Field of the invention

This invention concerns transgenic Brassica plants, plant material and seeds, particularly oilseed rape plants, characterized in that these plants harbor a combination of two specific transformation events, particularly by the presence of a male-sterility gene, at a specific location in the Brassica genome and a fertility-restoration gene at another specific location in the Brassica genome. The invention also concerns a pair of transgenic Brassica plants, particularly oilseed rape plants, which is particularly suited for the production of hybrid seed. More specifically, one of the plants is characterized by being male-sterile, due to the presence in its genome of a male-sterility gene, while the other is characterized by carrying a fertility-restorer gene, capable of preventing the activity of the male-sterility gene. The pair of Brassica plants of the invention combines the ability to form hybrid seed with optimal overall agronomic performance, genetic stability and adaptability to different genetic backgrounds.

Background of the invention

The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene (in a foreign DNA) at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps which include extensive genetic characterization, breeding, and evaluation in field trials. The term "rapeseed" covers every seed of the Brassica species. Brassica is cultivated from China and India to Finland and Canada as one of the most valuable oil crops. Most Brassica types belong to the family of Cruciferae. They originated as a diploid species having aneuploid chromosome numbers ranging from 7 {Brassica fruticulosa) to 12 (Sinapsis alba). The most extensively grown Brassica species in Canada is known as turnip rape, or Brassica campestris (aa, n=10). Brassica oleracea (cc, n=9) has diversified in recent evolutionary history into at least six major horticultural types, including broccoli, cauliflower and cabbage. Brassica nigra (bb, n= 8) or black mustard is a less important crop commercially and is mainly known for its seeds from which mustard was originally made. From these basic types, amphiploid hybrids have been derived in more recent evolutionary stages by intercrossing. The most important of these are Brassica napus (aacc), of which the winter types provide the highest rapeseed yields in northern Europe and Brassica juncea (aabb) or brown mustard which is one of the major oil crops of the Indian sub-continent. Though intercrossing between different Brassica species (particularly those with compatible genomes) is possible and often done for breeding purposes, not all traits (or genes) will be able to be transferred from one species to the other or, when transferred to a different species, will retain identical characteristics (or expression patterns). Thus, a genetic locus conferring optimal expression of a natural or chimeric gene in one Brassica species, will not necessarily have the same effect in another.

Brassica species are bisexual and typically 60-70% self pollinated. The production of hybrids and introduction of genetic variation as a basis for selection was traditionally dependent on the adaptation of natural occurring phenomena such as self-incompatibility and cytoplasmic male-sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides are not widely applied in Brassica breeding due to their limited practicability and high cost respectively.

Transgenic methods have been developed for the production of male or female-sterile plants, which provide interesting alternatives to the traditional techniques. EP 0,344,029 describes a system for obtaining nuclear male-sterility whereby plants are transformed with a male-sterility gene, which comprises for example a DNA encoding a barnase molecule under the control of a tapetum specific promoter TA29, which when incorporated into a plant ensures selective destruction of tapetum cells. Transformation of tobacco and oilseed rape plants with such a gene resulted in plants in which pollen formation was completely prevented (Mariani et al. 1990, Nature 347: 737-741).

Cytochemical and histochemical analysis of anther development of Brassica napus plants comprising the chimeric PTA29:barnase gene is described by De Block and De Brouwer (1993, Planta 189:218-225).

To restore fertility in the progeny of a male-sterile plant, a system was developed whereby the male-sterile plant is crossed with a transgenic plant carrying a fertility- restorer gene, which when expressed is capable of inhibiting or preventing the activity of the male-sterility gene (US 5,689,041; US 5,792,929; De Block and De Brouwer, supra). The use of coregulating genes in the production of male-sterile plants to increase the frequency of transformants having good agronomical performance is described in WO 96/26283. Typically, when the sterility DNA encodes a barnase, the coregulating DNA will encode a barstar.

Elite Event MS-B2, and male-sterile plants comprising this elite event conferring male- sterility have been extensively described in WO01/31042 (herein incorporated by reference) including characterization of the transgene and of the plant DNA sequences immediately flanking the inserted transgene.

Reference seed comprising elite event MS-B2 was deposited at the ATCC (10801 University Blvd., Manassas, VA 201 10-2209) on October 14, 1999, under ATCC accession number PTA-850. Another sample of the same seed was deposited under accession number PTA- 2485. An alternative name for MS-B2 is MS 1 1. Elite Event RF-BNl, and plants comprising this elite event conferring male-sterility have been extensively described in WOOl/41558 (herein incorporated by reference) including characterization of the transgene and of the plant DNA sequences immediately flanking the inserted transgene.

Reference seed comprising elite event RF-BNl was deposited at the ATCC (10801 University Blvd., Manassas, VA 20110-2209) on September 20, 1999, under ATCC accession number PTA-730. An alternative name for RF-BNl is Rf 3. WOOl/41558 also describes elite event MS-BNl, male sterile plants comprising the event, as well as methods to identify such plants and progeny thereof. An alternative name for MS- BNl is MS 8.

