WO2000052171A1 - Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de cdc25 - Google Patents

Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de cdc25 Download PDF

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WO2000052171A1
WO2000052171A1 PCT/AU2000/000134 AU0000134W WO0052171A1 WO 2000052171 A1 WO2000052171 A1 WO 2000052171A1 AU 0000134 W AU0000134 W AU 0000134W WO 0052171 A1 WO0052171 A1 WO 0052171A1
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promoter sequence
plant
gene
cdc25
cell
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PCT/AU2000/000134
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Kerong Zhang
Francis John Sek
Peter Crook Lloyd John
Wim Van Camp
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Cropdesign N.V.
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Priority to AU27859/00A priority Critical patent/AU2785900A/en
Publication of WO2000052171A1 publication Critical patent/WO2000052171A1/fr

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    • 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
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    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/09—Recombinant DNA-technology
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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

  • the present invention relates generally to a method of modifying plant morphological, biochemical and physiological properties or characteristics, such as one or more environmental adaptive responses and/or developmental processes, said method comprising expressing a cell cycle control protein, in particular Cdc25 phosphoprotein phosphatase, in the plant, operably under the control of a regulatable promoter sequence.
  • a cell cycle control protein in particular Cdc25 phosphoprotein phosphatase
  • the characteristics modified by the present invention are cytokinin-mediated and/or gibberellin-mediated characteristics.
  • the present invention extends to gene constructs which are useful for performing the inventive method and to transgenic plants produced therewith having altered morphological and/or biochemical and/or physiological properties compared to their otherwise isogenic counterparts.
  • cytokinins produces a variety of morphological, biochemical and physiological effects in plants, including the stimulation of organogenesis, shoot initiation from callus cultures, release of lateral buds from apical dominance, dwarf growth, alteration of source/sink relationships, stimulation of pigment synthesis, inhibition of root growth, and delay of senescence.
  • cytokinins have also been implicated in promoting the initiation of tuber formation in potato (International Patent Publication No. WO 93/07272) and in improving the resistance of potato plants to insects (United States Patent No. 5, 496, 732) and in inducing male sterility and partial female sterility in tobacco plants (European Patent No. EP-A- 334,383).
  • cytokinin on plant development and morphology may be attributed, at least in part, to modified biochemistry of the plant, such as a modification to the source/sink relationship in the plant or plant part.
  • Attempts to modify plant cytokinin-mediated and/or gibberellin-mediated growth and developmental responses employ the exogenous application of cytokinins and/or gibberellins respectively.
  • Such approaches are costly and produce undesirable pleiotropic side-effects on the plant tissue.
  • Modified plant growth or architecture has been shown to be effected by modifying expression of the G1 cyclin D3 (International Patent Publication No. W098/42851).
  • G1 cyclins are required for the induction DNA replication (S phase), and the induction of G1 cyclin expression is dependent on mitogenic signals such as, for example, cytokinin and sucrose (Riou-Khamlichi et al. 1999, Soni et al. 1995).
  • mitogenic signals such as, for example, cytokinin and sucrose (Riou-Khamlichi et al. 1999, Soni et al. 1995).
  • cytokinin and sucrose Rost al. 1999, Soni et al. 1995.
  • prior art methods for modifying plant growth and architecture using these gene include pleiotropic effects.
  • the present invention determined the significance of Cdc25 in the induction of DNA replication (S phase) under growth- limiting conditions. Based upon this determination, the present inventors sought to develop a method of producing specific targeted modifications to plant morphology, biochemistry and physiology, in particular specific target modifications to cytokinin- mediated and gibberellin-mediated plant growth and development or aspects thereof, thereby avoiding the problem of pleiotropy associated with the prior art.
  • the inventors discovered that the targeted ectopic expression of a cell cycle control protein in particular cells, tissues or organs of the plant would produce localised specific modifications to plant morphology, biochemistry and physiology, compared to otherwise isogenic non-transformed plants.
  • cytokinin-mediated or gibberellin-mediated induction of mitosis (M phase) in plants, or plant cells can be obtained by the expression of the yeast Cdc25 phosphoprotein phosphatase therein.
  • the inventors have also discovered that expression of the yeast Cdc25 phosphoprotein phosphatase in plants or plant cells can override the arrest of DNA replication (S phase) under growth-limiting conditions. Unexpectedly, the same process partially sustains endoreplication or eudoreduplication.
  • Example 2 The cytokinin-mediated induction of mitosis by Cdc25 is shown in Example 2.
  • Example 9 The induction of DNA replication and endoreduplication under growth-limiting conditions is shown in Example 9. Whilst not being bound by any theory or mode of action, it is likely that the ectopic expression of yeast Cdc25 phosphatase in plants releases the inhibition of Cdc2 activity, which is a key enzyme in the control of the cell cycle, and as a consequence, causes isolated cells to enter mitosis and/or S-phase DNA replication.
  • the Cdc25 phosphatase is an intracellular protein, which, unlike exogenously-applied cytokinins or cytokinins produced by ectopic expression of ipt or rolC genes, will only exert a localised effect at the site of protein synthesis.
  • This observation has led the present inventors to develop methods for controlled expression of yeast Cdc25 in particular cells, tissues and organs of plants, for the purposes of mimicking and/or modifying and/or overriding aspects of cytokinin-mediated plant morphology and/or biochemistry and/or physiology, and to facilitate the selection of specific cells, tissues and organs which exhibit cytokinin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics.
  • one aspect of the invention provides a method of modifying plant morphology and/or biochemistry and/or physiology comprising expressing in particular cells, tissues or organs of a plant, a genetic sequence encoding a cell cycle control protein operably under the control of a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, and cell cycle specific gene promoters.
  • the cell cycle control protein is the yeast Cdc25 phosphoprotein phosphatase or a biologically-active homologue, analogue or derivative thereof.
  • the present invention clearly contemplates the use of functional homologues of the fission yeast Cdc25 protein, based upon the evidence provided herein for the presence of Cdc25-like activity and Cdc25-like protein in plants, in particular tobacco (Example 3).
  • the present invention is not limited in application to the use of nucleotide sequences encoding the fission yeast Cdc25 protein, and clearly encompasses the use of Cdc25 polypeptides and genes of plants, preferably monocotyledonous and/or dicotyledonous plants, and more preferably the Cdc25 of a plant as listed or referred to herein.
  • the ectopic expression of a Cdc25 protein or a homologue, analogue or derivative thereof can produce a range of desirable phenotypes in plants, such as, for example, by modifying one or more morphological, biochemical, or physiological characteristics as follows: (i) modification of the initiation, promotion, stimulation or enhancement of cell division; (ii) modification of the initiation, promotion, stimulation or enhancement of DNA replication;(iii) modification of the initiation, promotion, stimulation or enhancement of seed set and/or size and/or development; (iv) modification of the initiation, promotion, stimulation or enhancement of tuber formation; (v) modification of the initiation, promotion, stimulation or enhancement of shoot initiation and/or development; (vi) modification of the initiation, promotion, stimulation or enhancement of root initiation and/or development; (vii) modification of the initiation, promotion, stimulation or enhancement of lateral root initiation and/or development; (viii) modification of the initiation, promotion, stimulation or enhancement of nodule formation and/or nodule function; (ix) modification
  • the term "modification of the initiation, promotion, stimulation or enhancement" in relation to a specified integer shall be taken as a clear indication that the integer is capable of being enhanced, increased, stimulated, or promoted, or alternatively, decreased, delayed, repressed, or inhibited.
  • Preferred embodiments of the invention relate to the effect(s) of cytokinins and/or gibberellins on plant morphology and architecture.
  • the present invention clearly contemplates the broad application of the inventive method to the modification of a range of cellular processes, including but not limited to the modification of the initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment synthesis, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence.
  • the present invention clearly contemplates the broad application of the inventive method to the modification of a range of cellular processes, including but not limited to the modification of the initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment synthesis, and/or the modification of source/sink relationships, and/or the modification of root growth and/or the inhibition of apical dominance and/or the delay of senescence.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed operably under the control of a promoter derived from a stem-expressible gene, to increase the strength and thickness of a plant stem to confer improved stability and wind-resistance on the plant.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed in a tuber-forming plant operably under the control of a promoter derived from a stem-expressible gene or tuber-expressible gene, to increase tuber production in the plant.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed in a tree crop plant such as, but not limited to, Eucalyptus spp. or Populus spp., operably under the control of a promoter derived from a gene that is expressed in vascular tissue and/or cambium cells, to increase lignin content therein.
  • a promoter derived from a gene that is expressed in vascular tissue and/or cambium cells
  • the ectopic expression of Cdc25 under control of a promoter that is operable in vascular tissue and preferably, in cambial cells will produce thick-stemmed plants and a higher ratio of vascular tissue-to-pith cells within the stem, thereby resulting in more lignin production.
  • cambial cells contain the highest levels of auxins and are therefore the preferential tissue for Cdc25 overproduction.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed operably under the control of a promoter derived from a seed-expressible gene, to increase seed production in plants, in particular to increase seed set, seed size and seed yield, such as, for example, by enhancing endoreduplication in the seed.
  • the promoter is operable in the endosperm of the seed, in which case the combination of the cell cycle-control protein and endosperm-expressible promoter provides the additional advantage of increasing the grain size and grain yield of the plant.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed operably under the control of a promoter derived from a meristem-expressible gene or a shoot-expressible gene or a root-expressible gene, to reduce apical dominance and/or to promote bushiness of the plant and/or to increase or enhance the production of lateral roots, and/or alter leaf shape.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed operably under the control of a promoter derived from a leaf-expressible gene, to prevent or delay or otherwise reduce leaf chlorosis and/or leaf necrosis.
