Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
  • C12N15/8275—Glyphosate

Definitions

• the present invention relates to a novel expression cassette comprising a nucleic acid sequence encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and to its use for obtaining plants resistant to herbicides which inhibit this enzyme, in particular herbicides of the phosphonic acid family, in particular of the N-phophonomethylglycine family.
• EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
• EPSPS is a plastid enzyme involved in the shikimate biosynthetic pathway, leading to the synthesis of aromatic amino acids.
• EPSPS is known to be the target enzyme for herbicides of the family of phosphonic acids of the phophonomethylglycine type.
• 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-inhibiting herbicides are well known as being highly effective foliar herbicides.
• the most well known herbicide of this herbicide class is glyphosate [N-(phosphonomethyl)glycine].
• Sulfonate or fosametine are also known.
• Glyphosate is characterized by a lack of selectivity for crop species and is, consequently, generally used under conditions in which there is no need for selectivity, for example as a total herbicide.
• plants tolerant to this herbicide have been developed by transformation of said plants with a gene encoding a glyphosate-tolerant EPSPS enzyme.
• Genes encoding glyphosate-tolerant EPSPS enzymes are in particular described in patent application EP 0837944.
• glyphosate-tolerant maize and soybean are sold respectively under the trade marks Roundup-Ready CornTM and Roundup-Ready SoybeanTM. In this way, glyphosate can be applied to crops without affecting the crop plants which have been made tolerant thereto.
• the success of this strategy is essentially based on the quality and the quantity of expression of the enzyme in the tissues of the plant intended to be made tolerant.
• These parameters of quality and quantity of expression are controlled by the regulatory elements introduced into the expression cassette with the nucleic acid sequence encoding said EPSPS enzyme.
• the regulatory elements essential to an expression cassette are the promoter regulatory sequence and the terminator regulatory sequence.
• the expression cassettes can also contain a signal peptide or a transit peptide, and also a transcription activator element or enhancer.
• the regulatory element which contributes most to the quality and the quantity of expression of a protein encoded by a nucleic acid sequence in an expression cassette is the promoter.
• Identification of the promoter suitable for expression of a given protein also depends largely on the nature of said protein, and in particular on the desired quantity and quality of expression of said protein.
• Associated with a given promotor is a quantity of expression of the product encoded by the nucleic acid sequence which it controls, and also a quality, in particular spatiotemporal quality, of this expression.
• some promoters are constitutive and others inducible.
• An important characteristic for a promoter used in an expression cassette intended for the expression of an EPSPS enzyme in a plant is that it should allow a quantitative expression sufficient to confer tolerance to glyphosate on all the tissues of the plant which may be affected by this herbicide.
• the technical problem of the present invention consists in obtaining an expression cassette in which the promoter is particularly suitable for the quantitative and qualitative expression of an EPSPS enzyme in transformed plants, said expression cassette then conferring effective tolerance on said plants, with respect to a herbicide which inhibits this enzyme, in particular a herbicide of the phophonomethylglycine family, in particular glyphosate.
• Promoters which allow a high level of expression are generally promoters of highly expressed proteins.
• promoters which satisfy these criteria, mention may be made, by way of example, of bacterial promoters, such as that of the octopine synthase gene or that of the nopaline synthase gene, viral promoters, such as that of the gene controlling transcription of cauliflower mosaic virus 35S or 19S RNAs (Odell et al., 1985, Nature, 313, 810-812), or promoters of the cassava vein mosaic virus (as described in patent application WO 97/48819).
• promoters of plant origin mention will be made of the promoter of the ribulose-biscarboxylase/oxygenase (RuBis CO) small subunit gene, the promoter of a histone gene described in application EP 0 507 698, or the promoter of a rice actin gene (U.S. Pat. No. 5,641,876).
• RuBis CO ribulose-biscarboxylase/oxygenase
• promoters are expressed specifically in the cells of certain tissues. Such promoters are generally promoters which regulate the expression of proteins involved in the function of a particular tissue or organ.
