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WO2001031041A2 - Modification de vegetaux - Google Patents

Modification de vegetaux Download PDF

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Publication number
WO2001031041A2
WO2001031041A2 PCT/EP2000/010662 EP0010662W WO0131041A2 WO 2001031041 A2 WO2001031041 A2 WO 2001031041A2 EP 0010662 W EP0010662 W EP 0010662W WO 0131041 A2 WO0131041 A2 WO 0131041A2
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plant
cell cycle
construct
cell
promoter
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PCT/EP2000/010662
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WO2001031041A3 (fr
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Justin Paul Sweetman
Herman Van Mellaert
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Cropdesign N.V.
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Priority to AU13898/01A priority Critical patent/AU1389801A/en
Publication of WO2001031041A2 publication Critical patent/WO2001031041A2/fr
Publication of WO2001031041A3 publication Critical patent/WO2001031041A3/fr

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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • 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
    • 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
    • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—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 biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to the modification of plants.
  • it relates to the identification of active genetic components within the plant genome which can be harnessed or trapped to drive expression or alter expression of desired coding sequences, so as thereby to modify the plants as required.
  • Gene expression is controlled by regions upstream (5') of the protein encoding region, commonly referred to as the 'promoter 1 . Promoters are located immediately upstream of transcriptional start sites and control transcriptional initiation by RNA polymerase. Such promoters could be constitutive (e.g. An ⁇ t al., 1996 Plant J. 10, 107-21), organ- specific (e.g. Rosahl et al., 1987 Mol. Gen. Genet. 203, 214-20), tissue-specific (e.g. Peleman et al., 1989 Plant Cell 1 , 81-93), light-regulated (e.g. Kuhlemeier et al., 1989 Plant Cell 1 , 471-78), wound-inducible (e.g.
  • promoters have been isolated from genomic libraries using cDNA probes to identify sequences upstream of the transcriptional start site and by testing nearby sequences for promoter activity.
  • isolating cellular promoters can be difficult because nearly full-length cDNA clones may be required to identify genomic sequences near the sites of transcriptional initiation, and transcribed genomic sequences may be hard to distinguish from untranscribed pseudogenes.
  • cDNA probes a preference for the isolation of highly transcribed gene promoters exists.
  • Promoters and their associated genes have also been isolated by insertional mutagenesis strategies involving transposon or T-DNA integration and analysis of resulting knock-out phenotypes to interpret gene function (Sheridan 1988 Ann. Rev. Genet. 22, 353-85; Feldman 1991 Plant J. 1, 71-82). The resulting insertion of foreign
  • DNA, into, or near the gene of interest causes a mutation that molecularly tags the gene and facilitates its cloning.
  • knock-out of non-essential genes will not produce a noticeable phenotype.
  • Transposon tagging has largely been confined to diploid self-fertilising species such as maize, which has a proven transposable element system.
  • various derivatives of the well-characterised maize Ac-Ds transposon system have been introduced into other plant species where they have been shown to be active, such as rice (Izawa et al., 1997 Plant Mol. Biol. 35: 219-229) and tomato (Carroll et al., 1995 Genetics 139: 407-420).
  • T-DNA tagging exploits the Ti-plasmid from Agrobact ⁇ rium tumefaciens, which inserts one or more copies of the T-DNA into the plant genome.
  • the major limitation in this approach is the large amount of effort required producing and propagating large numbers of independent T-DNA insertion plant lines. If the particular gene tag is not present in the resulting collection another library must be produced or an alternative method of cloning must be used.
  • reporter genes which possess either no promoter or a minimal promoter by integration into the plant genome via T-DNA or transposons (Lindsey et al., 1993 Trans. Res. 2, 33-47; Sundaresan et al., 1995 Genes Dev. 9, 1797-1810).
  • This approach can be used to identify promoter or enhancer sequences respectively by analysing reporter gene expression if integration occurs in a sense orientation to the regulatory sequences.
  • the expression pattern of the reporter gene would be expected to reflect the pattern of the native gene into which integration has occurred and this has been confirmed in Arabidopsis and tobacco (Kertbundit et al., 1991 PNAS 88, 5212-6; Koncz et al., 1989
  • the well known gus reporter gene from Escherichia coii encodes the stable and manipulation-tolerant enzyme ⁇ -glucuronidase, whose intracellular presence in a transgenic plant can be revealed by histochemical staining using 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-GlcA), a chromogen which is cleaved by the enzyme to release a blue dye.
  • X-GlcA 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid
  • the identification of a desirable promoter for example, by a trapping strategy generally will only be a first step, which will be followed by further manipulation to couple the promoter to a coding sequence of interest, whose expression in the plant will result in a desired phenotype.
  • the method provided by this invention provides a novel trapping strategy using cell cycle controlling genes which has an unexpected outcome in the generation of plants with altered phenotype and in particular modified architecture.
  • the invention enables from this modified architecture the identification of useful plant promoters, enhancers or genes in crops and other plants.
  • Such promoters, enhancers or genes can be trapped by directing or enhancing expression of, or by forming a functional fusion with, a ceil cycle controlling gene in particular tissues and thereby arrest or promote, respectively, cell division in those tissues and produce plants with modified architecture.
  • a method of modifying the architecture of a plant comprising: (i) transforming a plant cell with a construct comprising coding DNA which encodes a cell cycle controlling gene product, wherein the construct either
  • (a) does not comprise a promoter operatively coupled to the coding DNA and optionally comprises a splice acceptor site 3' to the coding DNA, or
  • (b) comprises a weak or minimal promoter operatively coupled to the coding DNA
  • architecture will be understood by those skilled in the art to refer to the appearance or morphology of a plant, including any one or more structural features or combination of structural features thereof.
