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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8217—Gene switch
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
  • C12N15/8289—Male sterility
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
  • C12N9/1037—Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase

Definitions

• the present invention relates to genetically transformed plants, methods for obtaining genetically transformed plants and recombinant DNA for use therein.
• the invention further relates to a method for restoring a plant phenotype previous- ly altered due to the expression of a transgene in that plant.
• the European Patent Application 344 029 A2 describes a method for restoring male-fertility in plants that are male- sterile due to the expression of a first transgene encoding Barnase in the tapetal cell layer of said plants, which method comprises the introduction into the same plant of a second transgene encoding Barstar which is expressed at least in all those cells wherein the first transgene is expressed.
• the Barnase/Barstar system for altering and restoring plant phenotype the first transgene, the barnase gene is believed to interfere with a large number of endogenous gene products in a non-specific way, rather than by interaction with a preselected endogenous gene product.
• the restoration of male-fertility is based on a direct interaction of Barstar with Barnase.
• fertility restoration accor ⁇ ding to this system is based on direct interaction of the restoration gene product with the sterility gene product in the plant cell.
• This is one of the best described phenotype restoration systems known in the art.
• a drawback of the Barnase/Barstar system is that its application is limited to phenotypes which allow disruption of cell structures by cell death. Phenotypes that require more subtle modification of plant cell functioning, such as alteration of flower colour, fruit ripening, and the like, are outside the scope of this system.
• Many systems for altering plant phenotypes are based on inhibition of endogenous plant genes. Examples thereof include but are not limited to disease-resistance, flower co- lour, fruit-ripening, male-sterility, and the like. It is an object of the invention to provide a phenotype restoration or moderation system that can be used when plant phenotypes ' have been altered due to the expression of a transgene capable of inhibiting expression of a particular endogenous gene.
• the present invention provides a process for the resto ration of a plant phenotype that is altered due to a first transgene which when expressed inhibits expression of an endogenous plant gene, by introducing into said plant, or progeny thereof, a second transgene which when expressed is capable of neutralising or partially neutralizing the effec caused by the first transgene, whereby said second transgen is expressed at least in those cells involved in the altere phenotype.
• a second transgene which encodes a protein or polypeptid gene product that is capable of substituting the function o the protein or polypeptide product encoded by the said endogenous gene and wherein the nucleotide sequence identit of the transcripts encoded by the second transgene and the first transgene is less than 90%, preferably less than 80%, yet more preferably said second transgene encodes a protein or polypeptide gene product that is not identical in amino acid sequence to the endogenous gene product and wherein th nucleotide sequence identity of the transcripts encoded by the second transgene and the first transgene is less than 75%.
• said secon transgene is obtainable from a different plant species.
• the invention further provides a process for the resto ration of fertility in a plant that is male-sterile due to first transgene which when expressed inhibits expression of an endogenous plant gene required for pollen development or functioning, by introducing into said plant a second trans ⁇ gene capable of neutralising the effect caused by the first transgene, whereby said second transgene is expressed in al cells in which the first transgene is expressed.
• said second transgene encodes a protein or polypeptide gene product that is capab of substituting the function of the protein or polypeptide product encoded by the said endogenous gene and wherein the nucleotide sequence identity of the transcripts encoded by the second transgene and the first transgene is less than 90% , preferably less than 80%, more preferably said second transgene encodes a protein or polypeptide gene product tha is not identical in its amino acid sequence to the endogeno gene product and wherein the nucleotide sequence identity o the transcripts encoded by the second transgene and the fir transgene is less than 75%.
• said secon transgene is obtainable from a different plant species.
• said first transgene is an antisense gene which when expressed inhibits expression of an endogenous flavonoid biosynthesis gene and said second transgene encod a flavonoid biosynthesis enzyme capable of substituting the function of the corresponding flavonoid biosynthesis enzyme encoded by the said endogenous gene.
• Preferred according to this embodiment is a first transgene which is an antisense gene inhibiting expression of an endogenous chalcone syntha gene and said second transgene encodes a chalcone synthase capable of substituting the function of the chalcone syntha encoded by the said endogenous gene.
• first transgenes and second transgenes for the restoration moderation of male-fertility are those obtainable from tabl 1 in this specification.
• Preferred in a process according to the invention is t process wherein said second transgene is introduced into th progeny of said plant by cross-pollination of a parent of said plant with pollen comprising said second transgene.