• root-specific promoters such as, for example, that described in patent application WO 00/29594
• flower-specific promoters such as those described in patent applications WO 98/22593, WO 99/15679 or WO 99/43818
• fruit-specific promoters in particular seed-specific promoters such as those described in patent applications WO 91/13993, WO 92/17580, WO 98/45460, WO 98/45461, or WO 99/16890, are known.
• the present invention relates to a novel expression cassette comprising, in the direction of transcription, functionally linked to one another, a promoter regulatory sequence which is functional in plant cells or plants, a nucleic acid sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and a terminator sequence which is functional in plant cells or plants, characterized in that the promoter regulatory sequence is a nucleic acid sequence chosen from the promoter regulatory sequences of the CsVMV (Cassaya Vein Mosaic Virus) plant virus.
• EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
• the expression “functionally linked to one another” means that said elements of the chimeric gene are linked to one another in such a way that their function is coordinated and allows expression of the coding sequence.
• a promoter is functionally linked to a coding sequence when it is capable of ensuring expression of said coding sequence.
• the construction of a chimeric gene according to the invention and the assembly of its various elements can be carried out using techniques well known to those skilled in the art, in particular those described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press).
• the expression “functional in plant cells and plants” is intended to mean capable of functioning in plant cells and plants.
• CsVMV promoter regulatory sequences are described in patent application WO 97/48819 (the content of which is incorporated herein by way of reference), in particular the promoter regulatory sequence comprising one of the nucleotide sequences represented by one of the sequence identifiers SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of patent application WO 97/48819, more particularly the nucleic acid sequence represented by the sequence identifier SEQ ID NO 3 of patent application WO 97/48819.
• the promoter regulatory sequence of the expression cassette comprises, in the direction of transcription, the nucleic acid sequences X, Y and Z as defined respectively by SEQ ID NOS 1, 2 and 3 of the present patent application.
• a preferred promoter regulatory sequence for the expression cassette is represented by SEQ ID NO 4 of the present patent application.
• the promoter regulatory sequence of the expression cassette comprises, in the direction of transcription, the nucleic acid sequences X, Y, Y and Z as defined above.
• the promoter regulatory sequence comprising the duplication of the nucleic acid sequence Y will be referred to as double CsVMV.
• the nucleic acid sequence of the double CsVMV is represented by SEQ ID NO 5 of the present application.
• the present invention also relates to the sequences capable of hybridizing selectively with the nucleic acid sequences above, the sequences homologous to the sequences above, and the functional fragments of said sequences.
• nucleic acid sequence is intended to mean a nucleotide or polynucleotide sequence which may be of the DNA or RNA type, preferably of the DNA type, in particular double-stranded.
• the expression “sequence capable of hybridizing selectively” is intended to mean the sequences which hybridize with the sequences above at a level significantly greater than the background noise.
• the background noise may be related to the hybridization of other DNA sequences present, in particular other cDNAs present in a cDNA library.
• the level of the signal generated by the interaction between the sequence capable of hybridizing selectively and the sequences defined by the SEQ IDs above according to the invention is generally 10 times, preferably 100 times, more intense than that of the interaction of the other DNA sequences generating the background noise.
• the level of interaction can be measured, for example, by labeling the probe with radioactive elements, such as 32 P.
• hybridization is generally obtained using very stringent medium conditions (for example 0.03 M NaCl and 0.03 M sodium citrate at approximately 50° C.-60° C.).
• very stringent medium conditions for example 0.03 M NaCl and 0.03 M sodium citrate at approximately 50° C.-60° C.
• the hybridization can of course be carried out according to the usual methods of the state of the art (in particular Sambrook & al., 1989, Molecular Cloning: A Labratory Manual).
• the term “homologue” is intended to mean a nucleic acid fragment exhibiting one or more sequence modifications relative to the nucleotide sequence encoding the fusion protein of the invention. These modifications may be obtained according to the usual mutation techniques, or else in choosing the synthetic oligonucleotides used in the preparation of said sequence by hybridization. With regard to the multiple combinations of nucleic acids which may lead to the expression of a same amino acid, the differences between the reference sequence according to the invention and the corresponding homologue may be considerable.
• the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications are generally and preferably neutral, i.e. they do not affect the primary sequence of the fusion protein.
