WO2004053135A2

WO2004053135A2 - Expression cassette encoding a hydroxyphenylpyruvate dioxygenase and herbicide-tolerant plants containing such a gene

Info

Publication number: WO2004053135A2
Application number: PCT/EP2003/015009
Authority: WO
Inventors: Jean-Marc Ferullo; Alain Sailland; Frédéric SCHMITT; Eric Paget
Original assignee: Bayer Cropscience S.A.
Priority date: 2002-12-12
Filing date: 2003-12-10
Publication date: 2004-06-24
Also published as: FR2848571A1; AR042442A1; WO2004053135A3; AU2003302810A1

Abstract

The present invention relates to a novel expression cassette comprising in the direction of transcription, functionally linked, a promoter regulatory sequence which is functional in plant cells or plants, a nucleic acid sequence encoding an HPPD and a terminator regulatory 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 ( Cassava Vein Mosaic Virus) plant virus and to its use for obtaining plants resistance to certain herbicides.

Description

Expression cassette encoding a hydroxyphenylpyruvate dioxygenase and herbicide- tolerant plants containing such a gene

The present invention relates to a novel expression cassette comprising a nucleic acid sequence encoding a hydroxyphenylpyruvate dioxygenase (HPPD) and to its use for obtaining plants resistant to certain herbicides.

Hydroxyphenylpyruvate dioxygenases are enzymes which catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is converted into homogentisate. This reaction takes place in the presence of iron (Fe²⁺) and in the presence of oxygen (Crouch N.P. et al., Tetrahedron, 53, 20, 6993-7010, 1997).

Some molecules which inhibit this enzyme, and which bind to the enzyme in order to inhibit the conversion of HPP to homogentisate are, moreover, known. Some of these molecules have been used as herbicides since inhibition of the reaction in plants leads to bleaching of the leaves of the treated plants, and to the death of said plants (Pallett K.E. et al., 1997 Pestic. Sci. 50 83-84). Such herbicides for which HPPD is the target, and which are described in the state of the art, are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276), in particular isoxaflutole (IFT), which is a selected herbicide for maize, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-l-(2-SO₂CH₃-4-CF₃- phenyl)propane-l ,3-dione and 2-cyano-3-cyclopropyl-l -(2-SO₂CH₃-4-2,3-Cl₂- phenyl)propane-l,3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione or mesotrione, or else pyrazolinates.

Three main strategies are available for making plants tolerant to herbicides, (1) detoxification of the herbicide with an enzyme which converts the herbicide, or its active metabolite, to nontoxic degradation products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP 242 236, EP 337 899); (2) mutation of the target enzyme into a functional enzyme which is less sensitive to the herbicide, or its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP 293 356, Padgette S.R. et al, J. Biol. Chem. 266,33,1991. FR 2 736 926); or (3) overexpression of the sensitive enzyme, so as to produce in the plants amounts of target enzyme which are sufficient with regard to the kinetic constants of this enzyme with respect to the herbicide, so as to have enough functional enzyme, despite the presence of its inhibitor.

It is this third strategy which has been described for successfully obtaining plants tolerant to HPPD inhibitors (WO 96/38567), it being understood that, for the first time, a strategy of simple overexpression of the sensitive (nonmutated) target enzyme was used successfully to confer on plants agronomic-level tolerance to a herbicide. An agronomic level of tolerance to HPPD inhibitors has also been obtained with chimeric HPPDs (WO 99/24586), and even improved with HPPDs which have been mutated in their C-terminal portion (WO 99/24586).

Although plant HPPDs are enzymes which are located in the cytoplasm (Garcia, I., et al., 1997, Biochem. J. 325: 761-769), it has also been shown that the best tolerance is obtained when the exogenous HPPD is accumulated in the plasts, particularly the chloroplasts.

Various expression cassettes capable of improving the level of tolerance of transformed plants have been described with various regulatory elements, and in particular "light-dependent" promoters (WO 99/25842) or, for monocotyledon plants, with a promoter regulatory sequence comprising the maize histone promoter H3C4 combined with the first intron of rice actin (WO 99/34005).

The present invention therefore relates to a novel expression cassette comprising, in the direction of transcription, functionally linked, a promoter regulatory sequence which is functional in plant cells or plants, a nucleic acid sequence encoding an HPPD and a terminator regulatory 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 CsNMN (Cassava Vein Mosaic Virus) plant virus.

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 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 SEQ ID No. 3 of patent application WO 97/48819.

According to a first preferred embodiment of the invention, the promoter regulatory sequence comprises, in the direction of transcription, the nucleic acid sequences X, Y and Z as defined respectively by SEQ IDs 1, 2 and 3 of the present patent application.

Preferably, the promoter regulatory sequence is represented by SEQ ID No.4 of the present patent application.

According to a second preferred embodiment of the invention, the promoter regulatory sequence 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.

Preferably, the nucleic acid sequence of the double CsNMV is represented by SEQ ID No.5 of the present patent application.

According to the invention, the term "HPPD" is intended to mean any native, mutated or chimeric HPPD enzyme exhibiting HPPD activity. Many HPPDs are described in the literature, in particular the HPPDs of bacteria such as Pseudomonas (Rύetschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), of plants such as Arabidopsis plants (WO 96/38567, Genebank AF047834) or carrot plants (WO 96/38567, Genebank 87257), of Coccicoides (Genebank COITRP), or of mammals such as mice or pigs.

According to the invention, the term "mutated HPPD" is intended to mean the HPPDs which have been mutated so as to obtain properties of tolerance to HPPD-inhibiting herbicides which are improved with respect to the corresponding native HPPD. Advantageously, the mutated HPPD is an HPPD which has been mutated in its C-teraiinal portion, as described in patent application WO 99/24585. The term "chimeric HPPD" is intended to mean an HPPD comprising elements originating from various HPPDs, in particular the chimeric HPPDs described in patent application WO 99/24586.