• the invention further provides a process for obtaining fertile hybrid seed of a self-fertilizing plant species, comprising the steps of cross-pollinating a plant S which i male-sterile due to a transgene which when expressed inhibi expression of an endogenous gene required for normal pollen development or functioning, with a plant R which is male- fertile and comprises a transgene that encodes a protein or polypeptide product capable of substituting the function of the protein or polypeptide product encoded by the said endogenous gene.
• Preferred according to this process is a first transgene which is an antisense chalcone synthase gen the endogenous gene is a chalcone synthase gene, and the second transgene encodes chalcone synthase, wherein the nucleic acid sequence identity of the transcripts encoded b the second transgene and the first transgene is less than 90%, preferably less than 80%, more preferably less than 75
• the invention also comprises fertile hybrid seed obtained by a process according to the invention, as well a plants obtained from fertile hybrid seed, as well as parts the plants, such as a bulb, flower, fruit, leaf, pollen, ro or root culture, seed, stalk, tuber or microtuber, and the like.
• the invention further comprises plants, as well as par thereof, which harbour a chimeric gene which when expressed produces a protein or polypeptide product capable of substi tuting the function of a polypeptide or protein encoded by endogenous gene of said plant, wherein the nucleotide sequence identity of the transcripts encoded by the transge and the endogenous gene is less than 90%, preferably less than 80%, more preferably less than 75%.
• FIGURES Figure 1 A representation of plasmid MIP289 harbourin an expression cassette with multiple cloning site, which ca be suitably used to insert foreign genes and antisense gene for expression in anthers of plant cells; CHI PB: chalcone iso erase B promoter; NOS tail: transcription termination signal derived from the nopaline synthase gene of Acrrobacte rium.
• Figure 2 Same plasmid as in figure 1, wherein the expression cassette contains a hybrid promoter based on the 35S promoter of cauliflower mosaic virus, and a so-called anther box (for details of promoter, vide Van der Meer, et al, 1992, sub) Figure 3.
• plant S maternal male-sterile line heterozygous for the sterility gene which when expressed inhibits expression of endogenous plant gene required for pollen development or functioning
• plant R pollinator line heterozygous for a restoration transgene capable of neutralising the effect caused by the first transgene.
• Binary vector pFBP125 This is a pBIN19 based vector with an insert comprising a chs gene from Arabidopsis thaliana between a hybrid promoter fragment comprising the CaMV 35S RNA promoter in which an anther-box (AB) has been inserted, and the nos-termination region of Agrobacterium tumefaciens.
• Binary vector pFBP130 This is a pBIN19 based vector with an insert comprising an chs gene from Arabidopsi thaliana between a promoter fragment of the chs-A gene of Petunia hvbrida and the nos-termination region of Agrobacte ⁇ rium tumefaciens.
• FIG. 7 Southern analysis of plant DNA of several petunia lines containing: (a) petunia anti-sense chs con- struct (T29) , (b) Arabidopsis sense chs gene construct (- T36004), (c) both constructs (a) and (b) (T38002 and T38007) and wild-type (W115) probed with 32 P-labelled Arabidopsis chs DNA (o/n exposure -80 degr. Celsius) .
• the Arabidopsis chs genes are clearly visible in T38002 (several strong bands) , T38007 (several strong bands) and T36004 (one strong upper band) , whereas there is only slight cross-hybridization with the endogenous petunia chs genes or antisense petunia chs genes (faint bands in the lanes of T38002, T38007, T29 and 115 and the antisense gene in T29) .
• FIG. 8 Northern analysis of messenger RNA of the same plants as in Fig. 7, including now T38005. Probed with petunia chs DNA; 6 days exposure -80 degr. Celsius) . The chs RNA are clearly visible in the lanes of T36004 and W115 as expected. In none of the antisense plant lines (T29, T38002, T38005, T38007) could a petunia RNA be detected, as could have been expected as well.
• FIG. 9 Northern analysis as in Figure 8, except that the blot was probed with Arabidopsis chs DNA, o/n exposure at - degrees Celsius. At o/n exposure the Arabidopsis chs MRNA i only detected in the lane of T36004. However, upon gross overexposure some very faint bands could be detected in the lanes of the double transgenic lines T38002, T38005 and T38007.
• male-sterile Petunia hybrida plant S which is transgenic fo an antisense CHS gene from Petunia hybrida under the contro of regulatory sequences that provide for expression of the transgene in anthers of the plants, is cross-fertilised wit a Petunia hybrida plant R that contains a transgene obtaina ble from the chs gene of Arabidopsis thaliana which is unde the control of regulatory sequences that provide for ex ⁇ pression of the transgene in anthers of the plants.