• fragments is intended to mean fragments of the DNA sequences according to the invention, i.e. the sequences above for which parts have been deleted but which conserve the function of said sequences.
• EPSPS is intended to mean any native or mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme, the enzymatic activity of which consists in synthesizing 5-O-(1-carboxyvinyl)-3-phosphoshikimate from phosphoenolpyruvate (PEP) and 3-phosphoshikimate (E.C. 2.5.1.19; Morell et al., 1967, J. Biol. Chem. 242, 82-90).
• said EPSPS enzyme may originate from any type of organism.
• An EPSPS enzyme according to the invention also has the property of being tolerant with respect to herbicides of the phophonomethylglycine family, in particular with respect to glyphosate.
• Sequences encoding EPSPSs which are naturally tolerant, or are used as such, with respect to herbicides of the phophonomethylglycine family, in particular glyphosate, are known.
• the sequence of the AroA gene of the bacterium Salmonella typhimurium (Comai et al., 1983, Science 221, 370-371)
• the sequence of the CP4 gene of the bacterium Agrobacterium sp. (WO 92/04449)
• sequences of the genes encoding Petunia EPSPS Shah et al., 1986, Science 233, 478-481
• Tomato EPSPS Garr et al., 1988, J. Biol. Chem. 263, 4280-4289
• Eleusine EPSPS (WO 01/66704).
• Sequences encoding EPSPSs made tolerant to glyphosate by mutation are also known.
• a sequence of a gene encoding a mutated plant EPSPS which is preferred according to the invention is that encoding the maize EPSPS described in patent application EP 0837944, comprising a first mutation replacing the threonine amino acid at position 102 with isoleucine, and a second mutation replacing the proline amino acid at position 106 with serine. Due to the strong sequence homology between EPSPSs, and more particularly between plant EPSPSs, a rice EPSPS carrying the same mutations have also been described in patent applications WO 00/66746 and WO 00/66747.
• any EPSPS, and the genes encoding them, carrying the threonine/isoleucine and proline/serine mutations described above, whatever the relative position of these amino acids with respect to positions 102 and 106 of maize EPSPS, can be used in the present invention.
• those skilled in the art will be readily able to find the two amino acids to be mutated in any EPSPS sequence by using standard techniques of sequence alignment.
• the nucleic acid sequence encoding an EPSPS included in the expression cassette is a sequence encoding an EPSPS which has been mutated at the amino acids corresponding to the threonine at position 102 and to the proline at position 106, said positions being relative with respect to the maize EPSPS sequence.
• the nucleic acid sequence encoding an EPSPS included in the expression cassette is a sequence encoding a mutated EPSPS comprising an isoleucine at position 102 and a serine at position 106, said positions being relative with respect to the maize EPSPS sequence.
• the nucleic acid sequence encoding an EPSPS included in the expression cassette is a sequence encoding maize mutated EPSPS comprising an isoleucine at position 102 and a serine at position 106.
• the expression cassette according to the invention may also comprise a subcellular addressing sequence encoding a signal peptide or transit peptide.
• a sequence located upstream or downstream of the nucleic acid sequence encoding the EPSPS, makes it possible to direct said EPSPS specifically into a cellular compartment of the host organism.
• the expression cassette may comprise a sequence encoding a signal peptide or a transit peptide for directing the EPSPS to a particular compartment of the cytoplasm, such as the mitochondria, the plasts, the endoplasmic reticulum or the vacuoles.
• the transit peptide may be a signal for chloroplastic or mitochondrial addressing, which is then cleaved in the chloroplasts or the mitochondria.
• the transit peptides may be either single or double transit peptides.
• the double transit peptides are optionally separated by an intermediate sequence, i.e. they comprise, in the direction of transcription, a sequence encoding a transit peptide of a plant gene encoding an enzyme which is located in plastids, a portion of sequence of the mature N-terminal portion of a plant gene encoding an enzyme which is located in plastids, and then a sequence encoding a second transit peptide of a plant gene encoding an enzyme which is located in plastids.
• Such double transit peptides are, for example, described in patent application EP 0 508 909.