  • Such structural features include the shape, size, number, position, texture, arrangement, and pattemation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, leaf, shoot, stem, petiole, trichome, flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre, cambium, wood, heartwood, parenchyma, aerenchyma, sieve element, phloem or vascular tissue, amongst others.
  • Modified architecture therefore includes all aspects of modified growth of the plant. Sometimes plants modify their architecture in response to certain conditions such as stress and pathogens (e.g. nematodes). Therefore, within the scope of the term "architecture" is included modified architecture under conditions such as stress and pathogens.
  • stress and pathogens e.g. nematodes
  • the present invention therefore aims to use DNA which encodes a ceil cycle controlling gene product - a "cell cycle controlling DNA" for brevity - which may have a modulating effect on the plant cell cycle for instance by having a promoting, increasing, inhibiting or abolishing etc. effect on the plant cell cycle, including such effects on the number, duration, or speed of a cell cycle, as part of a novel promoter, gene or enhancer trap vector.
  • the vector can be used to trap promoter, enhancer or gene sequences driving expression in tissues critical for the architecture of a plant by virtue of a modulation (for example, an interruption) of normal cell division in those tissues and the generation of novel growth phenotypes.
  • the cell cycle is central to the process of cell division.
  • the cell cycle controlling DNA could be expected to alter growth or architecture either of the entire plant or merely of target tissues.
  • Cell cycle means the cyclic biochemical and structural events associated with growth and with division of cells, and in particular with the regulation of the replication of DNA and mitosis.
  • Cell cycle includes phases called: Gap1 (G1 ), DNA synthesis (S), Gap2 (G2), and mitosis (M). Normally these four phases occur sequentially.
  • the term "cell cycle” as used herein also includes modified cycles in which one or more phases are absent, resulting in a modified cell cycle such as endomitosis, acytokinesis, polyploidy, polyteny and endoreduplication.
  • a “cell cycle controlling gene” or “cell cycle controlling DNA” refers to any DNA sequence, which may for example be a natural gene or mutant thereof, which exerts control on or regulates or is required for the cell cycle or part thereof of a cell, tissue, organ or whole organism and or DNA replication therein.
  • a “cell cycle controlling gene product protein” is produced by such a DNA sequence and includes peptides, polypeptides, oligopeptides, enzymes and other proteins.
  • Cell cycle controlling gene products active in S phase, at the G1 -to-S transition and at the G2-to-M transition are preferred for use in the invention.
  • the four phases of the cell cycle in eukaryotic cells are driven by a common class of heterodimeric serine/threonine protein kinases. These kinases consist of one catalytic subunit (cyclin dependent kinases or CDKs) and one activating subunit (cyciins).
  • CDKs cyclin dependent kinases
  • cyciins activating subunit
  • a large number of putative cell cycle genes have been cloned in plants over the last 7 years.
  • a last count identified 17 types of cyciins and 5 types of CDKs in the model plant Arabidopsis thaliana. Identifying which of these genes are actually involved in the cell cycle is a major objective of plant cell cycle work.
  • A-type classes of CDKs such as CDC2a, are homologues of yeast P 34 cdc2 CDC28 protein kinases and are characterised by a PSTAIRE motif in the N-terminal protein sequence. This motif is essential for cyclin binding (Ducommun et al., 1991 EMBO J. 10, 3311-19).
  • B-type CDKs such as CDC2b, are unique to plants in terms of their structure and function.
  • CDC2b is characterised by a PPTALRE cyclin binding motif (Segers et al., 1996 Plant J. 10, 601-12). Experimental evidence supports the role of CDC2a and CDC2b in cell division control in plants.
  • CDC2a-DN Down-regulation of CDC2a function by over-expressing a dominant negative mutant version of CDC2a (CDC2a-DN) in transgenic plants has the effect of abolishing the rate of cell division in Arabidopsis (Hemeriy et al., 1995 EMBO J. 14,
  • CDC2a Some tobacco plants constitutively producing CDC2a-DN were recovered and found to have considerably fewer but larger cells. As the relative duration of G1 and G2 is unaffected, CDC2a probably functions in the G1-to-S and G2-to-M transitions. In contrast, downregulation of CDC2b has the effect of lengthening the duration of G2, implicating these CDKs in progression through G2.
  • Gene expression studies using northern blots and GUS promoter fusions in transgenic plants has identified CDC2a expression in all meristematic (i.e. dividing) tissue throughout development, suggesting that CDC2a participates in the spatial and temporal control of cell divisions in the whole plant (Hemeriy et al., 1993 Plant Cell 5, 1711-23). CDC2a can therefore be seen as playing a central role in cell division in plants.
  • the catalytic activity and substrate specificity of CDKs is determined by cyclin subunits.
  • mammals at least 10 classes of cyciins (A to J) have been described (Pines 1995 Biochem. J. 308, 697-711). The majority of plant cyciins fall into classes A-, B- and D-type (Renaudin et al., 1996 Plant Mol. Biol. 32, 1003-18).
  • A-type cyciins are thought to activate CDC2a and CDC2b at the G 2 -to-M transition (Ito et al., 1997 Plant J.
  • B-type cyciins are thought to be involved specifically in the G 2 -to-M transition (Day et al., 1996 Plant Mol. Biol. 30, 565-575) whereas D-type cyciins are thought to interact with CDC2a in the d-to-S phase transition after their production by mitogenic stimulation (Soni et al., 1995 Plant Cell 7, 85-103).