  • the yeast Cdc25 protein or a homologue, analogue or derivative thereof is expressed operably under the control of a promoter derived from a leaf-expressible gene, to alter leaf shape.
  • the yeast Cdc25 protein or a homologue analogue or derivative thereof is expressed under the control of a promoter that is operative in meristem tissue of grain crops, to stimulate cell division in the intercalary meristem of the youngest stem internode and produce greater elongation of the stem and/or to generate a more extensive photosynthetic canopy.
  • a second aspect of the invention provides a gene construct or vector comprising a nucleotide sequence that encodes a Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a regulatable promoter sequence selected from the group consisting of: (i) a plant-expressible cell-specific promoter sequence;
  • a plant-expressible tissue-specific promoter sequence (ii) a plant-expressible tissue-specific promoter sequence; (iii) a plant-expressible organ-specific promoter sequence; (iv) a plant-expressible inducible promoter sequence; (v) a plant-expressible constitutive promoter sequence, wherein the nucleotide sequence encoding the Cdc25 protein and the plant-expressible constitutive promoter sequence are integrated into a transposable element; and (vi) a plant-expressible cell cycle specific gene promoter sequence.
  • the gene construct or vector according to this aspect of the invention is suitable for expression in a plant cell, tissue, organ or whole plant and more preferably, the subject gene construct or vector is suitable for introduction into and maintenance in a plant cell, tissue, organ or whole plant.
  • a third aspect of the invention provides a plant cell, tissue, organ or whole plant that has been transformed or transfected with an isolated nucleic acid molecule that comprises a nucleotide sequence which encodes a cell cycle control protein, wherein the expression of said nucleotide sequence is placed operably under the control of a plant-expressible cell-specific promoter sequence, plant-expressible tissue-specific promoter sequence, a plant-expressible organ-specific promoter sequence, plant- expressible cell cycle specific gene promoter or a plant-expressible constitutive promoter sequence such that said plant-expressible constitutive promoter sequence and said nucleotide sequence encoding a cell cycle control protein are integrated into a transposable genetic element.
  • Figure 1-1 is a copy of a photographic representation of a northern blot hybridisation showing the induction of Cdc25 mRNA in tobacco cells containing a dexamethasone- inducible Cdc25 gene, in the absence of exogenous cytokinin. Prior to induction, cells were brought to arrest at the cytokinin control point in late G2 phase by culture without hormone and then with auxin only.
  • RNA was extracted from tobacco cells either in the absence of added dexamethasone (lane 0), or after 12 h induction with 0.01 ⁇ M, or 0.10 ⁇ M, or 1.00 ⁇ M, or 10.00 ⁇ M dexamethasone and then loaded onto agarose gels (60 ⁇ g aliquots RNA per lane), transferred to membrane support and probed with a Cdc25-specific probe.
  • Figure 1-2 is a copy of a photographic representation of a western blot showing the induction of p67 Cdc25 protein in tobacco cells containing a dexamethasone-inducible
  • Cdc25 gene in the absence of exogenous cytokinin. Prior to induction, cells were brought to arrest at the cytokinin control point in late G2 phase by culture without hormone and then with auxin only. Total protein was extracted from tobacco cells either in the absence of added dexamethasone (lane 1), or after 12 h induction with
  • Figure 1-3 is a copy of a graphical representation showing the induction of cell division in culture, as measured by an increase in cell number, for tobacco cells transformed with a dexamethasone-inducible Cdc25 gene, in the absence of exogenous cytokinin.
  • Prior to induction cells were brought to arrest at the cytokinin control point in late G2 phase by culture without hormone and then with auxin only.
  • Cell numbers were determined either in the absence of added dexamethasone, or after 12 h induction with 0.01 -10.00 ⁇ M dexamethasone. Data were also obtained for both transformed cells (O) and for control non-transformed cells ( ⁇ ) grown under identical culture conditions.
  • FIG 2-1 is a copy of a photographic representation showing the activity of Cdc25 phosphatase (Cdc25) and Cdc2 histone kinase (Cdc2) in transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene and progressing from the late G2 phase hormonal control point into division, that have either not been induced with 0.1 ⁇ M dexamethasone (-D), or alternatively, that have been induced with 0.1 ⁇ M dexamethasone (+D).
  • the activity of Cdc25 was measured by activation of the tyrosine- phosphorylated Cdc2 enzyme substrate as determined by assaying for phosphorylation of H1 histone by H1 histone kinase.
  • the Cdc25 enzyme from cells induced for 6 hours with dexamethasone was purified using antibodies against authentic fission yeast Cdc25 protein, or alternatively, using preimmune serum (lane marked p-i) or an antibody that had been pre-competed with repeat-freeze-thaw inactivated GST-Cdc25 fusion protein (lane marked p-c).
  • the Cdc2 kinase from cells induced for 12 h with dexamethasone was purified with antibody, or antibody that had been pre-competed with 0.1 mM antigen (lane marked p-c), and assayed by phosphorylation of H1 histone.
  • Figure 2-2 is a graphical representation showing the change in activities of Cdc25 phosphatase ( ⁇ ) and Cdc2 histone kinase (O) in transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene progressing from the late G2 phase hormonal control point into division and following induction with 0.1 ⁇ M dexamethasone.
  • the activities of Cdc25 phosphatase and Cdc2 histone kinase were measured as described for Figure 2-1.
  • Figure 2-3 is a graphical representation showing the change in cell number (cells/ml x 10 6 ) of transgenic and non-transgenic tobacco cells containing a dexamethasone- inducible Cdc25 gene, progressing from the late G2 phase hormonal control point into division and following induction with 0.1 ⁇ M dexamethasone or cytokinin.
  • Data show cell number for both transgenic cells induced using dexamethasone (D) or cytokinin ( ⁇ ), and for non-transgenic cells induced using dexamethasone (O).
  • Figure 2-4 is a graphical representation showing the change in activities of Cdc25 phosphatase ( ⁇ ) and Cdc2 histone kinase (O) in transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene, progressing from the late G2 phase hormonal control point into division and following induction with cytokinin in the absence of added dexamethasone.
  • the activities of Cdc25 phosphatase and Cdc2 histone kinase were measured as described for Figure 2-1.
  • Figure 2-5 is a graphical representation showing the change in activity of Cdc2 histone kinase in transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene, progressing from the late G2 phase hormonal control point into division and following their stimulation with cytokinin.
  • the Cdc2 histone kinase was purified using p13 suc beads and treated with GST-Cdc25 fusion protein that had been produced in Escherichia coli cells. Data indicate the Cdc2 activity before Cdc25 treatment (O), and after treatment (•) with cytokinin.
  • Figure 2-6 is a copy of a photographic representation showing the activation of Cdc2 histone kinase by Cdc25 phosphatase in transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene, prior to stimulation with cytokinin (lanes 1-3) or following 3 hours stimulation with cytokinin (lanes 4-6).
  • Detectable Cdc2 activity was observed in control samples that had been incubated without added Cdc25 (lanes 1 and 4), or following incubation with (i) immunoprecipitated Cdc25 that had been derived from non-transgenic tobacco cells induced with cytokinin for 6 hours (lanes 2 and 5); or (ii) Cdc25 derived from transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene that had been induced with dexamethasone for 6 hours (lanes 3 and 6). The activity of Cdc2 histone kinase was measured as described for Figure 2-1. Detection of Cdc25 activity in the immuno-recovered fraction derived from non-transgenic cells indicates the presence of a plant-encoded Cdc25.
  • Figure 2-7 is a copy of a photographic representation showing the presence of phosphorylated tyrosine in Cdc2a (arrow) following induction of transgenic tobacco cells containing a dexamethasone-inducible Cdc25 gene with dexamethasone.
  • the Cdc2a protein was immuno-precipitated with purified antibody, or with antibody precompeted with repeat-freeze-thaw inactivated GST-Cdc25 (lane marked p-c).
  • the upper band indicated in the Figure represents excess IgG.
  • Figure 3 is a copy of a photographic representation of a western blot showing purified plant-derived Cdc25 protein.
  • the arrow indicates the plant Cdc25 polypeptide.
  • Anti- GST-Cdc25 antibody at a dilution of 1 :500 in buffered saline was used to probe affinity- purified plant Cdc25 protein alone (lane 1) or affinity-purified plant Cdc25 protein following incubation for 1 hour with 0.1 mM GST-Cdc25 fusion protein.
  • Molecular weight markers indicating the molecular mass (kDa) of proteins are indicated at the left of the Figure.
  • Figure 4 is a copy of a photographic representation showing the cytokinin-dependent proliferation of tobacco cells in culture.
  • Cell proliferation was detected by the incorporation of BrdU into nuclear DNA of excised tobacco pith tissue primary culture on MS medium either without added hormone (panels a,b), or supplemented with 5.4 ⁇ M NAA (panels c,d) or with 0.56 ⁇ M BAP (panels e,f) or 5.4 ⁇ M NAA plus 0.56 ⁇ M BAP (panels g,h).
  • Cell cultures shown in panels a, c, e, and g have been stained with DAPI, to detect nuclei.
