one of length between 2450 and 2838bp, preferably about 2477 bp;

v) a set of two NdeI fragments, both of length between 5077 and 14057bp, preferably one of approximately 6500bp and the other approximately 10 kbp;

wherein each restriction fragment hybridizes under standard stringency conditions to a 3942bp long fragment comprising the PTA 29-bacillus rnase sequence, wherein said 3942bp long fragment is obtainable by digesting the plasmid pTHW107 described herein with HindIII; and/or

b) The genomic DNA may produce at least two, preferably at least three, more preferably at least four sets of restriction fragments selected from the following sets of restriction fragments:

i) a set of three BamHI fragments, one of length between 805 and 1099bp, preferably about 814bp, one of length between 1700 and 1986bp, preferably about 1849bp, one of length between 2450 and 2838bp, preferably about 2607bp, and one of length between 5077 and 14057bp, preferably about 6500 bp;

ii) a set of four EcoRI fragments, one fragment between 805 and 1159bp, preferably about 1094bp, one between 1986 and 2450bp, preferably about 2149bp, and two between 5077 and 14057bp, preferably one about 7000bp, and one about 10 kbp;

iii) a set comprising two EcoRV fragments, both of which are between 5077 and 14057bp in length, preferably one of about 5.4kbp in length and the other of about 8kbp in length;

iv) a set of three HindIII fragments, wherein one fragment is between 1700 and 2140bp in length, preferably about 1969bp, and two are between 2450 and 2838bp in length, preferably one is about 2565bp in length, and one is about 2635bp in length;

wherein each restriction fragment hybridizes to a 2182bp long fragment comprising the PTA 29-Bacillus RNase inhibitor sequence under standard stringent conditions, wherein the 2182bp long fragment is obtainable by digestion of the plasmid pTHW118 described herein with HpaI.

The present invention relates to seeds of a WOSR plant or a plant grown from such seeds, or cells or tissues thereof, the genomic DNA of which has one or both of the following characteristics:

a) the genomic DNA may produce at least two, preferably at least three, e.g. at least four, more preferably five sets of restriction fragment sets selected from the restriction fragment sets described in a) above and comprised in a), ii), iii), iv) and v) above, thus selecting a set that may comprise any combination of i), ii), iii), iv) and v) described in a) above; and/or

b) The genomic DNA may produce at least two, preferably at least three, most preferably four sets of restriction fragments selected from the restriction fragment sets described in b) above and comprised in b) i), ii), iii) and iv) above, thus selecting any combination that may comprise i), ii), iii) and iv) described in b) above.

The invention further relates to WOSR seeds or plants that can grow from such seeds, whose genomic DNA has one or both of the following characteristics:

c) genomic DNA can be used to amplify DNA fragments of between 260 and 300bp, preferably about 280bp, by polymerase chain reaction, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively, and/or

d) A DNA fragment of between 195 and 235bp, preferably about 215bp in length can be amplified by polymerase chain reaction using genomic DNA, the nucleotide sequences of the two primers being SEQ ID No 23 and SEQ ID No 41, respectively.

The invention further relates to WOSR seeds or plants which can be grown from such seeds, whose genomic DNA has the features in a) and c) above and/or the features in b) and d) above.

The present invention relates to seeds of a WOSR plant or a plant which can grow from such seeds, the genomic DNA of which has one or both of the following characteristics:

c) A DNA fragment of between 260 and 300bp, preferably about 280bp in length can be amplified by polymerase chain reaction using genomic DNA, the two primers having the nucleotide sequences of SEQ ID No12 and SEQ ID No 19, respectively.

The present invention relates to seeds of a WOSR plant, preferably a male sterile plant, or a plant which can grow from such seeds, or cells or tissues thereof, having genomic DNA comprising: at least two, preferably at least three, more preferably five sets of restriction fragments selected from the restriction fragment sets described above and comprised in i), ii), iii), iv) and v) above may be generated, thus selecting a set that may comprise any combination of i), ii), iii), iv) and v) above.

The present invention further relates to seeds of a WOSR plant, or a plant which can grow from such seeds, whose genomic DNA has one or both of the following characteristics:

b) the genomic DNA may produce at least two, preferably at least three, more preferably four sets of restriction fragments selected from the following sets of restriction fragments:

wherein each restriction fragment hybridizes to a 2182bp long fragment comprising a PTA 29-Bacillus RNase inhibitor sequence under standard stringent conditions, wherein the 2182bp long fragment is obtainable by digestion of the plasmid pTHW118 described herein with HpaI; and/or

d) A DNA fragment of between 195 and 235bp, preferably about 215bp in length can be amplified by polymerase chain reaction using genomic DNA, the two primers having the nucleotide sequences of SEQ ID No 23 and SEQ ID No 41, respectively.

The present invention relates to a WOSR plant, preferably a seed of a fertility restorer plant, or a cell or tissue thereof, which genomic DNA can produce at least two, preferably at least three, most preferably four sets of restriction fragments selected from the restriction fragment sets described above and comprised in b) i), ii), iii) and iv) above, thus selecting a set which can comprise any one of the combinations of i), ii), iii) and iv) described above in b) above.

The present invention relates to a transgenic WOSR plant, cell, tissue or seed, preferably having both the above-mentioned properties b) and/or d), respectively.

The invention further relates to a transgenic plant, preferably a crossed fertility restored WOSR plant, cell, tissue or seed, obtained by crossing a male sterile plant with a fertility restored plant of the invention having the above characteristics, whereby the fertility restored plant, cell tissue or seed has the molecular characteristics of male sterility and the characteristics of the above fertility restored WOSR plant. The invention further relates to a transgenic plant, preferably a hybrid WOSR plant, cell, tissue or seed, obtained by crossing a male sterile plant with a fertility restorer plant of the invention having the above-mentioned molecular characteristics, whereby the hybrid plant, cell tissue or seed has the molecular characteristics of the above-mentioned fertility restorer WOSR plant.

The invention also relates to the seed deposited under ATCC accession number PTA-730, to plants grown from the seed, and to cells or tissues of plants grown from the seed. The present invention further relates to a plant obtainable by propagation and/or breeding of a WOSR plant grown from the seed deposited under ATCC accession No. PTA-730.

The present invention further relates to a method of producing hybrid WOSR seed comprising crossing a male sterile WOSR plant of the invention with a fertility restorer plant of the invention.

The invention further relates to a WOSR plant, plant cell, plant tissue or seed comprising a recombinant DNA comprising at least one transgene of sequence SEQ ID No 22 and/or at least one transgene of sequence SEQ ID No 34 integrated into part of the chromosomal DNA.

The invention further provides a method of producing a transgenic cell of a WOSR plant or a plant obtained therefrom, the method comprising inserting a recombinant DNA molecule having the sequence feature of SEQ ID No 22 into part of the chromosomal DNA of a WOSR cell, and optionally, regenerating a WOSR plant from the transformed WOSR cell.

The invention further provides a method of producing a transgenic cell of a WOSR plant or a plant obtained therefrom, the method comprising inserting a recombinant DNA molecule having the sequence feature of SEQ ID No 34 into part of the chromosomal DNA of a WOSR cell, and optionally, regenerating a WOSR plant from the transformed WOSR cell.

The present invention further relates to a method of identifying a transgenic plant, or a cell or tissue thereof, comprising elite event MS-BN1 according to the present invention, comprising establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or cell or tissue thereof:

c) Genomic DNA can be used to amplify DNA fragments between 260 and 300bp, preferably about 280bp, in length according to the PCR identification method described herein, and the nucleotide sequences of the two primers identifying the elite event are SEQ ID No12 and SEQ ID No 19, respectively.

The present invention further relates to a method of identifying a transgenic plant, or a cell or tissue thereof, comprising elite event RF-BN1 according to the present invention, which method comprises establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or cell or tissue thereof:

wherein each restriction fragment hybridizes to a 2182bp long fragment comprising a PTA 29-Bacillus RNase inhibitor sequence under standard stringent conditions, wherein the 2182bp long fragment is obtainable by digestion of the plasmid pTHW118 described herein with HpaI, and/or

d) A DNA fragment of between 195 and 235bp in length, preferably about 215bp, can be amplified using the PCR identification method described herein using genomic DNA, and the nucleotide sequences of the two primers identifying this elite event are SEQ ID No 23 and SEQ ID No 41, respectively.

The invention also relates to a kit for identifying a plant comprising elite event MS-BN1 of the invention, said kit comprising PCR probes having the nucleotide sequences SEQ ID No.12 and SEQ ID No. 19.

The invention further relates to a kit for identifying a plant comprising elite event RF-BN1 according to the invention, said kit comprising PCR probes having the nucleotide sequences SEQ ID No.23 and SEQ ID No. 41.

The invention also relates to a kit for identifying elite event MS-BN1 and/or RF-BN1 in biological samples, comprising at least one specific primer or probe having a sequence corresponding to (or complementary to) a sequence having 80% to 100% sequence identity with a specific region of MS-BN1 and/or at least one specific primer or probe having a sequence corresponding to (or complementary to) a sequence having 80% to 100% sequence identity with a specific region of RF-BN 1. Preferred probe sequences correspond to specific regions comprising portions of the MS-BN1 and/or RF-BN 15 'or 3' flanking regions. Most preferably, the specific probe has (or is complementary to) a sequence which has 80% to 100% sequence identity to the plant DNA sequence of SEQ ID No.36 or SEQ ID No.38 for MS-BN1 or 80% to 100% sequence identity to the plant DNA sequence of SEQ ID No.39 or SEQ ID No.40 for RF-BN 1.

Preferably, the kit of the present invention comprises, in addition to one primer specifically recognizing the 5 'or 3' flanking region of MS-BN1 and/or RF-BN1, a second primer specifically recognizing an exogenous DNA sequence of MS-BN1 and/or RF-BN1 for use in the PCR identification method. Preferably, the kit of the invention comprises two (or more) specific primers, one of which recognizes a sequence in the 5 'or 3' flanking region of MS-BN1 and/or RF-BN1, most preferably, a sequence of a plant DNA region of MS-BN1SEQ ID No.36 or SEQ ID No.38, or a sequence of a plant DNA region of SEQ ID No.39 or SEQ ID No.40 for RF-BN1, and the other of which recognizes a sequence in the foreign DNA of MS-BN1 and/or RF-BN 1. Particularly preferably, the primer recognizing the plant DNA sequence of the flanking region of MS-BN 15' comprises the nucleotide sequence of SEQ ID No. 19. In particular, the primer recognizing the plant DNA sequence of the flanking region of MS-BN 15' comprises the nucleotide sequence of SEQ ID No.19 and the primer recognizing the foreign DNA of MS-BN1 comprises the nucleotide sequence of SEQ ID No.12 as described herein. Particularly preferably, the primer recognizing the plant DNA sequence of the RF-BN 15' flanking region comprises the nucleotide sequence of SEQ ID No. 41. In particular, the primer recognizing the plant DNA sequence of the RF-BN 15' flanking region comprises the nucleotide sequence of SEQ ID No.41 and the primer recognizing the foreign DNA of RF-BN1 comprises the nucleotide sequence of SEQ ID No.23 described herein.

The methods and kits encompassed by the present invention may be used for different purposes, such as, but not limited to, the following: for identifying MS-BN1 and/or RF-BN1 in plants, plant materials or products, such as but not limited to food or feed products (fresh or processed) comprising or derived from plant materials; in addition, the methods and kits of the invention can be used to identify transgenic plant material with the aim of separating transgenic material from non-transgenic material; in addition, the methods and kits of the invention can be used to determine the mass (i.e., percentage of pure material) of plant material containing MS-BN1 and/or RF-BN 1.

It is to be understood that the dependent claims mentioned herein describe specific embodiments of the invention.

Brief Description of Drawings

The following detailed description, given by way of example and not intended to limit the invention to the particular embodiments described, may be understood in conjunction with the accompanying drawings, which are incorporated herein by reference, in which:

FIG. 1: pVE113 plasmid map

FIG. 2: restriction map obtained after digestion of MS-BN1 genomic DNA

The order of loading of the gel was analyzed by Southern blotting: lane 1, digestion of MS-BN1DNA with EcoRI, lane 2, digestion of MS-BN1DNA with EcoRV, lane 3, digestion of MS-BN1DNA with HpaI, lane 4, digestion of MS-BN1DNA with AflIII, lane 5, digestion of MS-BN1DNA with NdeI, lane 6, digestion of non-transgenic WOSR DNA with BamHI, lane 7, digestion of non-transgenic WOSR + digestion of the control plasmid pTHW1O7 DNA with BamHI.

FIG. 3 restriction map obtained after digestion of genomic DNA of RF-BN1

The order of loading of the gel was analyzed by Southern blotting: lane 1, RF-MS 1DNA digested with BamHI, lane 2, RF-BN 1DNA digested with EcoRI, lane 3, RF-BN 1DNA digested with EcoRV, lane 4, RF-BN 1DNA digested with HindIII, lane 5, non-transgenic WOSR DNA digested with BamHI, lane 6, non-transgenic WOSR + control plasmid pTHW118 DNA digested with BamHI.

FIG. 4 PCR analysis of different lines using MS-BN1PCR identification method

Loading sequence of gel: lane 1, DNA sample from OSR plant comprising transgenic event MS-BN1, lane 2, DNA sample from OSR plant comprising another transgenic event, lane 3, DNA sample from wild type OSR, lane 4, negative control (water), lane 5, molecular weight marker (100bp ladder).

FIG. 5 PCR analysis of different lines using the RF-BN1PCR identification method

Loading sequence of gel: lane 1, OSR plant with DNA sample containing transgenic event RF-BN1, lane 2, OSR plant with DNA sample containing another transgenic event, lane 3, wild type OSR, lane 4, negative control (water), lane 5, molecular weight marker (100bp ladder).

Detailed Description

The term "gene" as used herein refers to any DNA sequence comprising operably linked DNA segments such as a promoter and a 5 'untranslated region (5' UTR) which together form a promoter region, a coding region (which may or may not encode a protein) and a 3 'untranslated region (3' UTR) comprising a polyadenylation site. In plant cells usually the 5 'UTR, coding region and 3' UTR are transcribed into RNA, which in the case of protein-coding genes is translated into protein. The gene may include additional DNA segments, such as introns. As used herein, a locus is the location of a given gene in the genome of a plant.

The term "chimeric" when used in reference to a gene or DNA sequence refers to a gene or DNA sequence comprising at least two functionally related DNA segments (e.g., promoter, 5 'UTR, coding region, 3' UTR, intron) that are not naturally associated with each other, and which are derived, for example, from different sources. Reference to a "foreign" gene or DNA sequence of a plant species means that the gene or DNA sequence is not found in nature in that plant, or that the gene or DNA sequence is not found in nature in that locus of that plant species. The term "foreign DNA" as used herein refers to a DNA sequence that is incorporated into the genome of a plant as a result of transformation. "transforming DNA" as used herein refers to recombinant DNA molecules used for transformation. The transforming DNA typically comprises at least one "gene of interest" (e.g., a chimeric gene) that confers one or more specific properties to the transformed plant. The term "recombinant DNA molecule" is used for illustration and may comprise an isolated nucleic acid molecule, which may be DNA and which may be obtained by recombinant or other means.

The term "transgene" as used herein refers to a gene of interest that is incorporated into the genome of a plant. "transgenic plant" refers to a plant comprising at least one transgene in the genome of all of its cells.

The exogenous DNA present in the plants of the invention preferably comprises two genes of interest, more specifically either a male sterility gene and a herbicide resistance gene, or a fertility restorer gene and a herbicide resistance gene.

The term "male-sterile gene" as used herein refers to a gene which, when expressed in a plant, renders the plant incapable of producing fertile, viable pollen. An example of a male sterility gene is a gene comprising a DNA sequence encoding a bacillus rnase under the control of a promoter that mediates its expression in tapetum cells. More specifically, the male sterility gene of the present invention is "TA 29-Bacillus RNase" described herein.

As used herein, a "fertility restorer gene" refers to a gene which, when expressed in a plant containing a male sterility gene, prevents phenotypic expression of the male sterility gene, and restores fertility to the plant. More specifically, the fertility restorer gene comprises DNA encoding a protein or polypeptide which prevents the phenotypic expression of the male sterility gene under the control of a promoter which mediates its expression at least in cells expressing the male sterility gene. More specifically, the fertility restoration gene of the present invention is "TA 29-barnase inhibitor" as described herein.

Incorporation of recombinant DNA molecules into the genome of a plant typically results from transformation of a cell or tissue (or from another genetic manipulation). The specific site of incorporation is either random or at a predetermined location (if targeted integration methods are used).