  • CKS proteins cyclin-dependant kinase subunit
  • yeast In yeast high levels of CKS1 protein delays entry into mitosis (Dunby and Newport 1989 Cell 58, 181-91) whereas a depletion of the protein suppresses exit from mitosis (Basi and Draetta 1995 Mol. Cell Biol. 15, 2028-36).
  • CKSlAt was isolated (De Veylder et al., 1997 FEBS Letts. 412, 446-52). In situ hybridisation analysis revealed strong expression of CKSlAt in actively dividing tissues (like CDKs) and in a number of polyploid tissues (unlike CDKs), suggesting that CKS may interact with CDKs in plants and also play a role In the process of endoredupiication (Jacqmard etal., 1999 Planta 207, 496-504).
  • CKIs CDK inhibitor proteins
  • ICK1 was isolated (Wang et al., 1997 Nature 386, 451-52). Although only limited sequence homology to human cell cycle inhibitors was found this short region of 30 amino acids was found to be essential for interaction with CDC2a and CYCD3;1 (Wang et al., 1998 Plant J. 15, 501-10).
  • Cell cycle controlling gene products include cyclin dependant kinases (CDKs) such as CDC2a and CDC2b (Segers et al., 1997 Plant cell proliferation and its regulation in growth and development Chichester UK: John Wiley & Sons 1 -19), cyciins A, B, C, D and E including CYCA1 ;1 , CYCA2;1 , CYCA3;1 , CYCB;1 , CYCB;2, CYC B2;2,
  • CYCD1;1 , CYCD2;1 , CYCD3;1 , and CYCD4;1 (Evans ⁇ t al., 1983 Cell 33, 389-96; Labbe et al., 1989 EMBO J. 8, 3053-8; Murray and Kirschner 1989 Science 246, 614- 21 ; Renaudin et al., 1996 Plant Mol. Biol. 32, 1003-18; Soni et al., 1995 Plant Cell 7, 85-103; Sorrell et al., 1999 Plant Physiol. 119, 343-352; Swenson et al., 1986 Cell 47, 861-870) cyclin dependent kinase inhibitor (CKI) proteins such as ICK1 (Wang et al.,
  • Cdc2 such as Cdc2MsB (Hirt et al., 1993 Plant J. 4, 61-9) CdcMs kinase (Bogre et al., 1997 Plant Physiol. 113, 841-52) cdc2 T14Y15 phosphatases such as Cdc25 protein phosphatase or p80cdc25 (Bell et al., 1993 Plant Mol. Biol.
  • CDC45 Harmonic 1997 Gene 187, 239-246
  • RPA Carmichael et al., 1995 Mol. Cell Biol. 13, 408-20
  • MCM genes Sabelli et al., 1996 Mol. Gen. Genet. 252, 125-36 or in the regulation of formation of this pre-replicative complex, such as, but not limited to, the CDC7 (Sato et al., 1997 EMBO 16, 4340-51), DBF4 (Jackson et al., 1993 Mol. Cell. Biol. 13, 2899-2908) and MBF (Koch et al., 1993 Science 261 , 1551-
  • cell cycle controlling DNA shall further be taken to include any one or more of those genes or other DNA molecules whose protein products are involved in the turnover of a ceil cycle control protein, or in regulating the half-life of a cell cycle control protein, such as, but not limited to, genes that are involved in the proteolysis of one or more of the above-mentioned cell cycle control proteins.
  • genes that are involved in the proteolysis of one or more of the above-mentioned cell cycle control proteins include the yeast-derived and animal-derived proteins, Skp1, Skp2, Rub1, Cdc20, cullins, SCF ubiquitin ligase,
  • cell cycle controlling DNAs include any one or more of those genes or other DNAs whose protein products are involved in the transcriptional regulation of cell cycle gene expression such as transcription factors and upstream signal proteins. Additional cell cycle genes are not excluded.
  • the present invention encompasses the use of cell cycle controlling genes coding for cell cycle control proteins selected from the examples described above, such genes including sense, anti-sense, dominant negative, dominant positive, wild-type or mutant versions thereof and any homologous (including functionally homologous) gene related thereto. It also includes sequence of a cell cycle gene in a cosuppression inducing configuration.
  • Homology in the above definition may be based on amino acid or nucleotide matching performed using an alignment programme or algorithm such as the CLUSTAL programme (Higgins and Sharp 1989 Gene 73, 237-44) or BLAST or FASTA (Genetics Computer Group, Madison, Wl, USA) or experimentally such as hybridisation in high stringency conditions (20 mM sodium phosphate, 1 % SDS, 65°C).
  • Two nucleotide sequences may have sequence homology if the sequences have at least 60 percent, more preferably 70 percent, more preferably 80% and most preferably 90 percent sequence similarity between them.
  • Two amino acid sequences have sequence homology if they have at least 50 percent, preferably 70 percent, and most preferably 90 percent similarity between the active portion of the poiypeptides.
  • these values can be appropriately adjusted by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
  • Homology may also be considered with respect to those regions of the respective sequence essential for function.
  • Homology further means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent.
  • DNA sequences which code for the same proteins as those encoded by natural gene sequences are within the scope of the invention.
  • the present invention encompasses the use of homologues, analogues or derivatives of any of the above mentioned cell cycle control genes whose protein products function in DNA synthesis, mitosis, S phase, endomitosis, acytokinesis, polyploidy, polyteny and endoreduplication.
  • the cell cycle controlling gene is derived from a yeast cell or plant cell or animal cell, more preferably, from a plant cell, such as a monocotyledonous or dicotyledonous plant cell (Mironov et al., 1999 Plant Cell 11 , 509-22).