  • Cell cultures shown in panels b, d, f, and h have been incubated with BrdU, and BrdU-containing DNA has been detected by fluorescence of antibody specific for BrdU-containing DNA.
  • Figure 5 is a graphical representation showing enhanced cell endoreduplicafion in tobacco cells expressing Cdc25.
  • Data on the x-axis indicate nuclei having ploidies of 2n, 4n and 8n in cultures in which cell division is arresting.
  • Data on the y-axis indicates the frequency of nuclei for each ploidy.
  • Panel (A) shows nontransgenic cells after 6 days of culture.
  • Panel (B) shows transgenic cells after 6 days, in which the Cdc25 gene is expressed under the control of a glucocorticoid dexamethasone.
  • One aspect of the invention provides a method of modifying one or more plant morphological and/or biochemical and/or physiological characteristics comprising expressing in one or more particular cells, tissues or organs of a plant, a cell cycle control protein operably under the control of a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissue-specific promoter sequences, organ-specific promoter sequences, and cell cycle specific gene promoter sequences.
  • the plant morphological, biochemical or physiological characteristic which is modified is a cytokinin-mediated or a gibberellin-mediated characteristic.
  • modifying one or more plant morphological and/or biochemical and/or physiological characteristics is meant that one or more morphological and/or biochemical and/or physiological characteristics of a plant is altered by the performance of one or more steps pertaining to the invention described herein.
  • Plant morphology or the term “plant morphological characteristic” or similar term will be understood by those skilled in the art to refer to the external appearance 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, colour, texture, arrangement, and patternation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, stem, leaf, shoot, 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.
  • plants may modify one or more plant morphological characteristics in response to the external stimuli, such as, for example, a plant pathogenic infection, or an external stress (drought, flooding, salt stress, dehydration, heavy metal contamination, mineral deficiency, etc).
  • a plant morphological characteristic that has been modified in response to one or more external stimuli is within the scope of the inventive method described herein, notwithstanding that the imposition of said external stimuli is not an essential feature of the present invention.
  • Plant biochemistry or the term “plant biochemical characteristic” or similar term will be understood by those skilled in the art to refer to the metabolic and catalytic processes of a plant, including primary and secondary metabolism and the products thereof, including any small molecules, macromolecules or chemical compounds, such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibres, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.
  • small molecules, macromolecules or chemical compounds such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibres, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.
  • Plant physiology or the term “plant physiological characteristic” or similar term will be understood to refer to the functional processes of a plant, including developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fibre production, secondary growth or wood production, amongst others; responses of a plant to externally-applied factors such as metals, chemicals, hormones, growth factors, environment and environmental stress factors (eg. anoxia, hypoxia, high temperature, low temperature, dehydration, light, daylength, flooding, salt, heavy metals, amongst others), including adaptive responses of plants to said externally- applied factors.
  • developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fibre production, secondary growth or wood production, amongst others
  • the ectopic expression of a Cdc25 substrate or modified Cdc25 substrate protein produces a wide range of desirable phenotypes in the plant, selected from the group consisting of:(i) enhanced growth and/or enhanced vigour of the plant; (ii) increased total biomass of the plant; (iii) increased cell number; (iv) reduced flowering time; (v) increased inflorescence formation; (vi) reduced time to seed set (vii) enhanced seed set; (viii) enhanced seed size; (ix) enhanced grain yield; (x) enhanced stem strength; (xi) enhanced stem thickness; (xii) enhanced stem stability; (xiii) enhanced wind-resistance of the stem; (xiv) enhanced tuber formation; (xv) enhanced tuber development; (xvi) increased lignin content; (xvii) enhanced ploidy of the seed; (xviii) enhanced endosperm size; (xix) reduced apical dominance; (xx) increased bushiness; (xxi) enhanced
  • the plant morphological, biochemical or physiological characteristic which is modified is a cytokinin-mediated or a gibberellin-mediated characteristic selected from the group consisting of: (i) enhanced stem thickness; (ii) enhanced stem stability; (iii) enhanced wind-resistance of the stem; (iv) enhanced tuber formation; (v) enhanced tuber development; (vi) increased lignin content; (vii) enhanced seed set; (viii) enhanced seed production; (ix) enhanced grain yield; (x) enhanced ploidy of the seed; (xi) enhanced endosperm size; (xii) reduced apical dominance; (xiii) increased bushiness; (xiv) enhanced lateral root formation; (xv) enhanced rate of lateral root production; (xvi) enhanced nitrogen-fixing capability; (xvii) enhanced nodulation or nodule size; (xviii) reduced or delayed leaf chlorosis; (xix) reduced or delayed leaf necrosis; (xx) partial or complete inhibition of the arrest
  • the plant morphological, biochemical or physiological characteristic which is modified is selected from the group consisting of: (i) plant growth and vigour; (ii) total biomass; (iii) cell number; (iv) flowering time; (v) branching; (vi) inflorescence formation; and (vii) seed set and/or seed yield, and/or seed size.
  • expression of the cell cycle control protein preferably leads to increased plant productivity such as, for example, increased growth and vigour; increased total biomass; increased cell number; reduced flowering time; increased branching; increased inflorescence formation; and increased seed set; amongst others.
  • express or variations such as “expressing” and “expression” as used herein shall be taken in their broadest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • expression comprising the production of a sense mRNA transcript
  • the word “express” or variations such as “expressing” and “expression” may also be construed to indicate the combination of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localization, turnover or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • cell cycle shall be taken to include 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: GO (gap 0), G1 (gap 1), DNA replication (S), G2 (gap 2), and mitosis including cytokinesis (M). Normally these four phases occur sequentially.
  • the cell cycle also includes modified cycles such as endomitosis, acytokinesis, polyploidy, polyteny, endopolyploidisation and endoreduplication or endoreplication.
  • cell cycle interacting protein means a protein 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. It may also be capable of binding to, regulating or being regulated by cyclin dependent kinases or their subunits.
  • the term also includes peptides, polypeptides, fragments, variant, homologs, alleles or precursors (eg preproproteins or preproteins) thereof.
  • cell cycle control gene refers to any gene or mutant thereof which exerts positive or negative control on, or is required for, chromosomal DNA synthesis, mitosis (preprophase band, nuclear envelope, spindle formation, chromosome condensation, chromosome segregation, formation of new nuclei, formation of phragmoplast, etc) meiosis, cytokinesis, cell growth, or endoreduplicafion.
  • cell cycle control gene also includes any and all genes that exert control on a cell cycle protein as hereinbefore defined, including any homologues of CDKs, cyclins, E2Fs, Rb, CKI, Cks, cyclin D, Cdc25, Wee1 , Nim1 , MAP kinases, etc.
  • a cell cycle control gene will exert such regulatory control at the post-translation level, via interactions involving the polypeptide product expressed therefrom.
  • cell cycle control genes are all genes involved in the control of entry and progression through S phase. They include, not exclusively, genes expressing "cell cycle control proteins” such as cyclin dependent kinases (CDK), cyclin dependent kinase inhibitors (CKI), D, E and A cyclins, E2F and DP transcription factors, pocket proteins, CDC7/DBF4 kinase, CDC6, MCM2-7, Ore proteins, Cdc45, components of SCF ubiquitin ligase, PCNA, and DNA-polymerase, amongst others.
  • CDK cyclin dependent kinases
  • CKI cyclin dependent kinase inhibitors
  • D cyclin dependent kinase inhibitors
  • E2F and DP transcription factors DP transcription factors
  • pocket proteins CDC7/DBF4 kinase
  • CDC6, MCM2-7 or MCM2-7
  • Ore proteins Cdc45
  • components of SCF ubiquitin ligase PC
  • cell cycle control protein includes cyclins A, B, C, D and E, including CYCA1;1 , CYCA2;1 , CYCA3;1 , CYCB1 ;1, CYCB1 ;2, CYCB2;2, CYCD1 ;1 , CYCD2;1.
  • CYCD3;1 , and CYCD4;1 Evans et al. 1983; Francis et al. 1998; Labbe et al.
  • CKI cyclin dependent kinase inhibitor proteins
  • ICK1 Wang etal 1997), FL39, FL66, FL67 (PCT/EP98/05895), Sid, Far1 , Rum1 , p21 , p27, p57, p16, p15, p18, p19 (Elledge 1996; Pines 1995a, b), p14 and p14ARF; p13sud or CKSIAt (De Veylder et al 1997; Hayles and Nurse 1986) and nim-1 (Russell and Nurse 1987a; Russell and Nurse 1987b; Fantes 1989; Russell and Nurse 1986; Russell and Nurse 1987a; Russell and Nurse 1987b) homologues of Cdc2 such as Cdc2MsB (Hirt et al 1993) CdcMs
  • cell cycle control proteins are involved in cyclin D-mediated entry of cells into G1 from GO include pRb (Xie et al., 1996; Huntley et al., 1998), E2F, RIP, MCM7, and the pRb-like proteins p107 and p130.
  • cell cycle control proteins are involved in the formation of a pre-replicative complex at one or more origins of replication, such as, but not limited to, ORC, CDC6, CDC14, RPA and MCM proteins or in the regulation of formation of this pre-replicative complex, such as, but not limited to, the CDC7, DBF4 and MBF proteins.
  • cell cycle control protein shall further be taken to include any one or more of those proteins that are involved in the turnover of any other cell cycle control protein, or in regulating the half-life of said other cell cycle control protein.
