Method for obtaining male-sterile plants

Abstract

A recombinant polynucleotide which can be used for obtaining a male-sterile plant comprising an inhibitor gene capable of inhibiting the expression of a target gene encoding MS2 protein or a homologous target gene and a promoter that is active in the tapetum. A method for obtaining male-sterile plants. Cells, fruit, seeds and progeny of male-sterile plants are also described.

Description

FIELD OF THE INVENTION

The invention involves genetic manipulation of plants by the application of recombinant DNA technology. The invention describes a method for obtaining plants which display nuclear encoded male-sterility, due to expression of said recombinant DNA as well as parts of said plants which are either sexually or asexually reproducible, or both.

BACKGROUND OF THE ART

Heterosis is the effect that progeny obtained through cross-pollination show better agronomical properties than progeny derived through self-pollination. These better properties result in a higher profit which is the reason that the development of hybrid seed is one of the prime objectives for the seed industry.

To obtain hybrid seeds it is necessary that the female plants are unable to self-pollinate. Many crop plants accommodate both the female and male reproductive organs on the same individual and thus self-pollination is dominating cross-pollination. Introduction of male sterility in many crops is thus necessary for hybrid seed production.

In production fields the male-sterile acceptor and male-fertile donor plants are grown side by side and after cross-pollination the hybrid seed is harvested. Hybrid seed is separately collected from the non-hybrid seeds formed on the donor plants by destruction of these male-fertile plants before the harvest. This approach makes the discrimination between fertile and sterile plants necessary which can be accomplished by the appearance of different phenotypes. Also a selectable marker gene such as a gene coding for a herbicide resistance, closely linked to the male-sterility locus can be used for this purpose. Selection of the hybrid seeds can than be accomplished by spraying the herbicide, which will result in the destruction of the male-fertile plants. Alternatively the hybrid seeds can be selected after harvesting by a phenotypic marker expressed at the seed level.

Nowadays in agricultural practice male-sterile parental lines are obtained by physically emasculation of the plants or by the application of cytoplasmic or nuclear encoded male-sterile mutants. There are only a limited number of natural male-sterile mutants available in the commercial interesting crops. This renders the latter approach often not feasible. In addition naturally male-sterile plants have their disadvantages. There preparation is laborious and there maintenance and propagation difficult. They often show additional, detrimental traits and a difficult inheritance.

The development of nuclear encoded male-sterile plants by the application of recombinant DNA techniques has succeeded in new approaches which circumvent most of the disadvantages mentioned above.

STATE OF THE ART

The International Patent Application WO92/18625, MOGEN International N.V. proposes methods for the production of restorable male-sterile plants in general terms, essentially comprising integration of a recombinant polynucleotide into their genome, essentially comprising an inhibitory gene, which, upon proper expression in the anthers of the plant, is capable of inhibiting expression of one or more genes encoding an enzyme involved in the synthesis of chalcone, or one of its precursors.

DEFINITIONS

Antisense gene: a nucleotide sequence having a homology of more than 50%, preferably more than 80% with a target gene as defined herein, and which is linked to a promotor in 3' to 5' orientation with respect to the target gene and can be expressed as an RNA molecule.

Gene or sense gene: a nucleotide sequence that can be expressed as RNA molecule and/or polypeptide.

Promoter: a nucleotide sequence which directs the expression of a (sense-) gene or antisense gene, or nucleotide sequences derived thereof.

Inhibitor gene: a (sense-)gene or antisense gene, expression of which leads to prevention or inhibition of the expression of a target gene as defined herein.

Repair gene: a gene capable to prevent or sufficiently inhibit the action of an inhibitor gene thus repairing the activity of a target gene.

Target gene: a gene which activity is to be inhibited by proper expression of an inhibitor gene as herein defined.

SUMMARY OF THE INVENTION

The present invention describes the male-sterile Arabidopsis thaliana mutant PA and the molecular isolation and analysis of the gene, called MS2 (FIG. 1). Mutation of this gene is responsible for the male-sterile phenotype of PA. The only other male sterile mutant of Arabidopsis thaliana, ms1, has been reported by Van der Veen and Wirtz (1968). This mutant was obtained by classical mutagenesis and is the only other male sterility mutant described in the literature, which does not show pleiotropic effects. Further molecular characterization of ms1 has not been reported so no information is available on the nature of the male-sterility gene.

Accordingly, the present invention provides a gene isolated from the cDNA library of a plant which encodes a protein indicated as MS2 which is involved in pollen development, said gene having the nucleotide sequence as shown in FIG. 2 or a homologous gene. Further, this invention provides a promoter of the MS2 gene isolated from the chromosomal DNA of a plant, said promoter having the nucleotide sequence as shown in FIG. 3 or a homologous promoter.

Further, the present invention provides recombinant polynucleotides which can be suitably used for obtaining a male-sterile plant, essentially comprising:

a) an inhibitor gene capable of inhibiting expression of a target gene in the said plant, encoding the MS2 protein involved in pollen development said gene having the sequence as shown in FIG. 2 or a homologous target gene, and b) the promoter that is active in the tapetum of said plant, linked to said inhibitor gene as to achieve expression thereof in the tapetum of said plant.

In the preferred embodiment of the invention the inhibitor gene is a (sense-)gene or an antisense gene directed against the target gene. In another preferred embodiment according to the invention the promoter that is active in the tapetum of a plant comprises the promoter of the MS2 gene, said promoter having the sequence as shown in FIG. 3.

The present invention also provides a method for obtaining a male-sterile plants comprising the steps of:

a) transferring a recombinant polynucleotide according to the invention to cells of a male-fertile plant, b) generating whole new plants from cells having incorporated said recombinant polynucleotide, and c) selecting a plant that is male-sterile.

Yet another embodiment of the invention is a recombinant plant genome, comprising incorporated therein a recombinant polynucleotide according to the invention.

In a yet further preferred embodiment of the invention a method is provided for a cost effective production of hybrid seed, due to use of large numbers of heterozygous male-sterile plants, which have been obtained by crossing a homozygous male-sterile plant of the desired variety with a male-fertile plant of the same variety.

The invention further encompasses (hybrid) seed obtained through crossing or selfing of any of the plants according to the invention.

Other preferred embodiments of the invention are the plasmids pMS2 and pMS2as (see FIG. 6).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Southern analysis of the progeny of a cross between the male-sterile Arabidopsis thaliana mutant PA and male-fertile Arabidopsis thaliana ecotype Landsberg erecta.

FIG. 2: The nucleotide sequence of the MS2 mRNA.

FIG. 3: The nucleotide sequence of the promoter of the MS2 gene.

FIG. 4a: Northern analysis of the expression of the MS2 gene in different organs of Arabidopsis thaliana.

FIG. 4b: Northern analysis of the expression of the MS2 gene in different organs of Raphanus satirus (radish).