  • WO-A-9209685 (Australian National University) relates to a method for controlling cell division in plants either by modulating the levels of CDC2 directly or indirectly by the level of interacting proteins such as p13SUC1 , NIM-1 , WEE-1 , MIK-1 and CDC25.
  • WO-A-9841642 The alteration of cell division in cells of transgenic plants by modulating the activity of CDC2 is disclosed in WO-A-9841642 (Cr ⁇ pDesign NV.).
  • One such method utilises over-expression of the CKSlAt protein to restrain CDC2b activity and thus block progression through the cell cycle and cell division and increase endoreduplication.
  • Another method describes the over-expression of dominant negative mutations of CDC2b to by-pass mitosis and increase endoreduplication.
  • WO-A-9803631 (Salk Institute for Biological Studies) describes a method of increasing growth and yield in plants by using ectopic expression of B-type cyciins such as cydAt either from an endogenous promoter. Expression of cydAt from the CDC2a gene promoter had a marked effect on root growth.
  • WO-A-9312239 (ICI pic) describes the alteration of plant and plant cell growth characteristics using expression of the CDC25 gene from Schizosaccharomyces pombe (S. pombe) to increase the number of flowers and the precocity of flowering.
  • WO-A-9842851 (Cambridge University Technical Services Ltd.) describes a process for modifying the growth and architecture of plants by modulating the levels of D-type cyciins within the plant.
  • the invention can be applied to all plant species and varieties, whether dicotyledonous or monocotyledonous, but it is especially suited to plants with an agricultural or horticultural value.
  • plant species include: oil-seed rape (canola), sunflower, tobacco, sugar-beet, cotton, soya, maize, tree species, cassava, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, vegetables such as carrot, lettuce, cabbage, onion and ornamentals such as carnations, roses, tulips and chrysanthemums.
  • the invention is also relevant for "model” plants such as Arabidopsis thaliana.
  • the invention is particularly relevant for plants which are glasshouse grown, for example, to decrease the time for plants to reach a desired developmental stage such as fruit-set, seed-set or flowering or the time needed to reach an economically important developmental stage.
  • the invention is particularly applicable to the tomato, Lycopersicum esculentum.
  • plant cells are transformed with a DNA construct comprising coding DNA which encodes a cell cycle controlling gene product.
  • the coding DNA may be genomic, cDNA, a minigene (containing some, but not all, of any introns present) or wholly or partially synthetic.
  • This cell cycle controlling DNA may have a modulating effect on the plant cell cycle for instance by having a promoting, increasing, positive, inhibiting, negative or abolishing etc. effect on the plant cell cycle. Such effects could be on the number, duration, type or speed of a cell cycle.
  • the cell cycle controlling gene product is a cyclin dependent kinase (CDK) such as CDC2a, cyclin dependent kinase inhibitor (CKI), cyclin, cyclin dependent kinase subunit (CKS) or RepA.
  • CDK cyclin dependent kinase
  • CKI cyclin dependent kinase inhibitor
  • CKS cyclin dependent kinase subunit
  • RepA RepA
  • cell cycle controlling genes which could act to promote the cell cycle include the CYCD2;1 or CYCB1 ;1 gene expressed in a sense orientation or a CKI gene such as ICK1 from Arabidopsis (Wang et al., 1998 citation above) or FL39, FL66, FL67 (WO-A-9914331) expressed either in an anti-sense orientation to a TATA box or any cosuppression-inducing configuration.
  • Examples of cell cycle controlling genes which could act to abolish or otherwise have a negative effect on the cell cycle include a CKI, a CKS such as CKSlAt (De Veylder et al., 1997 citation above) or a dominant negative mutation of CDC2a (Hemeriy et al., 1995 citation above) in the sense orientation to a TATA box or a CDC2a gene in an anti-sense orientation to a TATA box.
  • the preferred cell cycle controlling gene would be a dominant negative mutation of CDC2a expressed in an sense orientation to a TATA box.
  • a “dominant negative mutation” is a mutation which has a negative effect on protein function (e.g. for CDC2a it affects its ability to kinase pRb proteins once bound with CycD, or in the case of a transcription factor it affects its ability to activate transcription once bound to its operator sequence) but is dominant over the endogenous wild type protein.
  • a “dominant positive mutation” has a positive effect on the protein function and is dominant over the endogenous wild type protein. Note, therefore, that the "dominant” effect of the cell cycle controlling DNA is on protein function and not necessarily the cell cycle; so, for example, a dominant negative mutation would negatively affect the protein and, depending on the function of that protein, may have a positive or negative effect on the cell cycle.
  • the preferred mutation for the creation of a dominant negative CDC2a cell cycle controlling gene in previous studies has been a residue change 147 from aspartate to asparagine, although the position of this mutation changes according to the specific CDC2a.
  • yeast humans and Arabidopsis this mutation has been shown to inactivate the kinase causing an arrest of the cell cycle (Mendenhall et al., 1988 PNAS 85, 4426- 4430; van den Heuvel and Hariow 1993 Science 262, 2050-54; Hemeriy et al., 1995 citation above).
  • the relevant mutation site is 146.
  • the preferred cell cycle gene is the CDC2a gene bearing a dominant negative mutation such as D147N (A. thaliana; Hemeriy et al., 1995 citation above), P162L, P162S or a deletion of residues L240, Q241 and D242 (S. pombe; Labib et al., 1995 EMBO J. 14, 2155-2165).
  • D147N A. thaliana; Hemeriy et al., 1995 citation above
  • P162L, P162S or a deletion of residues L240, Q241 and D242 S. pombe; Labib et al., 1995 EMBO J. 14, 2155-2165.