• hybrid seed SR 50% contains in addition to the endogenous chs gene an the Arabidopsis chs gene in the sense orientation, the antisense chs gene from Petunia hybrida. Contrary to expecta tion, it will be found, that a percentage of the progeny plants grown from the hybrid seed (50% SsRr; 50% ssRr) harbouring both the transgenes is again capable of self- fertilization in spite of the fact that about 50% also inherited the sterility gene.
• nucleic acid sequences of the Arabidopsis transgene and the Petunia gene transcripts diff at least 30% in the protein encoding region, presumably eve more if the non-translated regions of the transcript are taken into account.
• nucleic acid divergence of t transcript is deemed responsible for its translatability in the plant cell, thereby producing a fully active chalcone synthase which substitutes the endogenous chalcone synthase.
• male-fertility is restored in a percentage of t progeny plants despite the fact that about 50% thereof contain the sterility transgene.
• the invention can be worked with any phenotype alterati on system that involves an inhibitory gene of the antisense type, such as described in EP 240 208 A2, directed against a endogenous gene. Evenly so, it can be worked with an inhibi- tory gene of the sense type, which work by the as yet not fully understood mechanism referred to as co-suppression, disclosed in Napoli et al.. , 1990, supra.
• examples of such phenotypes include, but are not limited to disease-resis ⁇ tance, drought-resistance, flower colour, fruit ripening, a the like.
• the restoration gene must encode a transcript that is sufficiently divergent from both the endogenous gene trans ⁇ cript as well as the inhibitory transgene transcript and yet encodes a protein or polypeptide capable of substituting the function of the endogenous gene product.
• Phenotype resto ⁇ ration can be made absolute. Alternatively, phenotype resto ⁇ ration may be made not absolute; in this case it is preferre to speak of partial phenotype restoration or 'phenotype moderation'. If absolute phenotype restoration is aimed at. the divergence of the transcript must diverge preferably by more than 20%, that is the nucleic acid identity of the restoration transcript with either the inhibitory transgene transcript or the endogenous gene transcript does not exceed 80%, preferably it does not exceed 75%.
• optimal moderation can be achieved by making transgenes with different levels of divergence and selecting the desired phenotype.
• phenotype restoration is not required to be absolute, or desired to be not absolute, divergence of the restoration transgene transcript should not exceed 20%, preferably it should not exceed 10%. The latter is referred to as phenotyp moderation.
• phenotype alteration systems that involve inhibitory genes of the ribozyme type directed as sequence specific endo-ribonucleases against an endogenous gene trans cript, as disclosed in US Patent 4,987,071, may be restored with a transgene according to the invention, with the provis that the restoration gene encodes a transcript that is lacking the recognition and/or cleavage consensus of the ribozyme. Phenotype moderation should be possible using this kind of inhibitory transgenes as well, although manipulating the recognition and cleavage sequence of the restoration gen to affect its affinity for the ribozyme may require some trial and error.
• the restoration gene must not give rise to a transcript that is identical to the endogenous gene trans- cript.
• the restoration gene transcribed region i as much divergent from the transcribed region of the endoge ⁇ nous gene as possible, while the protein product encoded by said transcript is identical, or almost identical.
• each amino acid can be encoded by a more than one codon; this fact, referred to as the degenerac of the genetic code, stems from the fact that there are abou 20 different amino acids, which are encoded by triplets of four different bases, yielding a total of 64 possible codons
• Three codons comprise stop signals for translation, so that in actual fact 61 codon specify about 20 amino acids.
• every third base may be changed in a coding region without affecting the amino acid sequence of the protein.
• the transcribed region of a restoration gene can at least diverge 33% from the endogenous gene.
• sinc a gene transcript generally comprises non-translated regions flanking the coding region on both sides, even further nucleic acid divergence may be achieved in order to avoid interaction of the restoration gene transcript with the endogenous gene transcript or the first transgene transcript
• still greater divergence may be achieved i one takes into account the fact that two proteins may differ in their amino acid sequence, while retaining their physiolo gical activity in the plant cell.
• a restoration gene according to the present invention need not be more identical to its endogenous counterpart than about 40-50% on the nucleic acid level.
• Some aspects of the invention will be further illustra ⁇ ted with male-sterility as exemplifying phenotype.