FIG. 5: Southern analysis of the presence of MS2 homologues in radish and oilseed rape.

FIG. 6: A diagrammatic representation of the different cloning steps for obtaining the chimeric sense and antisense MS2 constructs and plasmids pMS2 en pMS2as.

DETAILED DESCRIPTION OF THE INVENTION

1. Isolation of the MS2 gene of Arabidopsis thaliana

The maize Enhancer-Inhibitor (En-I) (Peterson, 1953), also known as Suppressor-Mutator (Spm) (McClintock, 1954), transposable element system has been used for transposon tagging of a number of genes in maize and was shown to transpose when introduced by transformation in a heterologous plant species (Masson and Federoff, 1989; Pereira and Saedler, 1989; Frey et al., 1989). We constructed a transformation vector containing three units, namely: a) an immobile En transposase source (Masson and Federoff, 1987), under control of the strong Cauliflower Mosaic Virus 35S promotor; b) a mobile I element as insertion mutagen (Schwartz-Sommer et al., 1985); c) a hygromycin phosphotransferase gene (Van den Elzen et al., 1985) conferring resistance to the antibiotic hygromycin. This `in cis two element En-I` vector construct was used for Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana (Valvekens, 1988). A male sterile plant was found among the third generation (T3) progeny of a successively self-fertilized primary transformant (T1) with frequently transposing I elements. The plant was coded PA (Pollen Absent). As the mutation involved can be complemented by ms1, the only other male-sterile mutant without pleiotropic effects described in Arabidopsis (Van der Veen and Wirtz, 1968), we called this novel mutant ms2. Segregation analysis of the male-sterile phenotype and the I transposable element was performed as follows. DNA of F2 progenies from a cross PA x Landsberg erecta was Southern transferred and hybridized with an I element specific probe (FIG. 1). DNA was digested with HindIII which does not cut inside the I element. This F2 progeny lacked the En transposase genes and therefore all detected fragments denote stable I element inserts. A 6.6 kb I element containing fragment (.box-solid.), is homozygous only in plants displaying a male sterile (ms) phenotype. Plant F2 3-1 (*) has only this ms2 accompanying I element and was therefore used for the isolation of the transposon flanking DNA by Inverse Polymerase Chain Reaction (IPCR) (Masson and Federoff, 1989; Ochman et al. 1988). The IPCR fragment was cloned, sequenced and used as a probe to isolate clones from a flower specific cDNA (Weigel et al., 1992) as well as a genomic lambda library of Arabidopsis.

Analysis of the MS2 MRNA

The cDNA clone (largest out of 3 isolated) was double-stranded sequenced using the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an automated DNA sequencer (Applied Biosystems). Nucleotide and predicted amino-acid sequences of MS2 cDNA are shown in FIG. 2. Sequence analysis was performed using the Genetics Computer Group (GCG) software package (Devereux et.al. 1984). Nucleotides are numbered beginning with the first position of the ATG initiation triplet. A nonsense codon upstream (-36) indicates that this the translation start. The polyadenylation signal sequence (position 1967 underlined) precedes the poly(A) tail. The position of the I-element insertion (AAA) at position 1800 is underlined. The last three amino acids (Gly-Arg-Gly) contain a putative C-terminal microbody targeting signal (CMTS) suggesting a role of this part in the protein which is absent in male sterile mutant alleles due to excision footprints causing frameshifts. Data base searches (FAS-TA/BLAST) revealed homology to a DNA sequence (EMBL release 33.0, 12/92) located upstream of the wheat mitochondria 26S ribosomal RNA rrn26 gene. The region of homology in the MS2 cDNA (1040-1203) is underlined and shows 77.6% identity to the wheat mitochondrial nucleotide sequence. This region is located about 2 kbp upstream of the rrn26 gene at position 650-814 of the published sequence (Spencer et al., 1992). No function has been ascribed to this mitochondrial DNA segment which reveals an open reading frame whose predicted amino acids show 81.7% identity to the MS2 protein (55 amino-acids). The homology in the mitochondrial DNA segment does not extend beyond this region but is precisely flanked by intron-exon boundaries.

FIG. 3 shows the nucleotide sequence of the promoter of the MS2 gene. Said sequence is obtained from A. thaliana ecotype Landsberg erecta genomic clone of the MS2 gene. The TATA box of the MS2 gene is shown underlined (position 835) and the 5' end of the MS2 cDNA sequence is marked with an asterisk (position 957). A unique AseI restriction site (ATTAAT) at position 953 allows cloning of the promoter for further plasmid constructions described.

3. Analysis of the expression of the MS2 gene in Arabidopsis thaliana.

Northern analysis of the expression of the MS2 gene in different organs of Arabidopsis thaliana showed that MS2 MRNA could only be detected in flowers and siliques but not in seedlings and leaf or stem tissue (see FIG. 4a).

As shown in FIG. 4b MS2 RNA could also be detected in flowerbuds of radish. By Southern blot analysis we have confirmed that MS2 homologous genes are present in radish but also in oilseed rape (FIG. 5) which indicates that male sterility can be induced in oilseed rape by sense-MS2 or antisense-MS2 expression.

4. The present invention envisages the following steps towards obtaining male-sterile plants:

A sense-MS2 or antisense-MS2 gene of Arabidopsis thaliana or its homologous counterpart of other species is placed under the control of the MS2 promoter, which DNA sequence is shown in FIG. 3, and these constructs can be used for the transformation of fertile crop plants. After selection of transformed plants which express the construct, and after the plants are allowed to flower, transgenic plants showing disturbed pollen development can be selected.

The diagram of FIG. 6 shows the steps of the process for preparing the above sense and antisense constructs. A binary vector (origin pBIN19), containing a chimaeric NPT II gene conferring kanamycin resistance to plant cells, with unique SalI (S) and EcoRI (R) restriction sites serves as the basic vector unit. The MS2 gene promotor and CaMV 35S poly (A) addition signal/terminator (term) are cloned after Klenow polymerase treatment (Klen). The MS2 cDNA fragment is made blunt (with Klenow polymerase) and cloned into the SmaI restriction site in two orientations with respect to the MS2 promotor (prom). This results in the two plasmids designated pMS2 (sense) and pMS2as (anti-sense). Other abbreviations used: RB=T-DNA right border; LB=T-DNA left border; 5' and 3' refer to the ends of the cDNA (coding strand) defined by unique resistriction sites in the cDNA clone.

In general nuclear encoded male-sterile plants can thus be obtained and used for hybrid seed production. Plants of a selected variety have to be genetically transformed by introducing into cells of the said plant one or more recombinant polynucleotides, essentially comprising one or more inhibitor genes, which upon proper expression in the plant, are capable of inhibiting expression of a MS2 gene.