MS-BNl and RF-BNl events are comprised in hybrid B. napus plants sold under the brand name In Vigor® Canola.

These documents mentioned before do not describe the particular combination MS-B2 and RF-BNl in Brassica plants nor the use thereof in hybrid seed production. RF-BNl in Brassica juncea is also hitherto undescribed, as well as the use of MS-B2 to increase the yield in Brassica oilseed plants.

Summary of the invention

In one embodiment, the invention provides a method for producing hybrid seed from oilseed rape plants, such as Brassica napus or Brassica juncea comprising the steps of providing a male-sterile female parent oilseed rape plant comprising elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850; providing a male-fertile male parent oilseed rape plant comprising elite event RF-BNl, preferably in homozygous form, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730; allowing pollen from said male parent oilseed rape plant to pollinate said female parent oilseed rape plant; and harvesting hybrid seed from said female parent plant.

In another embodiment, the invention provides an oilseed rape plant, such as Brassica napus or Brassica juncea plant, comprising in its nuclear genome at least one copy of elite event elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850 and at least one copy of elite event RF-BN1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. Also provided are cells or tissues or seeds of such oilseed rape plants.

In yet another embodiment of the invention, a pair of oilseed rape plants is provided for use in production of hybrid seed wherein one of the oilseed rape plants comprises elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850 and the other of said oilseed rape plants comprises elite event RF-BN1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730.

It is also an object of the invention to provide genomic DNA of an oilseed rape plant, such as Brassica napus or Brassica juncea plant, comprising in its nuclear genome at least one copy of elite event elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA- 850 and at least one copy of elite event RF-BN1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730.

Also provided by the invention are Brassica juncea, as well as cells and seeds thereof, comprising elite event RF-BN1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. Such plants may further comprise elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850. The invention further provides use of elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850 to increase seed yield in a transgenic oilseed rape plant such as a Brassica juncea plant.

In yet another embodiment of the invention, a method is provided to increase yield in oilseed rape plants comprising the step of providing the oilseed rape plant with elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850.

Brief description of the drawings

Figure 1 : Comparison of the vigor after herbicide application of MS/RF hybrid B. napus lines. Panel A: no glufosinate ammonium application. Panel B: one glufosinate ammonium application. Panel C: two glufosinate ammonium applications. Squares: MS- BN1/RF-BN1 plants; dots: MS-B2/RF-BN1 plants.

Figure 2: Comparison of the yield after herbicide application of MS/RF hybrid B. napus lines. Panel A: no glufosinate ammonium application. Panel B: one glufosinate ammonium application. Panel C: two glufosinate ammonium applications. Dark gray bars: MS-BN1/RF-B 1 plants; light gray bars: MS-B2/RF-BN1 plants. Loci to Loc5: field trial locations; AVG: average over all locations. The yield is presented as % compared to MS-BN1/RF-BN1 plants set at 100%. Detailed description

The current invention describes a novel combination of a male sterility event MS-B2 and a fertility restoration event RF-B 1 in oilseed rape plants, particularly Brassica napus and Brassica juncea. The current invention is based inter alia on the unexpected finding that elite event RF- B 1 conferring fertility restoration is effective enough in Brassica oilseed rape plants to restore fertility when the plant further also contains elite event MS-B2 conferring male- sterility. Although RF-B 1 has previously successfully been used to restore fertility in oilseed rape plants, comprising elite event MS-BN1, it was unpredictable whether RF- BN1 could also been used to restore fertility in oilseed rape plants comprising elite event MS-B2. Different male-sterility comprising elite events have different expression levels of the barnase product expressed by the transgenic male-sterility gene and it cannot be predicted that the levels of barnase inhibitor, barstar, produced by transgenic fertility- restoration gene in RF-BN1 would match the expression of the barnase in other individual male-sterility events. As indicated in the examples below the combination of the current invention resulted in intrinsically higher yields that combination of the same male-sterility event MS-B2 with other fertility restoration events. Furthermore, RF-BN1 has been previously described to be located on the C-genome (see WO 01/31042; see also e.g. www.agbios.com/dbase.php?action=showProd&data=MS8xRF3. Accordingly, the introduction of RF-B 1 from B. napus (aacc) into B. juncea (aabb) which does not contain a C-genome would not be considered straightforward by the skilled artisan. The current specification provides data that RF-B 1 is however located on the A-genome, allowing introduction in B. juncea by crossing.

In addition, field trials have uncovered that the presence of MS-B2 increases the average yield (grain yield) when compared to an isogenic plant line not containing MS-B2.