  • the dominant mutations referred in the present invention can be preferably generated by substitution, deletion and/or addition of 1 to 5 or 5 to 10 amino acid residues in the amino acid sequence of the above-described wild type proteins.
  • the manipulation of DNA sequences to produce variant proteins which manifest as substitution, insertion or deletion variants is well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA having a known sequence such as by M13 mutagenesis or other site directed mutagenesis protocol, are well known to those skilled in the art.
  • Any suitable transformation method for the desired target plant or plant cells may be employed (see Hansen and Wright, Trends in Plant Science (1999) 4 (6) 226-231). Examples include infection by Agrobact ⁇ rium tumefaciens containing recombinant Ti plasmids. The use of right and left T-DNA borders from a binary plasmid such as pBIN19 (Bevan 1984 Nucl. Acids Res. 22, 8711-21), allows for stable insertion into the plant genome. Other methods of transformation include electroporation, microinjection of cells and protoplasts, bacterial microprojectiie bombardment, the glass fibre or 'whisker' method and pollen tube transformation. The transformed cells may then in suitable cases be regenerated into whole plants in which the new genetic material is stably incorporated into the genome. Reference made be made to the literature for full details of the known methods.
  • the construct may include a Ds (dissociation) transposable element which may then be activated to transpose in the genome by the action of an expressed Ac (activator) enzyme (Cocherel et al., 1996 Plant Mol. Biol. 30, 539-551).
  • Ds dissociation
  • Ac activator
  • the cell cycle controlling construct would be inserted into a T-DNA vector and be flanked by sequences from the Ds element. Such a construct can be transformed into plant cells to produce a small number of stock lines.
  • the Ac coding sequence under the control of the Ac promoter or of a plant constitutive promoter such as the Cauliflower Mosaic Virus 35S (CaMV35S) promoter would also be transformed into plants on a T-DNA vector to form a separate plant line.
  • the transposon approach has advantages over the T-DNA promoter trap insertion strategy in that the generation of large numbers of insertion plant lines using transposons is achieved by genetic crossing of a small number of Ac and Ds-cell cycle control gene master lines. Selection of plants that harbour unlinked transposition events could then be achieved using a suitable one-step plate selection (Sundaresan et al., 1995 Genes Dev. 9, 1797-1810; Fedoroff and Smith 1993 Plant J. 3, 273-289).
  • the construct apart from the cell cycle controlling DNA will depend on whether the construct is to be used in a method of trapping a promoter, gene or enhancer.
  • a method of identifying a promoter which is a preferred embodiment of the invention, the plant cell is transformed with a construct which does not comprise a promoter operatively coupled to the coding DNA.
  • the construct will therefore be "promoterless". While it is possible to envisage embodiments of the invention in which a promoter is present but not functional, or functional but not operatively coupled to the coding DNA, such embodiments are not perceived to have any particular advantage.
  • the preferred form of the promoter trap construct would comprise the cell cycle control DNA with its own ATG translational initiation codon, for example placed substantially adjacent to one T-DNA border so that integration of the T-DNA construct into the plant genome could place a plant promoter in a functional fusion to the cell cycle control DNA or substantially adjacent to one of the activatable transposon elements inverted repeats.
  • promoters which may be selected by such a promoter-trap construct, include those that are specific for particular cells, tissues or organs and/or those that are specific for particular stages in plant development as well as inducible and constitutive promoters.
  • Non-exclusive examples include seed-specific, root-specific, cortex-specific, fruit-specific, tuber-specific, meristem-specific, leaf-specific, endosperm-specific, phloem-specific, flower-specific, pollen-specific, anther-specific, tapetum-specific, stigma-specific and pathogen-inducible, nematode-inducible, stress- inducible, hormone-inducible and chemically-inducible promoters as well as constitutive promoters.
  • the construct may also comprise a splice acceptor site 3' to the coding DNA, so that a functional fusion may be formed if the coding DNA integrates within an intron.
  • the resulting chimeric transcripts are expressed in a pattern as directed by the gene into which insertion occurred.
  • More than one splice acceptor may be present, for example one for each of the three reading frames.
  • Such a triple splice acceptor site may be added to the gene trap construct as a synthetic oligonucleotide with the following sequence 5' GTTATATGCAG:GTTATATGCAG
  • the 3' splice acceptor site(s) may be placed substantially adjacent to one T-DNA border, if present.
  • the coding sequence is operatively coupled to a weak or minimal promoter.
  • the nucleic acid construct could comprise the coding sequence of a cell cycle control gene with its own ATG translational initiation codon, and TATA and CAAT boxes, which may be positioned adjacent to one T-DNA border, to provide the elements of a functional minimal promoter.
  • the TATA and CAAT boxes may be essentially the only functional promoter sequences present.
  • minimal promoters include the -45 CaMV 35S or the -60 CaMV 35S minimal promoters and promoters derived from the CHS promoter, PR1 promoter, nos promoter, Ac promoter (Fridlender, Mol. Gen. Genet.
  • Adh-1 promoter Ellis, EMBO J. 6 (1987), 11-16
  • Bz1 promoter Rh, Plant Cell 3 (1991), 317.
  • transcription directed by a minimal promoter is low and does not respond either positively or negatively to environmental or developmental signals in plant tissue. Integration of such a construct into the plant genome within range of the influence of an enhancer sequence could cause up-regulation of the transcription of the cell cycle controlling gene and the production of an altered architecture plant phenotype.
  • a minimal promoter such as one of those described above is not, however, the only promoter that could be used; weak promoters provide acceptable or even preferred alternatives.
  • Spm promoter from maize (Raina et al., 1993 PNAS USA 91 ,1706-10).