Inhibition of the expression of the MS2 gene will result in male-sterile plants. This can be accomplished by proper expression of an inhibitor gene directed against that target gene. Inhibitor genes can be suitably selected from a range of alternatives, including homologous or heterologous (i.e obtained from a different source) sense and antisense (synthetic)-genes or parts thereof with a suitable length and homology for proper inhibition, as illustrated in International Patent Application WO92/18625, MOGEN International NV and International Patent Application WO90/11682, DNA Plant Technology Inc.

Preferably the inhibitor gene is expressed in the tapetum according to the present invention. This can be accomplished by fusing the inhibitor gene under control of the promoter derived from the MS2 gene (see FIG. 4) from Arabidopsis or its heterologous counterpart (i.e obtained from another source).

Transfer of recombinant polynucleotides into plants or parts thereof can be achieved by numerous techniques. Some of them are listed here as illustration and comprise transformation of protoplasts using the calcium/polyethylene glycol method (Krens et al. 1982; Negrutiu et al., 1987), electroporation (Shillito et al., 1985), microinjection (Crossway et al., 1986), (DNA or RNA coated) particle bombardment (Klein et al., 1987), infection with viruses and the like, natural DNA transfer by Agrobacterium species preferably by the use of the so-called binary vector system (Bevan et al., 1984).

After identification of transformed plant material, whole plants are regenerated using well-known protocols described in literature (vide e.g. Horsch et al., 1985). There does not exist any restriction towards parts of the plants used.

After transformed plants have been obtained, they can be evaluated for the presence of the desired trait and/or the degree to which the desired traits are expressed. A first evaluation may include the level of expression of the inhibitor gene and the extent to which the transgenic plants are male-sterile. Subsequently transgenic plants can be selected that show stable and/or predictable inheritance of the male-sterile trait, and the like. Next the (heterozygous) male sterile plants can be used directly for the production of hybrid seed, or alternatively be selfed with rescued pollen in order for obtaining homozygous male-sterile plants. Clearly, the advantage of having a few homozygous male-sterile plants permits one to rapidly acquire large amounts of heterozygous male-sterile seed which can be directly used for large scale production of hybrid seed.

The present invention can be applied in any plant capable of self-pollination, for which the production of hybrid seeds is of commercial interest.

The invention also provides a method for obtaining homozygous male-sterile plants. This can be accomplished by selfing the heterozygous male-sterile plants according to the invention. The development of pollen of these plants can be rescued by overcoming the inhibition of in vivo MS2 protein synthesis. Inhibition of MS2 gene expression can be circumvented by allowing (temporary) in vivo production of MS2 protein in the tapetum of the plant. This can be achieved by introducing in addition to the inhibitor gene, a repair gene under control of an inducible promoter. In this configuration the expression of the repair gene can be externally controlled by adding an appropriate inducer. Preferably such a repair gene includes a gene at least containing part of the MS2 gene, that is capable upon expression of inhibiting the effect of expression of the antisense MS2 gene. Alternatively other approaches to inhibit the expression of an inhibitor gene as described in International Patent Application WO92/18625, MOGEN International N.V., can be used.

If hybrid crops are grown for their seed or fruit, inducible restoration of fertility is necessary. Male-sterility must then be eliminated to permit pollination and harvest of seed or fruit. Repairing male-fertility may be invoked by administration of an inducer to the crop in the field, causing a sufficiently high expression of the repair gene to neutralize the inhibition of the MS2 gene by the inhibitor gene.

ADVANTAGES

Former methods to provide nuclear male sterile plants involve expression of genes which encode products that are toxic to somatic cells, such as DNAses, RNAses, proteases or one of the enzymes involved in the chalcone biosynthesis pathway. Chalcone is a key compound in the flavonoid biosynthesis pathway. Flavonoids are secondary metabolites that are known to have a key-function in the pigmentation of flowers and fruit. In addition flavonoids appear to be involved in defense against phytopathogens (Lamb et al., 1989), the protection against UV-light (Schmelzer et al., 1988) and the induction of nodulation (Long, 1989). Flavonoids have also been implicated in the regulation of auxin transport (Jacobs and Rubery, 1988) and resistance to insects (Hedin and Waage, 1986). As a consequence of the properties mentioned above of the toxic products or the role of the product of the inhibited gene in the plant, strict developmental and tissue-specific expression limited to anthers only is required. The present method does not necessarily require such a strict developmental or tissue-specific expression.

Male-sterile plants according to the invention are highly sterile and appear entirely female-fertile.

The present invention also provides methods for obtaining homozygous male-sterile plants. Only a few are needed which can be multiplied in vitro by well-established techniques. The homozygous male-sterile acceptor plant is cross-fertilized with the male-fertile donor plant, and the heterozygous male-sterile seed used for hybrid crop production. Of the hybrid seed produced, 50% will be male-sterile and 50% male-fertile. In case this ratio is not sufficient for obtaining high yield of commercial product (i.e seed or fruit) by self-fertilization, this ratio can be ameliorated by partial restoration of male-sterility by administration of an inducer to activate expression of the restorer gene.

EXPERIMENTAL

Construction of plasmid cwEnN::I

The plasmid cwEnN::I used for Agrobacterium tumefaciens transformation was constructed with the aim of designing a phenotypic excision assay using kanamycin resistance, for the En-I transposon family, which can be used in a number of plant species. The binary vector pGDW3.1 (Wing et al., 1989) was the source of the chimeric nopaline synthase (nos) promoter-hygromycin phosphotransferase (HPT) gene used for selection during transformation. A chimeric neomycin phosphotransferase II (NPTII) gene was first inserted into pGDW3.1 which is similar to the NPTII gene used by Baker et al. (1987) except that a ClaI linker was introduced 5' to the unique BamHI present in the untranslated leader between the T.sub.R 1' promoter and the NPTII codogenic region. This introduced a translation start (GCGATGG) 5' to the BamHI site. In addition, the NPTII translation start was removed by exchanging the NPTII gene sequences from plasmid pBCK1 (Kaulen et al., 1986) downstream of the BamHI site. This generated the plasmid pBHN in which a translation start is present upstream of the unique BamHI site into which an I element, I-6078 of 2.2 kb with 56 bp flanking DNA (Pereira and Saedler, 1989), was inserted to generate plasmid pBHNI.

En-1 (Pereira et al., 1986) was digested with BssHII, which cuts at positions 399 and 8041, made blunt with the Klenow fragment of DNA polymerase I and cloned between the cauliflower mosaic virus (CaMV) 35S promoter-terminator cassette, originating from pD51 (Pietrzak et al., 1986) in pBR322. This clipped-wing (cwEn) under control of the 35S promoter was next cloned into the binary vector pBHNI to produce plasmid cwEnN::I which was mobilized into the Agrobacterium strain pGV3101-(pMP90RK) (Koncz and Schell, 1986).