• Any male-sterile plant phenotype that is due to expres ⁇ sion of an inhibitory gene of one of the types mentioned in the preceding paragraphs can be restored by a restoration gene according to the invention.
• genes can be identified that ar essential for pollen development or pollen functioning is given inter alia in WO89/10396 and WO90/08828. Once such genes are isolated they can be expressed or overexpressed in the sense or antisense orientation in those cells required for pollen development or functioning. In order to achieve expression in those cells that are necessary for pollen development, genes are placed under the control of promoters that are expressed in stamen cells (including filaments and anthers) , or more specifically in anthers, or even more specifically in tapetal cell layers thereof.
• sterility genes that are disruptive to general plant cell functioning or viability on the one hand, and genes that disrupt plant metabolism to the extent that i disrupt pollen development or functioning without drasticall affecting plant viability on the other hand.
• the antisense chalcone synthase gene is one of the latter category; conse ⁇ quently, it is not necessary for the latter type sterility gene to be expressed exclusively in stamen cells through the use of stamen-specific promoters.
• Sterility genes of the former type i.e. the general plant cell disrupters, must no be effective inside plant structures essential for survival of the plant. Methods for isolating promoters that provide for proper expression patterns of these genes are also described in both W089/10396 and WO90/08828, which are herewith deemed incorporated by reference.
• the seed obtained from this selfing can be grown into homozygous male-sterile maternal plant lines, which can optionally be propagated jLn vitro first, and then used as such in hybrid seed productio by cross-pollination with a pollinator line, which may be heterozygous or homozygous for the restoration gene accordi to the invention.
• sterility genes, herbicide resistance genes or restoration genes into plants, is achieved by a an one of the following techniques, the choice of which is not critical to the present invention.
• useful methods are the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al. , 1982, Nature 296. 72-74; Negrutiu I. gt al, June 1987, Plant Mol. Biol. 8., 363-373) , electroporation of protoplasts (Shillito R.D. et al. , 1985 Bio/Technol. 2, 1099-1102), microinjection into plant material (Crossway A. et al. , 1986, Mol. Gen. Genet. 202.
• Agrobacterium-mediated DNA transfer Especially preferred is the use of the so-called binary vector technology as disclo ⁇ sed in EP-A 120 516 and U.S. Patent 4,940,838).
• pollen cells are transformed, for instance by coated- particle acceleration, and used to pollinate receptive plants.
• monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells.
• preferred methods for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation (Shimamoto, et al. 1989, Nature 338. 274-276) .
• Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin) , into embryogeni cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm et al., 1990, Plant Cell, 2., 603- 618) . The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, 1989, Plant Mol. Biol. 13., 21-30).
• Wheat plants have been regenerated from embryogenic suspensi on culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil I., et al, 1990, Bio/Technol. 8., 429-434).
• Herbicide resistant fertile wheat plants were obtained by microprojectile bombardment of regenerable embryogenic callus (Vasil V. et al, 1992, Bi- o/technol. .10, 667-674) .
• the combination with transformation systems for these crops enables the application of the present invention to monocots.
• Monocotyledonous plants including commercially impor- tant crops such as corn are also amenable to DNA transfer by Agrobacterium strains (Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. 95, 426- 434) .
• Suitable marker genes that can be used to select or screen for transformed cells, can be selected from any one o the following non-limitative list: neomycin phosphotransphe- rase genes conferring resistance to kanamycin (EP-B 131 623) the hygromycin resistance gene (EP 186 425 A2) the
• Glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides EP-A 256 2273
• glutamine synthetase conferring upon overexpression resistan ce to glutamine synthetase inhibitors such as phosphinothri- cin (W087/05327)
• the acetyl transferase gene from Streptomy ces viridochromogenes conferring resistance to the selective agent phosphinothricin EP-A 275 957)
• the bar gene conferring resistance against Bialaphos e.g. W091/02071
• the actual choice of the marker is not crucial as long as it is functional (i.e. selective) in combination with the plant cells of choice.
• the marker gene and the gene of interest do not necessa rily have to be linked, since co-transformation of unlinked genes (U.S. Patent 4,399,216) is also an efficient process i plant transformation.
• the expression pattern required for the restoration gen depends on the expression pattern of the inhibitory transge ⁇ ne. The latter in its turn is dependent on the phenotype alteration aimed at. Thus, for modifying the fruit ripening phenotype in a plant, an inhibitory gene bringing about said alteration must at least be expressed in the fruits of said plant. Restoration or moderation can be achieved by an expression pattern that comprises at least the expression pattern of the inhibitory transgene.