  • a weak promoter is for example one which in a given context has five or even ten fold lesser activity than CaMV 35S in the same context.
  • the DNA construct may also contain a selectable marker, although if high transformation efficiencies are achieved a selectable marker may be neither necessary nor desirable.
  • Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, anti-metabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes have been described, namely trpB, which allows cells to utilise indole in place of tryptophan; hisD, which allows cells to utilise histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci.
  • mannose-6- phosphate isomerase which allows cells to utilise mannose
  • ODC ornithine decarboxylase
  • DFMO deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
  • the IAAH gene confers sensitivity of plants to NAM (naphthalene acetamide) by conversion of NAM to the potent auxin napthalene acetic acid (NAA), and thus can be used as a counter- selectable marker (Karlin-Neumann et al., 1991 Plant Cell 3, 573-582).
  • NAM naphthalene acetamide
  • NAA potent auxin napthalene acetic acid
  • a promoters such as the Nos, V or 2' from Agrobacteri ⁇ m tumefaciens could be used.
  • Other optional components to the DNA construct may be lox sites for example one either side of the marker gene(s) to enable their excision after stable integration of the cell cycle controlling gene trapping construct (Qin ⁇ t al., 1994 PNAS USA 91 ,1706-10).
  • phenotypic changes may not be identifiable, either readily or at all, by visual inspection, in which case another, suitable means of identification should be used.
  • the strategy for identification would include treatment of the transformed plant with the relevant pathogen, stress, hormone or chemical.
  • architecture traits which could be identified using a combination of organ/tissue specific promoters driving expression of the CDC2a-DN gene, include modifications in flowering, branching patterns, feeding structures or galls, fruit ripening, seed set, plant height, fruit shape, fruit size and root formation.
  • Such architecture traits could be used to improve growth and/or development in crop plant species, including monocotyledonous and dicotyledonous plants for agricultural applications.
  • the range of modified architectural phenotypes generated by a method of the first aspect of the invention implies that the invention can also provide a method for the identification of promoters, genes or enhancers.
  • a method of identifying a promoter, gene or enhancer in a plant comprising: (i) transforming a plant cell with a construct comprising coding DNA which encodes a cell cycle controlling gene product, wherein the construct either
  • (a) does not comprise a promoter operatively coupled to the coding DNA and optionally comprises a splice acceptor site 3' to the coding DNA, or
  • (b) comprises a weak or minimal promoter operatively coupled to the coding DNA
  • the promoter, gene or enhancer in question may be upstream, for example immediately upstream, of the site of integration of the construct. It may be identified by means of a suitable cloning method.
  • An example of a suitable cloning method is would be the use of IPCR (inverse PCR) to isolate flanking sequences adjacent to the tagging vector borders by using IPCR primers that correspond to T-DNA sequences (Topping et al., 1994 Plant J. 895-903).
  • IPCR inverse PCR
  • (a) does not comprise a promoter operatively coupled to the coding DNA and optionally comprises a splice acceptor site 3' to the coding DNA, or (b) comprises a weak or minimal promoter operatively coupled to the coding DNA.
  • a host cell comprising a construct as described above.
  • the host may be prokaryotic - for example E. coli or another suitable cloning host - or Agrobact ⁇ rium tumefaciens or another suitable plant transformation vehicle; or the host may be eukaryotic, typically a plant cell transformed in accordance with a method of the invention.
  • the construct will usually be stably maintained within the plant cell either integrated into the plant's genome or episomally.
  • Plant cells include those transformed with the construct by any suitable transformation method. Tissue cultures and cell lines comprising transformed cells, such as callus solid media cultures or liquid cultures are therefore within the aspect of this invention.
  • a plant, part of a plant, plant tissue or plant cell including a construct as described above.
  • the construct will usually be stably maintained within the plant either integrated into the plant's genome or episomally.
  • Parts of plants include tissues and organs generally, and more specifically seeds, fruits, roots, stems, pollen, leaves, flowers, tubers propagules and seedlings.
  • Plants in accordance with the invention include progeny of plants described above.
  • This invention also provides in a sixth aspect for a library of plants or parts of plants such as tissue cultures or cell lines each line with the cell cycle controlling gene construct stably maintained.
  • This library of plants or parts would form a resource that could be used to identify plant lines with a particular altered architecture phenotype.
  • Such libraries in crop plant species such as field crops, cereals, fruits and vegetables could lead to the identification of improved growth or other architecture characteristics of agronomic value.
  • the library of plants could be used to select tagged promoters which have identified to have useful expression for 'quality' genes conferring traits such as stress tolerance, disease resistance (such as virus and nematode resistance) and yield improvement.
  • the promoter-tagged library described in this application is a valuable resource which could be used to select promoters useful for expression of genes conferring quality traits in tomatoes such as stress resistance, disease tolerance (e.g. virus resistance and nematode resistance) and yield improvement.
  • the introduced DNA construct directly results in the observed phenotype, as a result of having trapped a particular promoter, gene or enhancer.
  • the invention encompasses a DNA construct comprising a tissue specific promoter, which may have been identified by means of a method as described above (and which may have been cloned by methods known in the art), operatively coupled to DNA encoding a cell cycle controlling gene product.
  • a DNA construct may be introduced into a plant, for example as previously described, to yield a plant with modified architecture.
  • the combination of a tissue specific promoter and a cell cycle controlling gene provides a means for the engineering of a broad range of targeted changes in plant architecture.
  • the modified architecture might involve the inhibition or inflation of specific tissues.
  • the cell cycle controlling gene product may be as described above, in that it may modify the cell cycle and preferably arrests or promotes the cell cycle or cell division of a plant cell - the end result being a plant with modified architecture.