Plant transformation, propagation and phenotypic excision assay

For plant transformation, root explants of ecotype Landsberg erecta were infected with the Agrobacterium strain containing the binary vector construct, according to a method described by Valvekens et al. (1988). Transformed calli were selected on medium containing hygromycin at 20 mg.l.sup.-1. Emerging T.sub.1 shoots were grown without selection and allowed to self-fertilize and set seed in vitro. Seedset of A. thaliana is very poor in vitro due to high humidity. This was solved by lifting the lid of the pots a little. Growth conditions were 12 to 16 hrs light at 22.degree. C. in growth chambers.

Seeds of T.sub.2 and subsequent generations were sterilized in Eppendorf tubes by treatment with ethanol 70% (2 min), 50% commercial bleach (5% sodium hypochlorite, 5 min) and five subsequent washes with sterile water. The seeds were sown either wet or dry on GM medium (Valvekens et al., 1988) or half strength MS (Murashige and Skoog, 1962) solidified with 0.8% purified agar. For determining the number of T-DNA loci, seeds were mostly sown on half strength MS with 20 mg.l.sup.-1 hygromycin. Using no sugar in the medium permitted sterilization with 70% ethanol for 2 min and only two to three washes with sterile water. Segregation of antibiotic resistance was scored 5 to 10 days after germination. If needed seedlings were transferred to soil (compost:vermiculite:sand=4:1:1) and grown in a climate controlled greenhouse at 20.degree. C. with additional light (16 hrs). To prevent cross-fertilization and spread of seeds, plants were grown in Aracon containers (Beta Developments, Gent, Belgium). For the phenotypic excision assay seeds were germinated on GM medium containing 100 mg.l.sup.-1 kanamycin sulphate. Resistance, variegation or sensitivity was scored 5 to 10 days after germination. Variegated and sensitive seedlings were transferred to GM without antibiotic to rescue them before transferring to soil.

Ploidy number was determined by counting chloroplasts in stomatal guard cells of the lower epidermis of young leaves as described by Detrez et al. (1989). At least 25 stomata of different T.sub.2 and T.sub.3 plants were regarded.

Genomic DNA isolation

Genomic DNA was isolated from young rosette leaves of individual greenhouse grown plants for both PCR and Southern analysis. The method we used was a modified version of the one described by Dellaporta et al. (1983). 100 mg of liquid N.sub.2 -frozen leaf tissue was grinded in a 1.5 ml Eppendorf tube and mixed with 300 .mu.l of extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 10 mM .beta.-mercaptoethanol) and 25 .mu.l of 20% SDS. The mix was incubated at 65.degree. C. for 10 min, after which 115 .mu.l 5 M potassium acetate was added, mixed and kept on ice for at least 20 min and then centrifuged. The DNA was precipitated from the resultant supernatant by adding 0.6 to 2 volumes of isopropanol and if needed kept at -20.degree. C. for a few hours. The pellet was redissolved in 200 .mu.l of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) centrifuged for 5 min to remove debris, transferred to a new tube and RNase A added to 20 .mu.g.ml.sup.-1. The reaction mix was incubated at 37.degree. C. for 15 min when Proteinase K was added upto 50 .mu.g.ml.sup.-1. After another 15 min at 37.degree. C. the solution was phenol/chloroform extracted. DNA was precipitated again by adding 0.25 volumes of 10 M ammonium acetate and 2 volumes of 100% ethanol. The pellet was dissolved in 20 .mu.l of TE. This procedure was either up or down scaled if different amounts of leaf tissue were used. It generally yielded about 1 to 2 .mu.g of DNA per 100 mg leaf tissue.

DNA-methodology

DNA subcloning, restriction analyses and sequencing were performed using standard procedures well know to persons skilled in the art, vide e.g. Maniatis et al., 1982.

PCR and sequence analysis

Inverse PCR analysis to amplify transposon-flanking DNA of transformants was performed with 100 to 200 ng of genomic DNA. For the reaction 15 pmole of each primer was used, 0.1 mM of dNTPs (equal amounts of each dNTP) and 0.1 unit of SuperTaq DNA Polymerase (HT Biotechnology Ltd., Cambridge, England) in a total volume of 50 .mu.l reaction buffer as supplied along with the enzyme (10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.01% (w/v) gelatin, 1.5 mM MgCl.sub.2 and 0.1% Triton X-100). 30 cycles of denaturation (94.degree. C., 30 sec), annealing (61.degree. C., 60 sec) and extension (72.degree. C., 60 sec) were carried out. One tenth volume of PCR product was checked on agarose gel and if of interest cloned in Bluescript SK.sup.+ as a blunt ended fragment after Klenow treatment and gel elution. Sequence analysis was performed on double stranded supercoiled plasmid using an automated sequencer (Applied Biosystems). Initially one strand, if needed both strands were sequenced.

Southern blot analysis

Genomic DNA was digested with both EcoRI and BglII and 0.5 to 1 .mu.g was separated on a 0.8% agarose gel in Tris-acetate running buffer. After electrophoresis the DNA was alkali transferred onto a Gene Screen Plus membrane by vacuum blotting. The blots were prehybridized and hybridized following the procedure recommended by the membrane manufacturer. Probes (FIG. 1) were �.sup.32 P! random prime labelled DNA fragments. A 0.27 kb fragment containing the left border of En (upto a SalI restriction site) was used to detect I elements. The T-DNA right border probe was a 1.1 kb fragment containing the HPT gene. A fragment of 1.8 kb containing the complete NPTII gene plus octopine synthase (ocs) terminator was used to detect excision fragments. In the experiment shown in FIG. 5, MS2 cDNA was used as probe. After hybridization the membranes were washed twice with 2.times.SSC, 1% SDS at 65.degree. C. for 30 minutes and autoradiographed to X-ray films (Fuji or Kodak) at -80.degree. C. using intensifying screens.

Isolation of RNA

Plant material was collected, frozen in liquid N.sub.2 and grinded using mortar and pestle. Equal volumes of extraction buffer (0.1 M Tris-HCl, pH 9.0, 1% SDS, 0.1 M LiCl, 0.01 M EDTA) and phenol were added (4 ml each) and the mixture incubated at 60.degree. C. After centrifugation (10 min, 3000 rpm) the aquaous layer was extracted with 5 ml chloroform, collected and after addition of 1/3 volume of 8 M LiCl, RNA was allowed to precipitate overnight at 4.degree. C. The RNA precipitate was collected by centrifugation at 4.degree. C. (10 min, 12000 rpm) and washed twice with 70% ethanol. The dried pellet was finally dissolved in 0.06 M phosphate buffer pH 6.5.