  • protein turnover is to include all biochemical modifications of a protein leading to the physical or functional removal of said protein. Although not limited to these, examples of such modifications are phosphorylation, ubiquitination and proteolysis.
  • Particularly preferred proteins which are involved in the proteolysis of one or more of any other of the above-mentioned cell cycle control proteins include the yeast-derived and animal-derived proteins, Skp1 , Skp2, Rub1 , Cdc20, cullins, CDC23, CDC27, CDC 16, and plant-derived homologues thereof (Cohen-Fix and Koshland 1997; Hochstrasser 1998; Krek 1998; Lisztwan et al 1998) and Plesse et al in (Francis et al 1998)).
  • cell cycle control genes shall further be taken to include any one or more of those gene that are involved in the transcriptional regulation of cell cycle control gene expression such as transcription factors and upstream signal proteins. Additional cell cycle control genes are not excluded.
  • cell cycle control genes shall further be taken to include any cell cycle control gene or mutant thereof, which is affected by environmental signals such as for instance stress, nutrients, pathogens, or by intrinsic signals such as the animal mitogens or the plant hormones (auxins, cytokinins, ethylene, gibberellic acid, abscisic acid and brassinosteroids).
  • the cell cycle control protein used in performing the invention is a Cdc25 protein or a homologue, analogue, or derivative thereof.
  • the switch that raises activity of Cdc2 at entry into mitosis is the Cdc25-catalysed removal of phosphate from theonine-14 and/or tyrosine-15 in Cdc2.
  • the activity of the Cdc2 protein involved in this process is inactivated by the wee-1 -mediated and/or mik-1- mediated phosphorylation of threonine-14 and/or tyrosine-15 in Cdc2.
  • CDK2 In yeasts there is only one CDK (Cdc2), and the Cdc25-catalysed removal of phosphate from tyrosine-15 in Cdc2 occurs only once in the cell cycle, at the G2/M phase transition. In contrast, higher eukaryotic cells contain several CDKs (animal and plant cells) and several Cdc25 proteins (animal cells). In mammals, the molecular switch of Cdc25-catalysed removal of phosphate from threonine-14 and/or tyrosine-15 in Cdc2 is also used at entry into the S phase, and a separate CDK (CDK2) and a separate Cdc25 (Cdc25A) perform this function.
  • CDK2 CDK2
  • Cdc25A separate Cdc25
  • CDK2 In plants, whilst it is known there are several CDKs, it is not known if there is a single CDK that is controlled at S phase, like CDK2 by the status of threonine-14 and/or tyrosine-15 phosphorylation.
  • the present invention encompasses altering the balance between Cdc25 dephosphorylation and wee-1/mik-1 phosphorylation of their substrates either by overexpression of Cdc25 and/or down-regulation of wee-1/mik-1.
  • the present invention provides evidence that the G2/M phase transition, and, surprisingly, the G1/S phase transition of plant cells can be modified by the expression of a single Cdc25 in plant cells.
  • “Homologues” of a Cdc25 protein are those peptides. oligopeptides, polypeptides, proteins and enzymes which contain amino acid substitutions, deletions and/or additions relative to the Cdc25 polypeptide without altering one or more of its cell cycle control properties, in particular without reducing the ability of the Cdc25 polypeptide to induce one or more aspects of cytokinin-mediated and/or gibberellin-mediated effects in a plant cell, tissue, organ or whole organism.
  • amino acids present in Cdc25 can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break ⁇ -helical structures or ⁇ -sheet structures, and so on.
  • Substitutional variants are those in which at least one residue in the Cdc25 amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the Cdc25 protein. Insertions can comprise amino- terminal and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.
  • Deletional variants are characterised by the removal of one or more amino acids from the Cdc25 sequence.
  • Amino acid variants of the Cdc25 polypeptide may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations.
  • the manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis or other site-directed mutagenesis protocol.
  • Analogues of a Cdc25 protein are defined as those peptides, oligopeptides, polypeptides, proteins and enzymes which are functionally equivalent to the Cdc25 polypeptide in inducing one or more cytokinin-mediated and/or gibberellin-mediated effects in plant cells, tissues, organs or whole organisms.
  • Preferred analogues of the Cdc25 protein are those peptides, polypeptide, proteins, and enzymes, that function as a substrate of Cdc25 or a modified substrate of Cdc25 in a plant cell and/or tissue and/or organ and/or whole plant.
  • the Cdc25 analogue is other than a Cdc2a protein or a cyclin B protein, or a homologue or derivative thereof, notwithstanding that such proteins are useful in performing the present invention.
  • the term "substrate of Cdc25” shall be taken to refer to any protein that interacts with Cdc25 in regulating the plant cell cycle, including, but not limited to cyclin-dependent kinases (CDKs), the most significant of which is Cdc2, which in all cells is the key enzyme driving entry into mitosis.
  • CDKs cyclin-dependent kinases
  • the substitution or deletion of the phosphorylation sites of a Cdc2 protein mimics the effect of a constitutive phosphatase activity, such as the effect of the Cdc25 protein phosphatase (p80 Cdc25 ) activity.
  • phosphorylation sites of a Cdc2a protein further mimics the effect of down-regulated kinase activity, such as a down- regulation of the wee-1 kinase and/or mik-1.
  • down-regulated kinase activity such as a down- regulation of the wee-1 kinase and/or mik-1.
  • wee-1 and mik-1 kinase adds the inhibitory phosphate on threonine-14 and/or tyrosine 15.
  • phosphorylated protein will not be produced at high steady state concentrations in either the absence of phosphorylation or when phosphatase(s) is(are) expressed at raised levels, or when kinase(s) is(are) expressed at lowered levels.
  • the modified Cdc2a activity effects described herein can also be obtained, albeit only in part and without the benefits derived from increasing the amount of active Cdc2a protein, by the regulated expression of Cdc25.
  • the modified Cdc2a activity effects described herein can also be obtained by down- regulating, or inhibiting, wee-1 and/or mik-1 kinase activity, such as, for example, by using antisense molecules, ribozymes, cosuppression molecules, gene targeting molecules, or gene silencing molecules, etc., which target the wee-1 and/or mik-1 genes or gene products, respectively.
  • modified substrate of Cdc25 refers to a homologue, analogue or derivative of a substrate of Cdc25 that mimics the effect of Cdc25 activity, in particular a non- phosphorylatable Cdc25 substrate that mimics the effect of Cdc25 activity or alternatively mimics the effects of down-regulated wee-1 and/or mik-1 kinase.
  • CDKs cyclin- dependent kinases
  • the present invention extends further to the co-expression of Cdc25 and one or more Cdc25 substrates and/or one or more modified Cdc25 substrates, operably under the control of a regulatable promoter that is selected for a particular application as described herein.
  • “Derivatives” of a Cdc25 protein are those peptides, oligopeptides, polypeptides, proteins and enzymes which comprise at least about five contiguous amino acid residues of a naturally-occurring Cdc25 polypeptide, in particular the fission yeast p80 Cd 25 polypeptide, but which retain activity in the induction of one or more cytokinin- mediated and/or gibberellin-mediated effects in a plant cell, tissue, organ or whole organism.
  • a “derivative” may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring Cdc25 polypeptide.
  • a derivative may comprise one or more non-amino acid substituents compared to the amino acid sequence of a naturally-occurring Cdc25 polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection.
  • recombinant or synthetic mutants and derivatives of the Cdc25 polypeptide include those incorporating single or multiple substitutions, deletions and/or additions therein, such as carbohydrates, lipids and/or proteins or polypeptides.
  • Naturally-occurring or altered glycosylated or acylated forms of the Cdc25 polypeptide are also contemplated by the present invention.
  • homopolymers or heteropolymers comprising one or more copies of the Cdc25 polypeptide are within the scope of the invention, the only requirement being that such molecules possess biological activity in inducing one or more cytokinin-mediated and/or gibberellin- mediated effects in plant cells, tissues, organs or whole organisms.
  • Preferred homologues, analogues and derivatives of the fission yeast Cdc25 polypeptide contemplated by the present invention are derived from plants. As exemplified herein, the present inventors have identified a Cdc25 activity in tobacco cells which is contemplated as being of particular use in performing the various embodiments described herein.
  • the cell cycle control protein is the yeast Cdc25 phosphoprotein phosphatase or a biologically-active homologue, analogue or derivative thereof and in particular, a plant-derived homologue of the yeast Cdc25 phosphoprotein phosphatase.
  • the present invention clearly contemplates the use of functional homologues of the fission yeast Cdc25 protein, based upon the evidence provided herein for the presence of Cdc25-like activity and Cdc25-like protein in tobacco (Example 3). Accordingly, the present invention is not limited in application to the use of nucleotide sequences encoding the fission yeast p80 Cdc25 protein.
  • either the protein may be introduced directly to said cell, such as by microinjection means or alternatively, an isolated nucleic acid molecule encoding said protein may be introduced into the cell, tissue or organ in an expressible format.
  • “expressible format” is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an intracellular or extracellular signal, such as an environmental stimulus or stress (anoxia, hypoxia, temperature, salt, light, dehydration, etc) or a chemical compound such as an antibiotic (tetracycline, ampicillin, rifampicin, kanamycin) hormone (eg.
  • gibberellin gibberellin, auxin, cytokinin, glucocorticoid, etc), hormone analogue (iodoacetic acid (IAA), 2,4-D, etc) , metal (zinc, copper, iron, etc), or dexamethasone, amongst others.