  • An illustration of this aspect of the invention includes the use of a tissue specific promoter in dividing cells in combination with a dominant negative CDK to suppress the development of one or more specific tissues.
  • a pollen- and/or tapetum-specific promoter in combination with DNA encoding a cell cycle controlling gene product, such as a dominant negative CDK, to suppress the proper development of pollen is one example, and the use of a lateral root-specific promoter in combination with such DNA to suppress development of lateral roots is another.
  • Another variation of the invention for the generation of architecture phenotypes involves cloning the CDC2a-DN or any other cell cycle gene downstream of a UAS-minimal promoter.
  • a plant line containing this UAS-CDC2a-DN could be crossed to an enhancer or promoter-trap plant line containing a transcription factor (e.g. GAL4-VP16, Haseloff WO-A-9730164) tagging construct.
  • Plants in the resulting F1 progeny could be identified which contained both the transcription factor tagging construct and UAS-CDC2a-DN construct.
  • These lines could be screened for an architecture phenotype as a result of activation of the CDC2a-DN gene in a promoter- specific fashion.
  • Figure 1 Schematic representation of a nucleic acid construct comprising a sequence in accordance with the invention.
  • Figure 2 Architag constructs for nematode induced promoter identification: (a) p0042 and (b) p0236.
  • the inventors have used a promoter tagging approach but adapted it to screen for plant promoter sequences which direct expression of a dominant negative cell cycle gene to plant tissues such that it causes cell cycle arrest in those tissues and results in an architecture alteration phenotype. Unless otherwise stated in the Examples, all recombinant DNA techniques are performed according to protocols as described in Sambrook et al., 1989 Molecular cloning: A Laboratory Manual. Cold Spring Harbour Press, NY or in Volumes 1 and 2 of Ausubel et al., 1994 Current Protocols in Molecular Biology, Current Protocols.
  • the CDC2A-1 cDNA (EMBL/Genbank Data Library accession number Y17225) from tomato (Lycopersicon esculentum Mill.) was PCR amplified from a tomato fruit cDNA library according to standard procedures.
  • Arabidopsis a mutation D146N (aspartate to asparagine), essential for binding ATP, was generated with a Quickchange PCR mutagenesis kit (Stratagene, UK) using the above overlapping oligonucleotides to amplify the whole cDNA according to the manufacturers instructions. The presence of the mutation and the absence of PCR errors were confirmed by cloning into the Xho ⁇ /EcoR ⁇ sites of pBluescript BS
  • the Nos terminator was transferred as a XbaVEcdrW fragment from p35SC1 into the Xba ⁇ IEcoR ⁇ sites of the binary vector pTHW136 (Plant Genetic Systems).
  • the CDC2a-DN sequence was cloned as a Hincti/Acd blunted fragment into the blunted Sa ⁇ site of construct the construct created in Example 1 above. Blunting of restriction fragment ends was carried out with Klenow enzyme (Pharmacia,
  • the Nos terminator was transferred as a Xba ⁇ /EcoR ⁇ fragment from p35SC1 into the Xbal/EcoRl sites of the binary vector pTHW136 (Plant Genetic Systems).
  • the CDC2a-DN sequence was cloned as a HincW/Acd blunted fragment from the construct created in Example 1 above into the blunted Sma ⁇ site of construct 2 above.
  • a CDC2a-DN construct described in Example 2 above can be transferred into Lycopersicon esculentum cv Mill by the Agrobacterium tumefaciens transformation and regeneration method described in Fillati et al., 1987 Biotech. 5, 726-30) to produce over 1000 transformed lines. Rooted shoots are transferred to soil and grown under glasshouse conditions.
  • Example 5 Production of a tomato architecture mutant
  • the T-DNA loci number of transgenic lines can be determined by segregation of the kanamycin resistance trait encoded by the npt-ll gene on the T-DNA using the kanamycin sulphate spray assay (Weide et al., 1989 Theor. Appl. Genet. 78, 169-72) and the T-DNA copy number determined by Southern blot analysis. The level of transgene expression will be determined by northern analysis.
  • the T1 plant lines will allowed to self for seed production and a study of architecture phenotypes made on homozygotes in the T2 population. Plant lines which show a stable pattern of inheritance of a novel architecture phenotype with the CDC2a-DN construct as determined by PCR to the CDC2a-DN coding region will form part of the promoter- trap library, described above.
  • Transgenic lines will be analysed under normal growth conditions and the effects of transgene expression on plant structure and productivity assessed according to: growth rate, total biomass production, root versus leaf biomass, number and size of roots, branching of roots and stems, leaf shape and microscopical analysis of tissue anatomy.
  • Example 6 Construction of transgenic tomato architag lines and identification of nematode induced promoters
  • CD0054 collection contains 165 lines. In a first experiment, four lines showed a reduction in nematode infection (Mi). The inoculation was repeated (exp2) after which line 36-172A still showed a 32% reduction in female development (see Table 1 ).
  • Nematode inoculations were performed as follows: 21 tomato seeds are first germinated and subsequently lined up on thin-medium plates ; 7 seeds per plate (30mL Knop per Greiner 688102 square plate). 1 week after germination, each main root is inoculated with 15 Meloidogyne incognita juveniles that were previously suspended in low-melting-point agarose. Scoring is performed by counting the number of induced galls per number of inoculated root tips at 5 days post inoculation (dpi) and 14 dpi.
  • galls can be harvested and hatched in order to analyze the content, i.e. the number of eggs and juveniles. Also the infectivity grade can be measured as an indicator for the viability of the eggs and juveniles.
  • CD0283 collection The second collection measures approximately 1000 lines.