In one embodiment of the invention, a method is thus provided to produce hybrid seed from oilseed rape plants, such as Brassica napus or Brassica juncea, comprising the steps of

providing a male-sterile female parent oilseed rape plant comprising elite event MS-B2 comprising a male sterility gene; providing a male-fertile male parent oilseed rape plant comprising elite event RF-BN1 comprising a fertility restoration gene, preferably in homozygous form;

allowing pollen from the male parent oilseed rape plant to pollinate the female parent oilseed rape plant; and

harvesting hybrid seed from the female parent plant.

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

The term "chimeric" when referring to a gene or DNA sequence is used to indicate that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5'UTR, coding region, 3'UTR, intron) that are not naturally associated with each other and originate, for example, from different sources. "Foreign" referring to a gene or a DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant species, or is not naturally found in that genetic locus in that plant species. The term "foreign DNA" will be used herein to refer to a DNA sequence as it has incorporated into the genome of a plant as a result of transformation. The "transforming DNA" as used herein refers to a recombinant DNA molecule used for transformation. The transforming DNA usually comprises at least one "gene of interest" (e.g. a chimeric gene) which is capable of conferring one or more specific characteristics to the transformed plant. The term "recombinant DNA molecule" is used to exemplify and thus can include an isolated nucleic acid molecule which can be DNA and which can be obtained through recombinant or other procedures. As used herein the term "transgene" refers to a gene of interest as incorporated in the genome of a plant. A "transgenic plant" refers to a plant comprising at least one transgene in the genome of all of its cells. The foreign DNA present in the plants of the present invention will preferably comprise 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.

A "male -sterility gene" as used herein refers to a gene which upon expression in the 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 barnase, under the control of a promoter directing expression in tapetum cells. More specifically, according to the present invention the male-sterility gene is "TA29-barnase" as described herein. A "fertility restorer gene" as used herein refers to a gene which upon expression in a plant comprising a male-sterility gene, is capable of preventing phenotypic expression of the male-sterility gene, restoring fertility in the plant. More specifically the fertility restorer gene will comprise a DNA encoding a protein or polypeptide capable of preventing phenotypic expression of the male -sterility gene, under the control of a promoter directing expression in at least the cells in which the male-sterility DNA is expressed. More specifically, according to the present invention, the fertility restorer gene is "TA29-barstar" as described herein.

The incorporation of a recombinant DNA molecule in the plant genome typically results from transformation of a cell or tissue (or from another genetic manipulation). The particular site of incorporation is either due to chance or is at a predetermined location (if a process of targeted integration is used).

The foreign DNA can be characterized by the location and the configuration at the site of incorporation of the recombinant DNA molecule in the plant genome. The site in the plant genome where a recombinant DNA has been inserted is also referred to as the "insertion site" or "target site". Insertion of the transgene into the plant genome can be associated with a deletion of plant DNA, referred to as "target site deletion". A "flanking region" or "flanking sequence" as used herein refers to a sequence of at least 20 bp, preferably at least 50 bp, and up to 5000 bp of the plant genome which is located either immediately upstream of and contiguous with or immediately downstream of and contiguous with the foreign DNA. Transformation procedures leading to random integration of the foreign DNA will result in transformants with different flanking regions, which are characteristic and unique for each transformant. When the transgene is introduced into a plant through traditional crossing, its insertion site in the plant genome, or its flanking regions will generally not be changed. An "insertion region" as used herein refers to the region corresponding to the region of at least 40 bp, preferably at least 100 bp, and up to more than 10000 bp, encompassed by the upstream and the downstream flanking regions of a transgene in the (untransformed) plant genome and including the insertion site (and possible target site deletion). Taking into consideration minor differences due to mutations within a species, an insertion region will retain at least 85%, preferably 90%, more preferably 95%, and most preferably 100% sequence identity with the sequence comprising the upstream and downstream flanking regions of the foreign DNA in a given plant of that species. Expression of a gene of interest refers to the fact that the gene confers on the plant one or more phenotypic traits (e.g. herbicide tolerance) that were intended to be conferred by the introduction of the recombinant DNA molecule - the transforming DNA - used during transformation (on the basis of the structure and function of part or all of the gene(s) of interest).

An "event" is defined as a (artificial) genetic locus that, as a result of genetic manipulation, carries a foreign DNA comprising at least one copy of the gene(s) of interest. The typical allelic states of an event are the presence or absence of the foreign DNA. As used herein an "MS" event and an "RF" event will refer to events carrying the "TA29-barnase" and "TA29-barstar" transgenes respectively. An event is characterized phenotypically by the expression of one or more transgenes. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, an event is characterized by the restriction map (e.g. as determined by Southern blotting) and/or by the upstream and/or downstream flanking sequences of the transgene, and/or the molecular configuration of the transgene. Usually transformation of a plant with a transforming DNA comprising at least one gene of interest leads to a multitude of events, each of which is unique.