  • expression of a functional protein may also require one or more post- translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others. All such processes are included within the scope of the term "expressible format".
  • expression of a cell cycle control protein in a specific plant cell, tissue, or organ is effected by introducing and expressing an isolated nucleic acid molecule encoding said protein, such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame, to said cell, tissue or organ, wherein said nucleic acid molecule is placed operably in connection with a suitable plant-expressible promoter sequence.
  • an isolated nucleic acid molecule encoding said protein such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame
  • promoter includes the transcriptional regulatory sequences derived from a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • promoter also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected.
  • copper-responsive, glucocorticoid-responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, glucocorticoid- inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule.
  • the promoter is a plant-expressible promoter sequence.
  • plant-expressible is meant that the promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, preferably a monocotyledonous or dicotyledonous plant cell and in particular a dicotyledonous plant cell, tissue, or organ. Accordingly, it is within the scope of the invention to include any promoter sequences that also function in non-plant cells, such as yeast cells, animal cells and the like.
  • a "regulatable promoter sequence” is a promoter that is capable of conferring expression on a structural gene in a particular cell, tissue, or organ or group of cells, tissues or organs of a plant, optionally under specific conditions, however does generally not confer expression throughout the plant under all conditions.
  • a regulatable promoter sequence may be a promoter sequence that confers expression on a gene to which it is operably connected in a particular location within the plant or alternatively, throughout the plant under a specific set of conditions, such as following induction of gene expression by a chemical compound or other elicitor.
  • the regulatable promoter used in the performance of the present invention confers expression in a specific location within the plant, either constitutively or following induction, however not in the whole plant under any circumstances.
  • promoters include cell-specific promoter sequences, tissue- specific promoter sequences, inducible promoter sequences, organ-specific promoter sequences, cell cycle specific gene promoter sequences, and constitutive promoter sequences that have been modified to confer expression in a particular part of the plant at any one time, such as by integration of said constitutive promoter within an excisable genetic element.
  • cell-specific shall be taken to indicate that expression is predominantly in a particular plant cell or plant cell-type, albeit not necessarily exclusively in that plant cell or plant cell-type.
  • tissue-specific shall be taken to indicate that expression is predominantly in a particular plant tissue or plant tissue-type, albeit not necessarily exclusively in that plant tissue or plant tissue-type.
  • organ-specific shall be taken to indicate that expression is predominantly in a particular plant organ albeit not necessarily exclusively in that plant organ.
  • an "inducible promoter” is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical, environmental, or physical stimulus.
  • cell cycle specific shall be taken to indicate that expression is predominantly cyclic and occurring in one or more, not necessarily consecutive phases of the cell cycle albeit not necessarily exclusively in cycling cells.
  • the present invention does not require the exclusive expression of the cell cycle control protein in a cell, tissue or organ of a plant, in order to induce one or more non-pleiotropic cytokinin-mediated and/or gibberellin-mediated effects therein, subject to the proviso that expression is at least predominantly localised in a particular cell, tissue or organ of the plant.
  • the promoter selected for regulating expression of the cell cycle control protein in the plant cell, tissue or organ will confer expression in a range of cell-types or tissue-types or organs, sufficient to produce the desired phenotype, whilst avoiding undesirable phenotypes produced in other cell-types or tissue-types or organs. More preferably, the promoter selected for regulating expression of the cell cycle control protein in the plant cell, tissue or organ, will confer expression in a limited number of cells or cell-types or tissues or tissue-types or organs of the plant.
  • the promoter selected for regulating expression of the cell cycle control protein in the plant cell, tissue or organ will confer expression in a single cell- type or tissue-type or organ of the plant.
  • constitutive promoters confer expression in a wide range of different tissues of plants, albeit not necessaryily all tissues.
  • tissue-specific, cell-specific, cell-cycle specific or organ-specific, regulatory elements from such regulated promoters may be added to the constitutive promoter sequence.
  • the otherwise constitutive expression activity of the promoter sequence may be regulated by integrating the promoter sequence and cell cycle control gene in one or more excisable genetic elements.
  • an excisable genetic element shall be taken to refer to any nucleic acid which comprises a nucleotide sequence which is capable of integrating into the nuclear, mitochondrial, or plastid genome of a plant, and subsequently being autonomously mobilised, or induced to mobilise, such that it is excised from the original integration site in said genome.
  • autonomously mobilised is meant that the genetic element is excised from the host genome randomly, or without the application of an external stimulus to excise.
  • the genetic element is preferably induced to mobilise, such as, for example, by the expression of a recombinase protein in the cell which contacts the integration site of the genetic element and facilitates a recombination event therein, excising the genetic element completely, or alternatively, leaving a "footprint", generally of about 20 nucleotides in length or greater , at the original integration site.
  • the excisable genetic element comprises a transposable genetic element, such as, for example, Ac, Ds, Spm, or En, or alternatively, on or more loci for interaction with a site-specific recombinase protein, such as, for example, one or more lox or fit nucleotide sequences.
  • a transposable genetic element such as, for example, Ac, Ds, Spm, or En, or alternatively, on or more loci for interaction with a site-specific recombinase protein, such as, for example, one or more lox or fit nucleotide sequences.
  • Known site-specific recombination systems for example the crellox system and the flp/frt system which comprise a loci for DNA recombination flanking a selected gene, specifically lox or frt genetic sequences, combination with a recombinase, ere or flp, which specifically contacts said loci, producing site-specific recombination and deletion of the selected gene.
  • European Patent No. 0228009 E.I.
  • Du Pont de Nemours and Company published 29 April, 1987 discloses a method for producing site-specific recombination of DNA in yeast utilising the crellox system, wherein yeast is transformed with a first DNA sequence comprising a regulatory nucleotide sequence and a ere gene and a second DNA sequence comprising a pre-selected DNA segment flanked by two lox sites such that, upon activation of the regulatory nucleotide sequence, expression of the ere gene is effected thereby producing site-specific recombination of DNA and deletion of the pre-selected DNA segment.
  • United States Patent No.4,959,317 (E.I. Du Pont de Nemours and Company) filed 29 April 1987 and International Patent Application No. PCT/US90/07295 (E.I. Du Pont de Nemours and Company) filed 19 December, 1990 also disclose the use of the crellox system in eukaryotic cells.
  • a requirement for the operation of site-specific recombination systems is that the loci for DNA recombination and the recombinase enzyme contact each other in vivo, which means that they must both be present in the same cell.
  • the prior art means for excising unwanted transgenes from genetically-transformed cells all involve either multiple transformation events or sexual crossing to produce a single cell comprising both the loci for DNA recombination and the site-specific recombinase.
  • a “site-specific recombinase” is understood by those skilled in the relevant art to mean an enzyme or polypeptide molecule which is capable of binding to a specific nucleotide sequence, in a nucleic acid molecule preferably a DNA sequence, hereinafter referred to as a "recombination locus” and induce a cross-over event in the nucleic acid molecule in the vicinity of said recombination locus.
  • a site-specific recombinase will induce excision of intervening DNA located between two such recombination loci.
  • recombination locus and “recombination loci” shall be taken to refer to any sequence of nucleotides which is recognized and/or bound by a site-specific recombinase as hereinbefore defined.
  • a number of different site specific recombinase systems can be used, including but not limited to the Cre/lox system of bacteriophage P1 , the FLP/FRT system of yeast, the Gin recombinase of phase Mu, the Pin recombinase of E.coli, the PinB, PinD and PinF from Shigella, and the R/RS system of the psR1 plasmid. Some of these systems have already been used with high efficiency in plants, such as tobacco, and A. thaliana.
  • Preferred site-specific recombinase systems contemplated for use in the gene constructs of the invention, and in conjunction with the inventive method are the bacteriophage P1 Cre/lox system, and the yeast FLP/FRT system.
  • the site specific recombination loci for each of these two systems are relatively short, only 34 bp for the lox loci, and 47 bp for the frt loci.
  • the recombination loci are lox sites, such as lox P, lox B, Lox L or lox R or functionally-equivalent homologues, analogues or derivatives thereof.
  • Lox sites may be isolated from bacteriophage or bacteria by methods known in the art (Hoess et al., 1982). It will also be known to those skilled in the relevant art that lox sites may be produced by synthetic means, optionally comprising one or more nucleotide substitutions, deletions or additions thereto.
  • the relative orientation of two recombination loci in a nucleic acid molecule or gene construct may influence whether the intervening genetic sequences are deleted or excised or, alternatively, inverted when a site-specific recombinase acts thereupon.
  • the recombination loci are oriented in a configuration relative to each other such as to promote the deletion or excision of intervening genetic sequences by the action of a site-specific recombinase upon, or in the vicinity of said recombination loci.
  • the present invention clearly encompasses the use of gene constructs which facilitate the expression of a site-specific recombinase protein which is capable of specifically contacting the excisable genetic element, in conjunction with the gene constructs containing the cell cycle control protein-encoding gene.
  • a single gene construct may be used to express both the site-specific recombinase protein and the cell cycle control protein, or alternatively, these may be introduced to plant cells on separate gene constructs.
  • the recombinase gene could already be present in the plant genome prior to transformation with the gene construct of the invention, or alternatively, it may be introduced to the cell subsequent to transformation with the gene construct of the invention, such as, for example, by a separate transformation event, or by standard plant breeding involving hybridisation or cross-pollination.