  • a first batch of 32 CD0283 lines was screened for reduced nematode infection levels or nematode resistance (Table 2).
  • Line 59-97B shows a remarkable reduction in nematode infection, the resistance test surely needs to be repeated on this line.
  • Nematode infections and scoring were performed as described above.
  • the collection of tomato tag lines is screened for nematode resistance and various scoring criteria are taken into account. Most important is a reduced nematode infection whereby the architectural characteristic of number of nematode feeding structures established per number of inoculated roots is significantly lower compared with the infection rate on wild type roots. Besides a significant infection reduction, the size of the feeding structures is also an important architectural characteristic. As an example, line 59-75A as shown in Table 2 does not show a reduced infection rate, however the size of the established gall structures is much reduced. Feeding structure size is involved in the later sex determination of the infective juveniles. Small feeding structures direct nematode development into males. As such reproduction will be highly reduced because of the lack of females. In case females do develop on reduced-size feeding structures, reproduction can be affected as in a reduced number of eggs and or reduced juvenile vitality.
  • the tagging collection or library of plants made according to the invention allows for a direct screening for an architectural change including that related to pathogen resistance and in particular promoters inducible by nematodes.
  • the sequence upstream of the promoter tag is isolated by means of inverse PCR.
  • suitable sized fragments are identified by restriction enzyme digests of the DNA from a particular interesting line through Southern hybridization with a labelled nucleotide sequence that is homologous to the integrated T-DNA sequence.
  • the actual iPCR comprises the circuiarisation of a suitable sized fragment followed by a standard PCR reaction on the self-ligated DNA that serves as template.
  • the iPCR fragment is then cloned into a suitable vector for transformation into any plant to confirm its promoter capacity and to monitor its activity.
  • the iPCR fragment can be blasted against a gene database (eg GenBank) in order to place this sequence in a genomic context; this data acquisition is valuable in order to identify the full promoter in case the iPCR fragment does not cover the full promoter.
  • a gene database eg GenBank
  • the promoter sequence identified in tomato according to this Example may be used to identify homologous promoter sequences in other plant species, including potato.
  • Those plant lines identified according to the invention with the modified architecture of reduced number and/or size of feeding structures or galls may used as nematode tolerant or resistant plants.

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Abstract

La présente invention concerne l'utilisation de gènes de cycle cellulaire de végétaux en tant qu'instruments génétiques pour isoler des portions de chromosome à activité transcriptionnelle telles que des protomères et des amplificateurs. L'insertion d'un gène de régulation de cycle cellulaire dominant négatif ou dominant positif ou d'un autre type d'ADN, qui peut être soit dépourvu de promoteur, soit couplé à un promoteur faible ou minimal, dans une orientation convenable dans une portion de chromosome de ce type, est susceptible d'entraîner une modification de l'architecture végétale résultant d'une modulation du cycle cellulaire ou de la division cellulaire dans des tissus ou cellules cibles.
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WO2006013010A2 (fr) 2004-07-31 2006-02-09 Metanomics Gmbh Preparation d'organismes a croissance plus rapide et/ou a meilleur rendement
WO2006058897A2 (fr) * 2004-12-01 2006-06-08 Cropdesign N.V. Plantes presentant des caracteristiques de croissance ameliorees et procede de fabrication de telles plantes
US7589256B2 (en) 2003-02-17 2009-09-15 Metanomics Gmbh Preparation of organisms with faster growth and/or higher yield
CN1950511B (zh) * 2004-03-10 2010-05-05 克罗普迪塞恩股份有限公司 产量增加的植物及制备其的方法
EP2272345A1 (fr) 2009-07-07 2011-01-12 Bayer CropScience AG Processus d'amélioration de la croissance de semis et/ou levée précoce de cultures
US7982095B2 (en) 2004-11-27 2011-07-19 Metanomics Gmbh Increase in yield by reducing gene expression

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Publication number Priority date Publication date Assignee Title
WO2002085104A3 (fr) * 2001-04-20 2003-04-17 Pioneer Hi Bred Int Techniques de transformation de vegetaux
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US7589256B2 (en) 2003-02-17 2009-09-15 Metanomics Gmbh Preparation of organisms with faster growth and/or higher yield
EP2322633A2 (fr) 2003-02-17 2011-05-18 Metanomics GmbH Préparation d'organismes dotés d'une croissance plus rapide et/ou d'un meilleur rendement
CN1950511B (zh) * 2004-03-10 2010-05-05 克罗普迪塞恩股份有限公司 产量增加的植物及制备其的方法
WO2006013010A2 (fr) 2004-07-31 2006-02-09 Metanomics Gmbh Preparation d'organismes a croissance plus rapide et/ou a meilleur rendement
US7982095B2 (en) 2004-11-27 2011-07-19 Metanomics Gmbh Increase in yield by reducing gene expression
WO2006058897A2 (fr) * 2004-12-01 2006-06-08 Cropdesign N.V. Plantes presentant des caracteristiques de croissance ameliorees et procede de fabrication de telles plantes
WO2006058897A3 (fr) * 2004-12-01 2006-09-21 Cropdesign Nv Plantes presentant des caracteristiques de croissance ameliorees et procede de fabrication de telles plantes
US7847157B2 (en) 2004-12-01 2010-12-07 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
EP2272345A1 (fr) 2009-07-07 2011-01-12 Bayer CropScience AG Processus d'amélioration de la croissance de semis et/ou levée précoce de cultures
WO2011003533A2 (fr) 2009-07-07 2011-01-13 Bayer Cropscience Ag Procédé permettant d'améliorer la croissance de semis et/ou l'émergence précoce de cultures

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