• transgenic plants harbouring more than one gene a number of alternatives are available, the actual choice of which is not material to the present invention: A. the use of one recombinant polynucleotide, e.g a plasmid, with a number of modified genes physically coupled to one selection marker gene.
• hybrid seed It is known in the art that, the need to separate hybri seed from non-hybrid seed can be avoided if the self-pollina tors can be destroyed, for example by using an antibiotic, preferably a herbicide. This requires that the maternal male sterile line is resistant to this antibiotic or herbicide du to the presence of transgene coding therefor.
• the herbicide resistance gene may be introduced into th maternal line simultaneously with the sterility gene accor ⁇ ding to the invention by genetic transformation with a ulti gene construct. However, the herbicide resistance gene may b introduced into the maternal line after the introduction of the sterility gene.
• herbicide resistance trait may be advantageous to introduce the herbicide resistance trait into the plant intended to use as maternal parent line prior to the introduction of the sterility gene. This simplifies the creation of plants that are homozygous for the herbicide resistance phenotype which may be advanta ⁇ geous. Then, plants provided subsequently with the sterility gene, may be cross-pollinated with a pollinator plant contai ning a restoration gene according to the invention. Suitable herbicides can be selected from any one listed under the heading marker genes.
• the process according to the invention is particularly useful for the production of hybrid progeny that is fully male-fertile.
• a transgenic (heterozygous) nuclear male-sterile plant line S may be crossed with a male- fertile plant line R (ssrr) to yield hybrids that are 50% fertile (ssrr) and 50% sterile (Ssrr) . Consequently, if such hybrid crops were grown in the field directly, 50% of the acreage would consist of plants that must be cross-fertilise in order to set seed, which may have significant yield reducing effects for those crops that rely on the setting o fruit or seed for their commercial value. Examples of such crops include but are not limited to cereals and oil seed rape.
• the present invention is especially suitable for the hybridization of naturally self-fertilizing crops by crossing a maternal line which is male-sterile due to the expression of a first transgene capable of inhibiting expres sion of an endogenous plant gene essential to normal pollen functioning, and a pollinator line containing a second transgene capable of neutralising the effect caused by the first transgene.
• a first transgene capable of inhibiting expres sion of an endogenous plant gene essential to normal pollen functioning
• a pollinator line containing a second transgene capable of neutralising the effect caused by the first transgene.
• this hybridization system can be used in combination with any sterility system that makes use of transgenes inhibitory to endogenous genes.
• the phenotype can be determined predominantly by the nature of the gene product, rather than the specificity of the expression pattern.
• the chiPB/as-chs construct comprises a chs cDNA fragmen from Petunia hybrida fused in the antisense orientation to a chalcone isomerase B promoter fragment.
• the chiPB/chs-At construct comprises a chs cDNA fragment from Arabidopsis thaliana fused in the sense orientation to a chalcone isomerase B promoter fragment.
• a 1.7 kb promoter fragment from the anther-specific chiP B promoter (Tunen, A.J. Van., Mur, L.A., Brouns, G.A. , Rienstra, J.D., Koes, R.E. and Mol J.N.M., 1990, The Plant Cell 2., 393-401) and a 0.2 kb NOS tail isolated from plasmid pBIlOl.l (Jefferson, R.A., Kavanagh, T.A. , and Bevan, M.W. (1987). EMBO J. 6_, 3901-3907) are cloned into the plasmid pUC19 (Messing, J. , 1978, Recombinant DNA Technical Bulletin NIH Publication No. 79-99, 2 , 43-48) yielding the recombinan plasmid MIP289 ( Figure 1) .
• a 1.4 kb BamHI chs fragment is isolated from plasmid pTS21 (Van der Meer et al. , 1992, supra) and cloned into plasmid MIP289 digested with BamHI.
• a clone with the chs fragment in an antisense orientation is selected on the basi of the asymmetric SstI restriction enzyme site. Subsequently this fragment is subcloned as a Hindlll/EcoRI fragment into the binary vector Binl9 (Bevan, M. (1984) Nucl. Acid Res. 12. 8711-8712) yielding plasmid pAS8.
• the PCR reaction is performed in 100 ⁇ l PCR buffer (10 mM Tris, pH 8.3, 50mM KC1, 2.5 mM MgCl 2 ) containing 50 pmole primers, and 200 ⁇ M of each deoxy nucleotide triphosphate.
• Amplification involved 30 cycles of a standard cycle for homologous primers.