• the expression cassette may also comprise other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators (enhancers), for instance the transcription activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, of the tobacco etch virus (TEV) described by Carrington & Freed, or of the figwort mosaic virus (U.S. Pat. No. 5,994,521), for example.
• transcription activators for instance the transcription activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, of the tobacco etch virus (TEV) described by Carrington & Freed, or of the figwort mosaic virus (U.S. Pat. No. 5,994,521), for example.
• the expression cassette according to the invention may also contain introns, in particular introns which promote gene expression in monocotyledon plants, such as intron 1 of the rice actin gene described in patent application WO 99/34005, or the maize adh1 intron, or in dicotyledon plants, such as the Arabidopsis histone intron (EP 0850311).
• introns in particular introns which promote gene expression in monocotyledon plants, such as intron 1 of the rice actin gene described in patent application WO 99/34005, or the maize adh1 intron, or in dicotyledon plants, such as the Arabidopsis histone intron (EP 0850311).
• the present invention also relates to a cloning an/or expression vector comprising an expression cassette according to the invention.
• the vector according to the invention is of use for transforming a host organism, in particular a plant, and expressing therein an EPSPS.
• This vector may be a plasmid, a cosmid, a bacteriophage or a virus.
• the vector for transforming plant cells or plants according to the invention is a plasmid.
• the main qualities of this vector should be an ability to maintain itself and to self-replicate in the cells of the host organism, in particular by virtue of the presence of an origin of replication, and to express therein an EPSPS.
• the vector used in the present invention also contains, in addition to the expression cassette according to the invention, another expression cassette containing a selection marker.
• This selection marker makes it possible to select the host organisms which have effectively been transformed, i.e. those which have incorporated the vector.
• the host organism to be transformed is a plant.
• markers containing genes resistant to antibiotics such as, for example, that of the hygromycin phosphotransferase gene (Gritz et al., 1983, Gene 25:179-188), but also markers containing genes for tolerance to herbicides, such as the bar gene (White et al., NAR 18:1062, 1990) for tolerance to bialaphos, the EPSPS gene (EP 0837944) for tolerance to glyphosate or else the HPPD gene (WO 96/38567) for tolerance to isoxazoles.
• GUS enzyme a readily identifiable enzyme
• pigments or enzymes which regulate the production of pigments in the transformed cells Such selection marker genes are in particular described in patent applications WO 91/02071, WO 95/06128, WO 96/38567, and WO 97/04103.
• the present invention also relates to plant cells transformed with a vector as described above.
• the term “transformed plant cell” is intended to mean a plant cell which has incorporated into its genome the expression cassette according to the invention, and consequently produces an EPSPS.
• those skilled in the art may use one of the many known methods of transformation.
• One of these methods consists in bringing the plant cells to be transformed into contact with polyethylene glycol (PEG) and the vectors of the invention (Chang and Cohen, 1979, Mol. Gen. Genet. 168(1), 111-115; Mercenier and Chassy, 1988, Biochimie 70(4), 503-517).
• Electroporation is another method, which consists in subjecting the plant cells or tissues to be transformed and the vectors of the invention to an electric field (Andreason and Evans, 1988, Biotechniques 6(7), 650-660; Shigekawa and Dower, 1989, Aust. J. Biotechnol. 3(1), 56-62).
• Another method consists in directly injecting the vectors into the plant cells or the plant tissues by microinjection (Gordon and Ruddle, 1985, Gene 33(2), 121-136).
• the “biolistic” method may be used. It consists in bombarding plant cells or plant tissues with particles onto which the vectors of the invention are absorbed (Bruce et al., 1989, Proc. Natl. Acad. Sci.
• the transformation of the plant cells will be carried out using bacteria of the genus Agrobacterium , preferably by infection of the cells or tissues of said plants with A. tumefaciens (Knopf, 1979, Subcell. Biochem. 6, 143-173; Shaw et al., 1983, Gene 23(3):315-330) or A. rhizogenes (Bevan and Chilton, 1982, Annu. Rev. Genet. 16:357-384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci.