An "elite event", as used herein, is an event which is selected from a group of events, obtained by transformation with the same transforming DNA or by back-crossing with plants obtained by such transformation, based on the expression and stability of the transgene and its compatibility with optimal agronomic characteristics of the plant comprising it. Thus the criteria for elite event selection are one or more, preferably two or more, advantageously all of the following:

a) That the presence of the transgene does not compromise other desired characteristics of the plant, such as those relating to agronomic performance or commercial value;

b) That the event is characterized by a well defined molecular configuration which is stably inherited and for which appropriate diagnostic tools for identity control can be developed;

c) That the gene(s) of interest in the transgene show(s) a correct, appropriate and stable spatial and temporal phenotypic expression, both in heterozygous (or hemizygous) and homozygous condition of the event, at a commercially acceptable level in a range of environmental conditions in which the plants carrying the event are likely to be exposed in normal agronomic use. It is preferred that the foreign DNA is associated with a position in the plant genome that allows introgression into desired commercial genetic backgrounds.

The status of an event as an elite event is confirmed by introgression of the elite event in different relevant genetic backgrounds and observing compliance with one, two or all of the criteria e.g. a), b) and c) above. Additionally, for the transgenes encoding male sterility and fertility restoration described herein, selection of the elite events will also be determined on the compatibility between these events, more specifically that the progeny resulting from a cross between a plant carrying a male -sterility event and a plant carrying a fertility restorer event, in which both events are present have the following characteristics:

a) adequate phenotypic expression of the fertility restored phenotype, i.e. male fertility; and

b) phenotypic expression at a commercially acceptable level in a range of environmental conditions in which plants carrying the two events are likely to be exposed in normal agronomic use.

An "elite event" thus refers to a genetic locus comprising a transgene, which answers to the above-described criteria. A plant, plant material or progeny such as seeds can comprise one or more elite events in its genome.

Elite event MS-B2 has been characterized extensively as described in WO 01/31042 (see particularly examples 1 and 3). The transforming DNA has been described in Example 1. The flanking plant DNA sequences after transgene insertion have been isolated and identified (see WO 01/31042, particularly examples 3.2 as well as SEQ ID Nos 1 and 2). A diagnostic PCR allowing identification of elite event MS-B2 in biological material has also been described in WO 01/31042, particularly Example 5. When elite event MS-B2 is present in Brassica plants, cells, seed or tissues, genomic DNA thereof can be used to amply a DNA fragment of between 160 and 200bp, particulary about 183 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No 3 and SEQ ID No. 4 respectively. Reference seed has been deposited at the ATCC under deposit number ATCC PTA-850 or PTA-2485. An alternative names for Elite event MS-B2 is MSI 1.

Elite event RF-BN1 has been characterized extensively as described in WO 01/41558 (see particularly examples lb and 4.2.2). The transforming DNA has been described in Example lb. The flanking plant DNA sequences after transgene insertion have been isolated and identified (see WO 01/31042, particularly examples 4.2.2 as well as SEQ ID Nos 5 and 6). A diagnostic PCR allowing identification of elite event RF-BN1 in biological material has also been described in WO 01/41558, particularly Example 5.2. When elite event RF-BN1 is present in Brassica plants, cells, seed or tissues, genomic DNA thereof can be used to amply a DNA fragment of between 195 and 235 bp, particulary about 215 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No 7 and SEQ ID No. 8 respectively. Reference seed has been deposited at the ATCC under deposit number ATCC PTA-730. Alternative names for RF-BN1 are RF3 or ACS-BN003-6.

Plants harboring RF-BN1 or MS-B2 can, for example, be obtained from the seeds deposited at the ATCC. Such plants can be further propagated and/or used in a conventional breeding scheme to introduce the elite event of the invention into other cultivars of the same plant species. The deposited seeds belong to the species Brassica napus. Nevertheless, methods to introduce alleles or transgenes located on the A-genome from B. napus to B.juncea are well known in the art and include repeated back-crossing.

The invention provides for the first time B. juncea plants, seeds, cells and tissues comprising in their nuclear genome elite event RF-BNl, comprising a fertility restoration genome. Previously available information indicated that the elite event RF-BNl was present on the C-genome; however information provided herein located RF-BNl insertion in the A-genome allowing transfer of the elite event to B.juncea.

Brassica plants harboring MS-B2 and/or RF-BNl are also characterized by their glufosinate tolerance, which in the context of the present invention includes that plants are tolerant to the herbicide Liberty™. Tolerance to Liberty™ is defined by the criterium that spraying of the plants in the three to four leaf stage (3V to 4V) with at least 200 grams active ingredient/hectare (g.a.i./ha), preferably 400 g.a.i./ha, and possibly up to 1600 g.a.i./ha, does not kill the plants. Plants harboring MS-B2 and/or RF-BNl can further be characterized by the presence in their cells of phosphinothricin acetyl transferase as determined by a PAT assay (De Block et al, 1987, EMBO J. 6: 2513-2518). The Brassica plants of this invention can be cultivated in a conventional way. The presence of the 35S-bar gene ensures that they are tolerant to glufosinate. Therefore, weeds in the fields where such Brassica plants are grown can be controlled by application of herbicides comprising glufosinate as an active ingredient (such as Liberty™).