Advantageously, the HPPD is a Pseudomonas fluorescens HPPD (WO 96/38567).

Advantageously, the mutated HPPD comprises the W336 mutation as described in patent application WO 99/24585.

It is known in the literature that the addition of a coding sequence corresponding to an additional peptide at the N- or C-terminal of the protein will allow the transport thereof to a cellular compartment other than the cytoplasm where the mRNA is translated. Depending on its sequence, this peptide, referred to as signal peptide or transit peptide as appropriate, will allow routing to this other compartment, which may, for example, be the plast (membrane or stroma), the thylakoid lumen or the thylakoid membrane, to the endoplasmic reticulum, to the vacuole, the wall, the mitochondria, without this list being exhaustive.

The transit peptide makes it possible to address the chimeric HPPD into the plasts, more particularly the chloroplasts, the transit peptide/HPPD fusion protein being cleaved between the transit peptide and the chimeric HPPD on passing through the plast membrane. The transit peptide may be a single transit peptide, such as an EPSPS transit peptide (described in US patent 5,188,642) or a transit peptide of that of the ribulose-biscarboxylase/oxygenase small subunit (RuBisCO ssu) of a plant, optionally comprising some amino acids from the N-terminal portion of the mature RuBisCO ssu (EP 189 707) or else a multiple transit peptide comprising a first plant transit peptide fused to a portion of the N-terminal sequence of a mature protein which is located in plastids, fused to a second plant transit peptide as described in patent EP 508 909, and more particularly the optimized transit peptide comprising a sunflower RuBisCO ssu transit peptide fused to 22 amino acids of the N-terminal end of the maize RuBisCO ssu fused to the maize RuBisCO ssu transit peptide as described with its coding sequence in patent EP 508 909. According to the invention, the term "protein signal sequence" is intended to mean any signal peptide or transit peptide which allows addressing of HPPD into cellular compartments other than the cytoplasm or the plasts (in particular chloroplasts).

The role of such protein sequences is in particular described in issue 38 of the review Plant molecular Biology (1998) devoted in large part to protein transport in the various compartments of the plant cell (Sorting of proteins to vacuoles in plant cells pp 127-144; the nuclear pore complex pp 145-162; protein translocation into and across the chloroplastic envelope membranes pp 91-207; multiple pathways for the targeting of thylakoid proteins in chloroplasts pp 209-221; mitochondrial protein import in plants pp 311-338).

Advantageously, the protein signal sequence allows addressing of HPPD to the vacuolar compartment (vacuole). Such peptides are widely described in the literature (Neuhaus J.M. and Rogers J.C. Sorting of proteins to vacuoles in plant cells Plant molecular Biology 38: 127-144, 1998).

Preferably, the vacuolaire peptide is the vacuolaire peptide of the protein described in J.M. FeruUo et al. (Plant Molecular Biology 33: 625-633, 1997), fused to the C-terminal portion of the HPPD.

The present invention also relates to the sequences capable of hybridizing selectively with the nucleic acid sequence above, to the sequences homologous to the sequence above, to the fragments of said sequences, and to the sequences which differ from the sequences above but which, due to the degenerescence of the genetic code, encode the same fusion protein.

According to the present invention, the term "nucleic acid sequence" is intended to mean a nucleotide or polynucleotide sequence which may be of DNA or RNA type, preferably of DNA type, in particular double-stranded. According to the invention, 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 sequence identifiers (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 ³²P. Selective 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). The hybridization may of course be carried out according to the usual methods of the state of the art (in particular Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual).

According to the invention, 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 according to 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 can bring about the expression of the same amino acid, the differences between the reference sequence according to the invention and the corresponding homologue may be considerable. Advantageously, 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.

The methods for measuring and identifying homologies between nucleic acid sequences are well known to those skilled in the art. Use may, for example, be made of the PILEUP or BLAST programs (in particular Altschul et al., 1993, J. Mol. Evol. 36: 290-300; Altschul et al., 1990, J. Mol. Biol. 215: 403-10). The methods for measuring and identifying homologies between polypeptides or proteins are also known to those skilled in the art. Use may, for example, be made of the UWGCG package and the BESTFITT program for calculating homologies (Deverexu et al., 1984, Nucleic Acid Res. 12, 387-395).

According to the invention, the term "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.

A subject of the invention is also the use of the expression cassette according to the invention, in a method for transforming plants, as a marker gene or as a coding sequence for conferring on the plant tolerance to HPPD-inhibiting herbicides. This expression cassette may of course also be used in combination with other marker gene(s) and/or coding sequence(s) for one or more agronomic properties.

According to the invention, the term "plant cell" is intended to mean any cell derived from a plant and which can constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.

According to the invention, the term "plant" is intended to mean any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants which may or may not be intended for animal or human food, such as maize, wheat, rapeseed, soybean, rice, sugar cane, beetroot, tobacco, cotton, etc.

According to the invention, use may also be made, in combination with the promoter regulatory sequence according to the invention, of other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators (enhancers), for instance the translation 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 (US 5 994 521), for example, or else introns, in particular introns which promote gene expression in monocotyledon plants, such as intron 1 of the rice actin gene as described in patent application WO 99/34005 or the maize adhl intron.

As terminator regulatory sequence or polyadenylation sequence, use may be made of any corresponding sequence of bacterial origin, such as, for example, the Agrobacterium tumefaciens nos terminator, of viral origin, such as, for example, the CaMV 35S terminator, or else of plant origin, such as, for example, a histone terminator as described in application EP 0 633 317.

The present invention also relates to a cloning and/or expression vector for transforming a host organism, containing at least one chimeric gene as defined above. This vector comprises, besides the chimeric gene above, at least one origin of replication. This vector may consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by introducing the chimeric gene according to the invention. Such vectors for transformation according to the host organism to be transformed are well known to those skilled in the art and widely described in the literature. For the transformation of plant cells or plants, it will in particular be a virus which can be used for transforming developed plants and which also contains its own replication and expression elements. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.