Exogenous DNA can be characterized by the location and configuration of the site of incorporation of the recombinant DNA molecule in the plant genome. The insertion site of a recombinant DNA in the plant genome is also referred to as the "insertion site" or "target site". The insertion of a transgene in the plant genome is associated with a deletion in the plant DNA, referred to as a "target site deletion". "flanking region" or "flanking sequence" as used herein refers to a sequence of at least 20bp, preferably at least 50bp, and up to 5000bp of the plant genome, located immediately upstream and adjacent to the foreign DNA or located immediately downstream and adjacent to the foreign DNA. Transformation steps that result in random integration of the exogenous DNA will produce transformants with different flanking regions that are unique and unique for each transformation. When a transgene is introduced into a plant by conventional crossing, its insertion site in the plant genome or its flanking regions are generally not altered. "insertion region" as used herein refers to a region corresponding to at least 40bp, preferably at least 100bp, and up to more than 10000bp, which is comprised in the flanking region upstream and downstream of the transgene in the (untransformed) plant genome and comprises the insertion site (and possibly the target site deletion). In view of the small differences due to intraspecies mutations, the insert region retains at least 85%, preferably 90%, more preferably 95%, and most preferably 100% sequence identity to the sequence comprising the flanking regions upstream and downstream of the exogenous DNA of the specified implant of that species.

Expression of the gene of interest refers to: by transformation, a recombinant DNA molecule-transforming DNA-is introduced (based on the structure and function of part or all of the gene of interest) to confer one or more phenotypic characteristics (e.g., herbicide resistance) to the plant.

An "event" is defined as a (artificial) locus that carries exogenous DNA comprising at least one copy of a gene of interest as a result of genetic manipulation. The general allelic state of an event is the presence or absence of exogenous DNA. As used herein, the "MS" event and the "RF" event refer to the expression of one or more transgenes characteristic of the RNA harboring "TA 29-Bacillus, respectively. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, the event is characterized by the restriction map of the transgene (e.g., as determined by Southern blotting) and/or the upstream and/or downstream flanking sequences of the transgene, and/or the molecular configuration of the transgene. Transformation of a plant with a transforming DNA comprising at least one gene of interest typically results in a plurality of events, each of which is unique.

As used herein, a "elite event" is an event selected from the group of events obtained by transformation with the same transforming DNA or by backcrossing with plants obtained by such transformation, based on the expression and stability of the transgene and compatibility with the optimal agronomic characteristics of the plant in which it is contained. The criteria for selection of elite events are therefore one or more, preferably two or more, advantageously all of the following:

a) the presence of the transgene does not detract from other desirable plant characteristics, such as those associated with agronomic or commercial value;

b) the event is characterized by a well-defined stably inherited molecular configuration and can be exploited as a suitable diagnostic tool for identifying controls;

c) in the case of heterozygous (or hemizygous) and homozygous events, the gene of interest in the transgene shows correct, suitable and stable spatial and temporal phenotypic expression at commercially acceptable levels under a range of environmental conditions, where the plant carrying the event may be in routine agricultural use;

preferably, the exogenous DNA is associated with a location in the genome of the plant that allows the gene to be incorporated into a desired commercial genetic background.

The situation of an event as a elite event is confirmed by incorporating the elite event into different relevant genetic backgrounds and observing e.g. one, two or all of the criteria a), b) and c) above.

In addition, for the transgenes described herein encoding male sterility and fertility restoration, the choice of elite event also depends on the compatibility between these events, more specifically the presence of two events in the progeny resulting from crossing a plant carrying a male sterility event with a plant carrying a fertility restoration event, said progeny having the following characteristics:

a) a fertility restoration phenotype, i.e., sufficient phenotypic expression of male fertility, an

b) Under a range of environmental conditions, the phenotype is expressed at a commercially acceptable level, where plants carrying both events may be in routine agricultural use;

thus, a "elite event" refers to a locus comprising a transgene that meets the criteria described above. A plant, plant material, or progeny, such as a seed, can comprise one or more elite events in its genome.

The development of "diagnostic tools" for identifying elite events or identifying plants or plant material comprising elite events is based on specific genomic characteristics of the elite event, such as a specific restriction map of the genomic region comprising the foreign DNA and/or the sequence of the transgenic flanking region. As used herein, a "restriction map" refers to a set of Southern blot patterns obtained by digesting plant genomic DNA with a specific restriction enzyme or set of restriction enzymes and hybridizing under standard stringency conditions to a probe that shares sequence similarity with a transgene. Standard stringency conditions as used herein refer to the conditions used for hybridization as described herein or conventional hybridization conditions as described by Sambrook et al (1989) (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring harbor Laboratory Press, N.Y.), which for example comprises the following steps: 1) immobilizing plant genomic DNA on a filter, 2) prehybridizing the filter with 50% formamide, 5X SSPE, 2X Denhardt's reagent and 0.1% SDS at 42 ℃ for 1 to 2 hours, or 6X SSC, 2X Denhardt's reagent and 0.1% SDS at 68 ℃ for 1 to 2 hours, 3) adding labeled hybridization probes, 4) incubating for 16 to 24 hours, 5) washing the filter with 1X SSC, 0.1% SDS at room temperature for 20 minutes, 6) washing the filter three times, 20 minutes, each at 68 ℃ for 0.2X SSC, 0.1% SDS, and 7) exposing the filter to X-ray film with an intensifying screen at-70 ℃ for 24 to 48 hours.

Since the (endogenous) restriction site is already present in the plant genome before the incorporation of the foreign DNA, the insertion of the foreign DNA will alter the specific restriction map of the genome. Thus, the particular transformants or progeny derived therefrom can be identified by one or more particular restriction patterns. The conditions for determining the restriction map of the event are given in the "restriction map identification method". In addition, once one or both flanking regions of a transgene have been sequenced, PCR-probes that specifically recognize such sequence(s) can be developed in a "PCR identification method". Plants or plant material containing elite events can be experimentally identified using these specific primers according to the PCR method.

As used herein, a "biological sample" is a sample of a plant, plant material, or product containing plant material. The term "plant" encompasses WOSR (brassica napus) plant tissue at any stage of maturity as well as any cell, tissue or organ taken from or derived from any such plant, including, but not limited to, any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture or protoplast. As used herein, "plant material" refers to material derived or derived from plants. Products comprising plant material relate to food, feed or other products produced with or contaminated with plant material. It will be appreciated that in the context of the present invention, such biological material is preferably used to test for the presence of MS-BN1 and/or RF-BN1 specific nucleic acids, suggesting the presence of nucleic acids in the sample. Thus the methods for identifying elite event MS-BN1 and/or RF-BN1 in biological samples referred to herein preferably relate to the identification of nucleic acids in biological samples comprising elite event.

As used herein, "kit" refers to: a set of reagents for the purpose of practicing the methods of the invention, and more specifically, a set of reagents for identifying elite event MS-BN1 and/or RF-BN1 in biological samples. More specifically, a preferred embodiment of the kit of the invention comprises at least one or two specific primers as described above. Optionally, the kit may further comprise any of the other reagents described in the PCR identification methods herein. In addition, according to another embodiment of the present invention, the kit may comprise a specific probe as described above, which specifically hybridizes to a nucleic acid of a biological sample to identify the presence of MS-BN1 and/or RF-BN1 therein. Optionally, the kit may further comprise any other reagent (e.g., without limitation, hybridization buffer, label) for identifying MS-BN1 and/or RF-BN1 in a biological sample with a specific probe.

The kit of the invention may be used, the components of which may be specifically adjusted for quality control (e.g. purity of seed lot), detection of elite events in plant material or material comprising or derived from plant material (such as, but not limited to, food or feed products).

The present invention relates to the generation of a set of elite events in WOSR, MS-BN1 and RF-BN1, plants comprising these events, progeny obtained from crossing these plants, plant cells or plant material derived from these events. Plants comprising elite event MS-BN1 were obtained by transformation with pTHW107 as described in example 1. Plants comprising elite event RF-BN1 were also obtained by transformation of DTHW118 as described in example 1.

The recombinant DNA molecule used to produce elite event MS-BN1 comprises a DNA sequence encoding a bacillus rnase molecule under the control of a promoter that mediates its expression selectively in tapetum cells (referred to as "TA 29-bacillus rnase"). The TA29 promoter has a "tapetum-selective" expression pattern in OSR (De Block and Debrower, Planta 189: 218-225, 1993). Expression of the TA 29-Bacillus RNAse gene in WOSR plants results in disruption of the tapetum and male sterility of the plant (Mariani et al, 1990, supra). The recombinant DNA molecule used to generate elite event RF-BN1 comprises a DNA sequence encoding a bacillus rnase inhibitor molecule under the control of a tapetum-specific promoter (referred to as "TA 29-bacillus rnase inhibitor"). In the presence of the "TA 29-barnase" gene in plants, expression of the TA 29-barnase inhibitor gene in WOSR plants prevents activity of the barnase in the tapetum cells of the plants, prevents disruption of the tapetum, and thus restores fertility in these plants (Mariani et al, 1992, supra).

Both recombinant DNA used to generate elite event MS-BN1 and RF-BN1 additionally comprise DNA sequences encoding phosphinothricin acetyltransferase as well as Cauliflower Mosaic Virus (Cauliflower Mosaic Virus)35S promoter, with the sequence encoding phosphinothricin acetyltransferase being under the control of the 35S promoter (referred to as "35S-bar"). The 35S promoter is "constitutive" in OSR, meaning that it is significantly expressed in most cell types during most plant life cycles. Expression of the 35S-bar gene in OSR plants confers resistance to herbicidal compounds, such as phosphinothricin or bialaphos or glufosinate, or more commonly glutamine synthetase inhibitors or salts or optical isomers thereof.

WOSR plants or plant material comprising MS-BN1 can be identified according to the MS-BN1 restriction map identification method described in example 5 herein. Briefly, WOSR genomic DNA is treated with the following restriction enzymes (preferably 2 to 5) selected: EcoRI, EcoRV, Ndel, Hpal, AflIII were digested and then transferred to a nylon membrane and hybridized with the 3942bp HindIII fragment of the plasmid pTHW107 (or the T-DNA contained therein). It was then determined whether each restriction enzyme used identified the following fragments:

-EcoRI: one fragment is between 2140 and 2450bp, preferably about 2266bp, and one fragment is greater than 14 kbp;

-EcoRV: one fragment is between 1159 and 1700bp, preferably about 1.4kbp, and one fragment is greater than 14kbp in length;

-Hpal: one fragment is between 1986 and 2140bp, preferably about 1990bp in length, and one fragment is between 2140 and 2450bp, preferably about 2229 bp;

-AflIII: one fragment is between 514 and 805bp, preferably about 522bp in length, one fragment is between 2140 and 2450bp, preferably about 2250bp, and one fragment is between 2450 and 2838bp, preferably about 2477 bp;

-Ndel: both fragments are between 5077 and 14057bp in length, preferably one fragment is about

6500bp and another approximately 10 kbp;

the length of the DNA fragments is determined by comparison with a set of DNA fragments of known length, in particular with the PstI fragment of bacteriophage lambda DNA. When DNA was extracted according to the method of Dellaporta et al (1983, plant molecular Biology Reporter, 1, Vol.3, pp.19-21), fragments of more than 14kbp were estimated to be between 14kbp and 40kbp in length.

A WOSR plant is determined to possess elite event MS-BN1 if after digestion of the plant material with at least two, preferably at least three, especially at least four, more especially with all these restriction enzymes, DNA fragments with the same length as described above are produced.

Plants or plant material comprising MS-BN1 can also be identified using the MS-BN1PCR identification method described in example 5 herein. Briefly, WOSR genomic DNA is PCR amplified with primers, one of which specifically recognizes the flanking sequence of MS-BN1, preferably the 5 'or 3' flanking sequence of MS-BN1 as described herein, in particular the primer having the sequence of SEQ ID No 19, and the other of which recognizes the sequence in the transgene, in particular the primer having the sequence of SEQ ID No 12. Endogenous WOSR primers were used as controls. The WOSR plant was determined to possess elite event MS-BN1 if the plant material produced fragments between 260 and 300bp, preferably about 280 bp.

The phenotypic characteristics of plants possessing MS-BN1 are: they are male sterile when they lack the restorer gene in their genome. Male sterile plants are defined as plants that are unable to produce viable, viable pollen.

Plants possessing MS-BN1, for example, can be obtained from seeds containing MS-BN1, which seeds were deposited at the ATCC under accession number PTA-730. Such plants can be further propagated to introduce the elite event of the invention into other cultivars of the same plant species

WOSR plants or plant material comprising RF-BN1 can be identified according to the identification method of RF-BN1 restriction map described in example 5 herein. Briefly, WOSR genomic DNA is treated with the following restriction enzymes (preferably 2 to 4) selected: BamHI, EcoRI, EcoRV and HindIII were digested, then transferred to a nylon membrane and hybridized with a 2182bp Hpal fragment of plasmid pTHW118 (or T-DNA contained therein). It was then determined whether each restriction enzyme used identified the following fragments:

-BamHI: one fragment between 805 and 1099bp, preferably about 814bp, one fragment between 1700 and 1986bp, preferably about 1849bp, one fragment between 2450 and 2838bp, preferably about 2607bp, and one fragment between 5077 and 14057bp, preferably about 6500 bp;

-EcoRI: one fragment between 805 and 1159bp, preferably about 1094bp, one fragment between 1986 and 2450bp, preferably about 2149bp, and two fragments between 5077 and 14057bp, preferably one about 7000bp, and one about 10kbp in length;

-EcoRV: both fragments are between 5077 and 14057bp, preferably one is about 5.4kbp in length and the other is about 8kbp in length;

-HindIII: one fragment is between 1700 and 2140bp in length, preferably about 1969bp, and two fragments are between 2450 and 2838bp in length, preferably one is about 2565bp in length and one is about 2635bp in length;

the length of the DNA fragments is determined by comparison with a set of DNA fragments of known length, in particular with the PstI fragment of bacteriophage lambda DNA.

A WOSR plant is determined to possess elite event RF-BN1 if after digestion of the plant material with at least two, preferably at least three, more particularly with all of these restriction enzymes, DNA fragments with the same length as described above are produced.

Plants or plant material comprising RF-BN1 can also be identified using the RF-BN1PCR identification method described in example 5 herein. Briefly, WOSR genomic DNA was PCR amplified with primers, one of which specifically recognizes the flanking sequence of RF-BN1, preferably the 5 'or 3' flanking sequence of RF-BN1 as described herein, in particular with the sequence of SEQ ID No 41, and the other of which recognizes the sequence in the transgene, in particular with the sequence of SEQ ID No 23. Endogenous WOSR primers were used as controls. The WOSR plant was determined to possess elite event RF-BN1 if the plant material produced a fragment between 195 and 235bp, preferably about 215 bp.

Plants possessing RF-BN1 are characterized by: expressing a barnase inhibitor in tapetum cells. Production of barnase inhibitors in tapetum cells of plants has been shown to be neither beneficial nor detrimental to pollen production (Mariani et al, 1992, supra). Thus, the TA 29-Bacillus RNase inhibitor gene will not cause an observable phenotype when the plant genome lacks a male sterility gene. When a male sterility gene is present in the plant genome, the TA 29-barnase inhibitor gene will result in restoration of fertility, i.e., a fertile phenotype. A plant having a fertility restoring phenotype is defined as a plant that produces fertile, viable pollen despite the presence of a male sterile gene in its genome.

The plant possessing RF-BN1 can be obtained, for example, from seed deposited at the ATCC under accession number PTA-730. Such plants may be further propagated and/or used in conventional breeding schemes to introduce elite events of the invention into other cultivated varieties of the same plant species.

Plants possessing MS-BN1 and/or RF-BN1 are also characterized by their glufosinate tolerance, which in the context of the present invention includes herbicide tolerance Liberty^TMThe plant of (1). Liberty^TMThe defined criteria for tolerance are: when the plants are sprayed with at least 200 g active ingredient/hectare (g.a.i./ha), preferably 400g.a.i./ha, possibly also up to 1600g.a.i./ha, at the 3 to 4 leaf stage (3V to 4V), the plants are not killed. Plants possessing MS-BN1 and/or RF-BN1 are also characterized by the presence of phosphinothricin acetyltransferase in their cells as determined by the PAT assay (DeBlock et al, 1987, supra).

The WOSR plants of the present invention can be cultivated by conventional methods. The presence of the 35S-bar gene makes them tolerant to glufosinate. Thus growing such WOSR plantsThe field of the object can be controlled by using a herbicide (e.g. Liberty) containing glufosinate-ammonium as an active ingredient^TM) Controlling weeds.

Plants possessing MS-BN1 and/or RF-BN1 are also characterized by agronomic characteristics comparable to the WOSR varieties marketed in the United states. The relevant agronomic characteristics are: plant height, stalk strength or stiffness, lodging tendency, winter stubborn strength, shatter resistance, drought resistance, disease resistance (Black leg, Light leaf spot, Sclerotinia), and grain production and yield.

It has been observed that the presence of foreign DNA at the insertion regions of the genome of a brassica napus WOSR plant as described herein, and more particularly at these insertion sites of the genome of a brassica napus WOSR plant, confers plants comprising these events a phenotypic and molecular feature of particular interest. More specifically, the presence of exogenous DNA in specific regions of the genome of these plants can result in stable phenotypic expression of the transgene without significant compromise of any other desirable agronomic characteristics of the plant, making them particularly suitable for the generation of hybrid WOSR. Thus, the insertion regions corresponding to SEQ ID No 22 and SEQ ID No 34, more specifically the MS-BN1 and RF-BN1 insertion sites therein, are shown to be particularly suitable for the introduction of a gene of interest. More specifically, the insertion regions of MS-BN1(SEQ ID No 22) and RF-BN1(SEQ ID No 34), or the respective insertion sites therein of MS-BN1 and RF-BN1, particularly suitable for the introduction of plasmids comprising a male-sterility gene and a fertility-restorer gene, respectively, can ensure optimal expression of each or both of these genes in plants without compromising the agronomic characteristics of the plants.