Field trials have further unveiled that the presence of MS-B2 in Brassica plants results in a seed or grain yield increase when compared to isogenic plant line without MS-B2. Accordingly, an embodiment of the current invention is a method to increase seed yield in oilseed rape plants comprising the step of providing the oilseed rape plant with elite MS-B2.

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

"Brassica" plants as used herein refers to plants of the family of the Brassicacea, preferably plants comprising an A genome. Preferably the Brassica plant will belong to one of the species Brassica napus, Brassica rapa (or campestris), or Brassica juncea. Alternatively, the plant can belong to a species originating from intercrossing of these Brassica species, such as B. napocampestris, or of an artificial crossing of one of these Brassica species with another species of the Cruciferacea. As used herein "oilseed plant" refers to any one of the species Brassica napus, Brassica rapa (or campestris), or Brassica juncea.

Oilseed plants according to the current invention can also be treated with herbicides including Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; with fungicides, including Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin or with insecticides, including Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, β- Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr or 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on.

As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA sequence which is functionally or structurally defined, may comprise additional DNA sequences, etc.

The following examples describe the characteristics of oilseed rape plants harboring the elite events MS-B2 and RF-BN1.

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

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

SEQ ID No 1 5' flanking sequence MS-B2

SEQ ID No 2 3' flanking sequence MS-B2

SEQ ID No 3 oligonucleotide primer 1 for detection of MS-B2 SEQ ID No 4: oligonucleotide primer 2 for detection of MS-B2 SEQ ID No 5 : 5' flanking sequence RF-BN1

SEQ ID No 6: 3' flanking sequence RF-BN1

SEQ ID No 7: oligonucleotide primer 1 for detection of RF-BN1

SEQ ID No 8 : oligonucleotide primer 2 for detection of RF-BN1

SEQ ID No 9: plasmid pTHW1 18

SEQ ID No 10: plasmid pTCOl 13

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

EXAMPLES

Example 1. Short description of MS-B2 and RF-BN1 1.1. Elite event MS-B2

Elite event MS-B2 was generated as by Agrobacterium-mediated transformation of B. napus plants with a chimeric DNA comprising the barnase gene under the control of a tapetum specific promoter (pTCOl 13).

Plasmid pTCOl 13 was essentially derived from the intermediate vector pGSVl . Vector pGSVl is itself derived from pGSC1700 (Comelissen and Vandewielle, 1989), but comprises an artificial T-region consisting of the left and right border sequences of the TL-DNA form pTiB6S3 and multilinker cloning sites allowing the insertion of chimeric genes between the T-DNA border repeats. The pGSVl vector is provided with a barstar gene on the plasmid mainframe, with regulatory signals for expression in E. coli.

A full description of the DNA comprised between the border repeats of pTC0113 is given in Table 1 (SEQ ID No. 10):

Table 1. Nucleotide positions of the DNA comprised between the T-DNA border repeats of pTCOl 13

Nt positions Orientation Description and references

1 -25 Right border repeat from the TL-DNA from pTiB6S3 (Gielen et al.

(1984) The EMBO Journal 3: 835-846).

26-53 Synthetic polylinker derived sequences

54-90 Residual sequence from the TL-DNA at the right border repeat

91 -97 Synthetic polylinker derived sequences

309-98 Counter The 3'untranslated end from the TL-DNA gene 7 (3'g7) of pTiB6S3 clockwise (Velten and Schell. (1985) Nucleic Acids Research 13: 6981 -6998;

Dhaese et al... (1983) The EMBO Journal 3 : 835-846).

310-331 Synthetic polylinker derived sequences

883-332 Counter The coding sequence of the bialaphos resistance gene {bar) of

clockwise Streptomyces hygroscopicus (Thompson et al... (1987) The EMBO

Journal 6: 2519-2523). The N-terminal two codons of the wild type bar coding region have been substituted for the codons ATG and GAC respectively.

2609-884 Counter The promoter from the atSIA ribulose-l,5-biphosphate carboxylase clockwise small subunit gene from Arabidopsis thaliana (PssuAra) (Krebbers et al... (1988) Plant Molecular Biology 11 : 745-759).

2610-2659 Synthetic polylinker derived sequences

2920-2660 Counter A 260 bp Taql fragment from the 3' untranslated end of the nopaline clockwise synthase gene (3"nos) from the T-DNA of pTiT37 and containing plant polyadenylation signals (Depicker et al.. (1982) Journal of

e ourna : - .

The flanking sequences were isolated as described in WO 01/31042.