According to another embodiment of the invention, the vector comprises, besides the chimeric gene according to the invention, another chimeric gene encoding another gene of interest. It may be a gene encoding a selection marker, such as a gene which confers on the transformed plant novel agronomic properties, or a gene for improving the agronomic quality of the transformed plant.

Selection markers

Among the genes encoding selection markers, mention may be made of the genes for resistance to antibiotics, the genes for tolerance to herbicides (bialaphos, glyphosate or isoxazoles), genes encoding readily identifiable reporter enzymes, such as the GUS enzyme, and genes encoding pigments or enzymes which regulate the production of pigments in the transformed cells. Such selection marker genes are in particular described in patent applications EP 242236, EP 242246, GB 2 197653, WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103.

Genes of interest

Among the genes which confer novel agronomic properties on the transformed plants, mention may be made of the genes which confer tolerance to certain herbicides, those which confer resistance to certain insects, those which confer tolerance to certain diseases, etc. Such genes are in particular described in patent applications WO 91/02071 and WO 95/06128.

Herbicidal tolerance

Among the genes which confer tolerance to certain herbicides, mention may be made of the Bar gene which confers tolerance to bialaphos, the gene encoding an appropriate EPSPS which confers resistance to herbicides for which EPSPS is the target, such as glyphosate and its salts (US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 5,633,435, FR 2 736 926), and the gene encoding glyphosate oxydoreductase (US 5,463,175).

Among the genes encoding an appropriate EPSPS which confer resistance to herbicides for which EPSPS is the target, mention will more particularly be made of the gene encoding a plant EPSPS, in particular a maize EPSPS, exhibiting two mutations 102 and 106, described in patent application FR 2 736 926, referred to below as EPSPS double mutant, or else the gene encoding an EPSPS isolated from Agrobacterium described by SEQ ID No.2 and SEQ ID No.3 of US patent 5,633,435, referred to below as CP4.

In the cases of the genes encoding an EPSPS, and more particularly encoding the genes above, the sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the transit peptide as defined above. Resistance to insects

Among the proteins of interest which confer novel properties of resistance to insects, mention will more particularly be made of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of the proteins extracted from bacteria such as Photorabdus (WO 97/17432 & WO 98/08932).

Resistance to diseases

Among the proteins or peptides of interest which confer novel properties of resistance to diseases, mention will in particular be made of chitinases, glucanases, oxalate oxydase, all these proteins and their coding sequences being widely described in the literature, or else antibacterial and/or antifungal peptides, in particular peptides of less than 100 amino acids rich in cysteines, such as plant thion ns or defensins, and more particularly lytic peptides of any origins comprising one or more disulfide bridges between the cysteines and regions comprising basic amino acids, in particular the following lytic peptides: androctonin (WO 97/30082 and WO 99/09189), drosomycin (WO 99/02717) or thanatin (WO 99/24594).

According to a particular embodiment of the invention, the protein or peptide of interest is chosen from fungal elicitor peptides, in particular elicitins (Kamoun et al., 1993; Panabiere et al., 1995).

Modification of quality

Mention may also be made of the genes which modify the constitution of the modified plants, in particular the content and the quality of certain essential fatty acids (EP 666 918) or else the content and the quality of the proteins, in particular in the leaves and/or the seeds of said plants. Mention will in particular be made of the genes encoding proteins enriched in sulfur-containing amino acids (Korit, A.A. et al., Eur. J. Biochem. (1991) 195. 329-334; WO 98/20133; WO 97/41239; WO 95/31554; WO 94/20828; WO 92/14822). These proteins enriched in sulfur-containing amino acids will also have the function of trapping and storing excess cysteine and/or methionine making it possible to avoid the possible problems of toxicity associated with overproduction of these sulfur-containing amino acids by trapping them. Mention may also be made of genes encoding peptides rich in sulfur-containing amino acids, and more particularly in cysteines, said peptides also having antibacterial and/or antifungal activity. Mention will more particularly be made of plant defensins, along with lytic peptides of any origin, and more particularly the following lytic peptides: androctonin, drosomicin or thanatin.

A subject of the invention is also a method for transforming host organisms, in particular plant cells, by integrating at least one nucleic acid sequence or a chimeric gene as defined above, which transformation can be obtained by any suitable known means widely described in the specialized literature, and in particular the references cited in the present application, particularly with the vector according to the invention.

A series of methods consists in bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached. Another series of methods consist in using, as means of transfer into the plant, a chimeric gene inserted into an Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri plasmid. Other methods can be used, such as microinjection or electroporation, or else direct precipitation with PEG. Those skilled in the art will choose the appropriate method as a function of the nature of the host organism, in particular of the plant cell or of the plant.

A subject of the present invention is also the transformed plant cells or plants containing a chimeric gene according to the invention. Preferably, the chimeric gene is stably integrated into the genome of the plant cells or of the plants.

According to another particular embodiment of the invention, the plant cells or the plants comprise, besides the chimeric gene according to the invention, another chimeric gene encoding another gene of interest as defined above. The various chimeric genes may be integrated into the genome of the host organism by transformation using a vector according to the invention comprising the various chimeric genes as defined above.

The various chimeric genes may also be stably integrated into the genome of the host organism by cotransformation using several vectors, each one comprising at least one chimeric gene which must be integrated into the genome of the host organism.

A subject of the present invention is also the transformed plants into the genome of which is stably integrated at least one chimeric gene according to the invention. The plants according to the invention contain transformed cells as defined above, in particular the plants regenerated from the transformed cells above. The regeneration is obtained by any suitable method, which depends on the nature of the species, as described, for example, in the references above. For the methods for transforming plant cells and for regenerating plants, mention will in particular be made of the following patents and patent applications: US 4,459,355, US 4,536,475, US 5,464,763,

US 5,177,010, US 5,187,073, EP 267,159, EP 604662, EP 672 752, US 4,945,050,

US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022,

US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233,

EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.