• the transformation of plant cells with Agrobacterium tumefaciens is carried out according to the protocol described by Ishida et al. (1996, Nat. Biotechnol. 14(6), 745-750). Those skilled in the art will choose the appropriate method according to the nature of the plant cells to be transformed.
• a subject of the present invention is a method for producing plants tolerant to EPSPS-inhibiting herbicides, in particular to herbicides of the phophonomethylglycine family, in particular glyphosate.
• This method consists in regenerating transformed plants from the transformed plant cells described above.
• the transformed plants according to the invention contain an expression cassette according to the invention in their genome and express an EPSPS in their tissues.
• the present invention therefore comprises transformed plants comprising an expression cassette according to the invention, parts of these plants, and the descendants of these plants.
• the expression “part of these plants” is intended to mean any organ of these plants, whether aerial or subterranean.
• the aerial organs are the stems, the leaves and the flowers.
• the subterranean organs are mainly the roots, but they may also be tubers.
• the term “descendants” is intended to mean mainly the seeds containing the embryos derived from the reproduction of these plants with one another. By extension, the term “descendants” applies to all the seeds formed at each new generation derived from crosses between a plant and the plant transformed by the method according to the invention.
• a subject of the present invention is therefore transformed plants into the genome of which there is integrated at least one expression cassette according to the invention in a stable manner.
• the plants thus transformed are tolerant to EPSPS-inhibiting herbicides, in particular herbicides of the phophonomethylglycine family, in particular to glyphosate.
• the transformed plants according to the invention also include the transformed plants derived from growing and/or crossing the plants above, and also the seeds of such plants.
• the transformed cells and plants according to the invention may comprise, in addition to an expression cassette according to the invention, at least one other expression cassette containing a polynucleotide encoding a protein of interest.
• polynucleotides encoding a protein of interest mention may be made of polynucleotides encoding another enzyme for resistance to a herbicide, for example the polynucleotide encoding the bar enzyme (White et al., NAR 18:1062, 1990) for tolerance to bialaphos, or the polynucleotide encoding the HPPD enzyme (WO 96/38567; WO 99/24585; WO 99/24586) for tolerance to isoxazoles.
• Other polynucleotides for resistance to diseases may also be contained in these plants, for example a polynucleotide encoding the oxalate oxydase enzyme as described in patent application EP 0 531 498 or U.S. Pat. No.
• the additional expression cassettes may be integrated by means of the vector according to the invention.
• the vector comprises the expression cassette according to the invention and at least one expression cassette encoding another protein of interest.
• the plants according to the invention may also be obtained by crossing parents, one carrying the expression cassette according to the invention, the other carrying another expression cassette encoding at least one other protein of interest.
• the transformed plants according to the invention may be monocotyledons or dicotyledons. Preferably, these plants are plants of agronomic interest.
• the monocotyledon plants are wheat, maize or rice, advantageously, the dicotyledon plants are oilseed rape, soybean, tobacco or cotton.
• the present invention also relates to a method for protecting crop plants with respect to EPSPS-inhibiting herbicides, in particular to herbicides of the phophonomethylglycine family, in particular to glyphosate, characterized in that said plants are transformed with a vector comprising an expression cassette according to the invention.
• the present invention also relates to a method for treating the plants according to the invention, characterized in that said plants are treated with EPSPS-inhibiting herbicide, in particular a herbicide of the phophonomethylglycine family, in particular glyphosate.
• EPSPS-inhibiting herbicide in particular a herbicide of the phophonomethylglycine family, in particular glyphosate.
• the present invention also relates to a method for controlling weeds in crops, characterized in that transformed plants comprising an expression cassette according to the invention are grown, and in that said plants are treated with an EPSPS-inhibiting herbicide, in particular a herbicide of the phophonomethylglycinse family, in particular glyphosate.
• an EPSPS-inhibiting herbicide in particular a herbicide of the phophonomethylglycinse family, in particular glyphosate.
• the present invention also relates to a method for growing transformed plants comprising an expression cassette according to the invention, characterized in that it consists in planting seeds of said transformed plants in an area of a field suitable for growing said plants, in applying to said area of said field an agrochemical composition, without substantially affecting said transformed seeds or said transformed plants, then in harvesting the plants grown when they have reached the desired maturity and, optionally in separating the seeds from the harvested plants.