  • the recombinase gene is supplied to the transgenic plants containing a vector backbone sequence flanked by recombination sites by sexual crossing with a plant containing the recombinase gene in it's genome.
  • Said recombinase can be operably linked to either a constitutive or an inducible promoter.
  • the recombinase gene can alternatively be under the control of single subunit bacteriophage RNA polymerase specific promoters, such as a T7 or a T3 specific promoter, provided that the host cells also comprise the corresponding RNA polymerase in an active form.
  • Yet another alternative method for expression of the recombinase consists of operably linking the recombinase open reading frame with an upstream activating sequence fired by a transactivating transcription factor such as GAL4 or derivatives (US5801027, WO97/30164, WO98/59062) or the Lac repressor (EP0823480), provided that the host cell is supplied in an appropriate way with the transcription factor.
  • a substantially purified recombinase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment.
  • the site-specific recombinase coding region will be operably linked to regulatory sequences enabling expression of the site-specific recombinase in the eukaryotic cell.
  • the site-specific recombinase sequences is operably linked to an inducible promoter.
  • Dual-specific recombinase systems can also be employed, which may employ a recombinase enzyme in conjunction with direct or indirect repeats of two different site- specific recombination loci corresponding to the dual-specific recombinase, such as that described in International Patent Publication No. WO99/25840.
  • transposase catalyzes a recombination reaction only between two transposon border sequences.
  • a number of different plant-operable transposon/transposase systems can be used including but not limited to the Ac/Ds system, the Spun system and the Mu system. All of these systems are operable in lea mays, and at least the Ac/Ds and the Spm system function in other plants.
  • Preferred transposon sequences for use in the gene constructs of the invention are the Ds-type and the Spm-type transposons, which are delineated by border sequences of only 11 bp and 13 bp in length, respectively.
  • the present invention clearly encompasses the use of gene constructs which facilitate the expression of a transposase enzyme which is capable of specifically contacting the transposon border sequence, in conjunction with the gene constructs containing thecell cycle control protein-encoding gene.
  • a single gene construct may be used to express both the transposase and the cell cycle control protein, or alternatively, these may be introduced to plant cells on separate gene constructs.
  • the transposase-encoding gene could already be present in the plant genome prior to transformation with the gene construct of the invention, or alternatively, it may be introduced to the cell subsequent to transformation with the gene construct of the invention, such as, for example, by a separate transformation event, or by standard plant breeding involving hybridisation or cross-pollination.
  • a substantially purified transposase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment.
  • the transposase coding region will be operably linked to regulatory sequences enabling expression of the transposase in the eukaryotic cell.
  • the transposase-encoding sequence is operably linked to an inducible promoter.
  • transposon border sequences are organized as inverted repeats flanking the excisable genetic element.
  • transposons often re-integrate at another locus of the host's genome, segregation of the progeny of the hosts in which the transposase was allowed to act might be necessary to separate transformed hosts containing only the gene(s) of interest and transformed hosts containing only the cell cycle control protein-encoding gene.
  • the site-specific recombinase gene or transposase gene present in the host's genome can be removed by segregation of the progeny of the hosts to separate transformed hosts containing only the gene(s) of interest and transformed hosts containing only the site-specific recombinase gene or transposase gene.
  • said site-specific recombinase gene or transposase gene are included in the same or in a different excisable genetic element as thecell cycle control protein-encoding gene.
  • Placing a nucleic acid molecule under the regulatory control of a promoter sequence, or in operable connection with a promoter sequence, means positioning said nucleic acid molecule such that expression is controlled by the promoter sequence.
  • a promoter is usually, but not necessarily, positioned upstream, or at the 5'-end, and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates.
  • heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting (i.e., the gene from which the promoter is derived). As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting (i.e., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in gene constructs of the present invention include those listed in Table 1 , amongst others.
  • the promoters listed in Table 1 are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention.
  • the promoter is a tissue-specific inducible promoter sequence, such as but not limited to a light-inducible rbcs-1A or rbcs-3A promoter, anoxia-inducible maize Adh1 gene promoter (Howard etal., 1987; Walker et al, 1987), hypoxia-inducible maize Adh1 gene promoter (Howard et al., 1987; Walker et al, 1987), and the temperature-inducible heat shock promoter.
  • tissue-specific inducible promoter sequence such as but not limited to a light-inducible rbcs-1A or rbcs-3A promoter, anoxia-inducible maize Adh1 gene promoter (Howard etal., 1987; Walker et al, 1987), hypoxia-inducible maize Adh1 gene promoter (Howard et al., 1987; Walker et al, 1987), and the temperature-inducible heat shock promoter.
  • the promoter is a chemically-inducible promoter, such 5 as the 3- ⁇ - indoylacrylic acid-inducible Tip promoter; IPTG-inducible lac promoter; phosphate-inducible promoter; L-arabinose-inducible araB promoter; heavy metal- inducible metallothionine gene promoter; dexamethasone-inducible promoter; glucocorticoid-inducible promoter; ethanol-inducible promoter (Zeneca); the N,N-diallyl- 2,2-dichloroacetamide-inducible glutathione-S-transferase gene promoter (Wiegand 10 et al, 1986); or any one or more of the chemically-inducible promoters described by Gatz et al. (1996), amongst others.
  • a chemically-inducible promoter such 5 as the 3- ⁇ - indoylacrylic acid-inducible Tip promoter; IP
  • the promoter is a wound-inducible or pathogen-inducible promoter, such as the phenylalanine ammonia lyase (PAL) gene promoter (Ebel etal, 15 1984), chalcone synthase gene promoter (Ebel et al, 1984) or the potato wound- inducible promoter (Cleveland et al, 1987), amongst others.
  • PAL phenylalanine ammonia lyase
  • the promoter is a hormone-inducible promoter, such as the abscisic acid-inducible wheat 7S globulin gene promoter and the wheat 20 Em gene promoter (Marcotte et a/.,1988);an auxin-responsive gene promoter, such as, for example, the SAUR gene promoter, the parAs and parAt gene promoters(van der Zaal et al, 1991 ; Gil et al, 1994; Niwa et al., 1994); or a gibberellin-inducible promoter such as the Amy32b gene promoter (Lanahan et al. 1992), amongst others.
  • a hormone-inducible promoter such as the abscisic acid-inducible wheat 7S globulin gene promoter and the wheat 20 Em gene promoter (Marcotte et a/.,1988)
  • an auxin-responsive gene promoter such as, for example, the SAUR gene promoter, the parAs and parAt gene promote
  • the promoter is a constitutive plant-expressible promoter sequence such as the CaMV 35S promoter sequence , CaMV 19S promoter sequence, the octopine synthase (OCS) promoter sequence , or nopaline synthase (NOS) promoter sequence (Ebert et al. 1987), amongst others.
  • constitutive plant-expressible promoter sequence such as the CaMV 35S promoter sequence , CaMV 19S promoter sequence, the octopine synthase (OCS) promoter sequence , or nopaline synthase (NOS) promoter sequence (Ebert et al. 1987), amongst others.
  • tissue-specific promoters listed in Table 1
  • nucleotide sequences derived from one or more of the above-mentioned tissue-specific inducible promoters to confer tissue-specificity thereon.
  • the CaMV 35S promoter may be modified by the addition of maize Adh1 promoter sequence, to confer anaerobically-regulated root-specific 5 expression thereon, as described previously (Ellis etal, 1987). Such modifications can be achieved by routine experimentation by those skilled in the art.
  • the promoter is a cell cycle specific gene promoter, such as, for example, the Cdc2a promoter sequence (Chung and Parish
  • Preferred embodiments of the invention relate to the effect(s) of cytokinins on plant morphology and architecture.
  • the present invention clearly contemplates the broad application of the inventive method to the modification of a range of cellular processes
  • the modification of initiation, promotion, stimulation or enhancement of cell division and/or seed development and/or tuber formation and/or shoot initiation and/or bushiness and/or dwarfism and/or pigment synthesis and/or the modification of source/sink relationships, and/or the inhibition of root growth and/or the inhibition of apical dominance and/or the delay of senescence.
  • a 25 method of increasing the strength and/or thickness and/or stability and/or wind- resistance of a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a stem-expressible promoter sequence.
  • the stem-expressible promoter sequence is derived from the rbcs-1A gene, the rbcs-3A gene, the AtPRP4 gene, the T. bacilliform virus gene, or the sucrose- binding protein gene set forth in Table 1 , or a stem-specific or stem-expressible homologue, analogue or derivative thereof.
  • a method of increasing tuber formation and/or development in a tuberous crop plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a tuber-specific promoter sequence.
  • the tuberous crop plant is potato and the tuber-specific promoter is the potato patatin gene promoter. Additional species and promoters are not excluded.
  • a method of modifying the lignin content of a woody crop plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a cambium-specific or vascular-tissue-specific promoter sequence.
  • the promoter is a cinnamoyl alcohol dehydrogenase (CAD) gene promoter, laccase gene promoter, cellulose synthase gene promoter and xyloglucan endotransglucosylase (XET) gene promoter sequences, amongst others.
  • CAD cinnamoyl alcohol dehydrogenase
  • laccase gene promoter laccase gene promoter
  • cellulose synthase gene promoter xyloglucan endotransglucosylase (XET) gene promoter sequences, amongst others.