The present invention also relates to the transformed plants derived from culturing and/or crossing the regenerated plants above, and also the seeds of transformed plants, said plants or seeds comprising at least one chimeric gene according to the invention.

According to a particular embodiment of the invention, the transformed plants comprise, besides the chimeric gene according to the invention, another chimeric gene encoding another gene of interest as defined above.

The various chimeric genes in the transformed plants according to the invention may come either from the same transformed parent plant and, in this case, the plant is derived from a single transformation regeneration process with the various chimeric genes contained in the same vector or by cotransformation using several vectors; or it may also be obtained by crossing parent plants each containing at least one chimeric gene, one of the parent plants comprising at least one chimeric gene according to the invention.

The transformed plants which can be obtained according to the invention may be of the monocotyledon type, such as, for example, cereals, sugar cane, rice and maize, or of the dicotyledon type, such as, for example, tobacco, soybean, rapeseed, cotton, beetroot, clover, etc.

A subject of the invention is also a method for selective weeding of plants, in particular crops, using an HPPD inhibitor, in particular a herbicide defined above, characterized in that this herbicide is applied to transformed plants according to the invention, equally in pre-sewing, in pre-emergence and in post-emergence of the crop.

The present invention also relates to a method of weed killing in an area of a field comprising seeds or plants transformed with the chimeric gene according to the invention, which method consists in applying, in said area of the field, a dose, which is toxic for said weeds, of an HPPD-inhibiting herbicide, without however substantially affecting the seeds or plants transformed with said chimeric gene according to the invention.

The present invention also relates to a method for growing the plants transformed according to the invention with a chimeric gene according to the invention, which method consists in sewing the seeds of said transformed plants in an area of a field suitable for growing said plants, in applying to said area of said field a dose, which is toxic for the weeds, of a herbicide for which the HPPD defined above is the target, in the event of weeds being present, 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. According to the invention, the expression "without substantially affecting said transformed seeds or said transformed plants" is intended to mean that the plants comprising the expression cassette according to the invention, subjected to application of a dose of herbicide which is toxic for the weeds, exhibit slight or zero phytotoxicity. According to the invention, the expression "dose which is toxic for the weeds" is intended to mean an applied dose of the herbicide for which the weeds are killed. According to the invention, the term "slight phytotoxicity" is intended to mean a percentage of bleached leaves of less than 25%, preferably less than 10%, more preferably less than 5%. It is also understood according to the present invention that application of the same toxic dose to a plant which is otherwise comparable but not transformed, i.e. which does not comprise the expression cassette according to the invention would lead to the observation on said plant of phytotoxicity symptoms greater than those observed for the transformed plant comprising the expression cassette according to the invention.

In the two methods above, the application of the herbicide for which HPPD is the target can be carried out according to the invention, equally in pre-sewing, in pre- emergence and in post-emergence of the crop.

For the purpose of the present invention, the term "herbicide" is intended to mean a herbicidal active material alone or combined with an additive which modifies its effectiveness, such as, for example, an agent which increases the activity (synergist) or which limits the activity (safener). The HPPD-inhibiting herbicides are in particular defined previously. Of course, for their practical application, the herbicides above are combined, in a manner known per se, with formulation adjuvants conventionally used in agrochemistry.

When the transformed plant according to the invention comprises another gene for tolerance to another herbicide (such as, for example, a gene encoding an EPSPS, which may or may not be mutated, conferring on the plant tolerance to glyphosate), or when the transformed plant is naturally insensitive to another herbicide, the method according to the invention may comprise the simultaneous application or the application at a different time of an HPPD inhibitor in combination with said herbicide, for example glyphosate.

Another subject of the invention is the use of the chimeric gene encoding a fusion protein according to the invention, as a marker gene during the cycle of "transformation-regeneration" of a plant species and selection on the herbicide above.

When the chimeric gene according to the invention is combined in the same vector with another chimeric gene encoding a protein of interest which confers novel agronomic properties, other than a gene for herbicidal tolerance (resistance to insects, resistance to diseases, modification of quality), application of the HPPD-inhibiting herbicide to the transformed plants and their descendants makes it possible to select, in the field, the plants derived from a cross between a parent plant comprising the two chimeric genes and a nontransformed plant, which have kept the chimeric genes.

The various aspects of the invention will be understood more clearly from the experimental examples below.

All the methods or operations described below in these examples are given by way of examples and correspond to a choice, made from the various methods available to achieve the same result. This choice has no bearing on the quality of the result and, consequently, any suitable method may be used by those skilled in the art to achieve the same result. Most of the methods for engineering DNA fragments are described in "Current Protocols in Molecular Biology" Volumes 1 and 2 Ausubel F.M. et al., published by Greene Publishing Associates and Wiley-Interscience (1989) or in Molecular Cloning, T. Maniatis, E.F. Fritsch, J. Sambrook, 1982.

The bibliographical references cited above are integrated into the present patent application by way of reference, in particular the bibliographical references defining the nucleic acid sequences encoding native, chimeric or mutated HPPDs, optionally combined with a signal peptide or transit peptide. Description of the figures:

Figure 1 : extract of the map of plasmid pILTAB 357

Figure 2: sequence of the CsVMV promoter (from Hind III to Xba I, 539 bp)

Figure 3 : map of plasmid pRD 254

Figure 4: map of plasmid pRD 257

Figure 5: map of plasmid pCH 10

Figure 6: map of plasmid pCH 27 Figure 7: map of plasmid pCH 27 PCR 1

Figure 8: map of plasmid pCH 27 pCR 2

Figure 9: map of plasmid pCH 45

Figure 10: map of plasmid pCH 13 D

Figure 11 : map of plasmid pCH 43 D Figure 12: map of plasmid pCH 55 D.