Right (5') flanking region

The 5' flanking region was amplified as ca. 415 bp fragment, the complete sequence of which was determined (SEQ ID No. 1). The sequence between nucleotide 1 and 234 corresponds to plant DNA, while the sequence between nucleotide 235 and 415 corresponds to T-DNA. Left (3') flanking region

The 3' flanking region was amplified as ca. 416 bp fragment, the complete sequence of which was determined (SEQ ID No. 2). The sequence between nucleotide 1 and 193 corresponds to T-DNA, while the sequence between nucleotide 194 and 416 corresponds to plant DNA. PCR Identification of MS-B2

As described in WO 01/31042, MS-B2 comprising biological material can be identified using the therein described PCR identification protocol.

The following primers, which specifically recognize the foreign DNA and a flanking sequence of MS-B2 may be used:

B01 : 5 '-gAA.ATC.CAT.gT A.AAg.CAg.CAg.gg-3 ' (SEQ ID No. 3)

(target: plant DNA)

B02: 5 '-gCT .Tgg.ACT.ATA. ATA.CTT.gAC-3 ' (SEQ ID No. 4)

(target: T-DNA) The expected amplified fragments in the PCR reaction are:

for primer pair B01 -B02: 183bp (MS-B2 Elite Event)

1.2. Elite event RF-BN1

Elite event RF-BN1 was generated as by Agrobacterium-mediated transformation of B. napus plants with a chimeric DNA comprising the barstar gene under the control of a TA29 promoter (pTHWl 18). Plasmid pTHW1 18 was also essentially derived from the intermediate vector pGSVl (described above). A full description of the DNA comprised between the border repeats of pTHWl 18 is given in Table 2 (SEQ ID No. 9): Table 2. T-DNA of plasmid pTHWl 18

The flanking sequences were isolated as described in WO 01/41558. Right (5') flanking region

The 5' flanking region was amplified as ca. 1077 bp fragment, the complete sequence of which was determined (SEQ ID No. 5). The sequence between nucleotide 1 and 881 corresponds to plant DNA, while the sequence between nucleotide 882 and 1077 corresponds to T-DNA. Left (3') flanking region

The 3' flanking region was amplified as ca. 1500 bp fragment, the complete sequence of which was determined (SEQ ID No. 6). The sequence between nucleotide 1 and 166 corresponds to T-DNA, while the sequence between nucleotide 167 and 1441 corresponds to plant DNA.

PCR Identification of RF-BNl

As described in WO 01/41558, RF-BNl comprising biological material can be identified using the therein described PCR identification protocol.

To identify plant material comprising RF-BNl, the following primers, which specifically recognize the transgene and a flanking sequence of RF-BNl are used:

BNA03: 5'-TCA.TCT.ACg.gCA.ATg.TAC.CAg-3' (SEQ ID 7)

(target: transgene)

BNA04: 5 '-Tgg.ACC.CCT.Agg.TAA.ATg.CC-3 ' (SEQ ID 8)

(target: plant DNA)

The expected amplified fragments in the PCR reaction are:

For primer pair BNA03-BNA04: 215bp (RF-BN 1 Elite Event)

Example 2. Identification of the genome on which the RF-BNl locus is located and introduction into Brassica iuiicea

To determine whether the RF-BNl locus is located on the A or C genome of Brassica napus, the co-heritage or linkage of RF-BNl with known markers on the Brassica genome was analyzed in 4 different segregating B. napus BC1 populations derived from a cross between the donor line containing the RF-BNl transgene in a homozygous state an a recurrent parent not containing the transgene. The AFLP method was used to generate genetic markers. AFLP analysis was adapted from Vos et al. (1995, NAR 23:4407-4414, EP0534858 and US 6,045,994). In order to identify AFLP markers linked to RF-BNl, bulked segregant analysis (BSA) was carried out according to Michelmore et al. (1991 , Proc. Natl. Acad. Sci. USA 88:9828-9823).

All the AFLP genetic markers that displayed differential amplification between the pools of RF-BNl positive plants (containing a visible AFLP marker) and RF-BNl negative plants (where the same marker was not visible) were analyzed on at least 46 individual samples of the BC1 population in which the AFLP marker has been shown to be potentially linked in the BSA analysis. Only markers that showed considerable amount of co -segregation with the RF-BNl PCR marker where retained, potential other markers not satisfying this criterium were discarded as false positives from the BSA analysis. Linkage analysis was carried out using data from the retained AFLP markers and the RF-BNl PCR marker data generated on single plants for each BC1 population separately using JoinMap Version 3.0 (Van Ooijen and Vorrips (2001) JoinMap Version 3.0, Software for the calculation of genetic linkage maps, Plant Research International, Wageningen, The Netherlands). As the 4 individual maps around the RF-BNl locus showed considerable agreement, these 4 BC1 maps could be integrated into one BC1 map representing the region around the RF-BNl locus using the same software, allowing local mapping of the RF-BNl locus.