Recombinant DNA molecules can be specifically inserted into an insertion region by targeted insertion methods. Such methods are well known to those skilled in the art and include, for example, homologous recombination with recombinases such as, but not limited to, FLP recombinase from Saccharomyces cerevisiae (Saccharomyces cerevisiae) (U.S. Pat. No. 5,527,695), CRE recombinase from Escherichia coli phage P1 (published PCT patent application WO 9109957), recombinase from Saccharomyces rouxii (Saccharomyces rouxii) pSRI (Araki et al, 1985, J. Mol Biol. 182: 191-203), or lambda recombination systems as described in U.S. Pat. No.4,673,640.

"sequence identity" as used herein with respect to a nucleotide sequence (DNA or RNA) refers to: the number of positions having the same nucleotide is divided by the number of nucleotides of the shorter of the two sequences. The alignment of the two nucleotide sequences was performed using the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window size of 20 nucleotides, a word length of 4 nucleotides and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including the sequence permutations described above, is conveniently performed, for example, using the intelligentization (TM) Suite program (intelligentization Inc., CA). Sequences are indicated as "substantially similar" when the sequence identity of such sequences is at least about 75%, particularly at least about 80%, more particularly at least about 85%, more particularly about 90%, particularly about 95%, more particularly about 100%, and more particularly is completely identical. Thus, when an RNA sequence is said to be substantially similar or have some degree of sequence identity to a DNA sequence, it should be understood that thymine (T) in the DNA sequence is considered to be identical to uracil (U) in the RNA sequence.

The word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the presence of stated features, integers, steps or components but not the exclusion of any other feature, integer, step, component or group of features, integers, steps, components or groups thereof. Thus, for example, a nucleic acid or protein comprising a nucleotide or amino acid sequence may comprise more nucleotides or amino acids than actually cited, i.e. be comprised in a larger nucleic acid or protein. Chimeric genes comprising functionally or structurally defined DNA sequences may comprise additional DNA sequences and the like.

The following examples describe the generation and characterization of WOSR plants possessing elite event MS-BN1 and RF-BN 1.

Unless otherwise indicated, all recombinant DNA techniques were performed according to standard procedures described in Sambrook et al (1989), molecular cloning: a laboratory Manual, second edition, Cold spring harbor laboratory Press, New York and Ausubel et al, (1994) Current protocols in Molecular Biology, volumes 1 and 2, Current protocols, USA. Standard materials and methods for Plant Molecular research are described in Plant Molecular Biology (Plant Molecular Biology) Labfax (1993) published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK R.D.D.Croy.

In the description and examples, reference is made to the following sequences:

SEQ ID No. 1: plasmid pTHW107

SEQ ID No. 2: plasmid pTHW118

SEQ ID No. 3: primer 248

SEQ ID No. 4: primer 249

SEQ ID No. 5: primer 247

SEQ ID No. 6: primer 250

SEQ ID No. 7: primer 251

SEQ ID No. 8: primer 254

SEQ ID No 9 primer 258

SEQ ID No. 10: primer SP6

SEQ ID No 11: primer T7

SEQ ID No 12: primer 201(BNA01)

SEQ ID No 13: sequences comprising the flanking region of MS-BN15

SEQ ID No 14: primer 611

SEQ ID No 15: primer 259

SEQ ID No 16: primer 260

SEQ ID No 17: primer 24

SEQ ID No 18: sequences comprising the flanking region of MS-BN 13

SEQ ID No 19: primer 51(BNA02)

SEQ ID No 20: primer 48

SEQ ID No 21: sequences comprising a deletion of the MS-BN1 target site

SEQ ID No 22: insertion region MS-BN1

SEQ ID No 23: primer 193(BNA03)

SEQ ID No. 24: sequences comprising the RF-BN 15' flanking region

SEQ ID No 25: primer 286

SEQ ID No 26: primer 314

SEQ ID No 27: primer 315

SEQ ID No 28: primer 316

SEQ ID No 29: primer 288

SEQ ID No. 30: sequences comprising the RF-BN 13' flanking region

SEQ ID No 31: primer 269

SEQ ID No 32: primer 283

SEQ ID No 33: primer 284

SEQ ID No 34: integration zone RF-BN1

SEQ ID No 35: primer 57

SEQ ID No 36: sequences comprising the MS-BN 15' flanking region in WOSR

SEQ ID No 37: primer 68

SEQ ID No 38: sequences comprising the MS-BN 13' flanking region in WOSR

SEQ ID No 39: sequences comprising the RF-BN 15' flanking region in WOSR

SEQ ID No. 40: sequences comprising the RF-BN 13' flanking region in WOSR

SEQ ID No 41: primer 268(BNA04)

SEQ ID No 42: primer BNA05

SEQ ID No 43: primer BNA06

Examples

Example 1 transformation of Brassica napus with Male sterile Gene and restorer Gene

a) Construction of chimeric DNA (pTHW107) comprising the RNase gene of Bacillus under the control of tapetum-specific promoter

The plasmid pTHW107(SEQ ID No.1) is essentially derived from the intermediate vector pGSV 1. PGSV1 was itself derived from pGSC1700(Cornelissen and Vanderielle, 1989), but contained an artificial T-region consisting of the left and right border sequences of the TL-DNA of pTiB6S3 and a polylinker cloning site, allowing insertion of a chimeric gene between the T-DNA border repeats. The pGSV1 vector provides the barnase inhibitor gene on a main frame of the plasmid and provides regulatory signals for expression in e.

Table 1 fully describes the DNA contained between the pTHW107 border repeats:

table 1: T-DNA of plasmid pTHW107

nt position	Direction of rotation	Description and reference
nt position	Direction of rotation	Description and reference	1-25		The right border repeat of TL-DNA from pTiB6S3 (Gielen et al (1984) The EMBO Journal 3: 835-846).
26-97		Synthetic polylinker derivative sequences	1-25
26-97		Synthetic polylinker derivative sequences	309-98	Counter clockwise	The 3 'untranslated end (3' g7) of the TL-DNA gene 7 of pTiB6S3 (Velten and Schell (1985) Nucleic acids research (Nucleic acids research) 13: 6981-698; Dhaese et al (1983) the EMBO Journal 3: 835-846).
310-330		Synthetic polylinker derivative sequences	309-98	Counter clockwise
310-330		Synthetic polylinker derivative sequences	882-331	Counter clockwise	The coding sequence of The bialaphos resistance gene (bar) of Streptomyces hygroscopicus (Thompson et al (1987) The EMBO Journal 6: 2519-2523). The two codons at the N-terminus of the coding region of the wild-type bar gene have been replaced with the codons ATG and GAC, respectively.

2608-883	Counter clockwise	atS1A promoter of the gene for the small subunit of ribulose-1, 5-biphosphate carboxylase of Arabidopsis thaliana (PssuAra) (Krebbers et al (1988) plant molecular biology 11: 745-759).
2608-883	Counter clockwise		2609-2658		Synthetic polylinker derivative sequences
2919-2659	Counter clockwise	A260 bp TaqI fragment from the 3 'untranslated end (3' nos) of the nopaline synthase gene from the T-DNA in pTiT37 and comprising a plant polyadenylation signal (Depicker et al (1982), Journal of Molecular and Applied Genetics 1: 561-.	2609-2658		Synthetic polylinker derivative sequences
2919-2659	Counter clockwise		2920-3031		3' untranslated region downstream of the barnase coding sequence of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
3367-3032	Counter clockwise	The coding region of the barnase gene from Bacillus amyloliquefaciens (Hartley (1988) J. mol. biol. 202: 913-.	2920-3031
3367-3032	Counter clockwise		4877-3368	Counter clockwise	The promoter region of the anther-specific gene TA29 from tobacco (Nicotiana tabacum). The promoter contained 1.5kb upstream sequence from the ATG start codon (Seurinck et al (1990) nucleic acids research 18: 3403).
4878-4921		Synthetic polylinker derivative sequences	4877-3368	Counter clockwise
4878-4921		Synthetic polylinker derivative sequences	4922-4946		The left-border repeat of TL-DNA from pTiB6S3 (Gielen et al (1984) The EMBO Journal 3: 835-846).

b) Construction of chimeric DNA (pTHW118) comprising the RNase inhibitor gene of Bacillus under the control of constitutive promoter

The plasmid pTHW118(SEQ ID No.2) is also essentially derived from the intermediate vector pGSV1 (see above for description). Table 2 gives a sufficient description of the DNA contained between the pTHW118 border repeats:

table 2: T-DNA of plasmid pTHW118

nt position	Direction	Description and reference
nt position	Direction	Description and reference	1-25		The right border repeat of TL-DNA from pTiB6S3 (Gielen et al (1984) The EMBO Journal 3: 835-846).
26-53		Synthetic polylinker derivative sequences	1-25
26-53		Synthetic polylinker derivative sequences	54-90		Residual sequence of TL-DNA at right border repeats.
91-97		Synthetic polylinker derivative sequences	54-90		Residual sequence of TL-DNA at right border repeats.
91-97		Synthetic polylinker derivative sequences	309-98	Counter clockwise	T of pTiB6S3The 3 'untranslated end of The L-DNA gene 7 (3' g7) (Velten and Schell (1985) nucleic acids Res 13: 6981-6998; Dhaese et al (1983) The EMBO Journal 3: 835-846).
310-330		Synthetic polylinker derivative sequences	309-98	Counter clockwise
310-330		Synthetic polylinker derivative sequences	883-331	Counter clockwise	The coding sequence of The bialaphos resistance gene (bar) of S.hygroscopicus (Thompson et al (1987) The EMBO Journal 6: 2519-2523). The two codons at the N-terminus of the wild-type bar coding region have been replaced by the codons ATG and GAC, respectively.
2608-883	Counter clockwise	The promoter of the atS1A ribulose-1, 5-biphosphate carboxylase small subunit gene of Arabidopsis thaliana (PssuAra) (Krebbers et al (1988) plant molecular biology 11: 745-759).	883-331	Counter clockwise
2608-883	Counter clockwise		2609-2658		Synthetic polylinker derivative sequences
2919-2659	Counter clockwise	A260 bp TaqI fragment from the 3 'untranslated region end (3' nos) of the nopaline synthase gene from the T-DNA in pTiT37 and comprising a plant polyadenylation signal (Depicker et al (1982) molecular and applied journal of genetics 1: 561-.	2609-2658		Synthetic polylinker derivative sequences
2919-2659	Counter clockwise		2920-2940		Synthetic polylinker derivative sequences
2941-2980		3' untranslated region downstream of the barnase inhibitor coding sequence of Bacillus amyloliquefaciens	2920-2940		Synthetic polylinker derivative sequences
2941-2980			3253-2981	Counter clockwise	The coding region of the barnase inhibitor gene from Bacillus amyloliquefaciens (Hartley (1988) J. mol. biol. 202: 913-915).
4762-3254	Counter clockwise	The promoter region of the tobacco anther-specific gene T29. The promoter contained 1.5kb upstream sequence starting from the ATG start codon (Seurinck et al (1990) nucleic acids research 18: 3403).	3253-2981	Counter clockwise
4762-3254	Counter clockwise		4763-4807		Synthetic polylinker-derived sequences
4808-4832		The left-border repeat of TL-DNA from pTiB6S3 (Gielen et al (1984) The EMBO Journal 3: 835-846).	4763-4807		Synthetic polylinker-derived sequences

c) Transformation of Brassica napus

Vector systems for the transformation of Brassica napus are described by Deblaere et al (1985, 1987). The vector system consists of an agrobacterium strain and two plasmid components: 1) non-tumorigenic Ti-plasmids (pGV4000) and 2) intermediate cloning vectors based on the plasmid pGSV 1. The non-tumorigenic Ti-plasmid, from which the T-region has been deleted, carries the vir gene, which is required for the transfer of the artificial T-DNA cloned on the second plasmid into the plant genome. Agrobacterium strains resulting from triparental crosses between these components can be used for plant transformation.

Except for plantlet regeneration, all stages were selected with phosphinothricin (PPT), which in the absence of PPT, promotes growth. This yielded a set of primary transformants (T)₀A progeny plant).

Example 2 Generation of events

2.1. Identification of transgenic events

Southern blot analysis of MS events

Standard Southern blot analysis was used to check for the presence of transgenes and the number of gene insertions. According to Dellaporta (1983, Plant Molecular biology reporter (Plant Molecular biology reporter), 1, Vol.3, pp.19-21) or Doyle et al, 1987, phytochemistry Communication (phytochem. Bull.) 19: 11) total genomic DNA was isolated from 1g of germinated tissue and digested with the restriction enzyme SacI. SacI has a unique restriction site in the T-DNA fragment, located between the barnase and the bar construct. Southern analysis was performed with the following two probes:

"Bacillus RNase" probe: a478 bp PstI-EcoRI fragment of plasmid pVE113

"bar" probe: 546bp NcoI-BgIII fragment of plasmid pDE110

Plasmids pVE113 and pDE110 are described in FIG. 1 and WO92/09696, respectively.

The MS event hybridized with the barnase probe to generate a 12Kb band and the bar probe to generate a 14Kb band.

The shade of the relevant band provides an indication of whether the plant is homologous or heterologous to the transgene location. These two events were found to be simple insertions. This can be confirmed by the fact that: the pattern of segregation of the transgene can be explained by mendelian inheritance of simple genomas.

Southern blot analysis of RF events

Standard Southern blot analysis was used to check for the presence of transgenes and the number of gene insertions. Total genomic DNA was isolated from 1g of germinated tissue (according to Doyle et al 1987, phytochemical letters 19: 11) and digested with the restriction enzyme SacI. SacI has a unique restriction site in the T-DNA fragment, located between the barnase and the bar construct. Southern analysis was performed with the following two probes:

"barstar" probe: 436bpHindIII-PstI fragment of plasmid pVE113

"bar" probe: 546bp NcoI-BglII fragment of plasmid pDE110

The RF event hybridized with the barnase inhibitor probe to generate a 3Kb band, and the bar probe to generate a 14Kb fragment.

The shade of the relevant band provides an indication of whether the plant is homologous or heterologous to the transgene location. Some events were found to be simple insertions. This can be confirmed by the fact that: the pattern of segregation of the transgene can be explained by mendelian inheritance of simple genomas.

2.1.3. General plant phenotype and agricultural characteristics

Evaluation of T with MS and RF events by a series of phenotypic traits₁Plants, including height, stalk strength or hardness, lodging tendency, winter stubborn strength, shatter resistance, drought resistance, disease resistance (Blackleg, Light leaf spot, sclerotiretinia), and grain production and yield.

The agronomic characteristics exhibited by this line were assessed as similar (or altered) compared to the untransformed line and a range of oilseed rape cultivars. In some cases, plant isolation is used for somaclonal variation of one or more of the above traits. Unless this results in the introduction of a phenotypic trait of commercial interest, these plants are discarded.

2.2. Generation of lines carrying MS or RF traits

Transition from tissue culture to different T₀Hemizygous plantlets ("Ms/-" or "R)f/- "), transferred to greenhouse soil. Southern blot analysis was used to check for the presence and copy number of the transgene (as described above). Plants are allowed to flower and the sterility or fertility of the flowers is assessed separately. T is₀Plants were crossed with wild type plants (-/-) to produce T1 seeds (MsT1 and RfT 1). T1 seeds were planted and grown in the greenhouse. Plants were evaluated for tolerance to glufosinate ammonium. The Ms-T1 plants were also evaluated for sterility/fertility segregation (in plants that were not sprayed) while the Rf-T1 plants were examined for fertility flowers.

Ms-T1 plants comprising the transgene are crossed with a test plant homozygous for the fertility restorer gene (Rf/Rf) to produce MsRf-F1 seeds. The seeds (Ms/-, Rf/-and-/-, Rf/-) were planted in the greenhouse and sprayed with Liberty^TM. The remaining F1 progeny fertility/sterility split was evaluated to test whether the male sterility trait could be sufficiently restored in brassica napus (fertility close to 100%).

The best event was selected for further testing. Ms-T₁The plants are crossed with homozygous fertility restorer plants and the seeds are planted in the field. Evaluation of plant Pair Liberty^TMTolerance to herbicides (at 800 grams active ingredient per hectare (g.a.i./ha), a recommended dose to farmers of 400g.a.i./ha), assessment of fertility/sterility segregation and assessment of overall phenotypic characteristics. Lines with 100% restored fertility and no negative penalty observed for their phenotypic or agronomic characteristics were selected as compared to wild type isogenic controls (see (d)).