• agrochemical composition is intended to mean any agrochemical composition comprising at least one active product having one of the following activities: herbicidal, fungicidal, bactericidal, virucidal or insecticidal.
• the agrochemical composition comprises at least one active product having at least one herbicidal activity, more preferably an EPSPS-inhibiting herbicide, in particular a herbicide of the phophonomethylglycine family, in particular glyphosate.
• the plasmid pILTAB 357 provided by The Scripps Research Institute (La Jolla, Calif., USA) contains the following elements in a pBIN 19 vector (Clontech):
• CsVMV promoter (sequence described by SEQ ID NO 4)
• CsVMV X Three regions have been defined in the CsVMV promoter sequence, called CsVMV X, CsVMV Y and CsVMV Z.
• CsVMV X from position 10 to position 227 (SEQ ID NO 1, length 218 bp)
• CsVMV Y from position 228 to position 394 (SEQ ID NO 2, length 167 bp)
• CsVMV Z from position 397 to position 522 (SEQ ID NO 3, length 126 bp)
• the X and Y regions are adjacent and the Y and Z regions are separated by the 2 bp sequence AT.
• the cloning vector pRD 254 corresponds to the commercial vector pBlueScript II SK ( ⁇ ) (Clontech) which has undergone mutagenesis so as to replace the unique Sca I site contained in the ApR gene with a Pvu II site.
• the 532 bp contained between the Hind III and Xba I sites of pILTAB 357 were cloned into the cloning vector pRD 254, so as to obtain the plasmid pRD 257.
• the pSF29 cassette comprises the CsVMV promoter as described by the sequence identifier SEQ ID NO 4, the sequence encoding the optimized transit peptide (OTP) as defined in patent application EP 0508909, the sequence encoding maize EPSPS comprising the mutations threonine 102 isoleucine and proline 106 serine as described in patent application EP 0837944, and the nos terminator as described in Bevan et al. (1983, Nucleic Acids Res. 11(2), 369-385).
• OTP optimized transit peptide
• a shuttle plasmid is used to be recombined in the superbinary plasmid pTVK 291 (Jun et al., 1987). Recombination between the unique COS sites present on the two plasmids produces a single circular molecule corresponding to fusion of the two plasmids.
• Recombination between pSF29 and the superbinary plasmid pTVK 291 was obtained by three-parent crossing with DH5 alpha [pSF29], C2110 [pTVK 291], and the JC2073 strain (“helper” strain).
• the resulting plasmid is called pSFK29.
• the strain obtained, C2110 [pSFK29] was selected on LB medium containing the 3 antibiotics gentamycin, kanamycin and nalidixic acid.
• the nalidixic acid allows the selection of C2110 against DH5 alpha or JC2073; since C2110 contains a chromosomal resistance to nalidixic acid which cannot be transferred to the other strains during crossing.
• pSF29 cannot replicate in C2110, unless it has been recombined with pTVK 291, since C2110 contains the origin of replication RK2 carried by pTVK 291, but not the origin of replication pBR 322 carried by pSF29.
• the recombinant plasmid was then transferred into the Agrobacterium strain LBA 4404 via a second three-parent cross.
• the resultant strain, LBA 4404 [pSFK29] was selected on AB medium (selective for Agrobacterium ) containing kanamycin and gentamycin.
• Transformation of the maize Zea mays by Agrobacterium is carried out according to the method described in Ishida, Y. et al., (1996, Nature Biotechnology, 14, 745-750).
• the disarmed Agrobacterium tumefaciens strain described in Example 3 is cocultured with immature maize embryos.
• a selection with 0.88 mM glyphosate is applied to the embryos.
• the transformation events obtained from the resistant calices are then back-crossed and their descendants are tested for glyphosate tolerance.
• a disparity in the number of tolerant plants for each event tested is due to the fact that these events are heterozygotes for the trait (the CsVMV-EPSPS expression cassette) and have one or more integration loci, and that some transformation events, by virtue of the position of insertion of the cassette, provide better expression of the EPSPS, and therefore better tolerance to glyphosate.

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