Northern blotting

5 .mu.l RNA (2 .mu.g/ul) is denatured in 5 .mu.l glyoxal and 10 .mu.l DMSO for 60 min at 50.degree. C. and separated according to length on a 1% agarose gel (3.5 h, 40 V) in 15 mM phosphatebuffer. Subsequently RNA is blotted to Hybond N.sup.+ membrane according to the manufacturer (Amersham). The blot was prehybridized and hybridized following the procedure recommended by the membrane manufacturer. The probe (MS2 cDNA) was �.sup.32 P! random prime labelled. After hybridization the membranes were washed twice with 2.times.SSC, 1% SDS at 65.degree. for 30 minutes and autoradiographed to X-ray films (Fuji or Kodak) at -80.degree. C. using intensifying screens.

REFERENCES

Baker, B., Coupland, G., Fedoroff, N., Starlinger, P. and Schell, J. (1987) Phenotypic assay for excision of the maize controlling element Ac in tobacco. EMBO J. 6, 1547-1554.

Bevan, M. A. (1984) Binary Agrobacterium vectors for plant transformation. Nucl Acids Res. 12, 8711-8712.

Crossway, A., Oakes, J. V., Irvine, J. M., Ward, B., Knauf, V. C., Shewmaker, C. K. (1986) Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol. Gen. Genet. 202, 179-185

Dellaporta, S. L., Woods, J. and Hicks, J. B. (1983) A plant DNA minipreparation version II. Plant Mol. Biol. Rep. 4, 19-21.

Detrez, C., Sangwan, R. S. and Sangwan-Norreel, B. S. (1989) Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture. Theor. Appl. Genet. 77, 462-468.

Devereux, J., Haeberli, P. and Smithies, O. (1992) Nucleic Acids Res. 12, 387-395

Frey, M., Travantzis, S. M., and Saedler, H. (1989) Mol. Gen. Genet. 217, 172-177

Hedin, P. A. Waage, S. K. (1986) Roles of flavonoids in plant resistance to insects. In: Progress in Clinical and Biological Research. Vol. 213: Plant Flavonoids in Biology and Medicine. V. Cody, E. Middleton Jr., and J. B. Harborne, weds. (New York: Alan R. Liss), pp. 87-100.

Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., Fraley, R. T. (1985) A simple and general method for tranferring genes into plants. Science 227, 1229-1231.

Jacobs, M., Rubery, P. H. (1988) Natural occuring auxin transport regulators. Science 241, 246-249.

Kaulen, H., Schell, J. and Kreuzaler, F. (1986). Light-induced expression of the chimeric chalcone synthase-NPTII gene in tobacco cells. EMBO J. 5, 1-8.

Klein, T. M., Wolf, E. D., Wu, R., Sanford, J. C. (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327, 70-73.

Koncz, C. and Schell, J. (1986). The promoter of T.sub.L -DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383-396.

Krens, F. A., Molendijk, L., Wullems, G. J., Schilperoort, R. A. (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296, 72-74

Lamb, C. J., Lawton, M. A., Dron, M., Dixon, R. A. (1989) Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56, 215-224.

Long, S. (1989) Rhizobium-legume nodulation: Life together in the underground. Cell 56, 203-214.

Masson, P. & Fedoroff, N. (1989) Proc. Natn. Acad. Sci. U.S.A. 86, 2219-2223

McClintock, B. (1954) Carnegie Inst. Washington Year Book 53, 254-260.

Negrutiu, I., Mouras, A., Horth, M., Jacobs, M. (1987) Direct gene transfer to plants: Present developments and some future prospectives. Plant Physiol. Biochem. 25, 493-503

Ochman, H., Gerber, A. S. & Hartl, D. L. (1988) Genetics 120, 621-623.

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).

Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

Pereira, A., Cuypers, H., Gierl, A., Schwartz-Sommer, Zs. and Saedler, H. (1986). Molecular analysis of the En/Spm transposable element system of Zea mays. EMBO J. 5, 835-841.

Pereira, A. and Saedler, H. (1989). Transpositional behavior of the maize En/Spm element in transgenic tobacco. EMBO J. 8, 1315-1321.

Peterson, P. A. Genetics 38, 682-683 (1953).

Pietrzak, M., Shillito, R. D., Hohn, T. and Potrykus, I. (1986). Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucl. Acids Res. 14, 5857-5868.

Schwartz-Sommer, Zs., Gierl, A., Berndtgen, R. & Saedler, H. EMBO J. 4, 2439-2443 (1985).

Schmelzer, E., Jahnen, W., Hahlbrock, K. (1988) In situ localization of light-induced chalcone synthase mRNA, chalcone synthase and flavonoid endproducts in epidermal cells of parsley leaves. Proc. Natl. Acad. Sci. USA 85, 2989-2993.

Shillito, R. D., Saul, M. W., Paszkowski, J., Muller, M., Potrykus, I. (1985) High frequency direct gene transfer to plants. Bio/Technology 3, 1099-1103

Spencer, D. F., Schnare, M. N., Coulthart, M. B. and Gray, M. W. (1992) Plant Mol. Biol. 20, 347-352.

Valvekens, D., Van Montagu, M. and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA. 85, 5536-5540

Van den Elzen, P., Townsend, J., Lee, K. & Bedbrook, J. Plant Mol. Biol. 5, 299-302 (1985)

Van der Veen, J. H. & Wirtz, P. Euphytica 17, 371-377 (1968).

Weigel, D., Alvarez, J., Smyth, D. R., Yanofsky, M. F. & Meyerowitz, E. M. Cell 69, 843-859 (1992).

Wing, D., Koncz, C. and Schell, J. (1989). Conserved function in Nicotiana tabacum of a single Drosophila hsp70 promoter heat shock element when fused to a minimal T-DNA promoter. Mol. Gen. Genet. 219, 9-16.