Rf-T1 plants containing the transgene were crossed with a test plant (Ms /) containing the male sterility gene to produce F1 seeds. Planting the seeds in a greenhouse, and spraying Liberty^TMAnd the restoration of fertility (close to 100%) was evaluated.

At the same time, Rf-T1 plants are self-pollinated to produce S1. The S1 plant was grown in a greenhouse and sprayed with Liberty^TMAnd self-pollinated again to produce S2; homozygous individuals were selected from S2.

Combining MS and RF events

In the greenhouse, selected Ms-T1 plants were combined with selected Rf-S2 plantsAnd (5) hybridizing the components, and performing a fertility restoration test. Planting the seeds in the greenhouse again, spraying the plants with Liberty^TMAnd the flower is checked for fertility.

2.4. Testing of MS and RF events in different genetic backgrounds and in different locations

Selected events were introduced into two different genetic backgrounds of heterozygous difference to confirm that both MS and RF events could function in either tested background with no negative penalties for yield and quality.

Selected MS and RF events are tested in four or five different environments simultaneously to ensure that there are no negative interactions between the environments and the MS or RF events.

In the next stage, more extensive experiments were performed in the field to produce hybrid seed with selected MS and RF events. Selected MS events are hybridized to selected RF events in their original context as well as in the context of two distinct and heterozygous differences. Evaluation of F1 hybrid Pair Liberty^TMResistance, fertility and overall agronomic characteristics (yield and quality).

2.5. Selection of elite events

In the process of generating transgenic MS lines, the selection step described above results in stock events that can show optimal expression of the transgene, i.e. resistance to glufosinate ammonium, a male sterile phenotype and susceptibility to full restoration of fertility with homozygous restorer lines, more particularly with selected RF stock events.

During the process of generating transgenic RF lines, the selection step described above produces elite events that can show optimal expression of the transgene, i.e., resistance to glufosinate ammonium and the ability of F1 to restore fertility after crossing with plants carrying a male sterility gene, more particularly a selected MS elite event.

Example 3: importing selected candidate elite event into WOSR

Some of the MS and RF elite events produced in brassica napus as described above were introduced into the WOSR cultivar by repeated backcrosses with the Drakkar variety plants.

The plants were examined and it was determined:

a) the presence of the exogenous DNA does not detract from other desirable plant characteristics, such as those associated with agronomic or commercial value;

b) the events are characterized by defined stably inherited molecular configurations;

c) in the case of event heterozygous (or hemizygous) and homozygous, the gene of interest in the exogenous DNA shows correct, suitable and stable spatial and temporal phenotypic expression at commercially acceptable levels under a range of environmental conditions, wherein the plant carrying the event may be in routine agricultural use;

further, agronomic characteristics and performance of the plants were evaluated in comparison to wild type WOSR species.

Extensive testing in the field showed that: when a candidate elite event for certain spring oilseed rape is introduced into a WOSR plant, the plant can exhibit sufficient expression of the gene of interest in the foreign DNA in combination with optimal agronomic performance. These events were selected as MS and RF elite events in WOSR and named MS-BN1 and RF-BN1, respectively.

Example 4 identification of elite events MS-BN1 and RF-BN1

Once the MS-BN1 and RF-BN1 events were identified as elite events with optimal expression and overall agronomic performance for each transgene, the location of the transgenes was analyzed in detail at the molecular level. This includes detailed Southern blot analysis (with multiple restriction enzymes) and sequencing of the transgenic flanking regions.

4.1. Southern blot analysis using multiple restriction enzymes

Leaf tissue was harvested from transgenic and control plants. Total genomic DNA was isolated from leaf tissue according to Dellaporta et al (1983, reporter in plant molecular biology, Vol.1, Vol.3, pp.19-21). The DNA concentration of each preparation was determined by measuring the optical density at a wavelength of 260nm using a spectrophotometer.

Mu.g of genomic DNA was digested with restriction enzymes and the final reaction volume was 40. mu.l, using the conditions recommended by the manufacturer. The digestion time and/or amount of restriction enzymes is adjusted to ensure complete digestion of the genomic DNA sample without non-specific degradation. After digestion, 4. mu.l of loading dye were added to the digested DNA samples and they were loaded on a 1% agarose gel.

The following control DNA was also loaded onto the gel:

negative control, genomic DNA prepared from non-transgenic brassica plants. This negative control was used to confirm the absence of background hybridization.

DNA positive control: by integrating the heterozygous single copy transgene into the Brassica napus genome, 10. mu.g of genomic DNA had the same number of molecular equivalents as the 1501bpPvuI-HindIII fragment from pTHW118 DNA at. + -.19 picograms (Brassica napus diploid genome size: 0.8X109 bp). An amount representing one plasmid copy per genome was added to 1. mu.g of digested non-transgenic Brassica napus DNA. The reconstituted sample is used to show that hybridization is performed under conditions that allow hybridization of the probe to the target sequence.

PstI digested phage lambda DNA (CIind 1 ts 857 Sam 7 strain, Life technologies) was used as a size standard.

After electrophoresis, DNA samples (digested brassica genomic DNA, control and size standard DNA) were transferred to nylon membranes by capillary blotting over 12 to 16 hours.

The DNA template used as a preparation of event probe for MS-BN1 was prepared by restriction digestion of PTW107 with HindIII. A3942 bp DNA fragment containing the relevant part of the transforming DNA was generated (part of PSSUARA, 3' nos, Bacillus RNase, PTA 29).

The DNA template used as a preparation of RF-BN1 event probe was prepared by restriction digestion of PTW118 with Hpal. A2182 bp DNA fragment containing the relevant portion of the transforming DNA was generated (PSSUARA part, 3' nos, barnase inhibitor, PTA 29).

After purification, the DNA fragments were labeled according to standard procedures and used for hybridization to membranes.

Hybridization was performed under standard stringency conditions: the labeled probe was denatured by heating in a water bath at 95 ℃ to 100 ℃ for 5 to 10 minutes, and cooled on ice for 5 to 10 minutes, and added to a hybridization solution of (6 XSSC (20 XSSC is 3.0M NaCl, 0.3M sodium citrate, pH7.0), 5 XDenhardt's (100 XDenhardt's ═ 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 0.5% SDS and 20. mu.g/ml denatured carrier DNA (single-stranded protamine DNA having an average length of 120-3000 nucleotides). The hybridization was performed overnight at 65 ℃ and the membrane was washed three times with a wash solution (2 XSSC, 0.1% SDS) at 65 ℃ for 20 to 40 minutes.

Electron scanning autoradiography.

4.1.1.MS-BN1

The restriction patterns obtained after digestion of the MS-BN1 genomic DNA with the different restriction enzymes are given in FIG. 2 and summarized in Table 3.

Table 3: restriction map of MS-BN1

Number of swimming strokes	Sample application of DNA	Hybridizing DNA fragments moving between size markers		Estimated length of hybridized DNA fragments
		Hybridizing DNA fragments moving between size markers			Is greater than	Is less than
		1	MS-BN1-EcoRI		Is greater than	Is less than	214014057	2450-	2266bp(^*)＞14kbp
2	MS-BN1-EcoRV	1	MS-BN1-EcoRI	115914057	1700-	1.4kbp(^*)＞14kbp	214014057	2450-	2266bp(^*)＞14kbp
2	MS-BN1-EcoRV	4	MS-BN1-HpaI	115914057	1700-	1.4kbp(^*)＞14kbp	19862140	21402450	1990bp2229bp
5	MS-BN1-AflIII	4	MS-BN1-HpaI	24502140514	28382450805	2477bp(^)2250bp552bp(^)	19862140	21402450	1990bp2229bp
5	MS-BN1-AflIII	6	MS-BN1-NdeI	24502140514	28382450805	2477bp(^)2250bp552bp(^)	50775077	1405714057	10kbp6510bp
7	Non-transgenic WOSR	6	MS-BN1-NdeI	-	-	-	50775077	1405714057	10kbp6510bp
7	Non-transgenic WOSR	8	Control plasmid DNA-BamHI	-	-	-	17002450	19862838	1966bp(^)2607bp(^)

(^*) The length of these fragments is the length predicted from the restriction map of plasmid pTHW 107.

4.1.2.RF-BN1

The restriction patterns obtained after digestion of the genomic DNA of RF-BN1 with different restriction enzymes are given in FIG. 3 and summarized in Table 4.

Table 4: restriction map of RF-BN1

Number of swimming strokes	Sample application of DNA	Hybridizing DNA fragments moving between size markers		Estimated length of hybridized DNA fragments
		Hybridizing DNA fragments moving between size markers			Is greater than	Is less than
		1	MS-BN1-BamHI		Is greater than	Is less than	805170024505077	10991986283814057	814bp1849bp(^)2607bp(^)6580bp
2	MS-BN1-EcoRI	1	MS-BN1-BamHI	805198650775077	115924501405714057	1094bp2149bp7000bp10kbp	805170024505077	10991986283814057	814bp1849bp(^)2607bp(^)6580bp
2	MS-BN1-EcoRI	3	MS-BN1-EcoRV	805198650775077	115924501405714057	1094bp2149bp7000bp10kbp	50775077	1405714057	5.4kbp8kbp
4	MS-BN1-HindIII	3	MS-BN1-EcoRV	170024502450	214028382838	1969bp2565bp2635bp	50775077	1405714057	5.4kbp8kbp
4	MS-BN1-HindIII	6	Non-transgenic WOSR	170024502450	214028382838	1969bp2565bp2635bp	-	-	-
5	Control plasmid DNA-BamHI	6	Non-transgenic WOSR	170024505077	1986283814057	1849bp(^)2607bp(^)8100bp	-	-	-

(^*) The length of these fragments was predicted from the restriction map obtained by digesting pTHW118 vector with BamHI.

4.2. Identification of flanking regions

First the flanking regions of these events in spring OSR, which had generated elite events MS-BN1 and RF-BN1, were identified and then examined for WOSR.

Identification of the flanking region of MS-BN1

4.2.1.1. Right (5') flank region

For the MS-BN1 right border flanking region sequence, the polymerase chain reaction mediated by a ligation reaction with extended capture (T * rmanen et al, 1993, NAR 20: 5487-.

Oligonucleotides used as linker preparations were:

MDB248：(SEQ ID No.3)

5′CAT.GCC.CTG.ACC.CAG.GCT.AAG.TAT.TTT.AAC.TTT.AAC.CAC.TTT.GCT.CCG.ACA.GTC.CCA.TTG

MDB249：(SEQ ID No.4)

5′CAA.TGG.GAC.TGT.CGG.AGG.ACT.GAG.GGC.CAA.AGC.TTG.GCT.CTT.AGC.CTG.GGT.CAG.GGC.ATG

after the joint is prepared, the joint is usedBiotinylated gene-specific primers, first strand synthesized from NcoI-digested genomic MS-BN1 DNA:

	sequence (5 '→ 3')	Position in pTHW107
	sequence (5 '→ 3')	Position in pTHW107	Biotinylated primer MDB247	CCG.TCA.CCG.AGA.TCT.GAT.CTC.ACG.CG(SEQ ID No.5)	322←347

The linker is then ligated to the first strand DNA, and the linker is coupled to magnetic beads from which the non-biotinylated strand is eluted. The DNA was amplified by large scale PCR using the following primers:

	sequence (5 '→ 3')	Position in pTHW107
	sequence (5 '→ 3')	Position in pTHW107	Adaptor primer MDB250	GCACTGAGGGCCAAAGCTTGGCTC(SEQ ID No.6)	-----
T-DNA primer MDB251	GGA.TCC.CCC.GAT.GAG.CTA.AGC.TAG.C(SEQ ID No.7)	293←317	Adaptor primer MDB250	GCACTGAGGGCCAAAGCTTGGCTC(SEQ ID No.6)	-----

The PCR produced a fragment of about 1150 bp. The right border fragment was eluted from the agarose gel,

nested PCR was performed on 100-fold dilutions of this DNA with the following primers:

	sequence (5 '→ 3')	Position in pTHW107
	sequence (5 '→ 3')	Position in pTHW107	Nested adaptor primer MDB254	CTTAGCCTGGGTCAGGGCATG(SEQ ID No.8)	-----
T-DNA primer MDB258	CTA.CGG.CAA.TGT.ACC.AGC.TG(SEQ ID No.9)	224←243	Nested adaptor primer MDB254	CTTAGCCTGGGTCAGGGCATG(SEQ ID No.8)	-----

This reaction produced an approximately 1000bp fragment which was eluted from agarose gel, purified, and ligated into pGem * -T vector. Recombinant plasmid DNA was screened by standard PCR reactions with the following primers:

	sequence (5 '→ 3')	Position in pTHW107
	sequence (5 '→ 3')	Position in pTHW107	SP6 primer	TAA.TAC.GAC.TCA.CTA.TAG.GGC.GA(SEQ ID No.10)	- (SP 6 promoter in pGem * -T vector)
T7 primer	TTT.AGG.TGA.CAC.TAT.AGA.ATA.C(SEQ ID No.11)	- (T7 promoter in pGem * -T vector)	SP6 primer	TAA.TAC.GAC.TCA.CTA.TAG.GGC.GA(SEQ ID No.10)	- (SP 6 promoter in pGem * -T vector)
T7 primer	TTT.AGG.TGA.CAC.TAT.AGA.ATA.C(SEQ ID No.11)	- (T7 promoter in pGem * -T vector)	T-DNA primer MDB201	gCT.TGG.ACT.ATA.ATA.CCT.GAC(SEQ ID No.12)	143←163

The above reaction produced the following fragments: SP 6-T7: 1224bp

SP6-MDB201：1068bp

T7-MDB201：1044bp

The right border fragment was purified and sequenced (SEQ ID No.13) yielding 953bp in which bp1-867 corresponds to plant DNA and bp868 to 953 to T-DNA of pTHW 107.

4.2.1.2. left (3') flanking region of MS-BN1

The sequences flanking The left border region of The transgene inserted in The MS-BN1 event were determined by The thermal asymmetric staggered (TAIL-) PCR method described by Liu et al (1995, Plant Journal 8 (3): 457-463). The method employs three nested specific primers that are reacted sequentially with a short Arbitrary Degenerate (AD) primer, thereby allowing thermal control of the relative amplification efficiency of specific and non-specific products. Specific primers were selected to anneal to the borders of the transgene and based on their annealing conditions. Small amounts (5. mu.l) of the second and third PCR products to be purified were analyzed on a 1% agarose gel. The third PCR product was used for preparative amplification, purified and sequenced on an automated sequencer using the DyeDeoxy Terminator cycle kit.

The primers used were as follows:

	sequence (5 '→ 3')	Position in pTHW107
	sequence (5 '→ 3')	Position in pTHW107	Degenerate primer MDB611	NgT.CgA.SWg.TNT.WCA.A(SEQ ID No.14)	-----
Initial TAILMDB259	gTg.Cag.ggA.AgC.ggT.TAA.CTg.g(SEQ ID No.15)	7164→4186	Degenerate primer MDB611	NgT.CgA.SWg.TNT.WCA.A(SEQ ID No.14)	-----
Initial TAILMDB259	gTg.Cag.ggA.AgC.ggT.TAA.CTg.g(SEQ ID No.15)	7164→4186	Second time TAILMDB260	CCT.TTg.gAg.TAA.ATg.gTg.TTg.g(SEQ ID No.16)	4346→4366
Third time TAILHCA24	gCg.AAT.gTA.TAT.TAT.ATg.CA(SEQ ID No.17)	4738→4757	Second time TAILMDB260	CCT.TTg.gAg.TAA.ATg.gTg.TTg.g(SEQ ID No.16)	4346→4366

Wherein: n ═ a, C, T, or g; s ═ C or g; w is A or T

The fragment amplified with HCA24-MDB611 was approximately 540bp, of which 537bp had been sequenced (3' flank: SEQ ID No 18). The sequence between bp1 and bp180 comprises pTHW107 DNA, while the sequence between bp181 and bp537 corresponds to plant DNA.

4.2.1.3. Identification of target site deletions

The insertion site of the transgene was identified using the sequence corresponding to the flanking region of the transgene as a primer and the wild type brassica napus species Drakkar as a template.

The primers used were as follows:

position in 5 'flank position in 3' flank position

Sequence (5 '→ 3') (SEQ ID No 13) (SEQ ID No 18)

VDS51 TgA.CAC.TTT.gAg.CCA.CTC.g 733→751 -----

(SEQ ID No. 19)

HCA48 GgA.ggg.TgT.TTT.Tgg.TTA.TC ----- 189←-208

(SEQ ID No. 20)

The reaction yielded a 178bp fragment (SEQ ID No 21) in which bp132 to 150 corresponded to the deletion of the target site.

Identification of the insertion region of MS-BN1

Based on the identification of the flanking regions and the deletion of the target site, the insertion region of MS-BN1(SEQ id no 22) can be determined:

1-822: bp46-867 of 5' flanking region SEQ ID No 13

823-841 target site deletion of bp132-150 of SEQ ID No 21

842-1198: 3' flanking region SEQ ID No 18 bp181 to 537

Identification of the flanking region of RF-BN1

The RF-BN1 boundary flanking region was determined by vector rette-PCR (using vector rette and subvectoret PCR to isolate the transgene flanking DNA, Maxine J.Allen, Andrew Collick and Alec J.Jeffreys PCR methods and applications-1994 (4), pp. 71-75) with HindIII digested RF-BN1 genomic DNA as template. The vector linker was prepared using the MDB248(SEQ ID No.3) primer and the MDB249(SEQ ID No.4) primer described above.