Figure 13: In vitro germination assay for TI descendants of tobacco lines transformed with pCH 43 K

Figure 14: In vitro germination assay for TI descendants of tobacco lines transformed with pCH 55 K.

All the plasmids described below were mobilized in the E. coli strain DH5 alpha, with the exception of pTVK 291 and its derivatives, which are mobilized in C2110.

Example 1: Cloning of the CsVMV promoter sequence in the multiple cloning vector pRD 254

The plasmid pILTAB 357, provided by The Scripps Research Institute, La Jolla, USA (figure 1), contains the following elements in a pBIN 19 vector (Clontech): CsVMV promoter (sequence given in figure 2, SEQ ID No.4) multiple cloning site

NOS terminator Three regions were defined in the CsVMV promoter sequence, called CsVMV X, CsVMV Y and CsVMV Z.

CsVMV X: from position 9 to position 227 (SEQ ID No. 1 , length 218 bp) CsVMV Y: from position 228 to position 395 (SEQ ID No.2, length 167 bp) CsVMV Z: from position 396 to position 522 (SEQ ID No. 3, length 126 bp).

In the original sequence of the CsVMV promoter, the regions X and Y are adjacent, as are the regions Y and Z.

The cloning vector pRD 254 corresponds to the commercial vector pBlueScript II SK (-) (Clontech) which has undergone mutagenesis to replace the unique Sea 1 site contained in the ApR gene with a Pvu II (figure 3).

The 532 bp contained between the Hind III and Xba l sites of pILTAB 357 were cloned into the cloning vector pRD 254, so as to obtain the plasmid pRD 257 (figure 4).

Example 2: Insertion of the CsVMV sequence into an OTP-HPPD expression cassette

pCH 10 is a pUC 19-based plasmid which contains an OTP/HPPD expression cassette. This cassette comprises the following components: maize H3C4 histone promoter first intron of the rice actin gene optimized transit peptide (OTP)

HPPD

NOS.

SEQ ID No.6 attached in the appendix describes the sequence of pCH 10 between the Ncol and Xbal sites. In pCHIO, the maize histone promoter + rice actin intron region was excised with the Xhol and Notl restriction enzymes, so as to be replaced with the CsVMV promoter sequence contained in pRD 257, as follows:

1. pRD 257 was opened with Notl, the Notl end was filled in with a Klenow polymerase, and the promoter fragment was excised with Xhol.

2. The 560 bp Xhol-Notl fragment containing the CsVMV promoter sequence was purified.

3. pCH 10 was opened with Ncol, the Ncol end was filled in with a Klenow polymerase, and the maize histone promoter + rice actin intron fragment was excised with Xhol.

4. The 4591 bp Xhol-Ncol (filled in) acceptor molecule was purified.

5. The two purified fragments were ligated so as to obtain pCH 27 (figure 6).

Example 3: Construction of a double-CsVMV chimeric promoter

A chimeric version of the CsVMV promoter, containing a duplication of the internal region Y, was prepared from pCH 27. For this purpose, two series (Setl and Set2) of oligonucleotides were prepared, corresponding to the following sequences: Setl

Universal Ml 3 primer: GAGGAAACAG CTATGACCAT GATT (SEQ ID NO 7)

CsVMV63: GGACTAGTGA CACGGAAAAA TATAAAAGG (SEQ ID NO 8) Set2

CsVMV222: GGACTAGTGA AGACGTAAGC ACTGACG (SEQ ID NO 9)

CsVMV3': AAGCCATGGG CCGCTTTAGA (SEQ ID NO 10)

A PCR reaction was carried out with pCH 27 as a matrix and the setl oligonucleotides as primers, so as to obtain a 493 bp PCR product corresponding to figure 7 (PCR CH27-l). A PCR reaction was carried out with pCH 27 as matrix and the set2 oligonucleotides as primers, so as to obtain a 332 bp PCR product corresponding to figure 8 (PCR pCH27-2).

A plasmid comprising the duplicated CsVMV Y region was obtained as follows.

1. PCR ρCH27-l was restricted with Xhol and Spel.

2. pCR pCH 27-2 was restricted with Spel and Ncol.

3. pCH 27 was restricted with Xhol and Spel and the 4591 bp acceptor plasmid was purified.

4. The three fragments were ligated so as to generate pCH 45 (figure 9).

Example 4: Integration of the expression cassette into an Agrobacterium tDNA- vector

pCH 13 D (figure 10) is a shuttle plasmid constructed in the laboratory 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 molecular corresponding to fusion of the two plasmids.

The 2791 bp PvuII-PvuII fragment containing the CsVMV-OTP-HPPD-NOS cassette was excised from pCH 27 and cloned into the Pmel site of pCH 13D, to give pCH 43 D (figure 11).

The 2821 bp Xbal-PvuII fragment containing the double CsVMV XYYZ-OTP-HPPD-NOS cassette was excised from pCH 45 and cloned between the Avrll and Pmel sites of pCH 13D, to give pCH 55 D (figure 12).

Recombination between pCH 43 D (or respectively pCH 55 D) and the superbinary plasmid pTVK 291 was obtained by three-parent crossing with DH5alpha [pCH 43 D] (respectively DH5alpha [pCH 55 D]), C2110 [pTVK 291] and the helper strain JC 2073. The resulting plasmid is called ρCH 43 K (respectively pCH 55 K). The strain obtained, C2110 [pCH 43 K] (respectively C2110 [pCH 55 K]), was selected on LB medium containing the 3 antibiotics gentamycin, kanamycin and nalidixic acid. The nalidixic acid allows the selection of C2110 against DH5alpha or JC2073, since C2110 contains a chromosomal resistance to nalidixic acid which cannot be transferred to the other strains during the crossing. Combining gentamycin and kanamycin allows selection of the bacteria containing pCH 43 D (respectively pCH 55 D) and pTVK 291. In addition, pCH 43 D (respectively pCH 55 D) cannot replicate in C2110, unless it is 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 pCH 43 D (respectively pCH 55 D).