• Amplified CDNA is fractionated on a 1% agarose gel and a 1.4 kb band is iso ⁇ lated and subcloned as a BamHI fragment (sites present in th 5' and 3' primers) in pAS8 after digestion with BamHI to remove the petunia chs CDNA.
• tumefaciens strains harbouring either pAS8 or pAS9 the tobacco leaf discs are grown on MS plates containing 3 ⁇ g/ml kinetin, 500 ⁇ g carbenicillin and 200 ⁇ g kanamycin. Plants obtained are checked for transformation on the basis of resistance for kanamycin and by Southern blot analysis using an npt fragment as a probe. After shoot and root induction plants are put on soil and transferred to the greenhouse. Plants are grown under in the greenhouse at 21* at a 16 hours light, 8 hours dark regime.
• Example 3 Analysis of transgenic plants expressing the antisense chs construct
• Transgenic tobacco plants containing the chimeric pA gene construct are investigated f fertility by self-pollination. At least one plant is almo completely sterile and shows a seed set of less than 1% selfings. Furthermore the pollen grains of this plant a morphologically aberrant, as was also published by Van d Meer et &!. (1991) and are not able to germinate in an vitro germination assay.
• This plant is designated SI a contains only one copy of construct pAS8 in its genome.
• transgenic plants expressing the chimeric Arabi dopsis chs construct From a number of 15 transgenic tobacco plants containi plasmid pAS9, one plant expressing the Arabidopsis chs cD in young anthers is selected by RNAse protection experiment using RNA isolated from young anthers. This plant is designa ted Rl.
• referenc sequence is Petunia hybrida V30 chalcone synthase gene. Bes match is given at a minimum sequence of 1000 bp.
• Boldface gene fragments that are used as sterility an restoration gene respectively, in this disclosure.
• Petunia W115 plants were transformed with a sterilit gene construct comprising the promoter region of the petuni chs gene linked to the coding region of the petunia chs gen in antisense orientation.
• This gene construct designate VIP176 (Krol A.R. van der et al. , 1990, Plant Molecula Biology .14., 457-466) was used to transform the petunia lin W115 and a self-sterile plant was selected and designate T17002.
• T17002 This self-sterile flavonoid depleted plant, T17002 was cross-pollinated with a W115 plant and among the progen a plant was selected, which was kanamycin sensitive but stil self-sterile and depleted for flavonoids; this plant wa designated T29.
• This sterile, kanamycin sensitive plant, T29 was used for a second transformation with pFBP125 (P C8MV35SAB
• Plant lines were tested for the presence of the con structs by Southern analysis. Expression of the genes wa verified by Northern analysis.
• Table 2 summarizes the results for 6 petunia lines: fro top to bottom are given Southern data, obtained by probin with petunia chs probes and Arabidopsis chs probes; Norther data, obtained by probing with both aforementioned probes corolla pigmentation (flavonol staining) ; and fertilit determination.
• the genetic backgrounds of the petunia line are as follows: W115 - wild-type petunia plants (non-trans genic) ; T29 - P CaHV35SAB -antisense petunia chs (transgenic fo sterility gene); T38002, T38005, T38002 - P chs -antisens petunia chs + P ch£ -A.thaiiana chs (transgenic for sterilit gene and restoration gene) ; T36004 - P C ⁇ MV35;: ⁇ D -A. thaliana ch (transgenic for the restoration gene only) .
• the Arabidopsis chs probe was only weakly capable o cross-hybridizing with the petunia chs gene and vice vers (Fig. 7).
• the Northern data on mRNA of corolla's corresponded wit the Southern data, except that the Arabidopsis chs-messenge RNA of plant lines T38002, T38005 and T38007, when probe with the Arabidopsis chs-probe. could only be detected afte gross over-exposure; this is probably due to weak expressio of the Pchs-Arabidopsis chs gene construct in corolla's.
• Selfed seed is used for linkage analysis in an out-crossing in order to establish agreement of the presence of the petunia antisense gene (self-sterile) , the sense Arabidopsis chs gene (male- fertile, both the petunia antisense chs and the Arabidopsis chs gene (partially male-fertile) , and no transgenes (fertile) .
• Flavonol staining is specific for quercetin and dihydro-kaempferol (aglykones) and is performed according to Sheahan J.J. and Rechnitz G.A., 1992, BioTechniques 1 , No. 6, 880-883.
• ORGANISM Arabidopsis thaliana
• ORGANISM Arabidopsis thaliana

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