The resulting local map of the RF-BNl region was then compared to a genetic reference map that represents all B. napus chromosomes. For this reference map, chromosome numbers had already been assigned according to Sharpe et al. (1995, Genome 38:1 12- 1121) and Parkin et al. (1995, Genome 38: 1122-1131). N01 to N10 are A-genome chromosomes, while Ni l to N19 are C-genome chromosomes. A very clear correlation was found between the RF-BNl region map and the N07 chromosome from the reference map, which is know to be an A-genome chromosome. RF-BNl is positioned on the A- genome of 5. napus. Event RF-BNl was introduced by repeated backcrossing from Drakkar variety plants comprising event RF-BNl into a Brassica juncea cultivar. After at least 4 generations of accelerated backcrosses, the B. juncea plants were examined and it was established that: a) the presence of the foreign DNA did not compromise other desired characteristics of the plant, such as those relating to agronomic performance or commercial value;

b) the event was characterized by a well defined molecular configuration which was stably inherited; and

c) the gene(s) of interest in the foreign DNA showed a correct, appropriate and stable spatial and temporal phenotypic expression, both in heterozygous (or hemizygous) and homozygous condition of the event, at a commercially acceptable level in a range of environmental conditions in which the plants carrying the event are likely to be exposed in normal agronomic use. Furthermore, the plants were evaluated for their agronomical characteristics and performance as compared with wild-type Brassica juncea species. Extensive testing in the field demonstrated that RF-BNl in Brassica juncea resulted in plants which showed adequate expression of the genes of interest in the foreign DNA, i.e. a male-sterile phenotype, combined with optimal agronomic performance. Thus, although originally developed in a B. napus, it was surprisingly found that RF-BNl was also an elite event in Brassica juncea. Moreover, RF-BNl could be efficiently used to restore fertility in B. juncea plants comprising MS-B2.

Example 3. Agronomic performance of MS-B2/RF-BN1 plants

Brassica oilseed rape plants comprising MS-B2/RF-BN1 events were field tested, together with isogenic not transgenic control checks as well as MS-B2 plants comprising other restorer events. Relevant yield data are summarized in the tables below.

Note that field trial data for additional plant lines were included in the analysis for calculation of mean, significant etc. It will be clear that plant lines comprising MS-B2 and RF-BNl yield consistently higher than plant lines comprising MS-B2 and other male-restoration events such as RF-BN2. It will further be clear that plant lines comprising MS-B2 yield higher than isogenic non- transgenic plant lines.

Table 3. Field trials at geographic location 1

Table 4. Field trials at geographic location 2

Table 5. Field trials at geographic location 3

YLD(9) YLD(9)

Variable description VIGAB

1 -9 kg kg

Variable code

Pedigree Mean Mean %Checks

MS-B2 BC4 4.91 1914.55 102.88

MS-B2 BC4 4.94 1932.70 103.85

MS-B2 BC4 4.89 1952.49 104.92

Isogenic line 7.34 1783.81 95.85

Isogenic line (50%seed density) 7.33 1784.18 95.87

Isogenic line 7.31 1786.08 95.98

Isogenic line (75%seed density) 7.37 1792.20 96.30

Isogenic line (50%seed density) 7.33 181 1.31 97.33

MS-B2 BC4 4.90 1946.01 104.57

MS-B2 BC4 x RF-BN1 + accBC3 5.56 2066.72 1 1 1.06

MS-B2 BC4 x RF-BN2+ BC4 5.23 1690.12 90.82

MS-B2 BC4 x RF-BN2+ BC4 5.19 1719.02 92.37

MS-B2 BC4 x RF-BN2+ BC5 5.22 1742.14 93.61

MS-B2 BC4 x RF-BN2+ BC5 5.20 1755.62 94.34

MS-B2 BC4 x RF-BN1 + accBC3 5.98 1954.28 105.01

MS-B2 BC4 x RF-BN1 RF-BN1

6.27 1965.55 105.62 BC5F2

MS-B2 BC4 x RF-BN1 + accBC3 6.01 1996.10 107.26

MS-B2 BC4 x RF-BN1 + accBC3 5.51 2062.47 1 10.83

MS-B2 BC4 x RF-BN1 RF-BN1

5.55 1921.95 103.28 BC5F2

Mean 5.93 1853.67 99.61

Check mean 6.91 1860.98 100.00

Signification ** ** **

P.p.d.s. 5% 0.42 140.25 7.54

P.p.d.s. 1% 0.55 185.37 9.96

P.p.e.s. 5% 0.56 188.1 1 10.1 1

P.p.e.s. 1% 0.63 21 1.95 1 1.39

C.V. 5.67% 6.17% 6.17%

Res. std. dev. 0.34 1 13.87 6.12

#reps analysed 4 4 4 Table 6. Field trials summary

Example 4. Agronomic performance of MS-B2/RF-BN1 B. nanus plants compared to MS-BN1/RF-BN1 B. napus plants

Field trials were performed on 5 locations (4 replications/full plots) to confirm restoration and evaluate herbicide tolerance and agronomic performance of MS-B2/RF-BN1 B. napus hybrids in comparison with MS-BN1/RF-BN1 hybrids in the same genetic background.