  • XET xyloglucan endotransglucosylase
  • Preferred target plant species according to this embodiment are woody plants of economic/ agronomic value, in particular hardwood crop plants such as, but not limited to Eucalyptus spp., Populus spp., Quercus spp., Acer spp., Juglans spp., Fagus spp., Acacia spp., or teak, amongst others. More preferably, this embodiment of the invention is applicable to modifying the lignin content of Eucalyptus spp., in particular E. globulus and E. robusta; or Quercus spp., in particular Q. dentata, Q. ilex, Q. incana, and Q.
  • Acacia spp. in particular A. brevispica, A. bussei, A. drepanolobium, A. nilotica, A. pravissima, and A. seyal; Acer spp., in particular A. pseudoplatanus and A. saccharum. Additional species are not excluded.
  • the ectopic expression of Cdc25 under control of a promoter that is operable in vascular tissue and preferably, in cambial cells will produce thick-stemmed plants and a higher ratio of vascular tissue- to-pith cells within the stem, thereby resulting in more lignin production.
  • cambial cells contain the highest levels of auxins and are therefore the preferential tissue for Cdc25 overproduction.
  • a method of increasing seed set and/or seed production and/or seed size and/or grain yield in a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a seed-specific promoter sequence.
  • the seed-specific promoter is operable in the seeds of monocotyledonous plants, for example the barley Amy32b gene promoter, Cathepsin ⁇ -like gene promoter, wheat ADP-glucose pyrophosphorylase gene promoter, maize zein gene promoter, or rice glutelin gene promoter.
  • the seed- specific promoter is operable in the seeds of dicotyledonous plant species, for example the legumin gene promoter, napA gene promoter, Brazil Nut albumin gene promoter, pea vicilin gene promoter and sunflower oleosin gene promoter, amongst others.
  • grain yield in crop plants is largely a function of the amount of starch produced in the endosperm of the seed.
  • the amount of protein produced in the endosperm is also a contributing factor to grain yield.
  • the embryo and aleurone layers contribute little in terms of the total weight of the mature grain.
  • endoreplication and endoreduplicafion are generally considered as an important factor for increasing yield (Traas et al 1998).
  • Enhancing, promoting or stimulating cell division during seed development is an alternative way to increase grain yield.
  • Cdc25 is involved both in the regulation of endoreplucation and in the stimulation of cell division.
  • the Cdc25 gene is placed operably in connection with a promoter that is operable in the endosperm of the seed, in which case the combination of the cell cycle-control protein and endosperm-expressible promoter provides the additional advantage of increasing the grain size and grain yield of the plant.
  • Endosperm-specific promoters that can be used to drive Cdc25 expression have been identified.
  • promoters can be used, including the barley blz2 gene promoter, the rice prolamin NRP33 promoter, the rice REB promoter, the zein (ZmZ27) gene promoter, the rice glutelin 1 gene (osGT1 ) promoter, the rice small subunit ADP-glucose pyrophosphorylase (osAGP) promoter, the maize granule-bound starch synthase (Waxy) gene (zmGBS) promoter surveyed by Russell and Fromm (1997), the Brazil Nut albumin gene promoter, and the pea vicilin gene promoter, amongst others.
  • Promoters derived from those genes that are expressed in the endosperm during nuclear proliferation are also useful for driving Cdc25 expression. Promoters derived from those genes that are expressed in the endosperm at the stage when nuclear proliferation is ending could be ideal for extending this period.
  • cytokinin level in the endosperm the number of endosperm cells formed during seed development and grain size, in which cytokinin activates Cdc25 enzyme which in turn activates Cdc2 kinase to drive nuclear division. Accordingly, ectopic expression of the Cdc25 gene in the endosperm enhances Cdc2 activation and nuclear proliferation, resulting in increased grain size, without incurring the non-specific side effects that application of cytokinin or expression of the ipt gene would produce in the plant.
  • a further advantage of the present inventive approach is that the activity of cytokinin metabolising enzymes is circumvented by the direct raising of Cdc25 activity in the endosperm, by the ectopic expression of Cdc25 therein.
  • the elevated cytokinin levels and nuclear division in the grain are curtailed by an increase in the activities of cytokinin degrading enzymes, including cytokinin oxidase (Chatfield and Armstrong 1987; reviewed by Morris et al 1993).
  • a method of inhibiting or reducing apical dominance or increasing the bushiness of a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a meristem-specific promoter sequence or a stem-specific promoter sequence.
  • a method of increasing lateral root production in a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a root-specific promoter sequence.
  • Preferred promoter sequences according to this embodiment of the present invention include any one of the root-expressible or root-specific promoters listed in Table 1 and in particular, the tobacco auxin-inducible gene promoter described by Van der Zaal et al (1991 ) that confers expression in the root tip of plants, in particular dicotyledonous plants.
  • a method of increasing the nitrogen-fixing capability of a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a nodule-specific promoter sequence.
  • Preferred nodule-specific promoter sequences according to this embodiment of the present invention are listed in Table 1. Additional promoters that are suited for this purpose include the hemoglobin gene promoters derived from Frankia spp., A. thaliana or other plants.
  • a method of preventing and/or delaying and/or otherwise reducing leaf chlorosis and/or leaf necrosis in a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a leaf-specific promoter sequence.
  • Preferred promoters for use according to this embodiment of the present invention include the SAM22 promoter, rbcs-1A and rbcs-3A gene promoters listed in Table 1.
  • the SAM22 gene promoter is particularly preferred in light of the developmental regulation of the SAM22 gene and its induction in senescent leaves.
  • the yeast Cdc25 protein or a homologue analogue or derivative thereof is expressed in one of the specialised minority of plant tissues in which the activation of cell cycle progression that is generally contributed by cytokinin is in part performed by other hormones.
  • An example of such a tissue is the youngest stem internode of cereal plants in which gibberellic acid stimulates cell division.
  • the present invention preferably provides a method of stimulating cell division in the intercalary meristem of the youngest stem internode to produce greater elongation of the stem and/or to generate a more extensive photosynthetic canopy of a plant comprising expressing the yeast Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a meristem specific promoter sequence.
  • Preferred promoters for use according to this embodiment of the invention include meristem promoters listed in Table 1 and in particular the Proliferating Cell Nuclear Antigen (PCNA) promoter of rice described by Kosugi et al (1991 ).
  • PCNA Proliferating Cell Nuclear Antigen
  • the cell cycle control protein is expressed under the operable control of a regulatable promoter sequence.
  • a gene construct or vector into plant cells by transformation or transfection means.
  • the nucleic acid molecule or a gene construct comprising same may be introduced into a cell using any known method for the transfection or transformation of said cell.
  • a cell is transformed by the gene construct of the invention, a whole organism may be regenerated from a single transformed cell, using any method known to those skilled in the art.
  • transfect is meant that the gene construct or vector or an active fragment thereof comprising the Cdc25 gene operably under the control of the regulatable promoter sequence is introduced into said cell without integration into the cell's genome.
  • transform is meant that the gene construct or vector or an active fragment thereof comprising the Cdc25 gene operably under the control of the regulatable promoter sequence is stably integrated into the genome of the cell.
  • the present invention provides a method of modifying one or more plant morphological and/or biochemical and/or physiological characteristics comprising
  • a gene construct or vector comprising a nucleotide sequence that encodes a cell cycle control protein, such as, for example, Cdc25 or a homologue, analogue or derivative thereof, operably in connection with a regulatable promoter sequence selected from the list comprising cell-specific promoter sequences, tissue-specific promoter sequences, inducible promoter sequences, organ-specific promoter sequences and cell cycle gene promoter sequences to produce a transformed or transfected cell; and
  • the inventive method comprises regenerating a whole plant from the transformed cell.
  • Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al, 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue expiants or cells (Christou et al, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et a/.(1985), Herrera-Estrella et al. (1983a, 1983b, 1985).
  • a microparticle is propelled into a cell to produce a transformed cell.
  • Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp etal. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050).
  • the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • microparticles suitable for use in such systems include 1 to 5 ⁇ m gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • a whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • existing meristematic tissue e.g., apical meristem, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centres.
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • the transformed plants are produced by a method that does not require the application of exogenous cytokinin and/or gibberellin during the tissue culture phase, such as, for example, an in planta transformation method.
  • plants are transformed by an in planta method using Agrobacterium tumefaciens such as that described by Bechtold et al., (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed without the exogenous application of cytokinin and/or gibberellin.
  • Agrobacterium tumefaciens such as that described by Bechtold et al., (1993) or Clough et al (1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspor
  • the regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed to give homozygous second generation (or T2) transformant, and the T2 plants further propagated through classical breeding techniques.
  • the regenerated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed root stock grafted to an untransformed scion ).
  • a further aspect of the present invention clearly provides the gene constructs and vectors designed to facilitate the introduction and/or expression and/or maintenance of the cell cycle control protein-encoding sequence and regulatable promoter into a plant cell, tissue or organ.
  • the gene construct of the present invention may further comprise one or more terminator sequences.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
  • terminators particularly suitable for use in the gene constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst others.
  • NOS nopaline synthase
  • OCS Agrobacterium tumefaciens octopine synthase
  • CaMV Cauliflower mosaic virus
  • t3'Bt2 Oryza sativa ADP-glucose pyrophosphorylase terminator sequence
  • Zea may
  • the gene constructs of the invention may further include an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.
  • an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.
  • Preferred origins of replication include, but are not limited to, the f1-o and co/E1 origins of replication.
  • the gene construct may further comprise a selectable marker gene or genes that are functional in a cell into which said gene construct is introduced.