__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 4- (2) INFORMATION FOR SEQ ID NO: 1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2126 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: YES- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE:#thaliana (A) ORGANISM: Arabidopsis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:72..1919 (D) OTHER INFORMATION:/cod - #on.sub.-- start= 72- (x) PUBLICATION INFORMATION: (A) AUTHORS: Stiekema D - #r., W. (G) DATE: 1993#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATTCTGTTCT TGGTTTACTT AATCTTCTTT CTAGTTAAGT ATATTCTTGT TG - #CTCATCAC 60#TCT TCT TCC TCC ATT 110TC TTC TTG AGT TCT#Leu Phe Leu Ser Ser Ser Ser Ser Ser Ile# 10- GTA GGG TCA AAC AAG CTT ACT AGG TTA CAC AA - #C CAT TGT GTC TGG TCT 158Val Gly Ser Asn Lys Leu Thr Arg Leu His As - #n His Cys Val Trp Ser# 25- ACA GTG ATT AGA GAT AAG AAA AGG TTC GGT CC - #C ACT TGG TGC CGT GTA 206Thr Val Ile Arg Asp Lys Lys Arg Phe Gly Pr - #o Thr Trp Cys Arg Val# 45- GGT GGT GGT GGT GAT GGT GGG AGA AAC AGT AA - #C GCA GAG AGT CCT ATT 254Gly Gly Gly Gly Asp Gly Gly Arg Asn Ser As - #n Ala Glu Ser Pro Ile# 60- CGG GTT TCT TCG CTT TTG AAA GAC AGA GGT CA - #G GTA CTG ATT AGG GAA 302Arg Val Ser Ser Leu Leu Lys Asp Arg Gly Gl - #n Val Leu Ile Arg Glu# 75- CAG AGT TCG CCG GCT ATG GAT GCT GAG ACA TT - #G GTT CTG TCT CCA AAC 350Gln Ser Ser Pro Ala Met Asp Ala Glu Thr Le - #u Val Leu Ser Pro Asn# 90- GGG AAT GGG AGA ACC ATT GAG ATC AAT GGA GT - #A AAG ACT TTG ATG CCT 398Gly Asn Gly Arg Thr Ile Glu Ile Asn Gly Va - #l Lys Thr Leu Met Pro# 105- TTT AGT GGC GCT TCT ATG GTG GGG ATG AAA GA - #A GGA CTT GGC ATA ATC 446Phe Ser Gly Ala Ser Met Val Gly Met Lys Gl - #u Gly Leu Gly Ile Ile110 1 - #15 1 - #20 1 -#25- AGT TTC CTC CAA GGG AAG AAG TTT CTA ATC AC - #T GGC TCG ACC GGT TTC 494Ser Phe Leu Gln Gly Lys Lys Phe Leu Ile Th - #r Gly Ser Thr Gly Phe# 140- TTA GCT AAA GTA CTG ATT GAG AAA GTC TTG AG - #A ATG GCT CCT GAT GTC 542Leu Ala Lys Val Leu Ile Glu Lys Val Leu Ar - #g Met Ala Pro Asp Val# 155- AGC AAG ATA TAT CTC TTG ATT AAA GCC AAA AG - #C AAA GAA GCT GCG ATC 590Ser Lys Ile Tyr Leu Leu Ile Lys Ala Lys Se - #r Lys Glu Ala Ala Ile# 170- GAG CGG CTA AAG AAC GAG GTG TTA GAT GCA GA - #G CTT TTT AAT ACT CTA 638Glu Arg Leu Lys Asn Glu Val Leu Asp Ala Gl - #u Leu Phe Asn Thr Leu# 185- AAA GAG ACT CAT GGA GCA TCT TAC ATG TCT TT - #C ATG TTA ACT AAA CTC 686Lys Glu Thr His Gly Ala Ser Tyr Met Ser Ph - #e Met Leu Thr Lys Leu190 1 - #95 2 - #00 2 -#05- ATC CCT GTG ACC GGA AAC ATT TGC GAT TCA AA - #C ATT GGG TTG CAA GCA 734Ile Pro Val Thr Gly Asn Ile Cys Asp Ser As - #n Ile Gly Leu Gln Ala# 220- GAT TCA GCT GAA GAG ATT GCG AAA GAA GTT GA - #T GTT ATA ATC AAT TCT 782Asp Ser Ala Glu Glu Ile Ala Lys Glu Val As - #p Val Ile Ile Asn Ser# 235- GCT GCT AAT ACA ACC TTC AAT GAA AGA TAC GA - #T GTT GCT CTG GAC ATC 830Ala Ala Asn Thr Thr Phe Asn Glu Arg Tyr As - #p Val Ala Leu Asp Ile# 250- AAC ACA AGA GGG CCC GGT AAT CTC ATG GGA TT - #C GCC AAG AAG TGC AAG 878Asn Thr Arg Gly Pro Gly Asn Leu Met Gly Ph - #e Ala Lys Lys Cys Lys# 265- AAA CTC AAA CTG TTC TTG CAA GTA TCC ACA GC - #T TAT GTG AAC GGA CAA 926Lys Leu Lys Leu Phe Leu Gln Val Ser Thr Al - #a Tyr Val Asn Gly Gln270 2 - #75 2 - #80 2 -#85- AGA CAA GGA AGG ATC ATG GAG AAG CCA TTT TC - #T ATG GGA GAT TGT ATA 974Arg Gln Gly Arg Ile Met Glu Lys Pro Phe Se - #r Met Gly Asp Cys Ile# 300- GCA ACA GAG AAC TTC CTC GAA GGA AAC AGA AA - #A GCA TTA GAT GTT GAT1022Ala Thr Glu Asn Phe Leu Glu Gly Asn Arg Ly - #s Ala Leu Asp Val Asp# 315- AGA GAG ATG AAG TTA GCT CTT GAA GCT GCT AG - #A AAA GGG ACT CAA AAT1070Arg Glu Met Lys Leu Ala Leu Glu Ala Ala Ar - #g Lys Gly Thr Gln Asn# 330- CAA GAT GAG GCA CCG AAG ATG AAG GAT CTC GG - #T CTA GAG CGG GCA AGA1118Gln Asp Glu Ala Pro Lys Met Lys Asp Leu Gl - #y Leu Glu Arg Ala Arg# 345- TCA TAT GGA TGG CAA GAC ACT TAT GTT TTC AC - #C AAA GCA ATG GGT GAG1166Ser Tyr Gly Trp Gln Asp Thr Tyr Val Phe Th - #r Lys Ala Met Gly Glu350 3 - #55 3 - #60 3 -#65- ATG ATG ATC AAT AGC ACT CGA GGA GAC GTA CC - #T GTT GTT ATT ATA AGG1214Met Met Ile Asn Ser Thr Arg Gly Asp Val Pr - #o Val Val Ile Ile Arg# 380- CCT AGC GTC ATC GAA AGC ACT TAC AAA GAT CC - #T TTC CCT GGA TGG ATG1262Pro Ser Val Ile Glu Ser Thr Tyr Lys Asp Pr - #o Phe Pro Gly Trp Met# 395- GAA GGA AAC AGG ATG ATG GAT CCT ATA GTT TT - #A TGT TAC GGG AAG GGG1310Glu Gly Asn Arg Met Met Asp Pro Ile Val Le - #u Cys Tyr Gly Lys Gly# 410- CAA CTC ACG GGG TTT TTG GTT GAT CCA AAA GG - #A GTT CTT GAT GTA GTT1358Gln Leu Thr Gly Phe Leu Val Asp Pro Lys Gl - #y Val Leu Asp Val Val# 425- CCT GCT GAT ATG GTT GTT AAT GCA ACG TTA GC - #T GCT ATA GCA AAG CAT1406Pro Ala Asp Met Val Val Asn Ala Thr Leu Al - #a Ala Ile Ala Lys His430 4 - #35 4 - #40 4 -#45- GGA ATG GCA ATG TCA GAT CCG GAA CCT GAA AT - #A AAC GTG TAT CAG ATC1454Gly Met Ala Met Ser Asp Pro Glu Pro Glu Il - #e Asn Val Tyr Gln Ile# 460- GCT TCT TCG GCG ATA AAC CCG CTG GTT TTC GA - #A GAC TTA GCG GAG CTT1502Ala Ser Ser Ala Ile Asn Pro Leu Val Phe Gl - #u Asp Leu Ala Glu Leu# 475- CTT TAT AAC CAC TAC AAA ACA TCC CCA TGC AT - #G GAC TCT AAA GGT GAT1550Leu Tyr Asn His Tyr Lys Thr Ser Pro Cys Me - #t Asp Ser Lys Gly Asp# 490- CCT ATT ATG GTG CGT TTG ATG AAA CTT TTC AA - #T TCC GTT GAT GAT TTC1598Pro Ile Met Val Arg Leu Met Lys Leu Phe As - #n Ser Val Asp Asp Phe# 505- TCG GAT CAT TTG TGG AGA GAT GCT CAA GAA CG - #G AGT GGG TTG ATG AGT1646Ser Asp His Leu Trp Arg Asp Ala Gln Glu Ar - #g Ser Gly Leu Met Ser510 5 - #15 5 - #20 5 -#25- GGT ATG AGT TCA GCG GAT AGT AAG ATG ATG CA - #G AAG CTA AAG TTT ATA1694Gly Met Ser Ser Ala Asp Ser Lys Met Met Gl - #n Lys Leu Lys Phe Ile# 540- TGC AAG AAA TCT GTT GAA CAA GCC AAA CAC CT - #T GCT ACT ATT TAT GAG1742Cys Lys Lys Ser Val Glu Gln Ala Lys His Le - #u Ala Thr Ile Tyr Glu# 555- CCA TAC ACT TTC TAT GGT