The recombined plasmid was then transferred into the Agrobacterium strain LBA 4404 via a second three-parent cross. The resulting strain LBA 4404 [pCH 43 K] (respectively LBA 4404 [pCH 55 K]) was selected on AB medium (selective for Agrobacterium) containing kanamycin and gentamycin.

Example 5: Transformation of tobacco plants by Agrobacterium tumefaciens with the expression cassettes

Transformation of the tobacco plant Nicotiana tabacum var. Petit Havana with Agrobacterium was carried out according to standard procedures of infection and regeneration of foliar disks and selection with kanamycin.

Ten tobacco lines were obtained with LBA 4404 [pCH 43 K] and 13 tobacco lines were obtained with LBA 4404 [pCH 55 K]. The regenerated plants were transferred into a greenhouse. After flowering and self-pollination, the TI seeds of each line were collected. The lines transformed with LBA 4404 [PCH 43 K] were then given the references 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9 and 43.11. The lines transformed with LBA 4404 [pCH 55 K] were then given the references 55.1, 55.2, 55.6, 55.8, 55.11, 55.12, 55.13, 55.14, 55.15, 55.16, 55.17, 55.18 and 55.19. Example 6: In vitro germination assay

In this assay, wild-type Petit Havana seeds were used as a negative control. As a positive control, T2 PBD6 transgenic line seeds were used as a reference. This transgenic line comprises an expression cassette encoding an HPPD under the control of a sunflower (Helianthus annuus) RuBisCO small subunit promoter. This line, given the reference 7.1., described in patent application WO 99/25842, has shown the best tolerance to IFT ever described to our knowledge in tobacco. Square Petri dishes containing 16 square wells 30 mm deep were filled with an autoclaved Murashige and Skoog half medium (Difco) containing 15 g/1 of phytoagar (Difco), 10 g/1 of sucrose, and either 0 or 8 ppm of DKN [which] (4 ml in each well). The TI -containing tobacco capsules were sprayed with 70% ethanol and then opened with sterile instruments in order to obtain sterile seeds. For each line and each concentration of DKN, 25 seeds were placed at the surface of a solid medium for germination in a culture chamber (24°C, 16 h day/8 h night). After a growth period of 10 days, a grade of 0 to 4 was attributed to each plantlet which had grown with 8 ppm of DKN, according to the following grading scale:

Size: - normal size (compared to untreated plantlets of the same line): 2 points

"dwarf size (cotyledons not developed): 0 points intermediate size: 1 point

Symptoms of phytotoxicity: - none: 2 points intermediate (a few green parts): 1 point completely burnt or bleached: 0 points no germination: not counted completely white (non transgenic plants): not counted

The sum of the scores of all the plantlets of the same line, expressed as a percentage of the total mark of the control lines 7.1, are reported in figure 13. The numbers in brackets indicate the number of plantlets graded for a given line after elimination of the transgenic plants or the seeds which had not germinated. The assay was repeated once by two different individuals. The grades observed with the transgenic lines transformed with LBA 44044 [pCH 55 K] are given in figure 14. An absence of data corresponds to contaminated cells. In this assay, the grades observed for all the lines tested were found to be between 2 and 9 times greater than those of the control line.

Example 7: Pre-emergence evaluation in a greenhouse

In this test, the same positive and negative controls from example 6 were used. Approximately 100 TI seeds of each line were sown on 3 trays (20 cm long x 15 cm wide x 5 cm deep) filled with vermiculite pre-soaked with a standard 20:20:20 nutritive solution (3 trays per line). Two trays were sprayed with a solution of IFT at two concentrations (0.1 g/1 and 0.2 g/1 respectively). The spraying device was calibrated so as to deliver a dose of IFT corresponding to 50 g/ha for the first tray and 100 g/ha for the second. The third tray remained untreated (control).

During the first 3 days following seeding, the trays were covered with a sheet of transparent plastic material covered with a sheet of white paper so as to reduce the light intensity. After the third day, the trays were uncovered and the plantlets were left to grow for a further 7 days with daily watering with a nutritive solution. For each tray, the level of phytotoxicity and the retarded growth compared to the non-treated tray was estimated and reported in the table below. The nontransgenic TI plantlets which appeared to be completely white on the treated trays are counted to determine the number of independent loci for integration of the tDNA in each line. The results are given in tables 1 and 2 below.

Table 1 : Lines transformed with PCH 43 K

Phytotoxicity: -: zero

+: slight (5-25% surface of leaves bleached) ++: intermediate (25-50% surface of leaves bleached) +++: intense (>50% surface of leaves bleached).

10 Retarded growth (compared to non treated controls): -: zero

+: slight (cotyledons completely developed, first leaves retarded) ++: intermediate (cotyledons developed, no first leaves visible) +++: intense (cotyledons not developed).

15 Example 8 : Insertion of the CsVMV sequence into an actin OS intron-OTP-HPPD expression cassette

pCH 10 (figure 5) is a pUC 19-based plasmid which contains an OTP/HPPD expression cassette. This cassette comprises the following components: maize H3C4 histone promoter (McElroy et al., 1990, Plant Cell 2(2) : 163- 171) ; first intron of the rice actin gene (McElroy et al., MGG 231(1) : 150-160) ; optimized transit peptide (OTP) ; HPPD ;

NOS.

SEQ ID NO 6 describes the sequence of pCH 10 between the Ncol and Xbal sites.

In pCHIO, the maize histone promoter region was excised with the Xhol and SacII restriction enzymes so as to be replaced with the CsVMV sequence contained in ρRD257. The CsVMV fragment was obtained by digestion of pRD257 with Xhol and SacII. Ligation of the 5107bp (Xhol- SacII) acceptor molecule and the 566bp (XhoI-SacII) CsVMV sequence gave pCH26 (figure 15).