Yield and vigor were determined in the absence of a glufosinate spray (A), or when treated once (B) or twice (C) with conventional applications of glufosinate (Liberty®). The results are summarized in Figure 1. The vigor of MS-B2/RF-B 1 hybrids is always greater than the vigor of MS-BN1/RF-BN1 , whether untreated (A), treated once (B) or treated twice (C) with glufosinate ammonium.

Overall MS-B2 RF-BN1 hybrids tended to flower (start and finish) earlier and matured earlier than the MS-B 1 /RF-B 1 hybrids .

Yield was also determined for field trials of the A, B, C type described above and the results are summarized in Figure 2 MS-B2 RF-BN1 hybrids have a 2 to 5% higher yield than MS-BN1/RF-BN1 hybrids. It was also observed that restoration on MS-B2 RF-B 1 was complete over all genotypes and locations.

In summary, the invention is drawn at the embodiments described in the following numbered paragraphs.

1. A method for producing hybrid seed from oilseed rape plants comprising the steps of

a. providing a male-sterile female parent oilseed rape plant comprising elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA- 850;

b. providing a male-fertile male parent oilseed rape plant comprising elite event RF-BN1, preferably in homozygous form, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730;

c. allowing pollen from said male parent oilseed rape plant to pollinate said female parent oilseed rape plant; and

d. harvesting hybrid seed from said female parent plant.

2. The method according to paragraph 1 , wherein said oilseed rape plants belong to the species Brassica napus or Brassica juncea.

3. An oilseed rape plant comprising in its nuclear genome at least one copy of elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850 and at least one copy of elite event RF-BN1 , reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. The oilseed rape plant of paragraph 3, wherein said oilseed rape plants belong to the species Brassica napus or Brassica juncea. A cell or tissue or seed of the oilseed rape plant of paragraph 3. A pair of oilseed rape plants for use in production of hybrid seed, one of said oilseed rape plants comprising elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850 and the other of said oilseed rape plants comprising elite event RF-B 1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. Genomic DNA of an oilseed rape plant according to paragraph 2. A Brassica juncea plant or plant cell comprising elite event RF-B 1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. Seed from the Brassica juncea plant according to paragraph 5 comprising elite event RF-B 1, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA-730. The plant or cell according to paragraph 8, further comprising elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850. Use of elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA- 850 to increase seed yield in a transgenic oilseed rape plant. Use according to paragraph 1 1, wherein said oilseed rape plant is Brassica juncea. A method to increase yield in oilseed rape plants comprising the step of providing said oilseed rape plant with elite event MS-B2, reference seed comprising said elite event being deposited at the ATCC under deposit number ATCC PTA 2485 or ATCC PTA-850. A method for producing hybrid B.juncea seeds and plants comprising a. Interplanting B. juncea plants comprising elite event MS-B2 with B. juncea plants comprising elite event RF-BNl

b. Allowing plants to cross-polinate

c. Harvest the seeds from the B.juncea plant comprising elite event MS-B2. Use of a B. juncea plant comprising elite event RF-BNl to produce progeny plants or to produce seeds. Use of RF-BNl to restore male fertility to a 5. juncea plant comprising MS-B2. Genomic DNA of B.juncea comprising RF-BNl . Genomic DNA according to paragraph 17, further comprising MS-B2. A B.juncea plant cell, plant or seed comprising in its genome a sequence having the nucleotide sequence of SEQ ID No. 5 and a sequence having the nucleotide sequence of SEQ ID No. 6, further comprising a barstar inhibitor coding sequence between said mentioned sequences. The plant cell, plant or seed of paragraph 19 further comprising in its genome a sequence having the nucleotide sequence of SEQ ID No. 1 and a sequence having the nucleotide sequence of SEQ ID No. 2, further comprising a barnase inhibitor coding sequence between said mentioned sequences.

A method for producing hybrid B. napus seeds and plants comprising a. Interplanting B. napus plants comprising elite event MS-B2 with B. napus plants comprising elite event RF-BN1 ;

b. Allowing plants to cross-polinate;

c. Harvest the seeds from the B. napus plants comprising elite event MS-B2. Genomic DNA of B. napus comprising RF-BN1 further comprising MS-B2. A B. napus plant cell, plant or seed comprising in its genome a sequence having the nucleotide sequence of SEQ ID No. 5 and a sequence having the nucleotide sequence of SEQ ID No. 6, further comprising a barstar inhibitor coding sequence between said mentioned sequences as well as a sequence having the nucleotide sequence of SEQ ID No. 1 and a sequence having the nucleotide sequence of SEQ ID No. 2, further comprising a barnase inhibitor coding sequence between said mentioned sequences.