4.2.2.1. Right (5') flanking region of BN-RF1

The primers used were as follows:

	sequence (5 '→ 3')	Position in pTHW118
	sequence (5 '→ 3')	Position in pTHW118	Vectorette primer MDB250	GCA.CTG.AGG.GCC.AAA.GCT.TGG.CTC(SEQ ID No.6)	-----
Vectorette primer MDB254	CTT.AGC.CTG.GGT.CAG.GGC.ATG(SEQ ID No.8)	-----	Vectorette primer MDB250	GCA.CTG.AGG.GCC.AAA.GCT.TGG.CTC(SEQ ID No.6)	-----
Vectorette primer MDB254	CTT.AGC.CTG.GGT.CAG.GGC.ATG(SEQ ID No.8)	-----	T-DNA primer MDB251	GGA.TCC.CCC.GAT.GAG.CTA.AGC.TAG.C(SEQ ID No.7)	293←317
T-DNA introduction of MDB193	TCA.TCT.ACG.GCA.ATG.TAC.CAG.C(SEQ ID No.23)	226←247	T-DNA primer MDB251	GGA.TCC.CCC.GAT.GAG.CTA.AGC.TAG.C(SEQ ID No.7)	293←317
T-DNA introduction of MDB193	TCA.TCT.ACG.GCA.ATG.TAC.CAG.C(SEQ ID No.23)	226←247	T-DNA primer MDB258	CTA.CGG.CAA.TGT.ACC.AGC.TG(SEQ ID No.9)	224←243
T-DNA primer MDB201	GCT.TGG.ACT.ATA.ATA.CCT.GAC(SEQ ID No.12)	143←163	T-DNA primer MDB258	CTA.CGG.CAA.TGT.ACC.AGC.TG(SEQ ID No.9)	224←243

The reaction yielded a 1077bp fragment (SEQ ID No.24) in which bp46-881 corresponds to plant DNA and bp882-1060 corresponds to T-DNA of pTHW 118.

4.2.2.2. left (3') flanking region of BN-RF1

To identify the 3' flanking region of elite event BN-RF1, a TAIL PCR was performed as described above, using one arbitrary degenerate primer and multiple primers located near the left border in T-DNA.

The primers used were:

any degenerate primer:

MDB286 NTg.CgA.SWg.ANA.WgA.A (SEQ ID No.25)

wherein: n ═ a, C, T, or g; s ═ C or g; w is A or T

T-DNA primer:

MDB314 gTA.ggA.ggT.Tgg.gAA.gAC.C(SEQ ID No.26)

MDB315 ggg.CTT.TCT.ACT.AgA.AAg.CTC.TCg.g (SEQ IDNo.27)

MDB316 CCg.ATA.ggg.AAg.TgA.TgT.Agg.Agg(SEQ ID No.28)

an approximately 2000bp fragment was obtained. This fragment was cloned into pGem * -T vector and used as template for PCR reaction using the following primers:

plant DNA primers:

MDB288 ATg.CAg.CAA.gAA.gCT.Tgg.Agg SEQ ID No.29)

T-DNA primer:

MDB314 gTA.ggA.ggT.Tgg.gAA.gAC.C (SEQ ID No.26)

the reaction produced a fragment of about 1500bp (SEQ ID No.30), where bp17-182 corresponds to the T-DNA of plasmid pTHW118 and bp183-1457 corresponds to the plant DNA.

4.2.2.3. Molecular analysis of target site deletions

Cloning of the target site deletion by TAIL-PCR (see above), with wild type genomic DNA specific primers directed to the T-DNA insert upstream of the insert and plant DNA specific primers:

any degenerate primer:

MDB286 NTg.CgA.SWg.ANA.WgA.A (SEQ ID No.25)

wherein: n ═ a, C, T, or g; s ═ C or g; w is A or T

Plant DNA primers:

MDB269 ggTTTTCggAggTCCgAgACg (SEQ ID No.31)

MDB283 CTTggACCCCTAggTAAATgC(SEQ ID No.32)

MDB284 gTACAAAACTTggACCCCTAgg(SEQ ID No.33)

a fragment of about 1068bp (SEQ ID No.34) was obtained, in which:

53-83: 5' flanking region

84-133: deletion of target site

134-1055: 3' flanking region

By inserting the T-DNA, the 51bp target site was deleted. Comparison of the wild-type genoma sequence with the Rf3 genoma sequence shows the presence of stuffer DNA at the right border junction. This filling at the right border

The 5 'end of the TCTCG sequence is flanked by TCA and the 3' end is flanked by CGA. These triplets were also found to exist at the breakpoint of the deletion at the target site and at the breakpoint of the T-DNA, respectively. Searching through distant plant sequences reveals the likely origin of the filler DNA. The tca.tctctcg.cga sequence is also located at the 3' end of the plant DNA from which the target site is deleted. It is the core sequence of two identical 13bp repeats located 209bp downstream of the breakpoint of the target site deletion.

An RF-BN1 insertion region can be defined as a left flanking region, a target site deletion and a right flanking region comprising:

1-836: 5' flanking region (bp 46-881 of SEQ ID No 24)

837-887: deletion of target site (bp 84-133 of SEQ ID No.34)

888 + 21623' flanking region (bp 183-1457 of SEQ ID No.30)

4.3. Genetic analysis of Geneoma

Molecular and phenotypic analysis was performed on multiple generations of progeny plants to check the genetic stability of the two event insertions.

T₀，T₁And T₂Southern blot analysis of progeny plants compared event MS-BN1 and RF-BN 1. The resulting pattern is the same in different generations for each event. This indicates that the molecular configuration of the transgene is stable in plants containing MS-BN1 and RF-BN 1.

The MS-BN1 and RF-BN1 events showed that their respective transgenes segregated as mendelian of a single genoma in at least the next three generations, indicating that the insertions were stable.

Based on the above results, MS-BN1 and MS-RF1 were identified as elite events.

4.4. Identification of flanking sequences of MS-BN1 and RF-BN1 in WOSR

Elite events MS-BN1 and RF-BN1 in WOSR were determined using primers developed based on the flanking sequences of these events in spring oilseed rape.

The right (5') flanking sequence of MS-BN1 WOSR was determined using a T-DNA primer (SEQ ID No.12) and a primer located in the plant DNA at the right border of MS-BN 1:

VDS57：5’-gCA.TgA.TCT.gCT.Cgg.gAT.ggC-3’(SEQ ID No.35)

the reaction produced an approximately 909bp fragment (SEQ ID No.36) having a sequence substantially similar to that of SEQ ID No.13 (starting at nucleotide 98).

The left (3') flanking sequence of MS-BN1 WOSR was determined using a T-DNA primer (SEQ ID No.17) and a primer located in plant DNA at the left border of MS-BN 1:

HCA68：5’-CCA.TAT.Acg.CCA.gAg.Agg.AC-3’(SEQ ID No.37)

the reaction produced an approximately 522bp fragment (SEQ ID No.38) having a sequence substantially similar to the sequence of SEQ ID No. 18.

The right (3') flanking sequence of RF-BN1 WOSR was determined using a T-DNA primer (SEQ ID No.12) and a primer (SEQ ID No.31) located in the right border plant DNA of RF-BN 1. The reaction produced an approximately 694bp fragment (SEQ ID No.39) whose sequence is essentially similar to that of SEQ ID No.24 (from nucleotide 293 to 980).

The left flank sequence of RF-BN1 WOSR was determined using a T-DNA primer (SEQ ID No.26) and a primer of plant DNA located at the left border of RF-BN1(SEQ ID No. 29). The reaction yielded an approximately 1450bp fragment of which 1279bp had been sequenced (SEQ ID No. 40). The sequence is substantially similar to the sequence of SEQ ID No.30 (from nucleotide 141 to nucleotide 1421).

It was thus confirmed that the left and right border sequences of the elite event MS-BN1 and RF-BN1 were substantially similar in SOSR and WOSR.

Example 5: development of diagnostic tools for identifying controls

The following method was developed to identify any WOSR plant material comprising elite event MS-BN 1.

MS-BN1 and RF-BN1 elite event restriction map identification method

WOSR plants comprising elite event MS-BN1 can be identified by Southern blotting using essentially the same procedures as described in example 4.1. Such WOSR genomic DNA, 1) was treated with the following restriction enzymes: at least two, preferably at least three, in particular at least four, more in particular all the restriction enzymes mentioned, of EcoRI, EcoRV, Ndel, Hpal, AflIII, 2) transfer to a nylon membrane, and 3) hybridization with the 3942bp HindIII fragment of the plasmid pTHW 107. If at least two restriction enzymes are used, the WOSR plant can be identified as possessing elite event MS-BN1, by identifying DNA fragments with fragments of the same length as listed in example 4.1.1. table 3.

WOSR plants comprising elite event RF-BN1 can be identified by Southern blotting using essentially the same procedures as described in example 4.1. Such WOSR genomic DNA, 1) was treated with the following restriction enzymes: BamHI, EcoRI, EcoRV, HindIII, preferably at least three, more preferably all digested with the restriction enzymes described, 2) transfer to nylon membranes, and 3) hybridization with a 2182bp HpaI fragment of plasmid pTHW 118. If at least two restriction enzymes are used, the WOSR plant can be identified as possessing elite event RF-BN1 by identifying DNA fragments with the same length as listed in example 4.1.2. Table 4.

MS-BN1 and RF-BN1 protospecies event polymerase chain reaction identification method

An assay with all appropriate controls was performed before attempting to screen unknown subjects. Existing methods may require optimization of components (template DNA preparations, taq DNA polymerase, primer properties, dNTP's, thermocyclers, etc.) that may differ between different laboratories.

Amplification of endogenous sequences plays a key role in the method. The PCR and thermocycling conditions that must be achieved are: for known transgenic genomic DNA templates, equimolar amounts of endogenous and transgenic sequences can be amplified. When the target endogenous fragment is not amplified or the target sequence is not amplified, it is necessary to optimize the PCR conditions as judged by agarose gel electrophoresis under the same ethidium bromide staining intensity.

5.2.1. Template DNA

Template DNA was prepared using a leaf drill or monoliths according to Edwards et al, (nucleic acids research, 19, p 1349, 1991). When the DNA used is prepared by other methods, it is necessary to perform the test with different amounts of template. Typically 50ng of genomic template DNA will produce the best results.

5.2.2. Positive and negative controls were assigned

The following positive and negative controls should be included in the PCR run:

basic mixture control (DNA negative control). This is a PCR in which no DNA was added to the reaction. When the expected result, i.e., no PCR product, was observed, it was indicated that the PCR mixture was not contaminated with the target DNA.

DNA positive control (genomic DNA sample known to contain transgene sequence). Successful amplification of the positive control indicates that the PCR was run under conditions that allow amplification of the target sequence.

Wild type DNA control. The template DNA provided for this PCR reaction is a PCR prepared from genomic DNA prepared from a non-transgenic plant. When the expected result is observed, i.e., the transgene PCR product is not amplified but the endogenous PCR product is amplified, it indicates that there is no detectable background amplification of the transgene in the genomic DNA sample.

5.2.3. Primer and method for producing the same

The following primers were used which specifically recognized the transgene and the flanking sequences of MS-BN 1:

BNA01：5’-gCT.Tgg.ACT.ATA.ATA.CCT.gAC-3’(SEQ ID 12)

(MDB201) (target: transgene)

BNA02：5’-TgA.CAC.TTT.gAg.CCA.CTC.g-3’(SEQ ID 19)

(VDS51) (target: plant DNA)

To identify plant material containing RF-BN1, the following primers were used that specifically recognized the transgene and the flanking sequences of RF-BN 1:

BNA03：5’-TCA.TCT.ACg.gCA.ATg.TAC.CAg.C-3’(SEQ ID 23)

(MDB193) (target: transgene)

BNA04：5’-Tgg.ACC.CCT.Agg.TAA.ATg.CC-3’(SEQ ID 41)

(MDB268) (target: plant DNA)

Primers targeting endogenous sequences are always included in the PCR mix. These primers can be used as internal controls in unknown samples as well as in DNA positive controls. A positive result for the endogenous primer pair indicates that there is sample DNA of sufficient quality in the genomic DNA preparation to produce a PCR product. The endogenous primers used were:

BNA05：5’-AAC.gAg.TgT.CAg.CTA.gAC.CAg.C-3’(SEQ ID42)

BNA06：5’-CgC.AgT.TCT.gTg.AAC.ATC.gAC.C-3’(SEQ ID 43)

5.2.4. amplification of fragments

The expected amplified fragments in the PCR reaction were:

primer pair BNA05-BNA 06: 394bp (endogenous control)

Primer pair BNA01-BNA 02: 280bp (MS-BN1 elite event)

Primer pair BNA03-BNA 04: 215bp (RF-BN1 elite event)

5.2.5.PCR conditions

The PCR mix of the 50. mu.l reaction system contained:

5. mu.l template DNA

Mu.l 10 Xamplification buffer (supplied with Taq polymerase)

1μl 10mM dNTP’s

1 μ l BNA01(MS-BN1) or BNA03(RF-BN1) (10pmoles/μ l)

1 μ l BNA02(RF-BN1) or BNA04(RF-BN1) (10pmoles/μ l)

0.5μl BNA05(10pmoles/μl)

0.5μl BNA06(10pmoles/μl)

0.2. mu.l Taq DNA polymerase (5 units/. mu.l)

Adding water to 50 μ l

For best results, the following thermal cycling parameters should be followed:

95 ℃ for 4 minutes

Then: 1 minute at 95 DEG C

1 minute at 57 DEG C

72 ℃ for 2 minutes

Performing 5 cycles

Then at 92 ℃ for 30 seconds

30 seconds at 57 DEG C

1 minute at 72 DEG C

Performing 22 to 25 cycles

Then: 5 minutes at 72 DEG C

5.2.6. Agarose gel analysis

10 to 20. mu.l of the PCR sample and the appropriate molecular weight marker (e.g.100 bp gradient PHARMACLA) are loaded onto a 1.5% agarose gel (Tris-borate buffer).

5.2.7. Result verification

Data for a single PCR cycle and a single PCR mixture of transgenic plant DNA samples cannot be used unless 1) the DNA positive control shows the expected PCR product (transgene and endogenous fragment), 2) the PCR amplified DNA negative control is negative (no fragment) and 3) the wild type DNA control shows the expected result (endogenous fragment amplification).

Lanes showing visible amounts of transgenic and endogenous PCR products of the expected size indicate that their corresponding plants (genomic template DNA prepared therefrom) have inherited the MS-BN1 and/or RF-BN1 elite events. Lanes showing no visible amount of one of the transgenic PCR products and showing a visible amount of the endogenous PCR product indicate that its corresponding plant (genomic template DNA prepared therefrom) does not contain the elite event. Lanes that do not show visible amounts of endogenous and transgenic PCR products indicate that the quality and/or amount of genomic DNA is not capable of producing PCR products. These plants were not evaluated. The preparation of genomic DNA should be repeated and a new round of PCR with appropriate controls must be performed.

5.2.8. Identification of MS-BN1 and RF-BN1 using discriminating PCR

WOSR leaf material from plants comprising MS-BN1, RF-BN1 or another transgenic event was tested as described above. Samples from WOSR wild-type served as negative controls.

The results of the PCR analysis are illustrated in FIGS. 4 and 5.

FIG. 4 illustrates the identification of MS-BN1 in two WOSR samples by elite event PCR identification (lanes 1 and 2). Lane 1 was considered to contain the elite event because a 280bp band was detected, whereas the sample of Lane 2 did not contain MS-BN 1.

FIG. 5 illustrates the identification of RF-BN1 in two WOSR samples by elite event PCR identification (lanes 1 and 2). Lane 1 was considered to contain the elite event because a 215bp band was detected, whereas the sample of Lane 2 did not contain RF-BN 1.

Example 6: production of hybrid seed with MS-BN1 and RF-BN1 in WOSR

A male sterile WOSR plant comprising MS-BN1 is crossed with a WOSR plant homozygous for RF-BN 1. Hybrid seed from MS-BN1 was collected and deposited with the ATCC under ATCC accession number PTA-730.

The hybrid seed is planted in a field. Plants were found to be 100% fertile and showed the best agronomic characteristics. The hybrid plants either contain both MS-BN1 and RF-BN1, or only the RF-BN1 event.

Example 7: introduction of MS-BN1 and RF-BN1 into preferred WOSR cultivars

Elite event MS-BN1 and RF-BN1, respectively, were introduced into a number of agriculturally important WOSR cultivars by repeated backcrossing of plants containing event MS-BN1 or RF-BN 1.