Example 9: Integration of the expression cassette into an Agrobacterium tDNA-vector

pCH 21 D (figure 16) is a shuttle plasmid constructed in the laboratory 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.

The 3197bp PacI-PvuII fragment containing the CsVMV-actin intron-OTP-HPPD-Nos cassette was excised from pCH26 and cloned into the Pacl-Mscl sites of ρCH21D, to give pCH31D (figure 17).

The 2679bp PacI-PvuII fragment containing the CsVMV- OTP- HPPD-Nos cassette was excised from ρCH27 and cloned into the Pacl-Mscl sites of ρCH21D, to give pCH32D (figure 18). Recombination between pCH 31 D (or respectively pCH 32 D) and the superbinary plasmid pTVK 291 was obtained by three-parent crossing with DH5 alpha [pCH 31 D] (respectively DH5 alpha [pCH 32 D]), C2110 [pTVK 291] and the helper strain JC 2073. The resulting plasmid is called pCH 31 K (respectively. pCH 32 K). The strain obtained, C2110 [pCH 31 K] (respectively C2110 [pCH 32 K]), was selected on LB medium containing the 3 antibiotics gentamycin, kanamycin and nalidixic acid. The nalidixic acid allows the selection of C2110 against DH5alpha or JC2073; since C2110 contains a chromosomal resistance to nalidixic acid which cannot be transferred to the others strains during the crossing. Combining gentamycin and kanamycin makes it possible to select the bacteria containing pCH 31 D (respectively pCH 32 D) and pTVK 291. In addition, pCH 31 D (respectively pCH 32 D) cannot replicate in C2110, unless it is 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 pCH 31 D (respectively pCH 32 D).

The recombined plasmid was then transferred into the Agrobacterium strain LBA 4404 by a second three-parent cross. The resulting strain LBA 4404 [pCH 31 K] (respectively LBA 4404 [pCH 32 K]) was selected on AB medium (selective for Agrobacterium) containing kanamycin and gentamycin.

The plasmid pCH 68 (figure 19) which was used as a control was constructed in a similar way (details not given) using the promoter element of the rice actin- 1 gene (McElroy et al., 1990) and the intron of this same gene (McElroy et al, 1991), and then integrated into an Agrobacterium t-DNA vector according to the method described in this example, to give the Agrobacterium strain LBA 4404 [pCH 68 k].

Example 10 : transformation of maize by Agrobacterium tumefaciens with the expression cassettes

Transformation of the A188 maize genotype was carried out according to the method described by Ishida, Y. et al., Nature Biotechnology, 1996, 14, 745-750. In summary, immature maize embryos are co-cultured in the presence of the agrobacteria and the calluses obtained are then selected in the presence of phosphinothricin (5 mg/1). Rooting of the plants is carried out in tubes on agar medium in the presence of DKN (100 mg/1) and the tolerant plans are acclimatized in a greenhouse and then pollinated with nontransgenic A188 maize in order to produce seeds. A sample of seeds from the descendants obtained is sown in a greenhouse (see protocol detailed in example YY) and treated at pre-emergence with IFT at 200 g/ha so as to select only transformed lines having minimum tolerance.

The lines C92A1, C99A1, L72A1, L74Q1, L78A2, L80A1, L80C3, L89B2, F85B3, L72H1 and L74A4 were thus obtained with LBA4404 [pCH31 K].

The lines C111D3, C111H1, C112A1, C112A3, F100A2, F100D7 and F102G3 were obtained with LBA4404 [pCH32 K].

The lines C62B2, F79-1A1, C60-1A1, L45B2, L54C1, C114B3M, C123B4M, C58C1F, C60-1B1M, L102F1M, L45B2M and L53A1M were obtained with LBA4404 [pCH68 K].

Exemple 11 : Measurement of the tolerance of maize to IFTt applied at pre-emergence in a greenhouse

In order to measure the tolerance to IFT applied at pre-emergence, maize lines produced from sowing in containers were prepared. Containers (15 x 20 x 5 cm) filled with sand (particle size 3) were prepared and the sand was wetted by watering. 15 seeds of each line were sown at a depth of 0.5 cm in each container. After sowing, the containers were covered with a glass plate in order to provide a degree of humidity conducive to emergence.

The day after sowing, a container of each line used was treated by means of a treatment rig calibrated in order to deliver a volume corresponding to 5001/ha onto each container. The following doses were sprayed: NT : pure water, 200 g/ha IFT (i.e. 200 g of active material per 500 litres), 400 g/ha IFT, 600 g/ha IFT, 800 g/ha IFT.

After treatment, the containers are again covered with a glass plate. The glass plate is removed after three days in order to allow plantlet development. The plantlets are watered in the trays containing the containers every two to three days depending on the plants' needs. Once a week, a complete nutritive solution (Fertigofol 313^® at 2.5 1/ha) is added to the water for watering.

The height of the plants is measured 30 days after treatment at the level of the cornet. Figure 20 shows the results obtained. The reduction in size is calculated for each dose relative to the non-treated control. The value represents, for each dose, the mean of the measurements made on the plants of the lines used for each construct (respectively pCH31D and pCH68), see table 3. The lines used are all heterozygous for the introduced HPPD gene.

Wild-type A188 maize plants were used as a negative control. From the dose of 200 g/ha IFT, all the wild-type plants were destroyed by the herbicide.

Table 3

Example 12 : Open-field treatment of IFT-tolerant lines

The lines transformed either with ρCH31D (lines C92A1, C99A1, L72A1, L74Q1, L78A2, L80A1, L80C3, L89B2), or with pCH32D (lines C111D3, C111H1, C112A1, C112A3, F100A2, F100D7 and F102G3), or with pCH68 (lines C114B3, C123B4, C58C1, C60-1B1, L102F1, L45B2, L53A1 and L54C1) were tested in the field. The seeds were planted at a depth of 2 cm in two rows (2 m long, 20 plants per row). Irrigation of the plants was carried out dropwise at the foot of the plants without wetting the foliar parts.