GGA AGA TTT GAT AA - #C AGC AAT ACA CAG AGA1790Pro Tyr Thr Phe Tyr Gly Gly Arg Phe Asp As - #n Ser Asn Thr Gln Arg# 570- TTA ATG GAG AAT ATG TCA GAG GAC GAG AAG AG - #A GAA TTT GGA TTT GAT1838Leu Met Glu Asn Met Ser Glu Asp Glu Lys Ar - #g Glu Phe Gly Phe Asp# 585- GTT GGA AGC ATT AAC TGG ACG GAC TAC ATT AC - #A AAC GTT CAC ATT CCC1886Val Gly Ser Ile Asn Trp Thr Asp Tyr Ile Th - #r Asn Val His Ile Pro590 5 - #95 6 - #00 6 -#05- GGT TTA AGA AGG CAT GTC TTG AAA GGA AGA GC - #T TAACTTTGAA TCTCACTAAA1939Gly Leu Arg Arg His Val Leu Lys Gly Arg Al - #a# 615- CCAGACCAAA CAGAATCGAT CCCTTCTTTT ATCTTTTTAT CTTTTTCTTT TT - #TCATTACG1999- TGTAATCGCG TTGTGTCTAA TATATCAGCT CGATTTGTAA TAATTTGAAA AA - #AACCGGAA2059- ATGTTGTTAT CTTTAAGTTT GCCCAAAATC TATAGTCATG TTCGATTCAA GA - #CAAAAAAA2119# 2126- (2) INFORMATION FOR SEQ ID NO: 2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 616 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Glu Ala Leu Phe Leu Ser Ser Ser Ser Se - #r Ser Ile Val Gly Ser# 15- Asn Lys Leu Thr Arg Leu His Asn His Cys Va - #l Trp Ser Thr Val Ile# 30- Arg Asp Lys Lys Arg Phe Gly Pro Thr Trp Cy - #s Arg Val Gly Gly Gly# 45- Gly Asp Gly Gly Arg Asn Ser Asn Ala Glu Se - #r Pro Ile Arg Val Ser# 60- Ser Leu Leu Lys Asp Arg Gly Gln Val Leu Il - #e Arg Glu Gln Ser Ser# 80- Pro Ala Met Asp Ala Glu Thr Leu Val Leu Se - #r Pro Asn Gly Asn Gly# 95- Arg Thr Ile Glu Ile Asn Gly Val Lys Thr Le - #u Met Pro Phe Ser Gly# 110- Ala Ser Met Val Gly Met Lys Glu Gly Leu Gl - #y Ile Ile Ser Phe Leu# 125- Gln Gly Lys Lys Phe Leu Ile Thr Gly Ser Th - #r Gly Phe Leu Ala Lys# 140- Val Leu Ile Glu Lys Val Leu Arg Met Ala Pr - #o Asp Val Ser Lys Ile145 1 - #50 1 - #55 1 -#60- Tyr Leu Leu Ile Lys Ala Lys Ser Lys Glu Al - #a Ala Ile Glu Arg Leu# 175- Lys Asn Glu Val Leu Asp Ala Glu Leu Phe As - #n Thr Leu Lys Glu Thr# 190- His Gly Ala Ser Tyr Met Ser Phe Met Leu Th - #r Lys Leu Ile Pro Val# 205- Thr Gly Asn Ile Cys Asp Ser Asn Ile Gly Le - #u Gln Ala Asp Ser Ala# 220- Glu Glu Ile Ala Lys Glu Val Asp Val Ile Il - #e Asn Ser Ala Ala Asn225 2 - #30 2 - #35 2 -#40- Thr Thr Phe Asn Glu Arg Tyr Asp Val Ala Le - #u Asp Ile Asn Thr Arg# 255- Gly Pro Gly Asn Leu Met Gly Phe Ala Lys Ly - #s Cys Lys Lys Leu Lys# 270- Leu Phe Leu Gln Val Ser Thr Ala Tyr Val As - #n Gly Gln Arg Gln Gly# 285- Arg Ile Met Glu Lys Pro Phe Ser Met Gly As - #p Cys Ile Ala Thr Glu# 300- Asn Phe Leu Glu Gly Asn Arg Lys Ala Leu As - #p Val Asp Arg Glu Met305 3 - #10 3 - #15 3 -#20- Lys Leu Ala Leu Glu Ala Ala Arg Lys Gly Th - #r Gln Asn Gln Asp Glu# 335- Ala Pro Lys Met Lys Asp Leu Gly Leu Glu Ar - #g Ala Arg Ser Tyr Gly# 350- Trp Gln Asp Thr Tyr Val Phe Thr Lys Ala Me - #t Gly Glu Met Met Ile# 365- Asn Ser Thr Arg Gly Asp Val Pro Val Val Il - #e Ile Arg Pro Ser Val# 380- Ile Glu Ser Thr Tyr Lys Asp Pro Phe Pro Gl - #y Trp Met Glu Gly Asn385 3 - #90 3 - #95 4 -#00- Arg Met Met Asp Pro Ile Val Leu Cys Tyr Gl - #y Lys Gly Gln Leu Thr# 415- Gly Phe Leu Val Asp Pro Lys Gly Val Leu As - #p Val Val Pro Ala Asp# 430- Met Val Val Asn Ala Thr Leu Ala Ala Ile Al - #a Lys His Gly Met Ala# 445- Met Ser Asp Pro Glu Pro Glu Ile Asn Val Ty - #r Gln Ile Ala Ser Ser# 460- Ala Ile Asn Pro Leu Val Phe Glu Asp Leu Al - #a Glu Leu Leu Tyr Asn465 4 - #70 4 - #75 4 -#80- His Tyr Lys Thr Ser Pro Cys Met Asp Ser Ly - #s Gly Asp Pro Ile Met# 495- Val Arg Leu Met Lys Leu Phe Asn Ser Val As - #p Asp Phe Ser Asp His# 510- Leu Trp Arg Asp Ala Gln Glu Arg Ser Gly Le - #u Met Ser Gly Met Ser# 525- Ser Ala Asp Ser Lys Met Met Gln Lys Leu Ly - #s Phe Ile Cys Lys Lys# 540- Ser Val Glu Gln Ala Lys His Leu Ala Thr Il - #e Tyr Glu Pro Tyr Thr545 5 - #50 5 - #55 5 -#60- Phe Tyr Gly Gly Arg Phe Asp Asn Ser Asn Th - #r Gln Arg Leu Met Glu# 575- Asn Met Ser Glu Asp Glu Lys Arg Glu Phe Gl - #y Phe Asp Val Gly Ser# 590- Ile Asn Trp Thr Asp Tyr Ile Thr Asn Val Hi - #s Ile Pro Gly Leu Arg# 605- Arg His Val Leu Lys Gly Arg Ala# 615- (2) INFORMATION FOR SEQ ID NO: 3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1077 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1057..1077 (D) OTHER INFORMATION:/par - #tial /codon.sub.-- - #start= 1057#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GATCTAAGAC AAAAACGTGG CCATTTGCTA ATTGTTGTTT TTGTTGTAGC AA - #TAACCTTA 60- GTCAAAGGAT TTTGTTTATT GCGGACCCAA GTTGGTTGGT CGGCTCTTGC TT - #AAACCACA 120- TTTGGAATTT GTTGTTCTGG AGTCTGGAGA TCATTGAAAC ACAACCAAGA AG - #ATAGCGCA 180- CGTGTTTTAA AGTCGTATGT GTAGTTCTTT GTTCACCACG AGTTTAAGGT TC - #TCTTTCAT 240- GTCTCATTGT TCTAAATATT CATCTTCGGT TGCATGTTTA ACTTCATAGT CC - #AGTTTATA 300- TTTTCCATCT AGATGATTGG GAACATTTTG CTTACTTTTA TGATCTTAAA CA - #GATGAACG 360- GTCTCATGTT AACAACATAG TACTGTTGAC TTCATGATAA TTTCATATCA TC - #TAATGACT 420- AAATTCTTTG CAGAGTTTAA TGGTGTTGAT TGTTGAAACA AGAGCAGATT GG - #TCAATCAC 480- TACAGAAAAA AAAAAGTTGG TAACATGTAA GTTTAACGTT ATTTAATAAA GG - #AGGATCTA 540- AGTTTTCTAC AAAAGCTATA ATTTTTATGA TGACCATATA ATCCTCAAAC CC - #TTCAAGAT 600- GTGATGTGAA TTATCTAAAT CCCAACACGA AGAAATGAGA TTTTTTAAAG TT - #AGCTATTT 660- ATCCTTAGTT GATTTCTTAA TTATAGGGTA ATGGCAATAT TTTTTGGAAC TG - #ATAATACG 720- TTTCTTTTTT TTTTCTGAAT TCTAGATGAT CACGTGTAGG AAACTGATAA AA - #TGTTGGAA 780- AGAATTCGTA AGGCAATCTT TTATTTCACT TGATTTTTAA AATATTTATT TG - #CCTATAAA 840- ACAGAGGAAG TTTTTCATCA TCTTTTGTCC TTAGAACTAA CCAATCTTTC AT - #TCCTCTTA 900- TAAAAACAAA ACCTACTTTA CTTGTCTCTT AACGATAACA AAATAACAAA TA - #ATTAATTC 960- TGTTCTTGGT TTACTTAATC TTCTTTCTAG TTAAGTATAT TCTTGTTGCT CA - #TCACCAAA1020#ACC AAC AAG 1074AG TATATTACAA GTCACC AAT TTC TTA# Asn Phe Leu Thr Asn Lys# 5 1# 1077Leu- (2) INFORMATION FOR SEQ ID NO: 4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 7 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Asn Phe Leu Thr Asn Lys Leu 1 5__________________________________________________________________________