It was observed that the incorporation of elite event genes into these cultivars did not significantly affect any of the latter's desired phenotypic or agronomic characteristics (non-linkage hindrance), whereas the expression of the transgene met commercially acceptable levels as determined by glufosinate tolerance. This confirms the status of events MS-BN1 and RF-BN1 as elite events.

The term "plant" as used in the following claims encompasses plant tissue at any stage of maturation, and any cell, tissue or organ taken from or derived from such a plant, including without limitation any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture or protoplast, unless specifically indicated otherwise.

Seeds comprising elite event MS-BN1 and elite event RF-BN1 or comprising elite event RF-BN1 alone were deposited at the American Tissue culture collection (American Tissue culture collection) under accession number PTA-730.

Sequence listing

<110> Arwinis crop science

<120> hybrid winter oilseed rape and production method thereof

<130>EE-BN1

<140>

<141>

<160>43

<170>PatentIn Ver.2.0

<210>1

<211>4946

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: T-DNA of plasmid pTHW107

<220>

<221> misc _ feature

<222>(964)..(4906)

<223> Hind III fragment

<400>1

aattacaacg gtatatatcc tgccagtact cggccgtcga actcggccgt cgagtacatg 60

gtcgataaga aaaggcaatt tgtagatgtt aattcccatc ttgaaagaaa tatagtttaa 120

atatttattg ataaaataac aagtcaggta ttatagtcca agcaaaaaca taaatttatt 180

gatgcaagtt taaattcaga aatatttcaa taactgatta tatcagctgg tacattgccg 240

tagatgaaag actgagtgcg atattatgtg taatacataa attgatgata tagctagctt 300

agctcatcgg gggatcctag acgcgtgaga tcagatctcg gtgacgggca ggaccggacg 360

gggcggtacc ggcaggctga agtccagctg ccagaaaccc acgtcatgcc agttcccgtg 420

cttgaagccg gccgcccgca gcatgccgcg gggggcatat ccgagcgcct cgtgcatgcg 480

cacgctcggg tcgttgggca gcccgatgac agcgaccacg ctcttgaagc cctgtgcctc 540

cagggacttc agcaggtggg tgtagagcgt ggagcccagt cccgtccgct ggtggcgggg 600

ggagacgtac acggtcgact cggccgtcca gtcgtaggcg ttgcgtgcct tccaggggcc 660

cgcgtaggcg atgccggcga cctcgccgtc cacctcggcg acgagccagg gatagcgctc 720

ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc tgcggctcgg tacggaagtt 780

gaccgtgctt gtctcgatgt agtggttgac gatggtgcag accgccggca tgtccgcctc 840

ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggtcc attgttcttc tttactcttt 900

gtgtgactga ggtttggtct agtgctttgg tcatctatat ataatgataa caacaatgag 960

aacaagcttt ggagtgatcg gagggtctag gatacatgag attcaagtgg actaggatct 1020

acaccgttgg attttgagtg tggatatgtg tgaggttaat tttacttggt aacggccaca 1080

aaggcctaag gagaggtgtt gagaccctta tcggcttgaa ccgctggaat aatgccacgt 1140

ggaagataat tccatgaatc ttatcgttat ctatgagtga aattgtgtga tggtggagtg 1200

gtgcttgctc attttacttg cctggtggac ttggcccttt ccttatgggg aatttatatt 1260

ttacttacta tagagctttc ataccttttt tttaccttgg atttagttaa tatataatgg 1320

tatgattcat gaataaaaat gggaaatttt tgaatttgta ctgctaaatg cataagatta 1380

ggtgaaactg tggaatatat atttttttca tttaaaagca aaatttgcct tttactagaa 1440

ttataaatat agaaaaatat ataacattca aataaaaatg aaaataagaa ctttcaaaaa 1500

acagaactat gtttaatgtg taaagattag tcgcacatca agtcatctgt tacaatatgt 1560

tacaacaagt cataagccca acaaagttag cacgtctaaa taaactaaag agtccacgaa 1620

aatattacaa atcataagcc caacaaagtt attgatcaaa aaaaaaaaac gcccaacaaa 1680

gctaaacaaa gtccaaaaaa aacttctcaa gtctccatct tcctttatga acattgaaaa 1740

ctatacacaa aacaagtcag ataaatctct ttctgggcct gtcttcccaa cctcctacat 1800

cacttcccta tcggattgaa tgttttactt gtaccttttc cgttgcaatg atattgatag 1860

tatgtttgtg aaaactaata gggttaacaa tcgaagtcat ggaatatgga tttggtccaa 1920

gattttccga gagctttcta gtagaaagcc catcaccaga aatttactag taaaataaat 1980

caccaattag gtttcttatt atgtgccaaa ttcaatataa ttatagagga tatttcaaat 2040

gaaaacgtat gaatgttatt agtaaatggt caggtaagac attaaaaaaa tcctacgtca 2100

gatattcaac tttaaaaatt cgatcagtgt ggaattgtac aaaaatttgg gatctactat 2160

atatatataa tgctttacaa cacttggatt tttttttgga ggctggaatt tttaatctac 2220

atatttgttt tggccatgca ccaactcatt gtttagtgta atactttgat tttgtcaaat 2280

atatgtgttc gtgtatattt gtataagaat ttctttgacc atatacacac acacatatat 2340

atatatatat atatattata tatcatgcac ttttaattga aaaaataata tatatatata 2400

tagtgcattt tttctaacaa ccatatatgt tgcgattgat ctgcaaaaat actgctagag 2460

taatgaaaaa tataatctat tgctgaaatt atctcagatg ttaagatttt cttaaagtaa 2520

attctttcaa attttagcta aaagtcttgt aataactaaa gaataataca caatctcgac 2580

cacggaaaaa aaacacataa taaatttgaa tttcgaccgc ggtacccgga attcgagctc 2640

ggtacccggg gatcttcccg atctagtaac atagatgaca ccgcgcgcga taatttatcc 2700

tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta 2760

atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta 2820

acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt 2880

aagaaacttt attgccaaat gtttgaacga tctgcttcgg atcctctaga gccggaaagt 2940

gaaattgacc gatcagagtt tgaagaaaaa tttattacac actttatgta aagctgaaaa 3000

aaacggcctc cgcaggaagc cgtttttttc gttatctgat ttttgtaaag gtctgataat 3060

ggtccgttgt tttgtaaatc agccagtcgc ttgagtaaag aatccggtct gaatttctga 3120

agcctgatgt atagttaata tccgcttcac gccatgttcg tccgcttttg cccgggagtt 3180

tgccttccct gtttgagaag atgtctccgc cgatgctttt ccccggagcg acgtctgcaa 3240

ggttcccttt tgatgccacc cagccgaggg cttgtgcttc tgattttgta atgtaattat 3300

caggtagctt atgatatgtc tgaagataat ccgcaacccc gtcaaacgtg ttgataaccg 3360

gtaccatggt agctaatttc tttaagtaaa aactttgatt tgagtgatga tgttgtactg 3420

ttacacttgc accacaaggg catatataga gcacaagaca tacacaacaa cttgcaaaac 3480

taacttttgt tggagcattt cgaggaaaat ggggagtagc aggctaatct gagggtaaca 3540

ttaaggtttc atgtattaat ttgttgcaaa catggactta gtgtgaggaa aaagtaccaa 3600

aattttgtct caccctgatt tcagttatgg aaattacatt atgaagctgt gctagagaag 3660

atgtttattc tagtccagcc acccacctta tgcaagtctg cttttagctt gattcaaaaa 3720

ctgatttaat ttacattgct aaatgtgcat acttcgagcc tatgtcgctt taattcgagt 3780

aggatgtata tattagtaca taaaaaatca tgtttgaatc atctttcata aagtgacaag 3840

tcaattgtcc cttcttgttt ggcactatat tcaatctgtt aatgcaaatt atccagttat 3900

acttagctag atatccaatt ttgaataaaa atagctcttg attagtaaac cggatagtga 3960

caaagtcaca tatccatcaa acttctggtg ctcgtggcta agttctgatc gacatggggt 4020

taaaatttaa attgggacac ataaatagcc tatttgtgca aatctcccca tcgaaaatga 4080

cagattgtta catggaaaac aaaaagtcct ctgatagaag tcgcaaagta tcacaatttt 4140

ctatcgagag atagattgaa agaagtgcag ggaagcggtt aactggaaca taacacaatg 4200

tctaaattaa ttgcattcgc taaccaaaaa gtgtattact ctctccggtc cacaataagt 4260

tattttttgg cccttttttt atggtccaaa ataagtgagt tttttagatt tcaaaaatga 4320

tttaattatt tttttactac agtgcccttg gagtaaatgg tgttggagta tgtgttagaa 4380

atgtttatgt gaagaaatag taaaggttaa tatgatcaat ttcattgcta tttaatgtta 4440

aaatgtgaat ttcttaatct gtgtgaaaac aaccaaaaaa tcacttattg tggaccggag 4500

aaagtatata aatatatatt tggaagcgac taaaaataaa cttttctcat attatacgaa 4560

cctaaaaaca gcatatggta gtttctaggg aatctaaatc actaaaatta ataaaagaag 4620

caacaagtat caatacatat gatttacacc gtcaaacacg aaattcgtaa atatttaata 4680

taataaagaa ttaatccaaa tagcctccca ccctataact taaactaaaa ataaccagcg 4740

aatgtatatt atatgcataa tttatatatt aaatgtgtat aatcatgtat aatcaatgta 4800

taatctatgt atatggttag aaaaagtaaa caattaatat agccggctat ttgtgtaaaa 4860

atccctaata taatcgcgac ggatccccgg gaattccggg gaagcttaga tccatggagc 4920

catttacaat tgaatatatc ctgccg 4946

<210>2

<211>4832

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: T-DNA of plasmid pTHW118

<220>

<221> misc _ feature

<222>(1883)..(4065)

<223> HpaI restriction fragment

<400>2

aattacaacg gtatatatcc tgccagtact cggccgtcga actcggccgt cgagtacatg 60

gtcgataaga aaaggcaatt tgtagatgtt aattcccatc ttgaaagaaa tatagtttaa 120

atatttattg ataaaataac aagtcaggta ttatagtcca agcaaaaaca taaatttatt 180

gatgcaagtt taaattcaga aatatttcaa taactgatta tatcagctgg tacattgccg 240

tagatgaaag actgagtgcg atattatgtg taatacataa attgatgata tagctagctt 300

agctcatcgg gggatcctag acgcgtgaga tcagatctcg gtgacgggca ggaccggacg 360

gggcggtacc ggcaggctga agtccagctg ccagaaaccc acgtcatgcc agttcccgtg 420

cttgaagccg gccgcccgca gcatgccgcg gggggcatat ccgagcgcct cgtgcatgcg 480

cacgctcggg tcgttgggca gcccgatgac agcgaccacg ctcttgaagc cctgtgcctc 540

cagggacttc agcaggtggg tgtagagcgt ggagcccagt cccgtccgct ggtggcgggg 600

ggagacgtac acggtcgact cggccgtcca gtcgtaggcg ttgcgtgcct tccaggggcc 660

cgcgtaggcg atgccggcga cctcgccgtc cacctcggcg acgagccagg gatagcgctc 720

ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc tgcggctcgg tacggaagtt 780

gaccgtgctt gtctcgatgt agtggttgac gatggtgcag accgccggca tgtccgcctc 840

ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggtcc attgttcttc tttactcttt 900

gtgtgactga ggtttggtct agtgctttgg tcatctatat ataatgataa caacaatgag 960

aacaagcttt ggagtgatcg gagggtctag gatacatgag attcaagtgg actaggatct 1020

acaccgttgg attttgagtg tggatatgtg tgaggttaat tttacttggt aacggccaca 1080

aaggcctaag gagaggtgtt gagaccctta tcggcttgaa ccgctggaat aatgccacgt 1140

ggaagataat tccatgaatc ttatcgttat ctatgagtga aattgtgtga tggtggagtg 1200

gtgcttgctc attttacttg cctggtggac ttggcccttt ccttatgggg aatttatatt 1260

ttacttacta tagagctttc ataccttttt tttaccttgg atttagttaa tatataatgg 1320

tatgattcat gaataaaaat gggaaatttt tgaatttgta ctgctaaatg cataagatta 1380

ggtgaaactg tggaatatat atttttttca tttaaaagca aaatttgcct tttactagaa 1440

ttataaatat agaaaaatat ataacattca aataaaaatg aaaataagaa ctttcaaaaa 1500

acagaactat gtttaatgtg taaagattag tcgcacatca agtcatctgt tacaatatgt 1560

tacaacaagt cataagccca acaaagttag cacgtctaaa taaactaaag agtccacgaa 1620

aatattacaa atcataagcc caacaaagtt attgatcaaa aaaaaaaaac gcccaacaaa 1680

gctaaacaaa gtccaaaaaa aacttctcaa gtctccatct tcctttatga acattgaaaa 1740

ctatacacaa aacaagtcag ataaatctct ttctgggcct gtcttcccaa cctcctacat 1800

cacttcccta tcggattgaa tgttttactt gtaccttttc cgttgcaatg atattgatag 1860

tatgtttgtg aaaactaata gggttaacaa tcgaagtcat ggaatatgga tttggtccaa 1920

gattttccga gagctttcta gtagaaagcc catcaccaga aatttactag taaaataaat 1980

caccaattag gtttcttatt atgtgccaaa ttcaatataa ttatagagga tatttcaaat 2040

gaaaacgtat gaatgttatt agtaaatggt caggtaagac attaaaaaaa tcctacgtca 2100

gatattcaac tttaaaaatt cgatcagtgt ggaattgtac aaaaatttgg gatctactat 2160

atatatataa tgctttacaa cacttggatt tttttttgga ggctggaatt tttaatctac 2220

atatttgttt tggccatgca ccaactcatt gtttagtgta atactttgat tttgtcaaat 2280

atatgtgttc gtgtatattt gtataagaat ttctttgacc atatacacac acacatatat 2340

atatatatat atatattata tatcatgcac ttttaattga aaaaataata tatatatata 2400

tagtgcattt tttctaacaa ccatatatgt tgcgattgat ctgcaaaaat actgctagag 2460

taatgaaaaa tataatctat tgctgaaatt atctcagatg ttaagatttt cttaaagtaa 2520

attctttcaa attttagcta aaagtcttgt aataactaaa gaataataca caatctcgac 2580

cacggaaaaa aaacacataa taaatttgaa tttcgaccgc ggtacccgga attcgagctc 2640

ggtacccggg gatcttcccg atctagtaac atagatgaca ccgcgcgcga taatttatcc 2700

tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta 2760

atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta 2820

acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt 2880

aagaaacttt attgccaaat gtttgaacga tctgcttcgg atcctctaga ccaagcttgc 2940

gggtttgtgt ttccatattg ttcatctccc attgatcgta ttaagaaagt atgatggtga 3000

tgtcgcagcc ttccgctttc gcttcacgga aaacctgaag cacactctcg gcgccatttt 3060

cagtcagctg cttgctttgt tcaaactgcc tccattccaa aacgagcggg tactccaccc 3120

atccggtcag acaatcccat aaagcgtcca ggttttcacc gtagtattcc ggaagggcaa 3180

gctccttttt caatgtctgg tggaggtcgc tgatacttct gatttgttcc ccgttaatga 3240

ctgctttttt catcggtagc taatttcttt aagtaaaaac tttgatttga gtgatgatgt 3300

tgtactgtta cacttgcacc acaagggcat atatagagca caagacatac acaacaactt 3360

gcaaaactaa cttttgttgg agcatttcga ggaaaatggg gagtagcagg ctaatctgag 3420

ggtaacatta aggtttcatg tattaatttg ttgcaaacat ggacttagtg tgaggaaaaa 3480

gtaccaaaat tttgtctcac cctgatttca gttatggaaa ttacattatg aagctgtgct 3540

agagaagatg tttattctag tccagccacc caccttatgc aagtctgctt ttagcttgat 3600

tcaaaaactg atttaattta cattgctaaa tgtgcatact tcgagcctat gtcgctttaa 3660

ttcgagtagg atgtatatat tagtacataa aaaatcatgt ttgaatcatc tttcataaag 3720

tgacaagtca attgtccctt cttgtttggc actatattca atctgttaat gcaaattatc 3780

cagttatact tagctagata tccaattttg aataaaaata gctcttgatt agtaaaccgg 3840

atagtgacaa agtcacatat ccatcaaact tctggtgctc gtggctaagt tctgatcgac 3900

atggggttaa aatttaaatt gggacacata aatagcctat ttgtgcaaat ctccccatcg 3960

aaaatgacag attgttacat ggaaaacaaa aagtcctctg atagaagtcg caaagtatca 4020

caattttcta tcgagagata gattgaaaga agtgcaggga agcggttaac tggaacataa 4080

cacaatgtct aaattaattg cattcgctaa ccaaaaagtg tattactctc tccggtccac 4140

aataagttat tttttggccc tttttttatg gtccaaaata agtgagtttt ttagatttca 4200

aaaatgattt aattattttt ttactacagt gcccttggag taaatggtgt tggagtatgt 4260

gttagaaatg tttatgtgaa gaaatagtaa aggttaatat gatcaatttc attgctattt 4320

aatgttaaaa tgtgaatttc ttaatctgtg tgaaaacacc aaaaaatcac ttattgtgga 4380

ccggagaaag tatataaata tatatttgga agcgactaaa aataaacttt tctcatatta 4440

tacgaaccta aaaacagcat atggtagttt ctagggaatc taaatcacta aaattaataa 4500

aagaagcaac aagtatcaat acatatgatt tacaccgtca aacacgaaat tcgtaaatat 4560

ttaatataat aaagaattaa tccaaatagc ctcccaccct atkacttaaa ctaaaaataa 4620

ccagcgaatg tatattatat gcataattta tatattaaat gtgtataatc atgtataatc 4680

aatgtataat ctatgtatat ggttagaaaa agtaaacaat taatatagcc ggctatttgt 4740

gtaaaaatcc ctaatataat cgcgacggat ccccgggaat tccggggaag cttagatcca 4800

tggagccatt tacaattgaa tatatcctgc cg 4832

<210>3

<211>60

<212>DNA

<213> Artificial

<220>

<223> description of artificial sequences: primer 248

<400>3

catgccctga cccaggctaa gtattttaac tttaaccact ttgctccgac agtcccattg 60

<210>4

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 249

<400>4

caatgggact gtcggaggac tgagggccaa agcttggctc ttagcctggg tcagggcatg 60

<210>5

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 247

<400>5

ccgtcaccga gatctgatct cacgcg 26

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 250

<400>6

gcactgaggg ccaaagcttg gctc 24

<210>7

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 251

<400>7

ggatcccccg atgagctaag c tagc 25

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 254

<400>8

cttagcctgg gtcagggcat g 21

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 258

<400>9

ctacggcaat gtaccagctg 20

<210>10

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer SP6

<400>10

taatacgact cactataggg cga 23

<210>11

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer T7

<400>11

tttaggtgac actatagaat ac 22

<210>12

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 201

<400>12

gcttggacta taatacctga c 21

<210>13

<211>953

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the 5' flanking region of MS-BN1