The plants were treated at post-emergence, at the N3-N4 (3- to 4-leaf) stage, with IFT at 250 g/ha supplemented with one percent rapeseed oil. The results were measured 14 days after treatments.

The phytotoxicity was evaluated by measuring the foliar surface exhibiting symptoms of bleaching and/or of necroses. The results are given in table 4. Table 4 : Results of open-field treatment with EFT

Claims

1. Expression cassette comprising, in the direction of transcription, functionally linked, a promoter regulatory sequence which is functional in plant cells or plants, a nucleic acid sequence encoding an HPPD and a terminator regulatory 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 (Cassava Vein Mosaic Virus) plant virus.

2. The expression cassette according to claim 1, characterized in that the promoter regulatory sequence comprises, in the direction of transcription, the nucleic acid sequences X, Y and Z as defined respectively by SEQ ID NO 1 , 2 and 3.

3. The expression cassette according to claim 1 or 2, characterized in that the promoter regulatory sequence is represented by SEQ ID NO 4.

4. The expression cassette according to claim 1 or 2, characterized in that the promoter regulatory sequence comprises, in the of transcription direction, the nucleic acid sequences X, Y, Y and Z (double CsVMV).

5. The expression cassette according to claim 4, characterized in that the double CsVMV promoter regulatory sequence is represented by SEQ ID NO 5.

6. The expression cassette according to one of claims 1 to 5, characterized in that the nucleic acid sequence encoding an HPPD is a sequence encoding an HPPD enzyme chosen from native, mutated or chimeric HPPDs.

7. The expression cassette according to claim 6, characterized in that the HPPD is chosen from the HPPDs of bacteria such as Pseudomonas, of plants such as Arabidopsis plants or carrot plants, of Coccicoides or of mammals such as mice or pigs. 8. The expression cassette according to claim 7, characterized in that the

HPPD is a Pseudomonas fluorescens HPPD.

9. The expression cassette according to one of claims 6 to 8, characterized in that the mutated HPPD is chosen from HPPDs mutated in their C-terminal portion.

10. The expression cassette according to claim 9, characterized in that the mutated HPPD comprises the W336 mutation.

11. The expression cassette according to one of claims 6 to 8, characterized in that the chimeric HPPD is an HPPD comprising elements originating from various HPPDs.

12. The expression cassette according to one of claims 1 to 11, characterized in that the nucleic acid sequence encoding an HPPD comprises a sequence encoding a signal peptide or transit peptide.

13. The expression cassette according to claim 12, characterized in that the transit peptide is a chloroplastic transit peptide.

14. The expression cassette according to claim 13, characterized in that the transit peptide is an optimized transit peptide.

15. The expression cassette according to one of claims 1 to 14, characterized in that it comprises a transcription activator (enhancer) or an intron between the promoter regulatory sequence and the sequence encoding an HPPD.

16. The expression cassette according to claim 15, characterized in that the transcription activator is chosen from the transcription activators of the tobacco mosaic virus (TMV), of the tobacco etch virus (TEV) or of the fϊgwort mosaic virus.

17. The expression cassette according to claim 15, characterized in that the introns are chosen from intron 1 of the rice actin gene or the maize adhl intron.

18. The expression cassette according to one of claims 1 to 17, characterized in that the terminator regulatory sequence is chosen from the sequences encoding the Agrobacterium tumefaciens nos terminator, the CaMV 35S terminator and the plant histone terminator.

19. A cloning and/or expression vector for transforming plant cells or plants, characterized in that it comprises at least one expression cassette according to one of claims 1 to 18.

20. Plant cells, characterized in that they comprise an expression cassette according to one of claims 1 to 19.

21. The plant cells according to claim 20, characterized in that the expression cassette is stably integrated into their genome. 22. Plant, characterized in that it comprises cells according to claim 20 or

21.

23. Tansformed plant, characterized in that an expression cassette according to one of claims 1 to 19 is stably integrated into its genome.

24. Transformed plant according to claim 22 or 23, characterized in that it is chosen from monocotyledon plants, such as cereals, sugar cane, rice or maize, and dicotyledon plants, such as tobacco, soybean, rapeseed, cotton, beetroot or clover.

25. Seed from a transformed plant according to one of claims 22 to 24, characterized in that it comprises an expression cassette according to claims 1 to 19.

26. Method of selective herbicidal treatment of plants using an HPPD- inhibiting herbicide, characterized in that this herbicide is applied to plants according to one of claims 22 to 24.

27. Method of controling weed development in an area of a field comprising seeds according to claim 25 or plants according to claim 23 or 24, characterized in that it consists in applying, in said area of the field, a dose, which is toxic for said weeds, of an HPPD-inhibiting herbicide, without however substantially affecting the seeds or plants transformed with said chimeric gene as claimed in the invention. 28. A method for growing plants according to one of claims 22 to 24, characterized in that it consists in sowing the seeds of said plants in an area of a field suitable for growing said plants, in applying to said area of said field a dose, which is toxic for the weeds, of a herbicide for which HPPD is the target, in the event of weeds being present, 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.

29. The method according to one of claims 26 to 28, characterized in that the application of the herbicide for which HPPD is the target is carried out in pre- sowing, in pre-emergence and/or in post-emergence of the crop. 30. The method according to one of claims 26 to 29, characterized in that the HDDP-inhibiting herbicides are chosen from isoxazoles, in particular isoxaflutole, diketonitriles, in particular 2-cyano-3-cyclopropyl-l-(2-Sθ2CH3-4-CF3- phenyl)propane-l ,3-dione and 2-cyano-3-cyclopropyl-l-(2-SO2CH3-4-2,3Cl2_ phenyl)propane-l, 3-dione, triketones, in particular sulcotrione or mesotrione, or pyrazolinates.