Claims

1. A recombinant polynucleotide for use in obtaining a male-sterile plant, comprising:

a) a MS2 sense gene or a MS2 antisense gene capable of inhibiting the expression of a target gene, which target gene is present in the plant and encodes a protein indicated as MS2 which is involved in pollen development, said target gene having the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence coding for the same protein product as that encoded by SEQ ID NO:1, and

b) a promoter that is active in the tapetum of said plant, operably linked to said MS2 sense gene or MS2 antisense gene as to achieve expression thereof in the tapetum of said plant.

2. A recombinant polynucleotide according to claim 1, wherein the promoter that is active in the tapetum of the plant comprises a MS2 promoter.

3. A method for obtaining a male-sterile plant, comprising the steps of

a) transferring a recombinant polynucleotide according to claim 1 to cells of a male-fertile plant,

b) generating whole new plants from cells having incorporated said recombinant polynucleotide and,

c) selecting a plant that is male-sterile.

4. A recombinant plant genome comprising the recombinant polynucleotide of claim 1.

5. A male-sterile plant comprising the recombinant plant genome of claim 4.

6. A cell, fruit, seed or progeny each obtained from a male-sterile plant according to claim 5.

7. A seed of a male-sterile plant according to claim 5.

8. A homozygous male-sterile plant obtainable from a seed according to claim 7.

9. A cell, fruit, seed or progeny each obtained from plant of claim 8.

10. A method for the production of heterozygous male-sterile plants comprising fertilizing a homozygous male-sterile plant of claim 8 with a male-fertile plant, harvesting the seed and growing the heterozygous male-sterile plant from said seed.

11. A method for obtaining hybrid seed comprising the steps of crossing a male-sterile plant of claim 5 with a male-fertile plant and collecting the hybrid seed.

12. A method for obtaining hybrid seed comprising the steps of crossing a male-sterile plant of claim 8 with a male-fertile plant and collecting the hybrid seed.

13. The recombinant polynucleotide according to claim 2, wherein said MS2 promoter has the sequence of SEQ ID NO:3.