<220>

<221> misc _ feature

<222>(1)..(24)

<223> pGEM * -T vector

<400>13

cccngccgcc atggccgcgg gattcttagc ctgggtcagg gcatgcatgg tgtgatccaa 60

agactttctc ggcccaaata ctaatcatca caagtcatgc atgatctgct cgggatggcc 120

aagaaaaatc gaacccatga caatattcac agttgtaagt tttttaccag tagacaaata 180

ccacttggtt taacatattg taaacttaat atatagaaga tgttcctatt cagaaaataa 240

tatatgtata tatataaaat tttattggcg actcgaggat gcacagaaat ataaaatgtt 300

ggtcgcttag accatctcca atgtatttct ctatttttac ctctaaaata aaggagctct 360

ataatagagg tgggttttgc tccaatgtat ttctttaaaa tagagatctc tacatataga 420

gcaaaatata gaggaatgtt atttcttcct ctataaatag aggagaaaat agcaatctct 480

attttagagg caaaaataga gatbsgttgg agtgattttg cctctaaatg ctattataga 540

ggtagaaata gaggtgggtt ggagatgctc ttactatttt catagtaggt gaaaacttga 600

aactagaaag ctttggagtg tacgagtgga aaacctctct ttgtagaaac atacacatgc 660

catttagtta actagttgac atagattttt gagtcagata actttaagaa tatatatgtt 720

tggatgagag tttgacactt tgagccactc gaaggacaaa ttttaaaaac ttgtgggatg 780

ctgtggccat aaaccttgag gacvstttga tcatattcta ttaactacag tacgaatatg 840

attcgacctt tgcaattttc tcttcaggta ctcggccgtc gaactcggcc gtcgagtaca 900

tggtcgataa gaaaaggcaa tttgtagatg ttaattccca tcttgaaaga aat 953

<210>14

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 611

<400>14

ngtcgaswgt ntwcaa 16

<210>15

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 259

<400>15

gtgcagggaa gcggttaact gg 22

<210>16

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 260

<400>16

cctttggagt aaatggtgtt gg 22

<210>17

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 24

<400>17

gcgaatgtat attatatgca 20

<210>18

<211>537

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the flanking region of MS-BN 13

<400>18

gcgaatgtat attatatgca taatttatat attaaatgtg tataatcatg tataatcaat 60

gtataatcta tgtatatggt tagaaaaagt aaacaattaa tatagccggc tatttgtgta 120

aaaatcccta atataatcga cggatccccg ggaattccgg gggaagctta gatccatgga 180

tttgttatga taaccaaaaa caccctcctt tttattataa aggtagggat agctaatctg 240

ttattcggtt ttgattagag atattaatcc cgttttatca agtacagttt gatgtatttt 300

tttgttcgtt ttcattacaa tccaagacaa gttaggttta ttacatttta ccaaaaaaaa 360

aggtttggtt tattgtgaac attgctgcgg tttatttaaa tttgattcta ttcaaaggtc 420

aatccgtatt taacaagtaa actagtcttt atataatctt aaatctaacg atctttgatt 480

tttaaattgc atttanctat gtcctctctg gcgtatatgg tctctttgaa aacactc 537

<210>19

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 51

<400>19

tgacactttg agccactcg 19

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 48

<400>20

ggagggtgtt tttggttatc 20

<210>21

<211>178

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequence comprising deletion of MS-BN1 target site

<400>21

gacactttga gccactcgaa ggacaaattt taaaaacttg tgggatgctg tggccataaa 60

ccttgaggac gctttgatca tattctatta actacagtac gaatatgatt cgacctttgc 120

aattttctct tgttttctaa ttcatatgga tttgttatga taaccaaaaa caccctcc 178

<210>22

<211>1198

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: MS-BN1 insertion region

<400>22

catggtgtga tccaaagact ttctcggccc aaatactaat catcacaagt catgcatgat 60

ctgctcggga tggccaagaa aaatcgaacc catgacaata ttcacagttg taagtttttt 120

accagtagac aaataccact tggtttaaca tattgtaaac ttaatatata gaagatgttc 180

ctattcagaa aataatatat gtatatatat aaaattttat tggcgactcg aggatgcaca 240

gaaatataaa atgttggtcg cttagaccat ctccaatgta tttctctatt tttacctcta 300

aaataaagga gctctataat agaggtgggt tttgctccaa tgtatttctt taaaatagag 360

atctctacat atagagcaaa atatagagga atgttatttc ttcctctata aatagaggag 420

aaaatagcaa tctctatttt agaggcaaaa atagagatbs gttggagtga ttttgcctct 480

aaatgctatt atagaggtag aaatagaggt gggttggaga tgctcttact attttcatag 540

taggtgaaaa cttgaaacta gaaagctttg gagtgtacga gtggaaaacc tctctttgta 600

gaaacataca catgccattt agttaactag ttgacataga tttttgagtc agataacttt 660

aagaatatat atgtttggat gagagtttga cactttgagc cactcgaagg acaaatttta 720

aaaacttgtg ggatgctgtg gccataaacc ttgaggacvs tttgatcata ttctattaac 780

tacagtacga atatgattcg acctttgcaa ttttctcttc aggttttcta attcatatgg 840

atttgttatg ataaccaaaa acaccctcct ttttattata aaggtaggga tagctaatct 900

gttattcggt tttgattaga gatattaatc ccgttttatc aagtacagtt tgatgtattt 960

ttttgttcgt tttcattaca atccaagaca agttaggttt attacatttt accaaaaaaa 1020

aaggtttggt ttattgtgaa cattgctgcg gtttatttaa atttgattct attcaaaggt 1080

caatccgtat ttaacaagta aactagtctt tatataatct taaatctaac gatctttgat 1140

ttttaaattg catttancta tgtcctctct ggcgtatatg gtctctttga aaacactc 1198

<210>23

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 193

<400>23

tcatctacgg caatgtacca gc 22

<210>24

<211>1077

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the RF-BN 15' flanking region

<220>

<221> misc _ feature

<222>(1)..(45)

<223> pGEM * -T vector

<220>

<221> misc _ feature

<222>(1061)..(1077)

<223> pGEM * -T vector

<400>24

gagctctccc atatggtcga cctgcaggcg gccgcactag tgattcttag cctgggtcag 60

ggcatggcat gtctgatggt acatgctaaa tgctatattt cctgtttaaa gtgttaaaat 120

cattttctga tggaactaaa tccagtttta agagtaactg acaagtacaa ttaagcacaa 180

caatataata gtagtaattg gcatctttga ttgttaaata tcaaaacagt aaagttacaa 240

aaaaaaatac caaaccaata atgaagactt ggcggagaca gtgccgtgcg aaggttttcg 300

gaggtccgag acgagttcaa aaatacattt tacataatat atttttcata tatatatata 360

tataacattc aaaagtttga attattacat aaacgttttc taaattttct tcaccaaaat 420

tttataaact aaatttttaa atcatgaaca aaaagtatga atttgtaata taaatacaaa 480

gatacaaatt tttgattgaa atattggtag ctgtcaaaaa agtaaatctt agaatttaaa 540

ttaactatag taaactatat attgaaaata ttataaattt ttatcaaatt ctcataaata 600

tataaaataa atctaactca tagcatataa aaagaagact aatgtggatc aaaatattta 660

cagtttttta gaagtagaat ctttatagtt ttatttaaaa tatagcaaaa atgatcacaa 720

acctagttay ttaaggagaa gtccaattca aaatcaaata aaaataaaat ctatctaaaa 780

aaatatgtta actaccatgc aaaagtattt tttttgtaat tagaaaccct gaaatttgta 840

caaaacttgg acccctaggt aaatgccttt ttcatctcgc gataagaaaa ggcaatttgt 900

agatgttaat tcccatcttg aaagaaatat agtttaaata tttattgata aaataacaag 960

tcaggtatta tagtccaagc aaaaacataa atttattgat gcaagtttaa attcagaaat 1020

atttcaataa ctgattatat cagctggtac atcgccgtag aatcccgcgc catggcg 1077

<210>25

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 286

<400>25

ntgcgaswga nawgaa 16

<210>26

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 314

<400>26

gtaggaggtt gggaagacc 19

<210>27

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 315

<400>27

gggctttcta ctagaaagct ctcgg 25

<210>28

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 316

<400>28

ccgataggga agtgatgtag gagg 24

<210>29

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 288

<400>29

atgcagcaag aagcttggag g 21

<210>30

<211>1501

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the RF-BN 13' flanking region

<220>

<221> misc _ feature

<222>(1)..(16)

<223> pGEM * -T vector

<220>

<221> misc _ feature

<222>(1458)..(1501)

<223> pGEM * -T vector

<400>30

ccatggccgc gggattgtag gaggttggga agacaggccc agaaagagat ttatctgact 60

cgttttgtgt atagttttca atgttcataa aggaagatgg agacttgaga agtttttttt 120

ggactttgtt tagctttgtt gggcgttttt tttttttgat caataacttt gttgggctta 180

tggtcgataa gcgtgcgcat gtctgatggt acatgctaaa tgctatattt ctgtttaaag 240

tgttaaaatc attttctgat ggaactaaat ccagttttaa gagtaactga caagtacaat 300

taagcacaac aataaaatag tagtaattgg catctttgat tgttaaatat caaaacaata 360

aagttacaaa aaaaaatacc aaaccaataa tgaagacttg gcggagacag tgccgtgcga 420

aggttttcgg aggtccgaga cgagttcaaa aatacatttt acataatata tttttcatat 480

atatatatat atataacatt caaaagtttg aattattaca taaacgtttt ctaaattttc 540

ttcaccaaaa ttttataaac taaaattttt maatcatgaa caaaaagtat gaatttgtaa 600

tataaatacm aagatacaaa tttttgattg aaatattggt agctgtcaaa aaagtaaatc 660

ttagaattta aattaactat agtaaactat atatggaaaa tattataaat ttttatcaaa 720

ttctcataaa tatataaaat aaatctaact catagcatat aaaaagaaga ctaatgtgga 780

tcaaratatt tacagttttt tagaagtaga atctctatag ttttatttaa aatatagcaa 840

aaatgatcac aaacctagtt actttaacca gaagtccaat tcaaaatcaa ataaaaataa 900

aaatctatct aaaaaaatat gttaactacc atgcaaaagt attttttttt gtaattagaa 960

accctgaaat ttgtacaaaa cttggacccc taggtaaatt ccctagaaag tatcctatta 1020

gcgtcgacaa actgttgctc atatttttct ctccttactt tatatcatac actaatatan 1080

gnagatgatc taattaatta ttcatttcca tgctagctaa ttcaagaaaa agaaaaaaaa 1140

ctattatcta aacttatatt cgagcaacac ctcggagata acaggatata tgtcattaat 1200

gaatgcttga actcatctcg cgaactcatc tcgcatcgct tatagccaca aagatccaac 1260

ccctctcttc aatcatatat cagtagtaca atacaaatag atattgtgag cacatatgcc 1320

gtctagtact gatgtgtaca tgtagaggag ccgcaaatgt ttagtcactc caacaaatga 1380

gcatgaccac gcatcttctg atgatgtaca gccgtccctt ttgctctctc aaatatcctc 1440

caagcttctt gctgcataaa tcactagtgc ggccgcctgc aggtcgacca tatgggagag 1500

c 1501

<210>31

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 269

<400>31

ggttttcgga ggtccgagac g 21

<210>32

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 283

<400>32

cttggacccc taggtaaatg c 21

<210>33

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer 284 for description of artificial sequence

<400>33

gtacaaaact tggaccccta gg 22

<210>34

<211>1068

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising a deletion of the RF-BN1 target site

<400>34

cgcgttggga gctctcccat atggtcgacc tgcaggcggc cgcactagtg attcttggac 60

ccctaggtaa atgccttttt caaaagcctc taagcacggt tctgggcggg gagtcagcga 120

gaaaaaaaga tatttcccta gaaagtatcc tattagcgtc gacaaactgt tgctcatatt 180

tttctctcct tactttatat catacactaa tataaaaaga tgatctaatt aattattcat 240

ttccatgcta gctaattcaa gaaaaagaaa aaaactatta tctaaactta tattcgagca 300

acacctcgga gataacagga tatatgttat taatgaatgc ttgaactcat ctcgcgaact 360

catctcgcat cgcttatagc cacaaagatc caacccctct cttcaatcat atatcagtag 420

tacaatacaa atagatattg tgagcacata tgccgtctag tactgatgtg tatatgtaga 480

gganngcaaa tgtttagtca ctccaacaaa tgagcatgac nacgcatctt ctgatgatgt 540

acagccgtcc cttttgctct ctcaaatatc ctccaagctt cttgctgcat ggaatcttct 600

tcttggtgtc tttcatgata acaaaatcta acgagagaga aacccttagt caagaaaaaa 660

caaataaaac tctaacgaga gtgtgtgaga aagtagagag tatgtgtgag tgacggagag 720

aaagtgagac cataaagatg ttgtgcaaag agagcaagac ttaacctata tatactcaca 780

tacacgtaca catcataccc attanagata ataaaaagga aaaaggaaca actaacaagg 840

gaactgtatc ccatacttta tctcatcata catgatgcat aatatattct ttcgtatatc 900

aagaaaaatg agcctgatat ttttttattt cgaaactaaa agagtgtcta tttctctctc 960

ttagagatag tgccatgtca aatttctaag aagtagcaag atttacaaag gaatctaaag 1020

caaccccacg cgcattgtgt tcatttctct cgaccatccc gcggccat 1068

<210>35

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 57

<400>35

gcatgatctg ctcgggatgg c 21

<210>36

<211>909

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the MS-BN 15' flanking region in WOSR

<400>36

tgcatgatct gctcgggatg gccaagaaaa atcgaaccca tgacaatatt cacagttgta 60

agttttttac cagtagacaa ataccacttg gtttaacata ttgtaaactt aatatataga 120

agatgttcct attcagaaaa taatatatgt atatatataa aattttattg gcgactcgag 180

gatgcacaga aatataaaat gttggtcgct tagaccatct ccaatgtatt tctctatttt 240

tacctctaaa ataaaggaac tctataatag aggtgggttt tactccaatg tatttcttta 300

aaatagagat ctctacatat agagcaaaat atagaggaat gttatttctt cctctataaa 360

tagaggagaa aatagcaatc tctattttag aggcaaaaat agagatgggt tggagtgatt 420

ttgcctctaa atgctattat agaggtagaa atagaggtgg gttggagatg ctcttactat 480

tttcatagta ggtgaaaact tgaaactaga aagctttgga gtgtacgagt ggaaaacctc 540

tctttgtaga aacatacaca tgccatttag ttaactagtt gacatagatt tttgagtcag 600

ataactttaa gaatatatat gtttggatga gagtttgaca ctttgagcca ctcgaaggac 660

aaattttaaa aacttgtggg atgctgtggc ccataaacct tgaggacgct ttgatcatat 720

tctattaact acagtacgaa tatgattcga cctttgcaat tttctcttca gtactcggcc 780

gtcgaactcg gccgtcgagt acatggtcga taagaaaagg caatttgtag atgttaattc 840

ccatcttgaa agaaatatag tttaaatatt tattggataa aataacaagt caggtattat 900

agtccaagc 909

<210>37

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 68

<400>37

ccatatacgc cagagaggac 20

<210>38

<211>522

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the MS-BN 13' flanking region in WOSR

<400>38

gcgaatgtat attatatgca taatttatat attaaatgtg tataatcatg tataatcaat 60

gtataatcta tgtatatggt tagaaaaagt aaacaattaa tatagccggc tatttgtgta 120

aaaatcccta atataatcga cggatccccg ggaattccgg gggaagctta gatccatgga 180

tttgttatga taaccaaaaa caccctcctt tttattataa aggtagggat agctaatctg 240

ttattcggtt ttgattagag atattaatcc cgttttatca agtacagttt gatgtatttt 300

tttgttcgtt ttcattacaa tccaagacaa gttaggttta ttacatttta ccaaaaaaaa 360

aggtttggtt tattgtgaac attgctgcgg ttttatttaa atttgattct attcaaaggt 420

caatccgtat ttaacaagta aactagtctt tatataatct taaatctaac gatacttgga 480

tttttaaatt gcatttagct atgtcctctc tggcgtatat gg 522

<210>39

<211>694

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the flanking region of WOSR RF-BN 15

<400>39

ggttttcgga ggtccgagac gagttcaaaa atacatttta cataatatat ttttcatata 60

tatatatata tataacattc aaaagtttga attattacat aaacgttttc taaattttct 120

tcaccaaaat tttataaact aaaattttta aatcatgaac aaaaagtatg aatttgtaat 180

ataaatacaa agatacaaat ttttgattga aatattggta gctgtcaaaa aagtaaatct 240

tagaatttaa attaactata gtaaactata tattgaaaat attataaatt tttatcaaat 300

tctcataaat atataaaata aatctaactc atagcatata aaaagaagac taatgtggat 360

caaaatattt acagtttttt agaagtagaa tctttatagt tttatttaaa atatagcaaa 420

aatgatcaca aacctagtta ctttaaccag aagtccaatt caaaatcaaa taaaaataaa 480

aatctatcta aaaaaatatg ttaactacca tgcaaaagta tttttttttg taattagaaa 540

ccctgaaatt tgtacaaaac ttggacccct aggtaaatgc ctttttcatc tcgcgataag 600

aaaaggcaat ttgtagatgt taattcccat cttgaaagaa atatagttta aatatttatt 660

gataaaataa caagtcaggt attatagtcc aagc 694

<210>40

<211>1279

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: sequences comprising the RF-BN 13' flanking region in WOSR

<400>40

gggggttttt ttttttgatc aataactttg ttgggcttat ggtcgataag cgtgcgcatg 60

tctgatggta catgctaaat gctatatttc tgtttaaagt gttaaaatca ttttctgatg 120

gaactaaatc cagttttaag agtaactgac aagtacaatt aagcacaaca ataaaatagt 180

agtaattggc atctttgatt gttaaatatc aaacaataaa gttcaaaaaa aaataccaac 240

ccaataatga agacttggcg gagacagtgc cgtgcgaagg ttttcggagg tccgagacga 300

gttcaaaaat acattttaca taatatattt ttcatatata tatatatata taacattcaa 360

aagtttgaat tattacataa acgttttcta aattttcttc accaaaattt tataaactaa 420

aatttttaaa tcatgaacaa aaagtatgaa tttgtaatat aaatacaaag atacaaattt 480

ttgattgaaa tattggtagc tgtcaaaaaa gtaaatctta gaatttaaat taactatagt 540

aaactatata ttgaaaatat tataaatttt tatcaaattc tcataaatat ataaaataaa 600

tctaactcat agcatataaa aagaagacta atgtggatca aaatatttac agttttttag 660

aagtagaatc tttatagttt tatttaaaat atagcaaaaa tgatcacaaa cctagttact 720

ttaaccagaa gtccaattca aaatcaaata aaaataaaaa tctatctaaa aaaatatgtt 780

aactaccatg caaaagtatt tttttttgta attagaaacc ctgaaatttg tacaaaactt 840

ggacccctag gtaaattccc tagaaagtat cctattagcg tcgacaaact gttgctcata 900

tttttctctc cttactttat atcatacact aatataaaaa gatgatctaa ttaattattc 960

atttccatgc tagctaattc aagaaaaaga aaaaaaactt attatctaaa cttatattcg 1020

agcaacacct cggagataac aggatatatg tcattaatga atgcttgaac tcatctcgcg 1080

aactcatctc gcatcgctta tagccacaaa gatccaaccc ctctcttcaa tcatatatca 1140

gtagtacaat acaaatagat attgtgagca catatgccgt ctagtactga tgtgtatatg 1200

tagaggagcc gcaaatgttt agtcactcca acaaatgagc atgaccacgc atcttctgat 1260

gatgtacagc cgtcccttt 1279

<210>41

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer 268

(BNA04)

<400>41

tggaccccta ggtaaatgcc 20

<210>42

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer BNA05

<400>42

aacgagtgtc agctagacca gc 22

<210>43

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer BNA06

<400>43

cgcagttctg tgaacatcga cc 22

Claims

A WOSR plant cell characterized in that a DNA fragment between 195 and 235bp in length can be amplified by polymerase chain reaction using genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No 23 and SEQ ID No 41, respectively.
2. Plant cell according to claim 1, characterized in that a DNA fragment of 215bp in length can be amplified by polymerase chain reaction using the genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No 23 and SEQ ID No 41, respectively.
3. The plant cell of claim 1, characterized in that the genomic DNA of said plant cell produces at least two sets of restriction fragments selected from the group consisting of:

i) a set of BamHI fragments, one between 805 and 1099bp long, one between 1700 and 1986bp long, one between 2450 and 2838bp long, and one between 5077 and 14057bp long;

ii) a set of EcoRI fragments, one of which is between 805 and 1159bp, one of which is between 1986 and 2450bp in length, and two of which are between 5077 and 14057bp in length;

iii) a set of EcoRV fragments, both of which are between 5077 and 14057bp in length;

iv) HindIII fragment group, wherein one fragment is between 1700 and 2140bp long and two fragments are between 2450 and 2838bp long;

wherein each restriction fragment hybridizes to a 2182bp long fragment comprising the PTA 29-Bacillus RNase inhibitor sequence under standard stringent conditions, wherein the 2182bp long fragment is obtainable by digestion of the plasmid pTHW118 with HpaI.
4. The plant cell of claim 3, characterized in that the genomic DNA of said plant cell produces at least three groups selected from said group of restriction fragments.
5. The plant cell of claim 4, characterized in that the genomic DNA of said plant cell produces at least four groups selected from said group of restriction fragments.
6. The plant cell of claim 1, further characterized in that the genomic DNA of said plant cell produces at least three sets selected from the group of restriction fragments consisting of:

i) an EcoRI fragment group, wherein one of the EcoRI fragment groups is between 2140 and 2450bp in length, and the length of one of the EcoRI fragment groups is more than 14 kbp;

ii) a set of EcoRV fragments, one of which is between 1159 and 1700bp in length and the other of which is greater than 14kbp in length;

iii) a set of Hpal fragments, one of which is between 1986 and 2140bp in length and one of which is between 2140 and 2450bp in length;

iv) AflIII fragment group, wherein one is between 514 and 805bp in length, one is between 2140 and 2450bp in length, and one is between 2450 and 2838bp in length;

v) NdeI fragment group, both of which are between 5077 and 14057bp in length;

wherein each restriction fragment hybridizes under standard stringency conditions to a 3942bp long fragment comprising the PTA 29-Bacillus RNase sequence, wherein said 3942bp long fragment is obtainable by digesting plasmid pTHW107 with HindIII.
7. The plant cell of claim 6, characterized in that the genomic DNA of said plant cell produces at least four sets selected from said set of restriction fragments.
8. The plant cell of claim 7, characterized in that the genomic DNA of said plant cell produces all five of said groups of restriction fragments.
9. The plant cell of claim 1, further characterized in that a DNA fragment between 260 and 300bp in length can be amplified by polymerase chain reaction using genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively.
10. Plant cell according to claim 9, characterized in that a DNA fragment of 280bp in length can be amplified by polymerase chain reaction using the genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively.
11. The plant cell of claim 1, obtainable by crossing a plant with a WOSR plant, wherein said WOSR plant is obtainable from seed deposited with ATCC as accession number PTA-730.
12. The plant cell of any one of claims 1 to 11, which is a hybrid plant cell.
A WOSR plant cell, characterized in that a DNA fragment of between 260 and 300bp in length can be amplified by polymerase chain reaction using genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively.
14. Plant cell according to claim 13, characterized in that a DNA fragment of 280bp in length can be amplified by polymerase chain reaction using the genomic DNA of said plant cell, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively.
15. The plant cell of claim 13, further characterized in that said plant cell produces at least three sets selected from the following sets of restriction fragments:

i) an EcoRI fragment group, wherein one of the EcoRI fragment groups is between 2140 and 2450bp in length, and the length of one of the EcoRI fragment groups is more than 14 kbp;

ii) a set of EcoRV fragments, one of which is between 1159 and 1700bp in length and the other of which is greater than 14kbp in length;

iii) a set of Hpal fragments, one of which is between 1986 and 2140bp in length and one of which is between 2140 and 2450bp in length;

iv) AflIII fragment group, wherein one is between 514 and 805bp in length, one is between 2140 and 2450bp in length, and one is between 2450 and 2838bp in length;

v) NdeI fragment group, both of which are between 5077 and 14057bp in length;

wherein each restriction fragment hybridizes under standard stringency conditions to a 3942bp long fragment comprising the PTA 29-Bacillus RNase sequence, wherein said 3942bp long fragment is obtainable by digesting plasmid pTHW107 with HindIII.
16. The plant cell of claim 15, characterized in that the genomic DNA of said plant, seed, plant cell or tissue can produce at least four groups selected from said group of restriction fragments.
17. The plant cell of claim 16, characterized in that the genomic DNA of said plant cell produces all five of said sets of restriction fragments.
18. A method of producing hybrid seed, the method comprising:

a) hybridizing a transgenic and male sterile WOSR plant with a fertility restorer WOSR plant, wherein the transgenic and male sterile WOSR plant is characterized in that a DNA fragment with the length of between 260 and 300bp can be amplified by using genome DNA of the plant through polymerase chain reaction, and nucleotide sequences of two primers are respectively SEQ ID No12 and SEQ ID No 19; wherein the fertility restorer WOSR plant comprises a fertility restorer gene stably integrated into the genome, wherein the fertility restorer gene comprises:

-DNA encoding a ribonuclease inhibitor, and

-a constitutive promoter; wherein said DNA is in the same transcription unit and under the control of said constitutive promoter; and is

b) Harvesting the hybrid seed from the male-sterile WOSR plant.
19. The method according to claim 18, wherein said fertility restorer plant is characterized in that a DNA fragment of 215bp in length can be amplified by polymerase chain reaction using genomic DNA of said plant, and the nucleotide sequences of the two primers are SEQ ID No 23 and SEQ ID No 41, respectively.
20. A method of identifying a transgenic plant, or cell or tissue thereof, comprising elite event MS-BN1, the method comprising determining whether genomic DNA of said plant, or cell or tissue thereof, can be used to amplify a DNA fragment of between 260 and 300bp in length by polymerase chain reaction, the nucleotide sequences of the two primers being SEQ ID No12 and SEQ ID No 19, respectively.
21. The method of claim 20, which comprises determining whether a DNA fragment of 280bp in length can be amplified by polymerase chain reaction using genomic DNA from a transgenic plant, or a cell or tissue thereof, using the two primers of SEQ ID No12 and SEQ ID No 19, respectively.
22. A kit for identifying a transgenic plant, cell or tissue thereof comprising elite event MS-BN1, said kit comprising PCR probes for use in a PCR identification method, wherein one PCR probe recognizes a sequence in the transgene and the other PCR probe recognizes a plant DNA sequence within SEQ ID No.13 or 5 'flanking sequence SEQ ID No.18 of the MS-BN 13' flanking region.
23. A kit according to claim 22 for identifying a transgenic plant, cell or tissue thereof comprising elite event MS-BN1, said kit comprising PCR probes having the nucleotide sequences of SEQ ID No.12 and SEQ ID No.19, respectively.
24. A method of identifying a transgenic plant, cell or tissue thereof comprising elite event RF-BN1, the method comprising determining whether genomic DNA of said plant, or cell or tissue thereof can be used to amplify a DNA fragment between 195 and 235bp in length by polymerase chain reaction, the nucleotide sequences of the two primers being SEQ ID No 23 and SEQ ID No 41, respectively.
25. The method of claim 24, which comprises determining whether a genomic DNA of the transgenic plant, or cells or tissues thereof, can be used to amplify a DNA fragment of 215bp in length by polymerase chain reaction using the nucleotide sequences of SEQ ID No 23 and SEQ ID No 41, respectively.
26. A kit for identifying a transgenic plant, cell or tissue thereof comprising elite event RF-BN1, said kit comprising PCR probes for use in a PCR identification method, wherein one PCR probe recognizes a sequence in the transgene and the other PCR probe recognizes a plant DNA sequence within SEQ ID No.24 or 5 'flanking sequence SEQ ID No.30 of the RF-BN 13' flanking region.
27. A kit according to claim 26 for identifying a transgenic plant, cell or tissue thereof comprising elite event RF-BN1, said kit comprising PCR probes having the nucleotide sequences of SEQ ID No.23 and SEQ ID No. 41.
28. A kit for identifying elite event MS-BN1 in biological samples, the kit comprising at least one PCR primer or probe recognizing a sequence located in the 3 'or 5' flanking region of MS-BN 1.
29. The kit of claim 28, wherein the sequence recognized by the at least one PCR primer is located on the plant DNA in SEQ ID No. 13.
30. The kit of claim 29, wherein the primer that recognizes a sequence within the plant DNA located in SEQ ID No.13 comprises the sequence SEQ ID No. 19.
31. The kit of any one of claims 28 to 30, further comprising at least a second PCR primer or probe that recognizes a sequence located within the exogenous DNA of MS-BN 1.
32. The kit of claim 31, wherein the primer that recognizes the foreign DNA sequence located in MS-BN1 comprises the sequence SEQ ID No. 12.
33. A method for confirming seed purity, the method comprising detecting a MS-BN1 specific DNA sequence in a seed sample with a specific primer or probe, wherein the specific primer or probe specifically recognizes a MS-BN15 'or 3' flanking region.
34. A method of screening for the presence of MS-BN1 in a seed, the method comprising detecting a MS-BN1 specific DNA sequence in a batch sample of seed with a specific primer or probe, wherein the specific primer or probe specifically recognizes the MS-BN15 'or 3' flanking region.
35. A kit for identifying elite event RF-BN1 in biological samples, the kit comprising at least one PCR primer or probe recognizing a sequence located in the 3 'or 5' flanking region of RF-BN 1.
36. The kit of claim 35, wherein the sequence recognized by the at least one PCR primer is located on the plant DNA in SEQ ID No. 24.
37. The kit of claim 36, wherein the primer that recognizes a sequence within the plant DNA located in SEQ ID No.24 comprises the sequence SEQ ID No. 41.
38. The kit of any one of claims 35 to 37, further comprising at least a second PCR primer or probe that recognizes a sequence located within the exogenous DNA of MS-BN 1.
39. The kit of claim 38, wherein the primer that recognizes a sequence located in the foreign DNA of RF-BN1 comprises the sequence SEQ ID No. 23.
40. A method for confirming seed purity in a seed sample, the method comprising detecting a DNA sequence specific for RF-BN1 with a specific primer or probe that specifically recognizes a sequence located in the RF-BN 15 'or 3' flanking region.
41. A method of screening seeds for the presence of MS-BN1 in a seed lot sample, the method comprising detecting a DNA sequence specific for RF-BN1 with a specific primer or probe, wherein the specific primer or probe specifically recognizes the RF-BN 15 'or 3' flanking region.
42. A DNA molecule amplifiable from a WOSR nucleic acid sample by using a set of primers, wherein a first of said primers comprises a sequence identical to SEQ ID NO: 13 or SEQ ID NO: 18 and a second comprising a sequence complementary to the exogenous DNA present in the WOSR nucleic acid sample.
43. The DNA molecule of claim 42, which is amplifiable from a nucleic acid sample of WOSR by using a set of primers, wherein a first one of said primers comprises the nucleotide sequence of SEQ ID NO: 19, the second comprising SEQ ID NO: 12.
44. a DNA molecule amplifiable from a WOSR nucleic acid sample by using a set of primers, wherein a first of said primers comprises a sequence identical to SEQ ID NO: 24 or SEQ ID NO: 30 and the second comprises a sequence complementary to the exogenous DNA present in the WOSR nucleic acid sample.
45. The DNA molecule of claim 44, which is amplifiable from a nucleic acid sample of WOSR by using a set of primers, wherein a first one of said primers comprises the nucleotide sequence of SEQ ID NO: 41, the second comprising SEQ ID NO: 23.