  • selectable marker gene includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp r ), tetracycline resistance gene (Tc r ), bacterial kanamycin resistance gene (Kan r ), phosphinothricin resistance gene, neomycin phosphotransferase gene ( ⁇ pfll), hygromycin resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene, amongst others.
  • Amicillin resistance Amicillin resistance
  • Tc r tetracycline resistance gene
  • Kan r bacterial kanamycin resistance gene
  • phosphinothricin resistance gene phosphinothricin resistance gene
  • ⁇ pfll neomycin phosphotransferase gene
  • hygromycin resistance gene ⁇ -glucuroni
  • the present invention is applicable to any plant, in particular a monocotyledonous plants and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp.,
  • the present invention clearly extends to any plant produced by the inventive method described herein, and any and all plant parts and propagules thereof.
  • the present invention extends further to encompass the progeny derived from a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by the inventive method, the only requirement being that said progeny exhibits the same genotypic and/or phenotypic characteristic(s) as that (those) characteristic(s) that has (have) been produced in the parent by the performance of the inventive method.
  • genotypic characteristic is meant the composition of the genome and, more particularly, the introduced gene encoding the cell cycle control protein.
  • phenotypic characteristic is meant one or more plant morphological characteristics and/or plant biochemical characteristics and/or plant physiological characteristics that are produced by ectopic expression of a cell cycle control protein in a plant.
  • the plant is produced according to the inventive method is transfected or transformed with a genetic sequence, or amenable to the introduction of a protein, by any art-recognised means, such as microprojectile bombardment, microinjection, Agrobacterium-me ⁇ i ' aied transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others.
  • any art-recognised means such as microprojectile bombardment, microinjection, Agrobacterium-me ⁇ i ' aied transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others.
  • Suspension cultured cells of Nicotiana plumbiginifolia were grown in CS V medium supplemented with 9 ⁇ M 2,4-dichlorophenoxyacetic acid and 0.23 ⁇ M kinetin, and were brought to arrest at the cytokinin control point by the omission of kinetin from the culture medium. Arrest of cell cultures was confirmed by cell counting.
  • Cdc2a carboxy terminal amino acid sequence of the tobacco Cdc2 protein
  • This peptide has the amino acid sequence KRITARNALEHEYFKDIGYVP and has been demonstrated by complementation analyses in yeast to be a functional homologue of Cdc2.
  • the Cdc2a peptide was synthesised chemically, purified by HPLC and conjugated to keyhole limpet haemocyanin.
  • Antibodies were also prepared against a recombinant GST-Cdc25 catalytic core fusion protein, that had been synthesised in Escherichia coli.
  • Cdc2 and Cdc25 enzyme activities were extracted from tobacco cells, by grinding the cells in liquid nitrogen.
  • NDE buffer containing 25 mM HEPES (pH 7.2) with protease and phosphatase inhibitors was used.
  • Cdc25 extraction PDE buffer, containing 25 mM MOPS (pH 7.2), 100 mM NaCl, 10 mM DTT, 5mM EDTA, 1 mM EGTA, 1 % NP-40, 50 mM NaF, 0.5 mM PMSF, 3 ⁇ g ml "1 leupeptin, and 20 ⁇ g ml "1 aprotinin, was used.
  • Immunoprecipitates of Cdc2 and Cdc25 were obtained by reaction with 25 ⁇ l protein A-purified antibodies against Cdc2 and Cdc25 respectively, for 3 h at 4°C, followed by sedimentation of the antigen-antibody complexes using 35 ⁇ l protein A beads per sample. The immunoprecipitates were then washed three times, for 10 min per wash, using HDW buffer, followed by similar washing using HBK buffer. In the case of Cdc25 immunoprecipitates, the HBK buffer was supplemented with 2 ⁇ M spermidine.
  • Cdc25 immunoprecipitates from 500 ⁇ g total soluble plant protein were incubated for 30 min at 30 °C in Cdc25 assay buffer with 0.25 ⁇ g tyrosine phosphorylated Cdc2 substrate that had been purified with p13 suc1 -beads from 500 ⁇ g protein of arrested Cdc25-22 mutant fission yeast.
  • the phosphatase reaction was stopped by removing the complexed Cdc25/Cdc2 by sedimentation.
  • the supernatant was assayed for yeast Cdc2 kinase that had been activated. Assays to be compared directly were run and exposed together in a Phosphorimager.
  • Cdc2a phosphotyrosine assay To assay phosphotyrosine in Cdc2a, the Cdc2a enzyme fraction was recovered essentially as described supra for the Cdc2 activity assay, except that 5 mg of extracted plant protein was used as starting material, and the NDE buffer was modified to include 2.5 mM sodium vanadate and 1 mM phosphotyrosine, and the immune complexes were washed with HDW buffer supplemented with 1 mM with sodium vanadate.
  • EXAMPLE 2 Expression of yeast Cdc25 makes cell division in plant cells independent of cell cytokinin
  • yeast Cdc25 The effect of ectopic expression of yeast Cdc25 in plant s was investigated because cells arrested by lack of cytokinin, whether derived from suspension culture or excised freshly from the plant, have abundant Cdc2 protein that is enzymically inactive because phosphorylated at tyrosine.
  • Latent Cdc2 protein kinase activity can be released in vitro by incubation with the phosphoprotein phosphatase Cdc25 that is specific for Cdc2.
  • Cdc25 phosphoprotein phosphatase
  • dephosphorylation of Cdc2 is one of the events that occur, but it was uncertain whether the hormone might have several effects in the cell cycle.
  • cytokinin in sustaining cell division is activation of Cdc25 since the hormone can be substituted by expression of this gene.
  • Levels of the fission yeast enzyme Cdc25 that removes inhibitory phosphate from tyrosine in Cdc2 kinase were brought under genetic control in the plant by joining the yeast Cdc25 gene to a modified plant promoter that contained rat glucocorticoid response elements (GREs), which are responsive to rat glucocorticoid receptor protein (GR) in the presence of dexamethasone and therefore allowed induction without interference from plant hormones.
  • GREs rat glucocorticoid response elements
  • GR rat glucocorticoid receptor protein
  • the GRE-Cdc25 together with the constitutively- expressed NOS promoter-GR construct, were inserted into the vector pBin19, which contains pnos:nptll for kanamycin resistance, and introduced into cells of N. plumbaginifolia by electroporation into protoplasts. Clones resistant to kanamycin were tested for ability to form a colony on solid medium containing dexamethasone and auxin but no cytokinin.
  • Inducible cell lines contained yeast Cdc25 DNA (detected in Southern blots, not shown) and in 0.01-10 ⁇ M dexamethasone they accumulated Cdc25 mRNA and protein ( Figure 1-1 ; Figure 1-2). Effects on division were tested in cells that had been arrested at the G2 phase hormonal control point by depletion of auxin and cytokinin followed by provision of auxin only. Dexamethasone at 0.01-10 ⁇ M induced division ( Figure 1-3) and a sharp optimum concentration of 0.1 ⁇ M dexamethasone was observed in independent clones, consistent with requirement for a critical optimum Cdc25 activity. No cell division was observed without inducer, or in untransformed cells treated with dexamethasone ( Figure 1-3). Three independent lines were analysed biochemically and had similar properties. Results from one line are shown.
  • the experimental system used for subsequent experiments involved the prior arrest of suspension culture cells, at the cytokinin control point in late G2 phase. Arrest at this point was obtained by incubation without hormone and then with auxin (2,4-D) without cytokinin. Mitosis could then be induced by addition of cytokinin, or alternative potentially mitogenic treatments could be tested. Progress through prophase is a little slower after this arrest than in cells not emerging from hormonal block and is very suitable for study of the succession of biochemical events in plant mitosis.
  • the resulting daughter cells were viable; indicating that mitosis driven by induced Cdc25 is functionally normal.
  • These daughter cells could proliferate indefinitely with dexamethasone replacing cytokinin and required nine-fold dilution every 7 days precisely as in control cultures provided with auxin and cytokinin. They are routinely maintained in dexamethasone without cytokinin.
  • the data provided herein reveal that the sole essential action of cytokinin in sustaining cell division is activation of Cdc25 and the hormone can be substituted by expression of the Cdc25 gene.
  • yeast Cdc25 to influence cytokinin-mediated cell division in plants suggested to the present inventors that the yeast protein replaces the activity of an endogenous plant Cdc25 enzyme that is activated by cytokinin. To demonstrate that this is the case, the effectiveness of induced yeast Cdc25 produced in transformed plant cells to activate Cdc2, was compared to the effectiveness of a putative plant- derived Cdc25 from genetically unmodified cells to activate Cdc2.
  • the yeast and putative plant Cdc25 enzymes recovered by immunoprecipitation using anti-Cdc25 antibody were compared in reaction with tyrosine phosphorylated plant Cdc2 enzyme taken from cells arrested at the G2 control point ( Figure 2-6, lanes 1-3).
  • Substrate Cdc2 was also taken from cells after 3 hours stimulation with cytokinin ( Figure 2-6, lanes 4-6), when partial Cdc2 activation had occurred ( Figure 2-5; Figure 2-6, lanes 1 and 4).
  • the activation of plant Cdc2 by yeast Cdc25 expressed in plant cells ( Figure 2-6, lane 6) demonstrates a mechanism by which Cdc25 can substitute for cytokinin. Furthermore this activation mechanism is a normal part of plant mitosis, because non- transgenic plant cells also contain a Cdc25 activity, unambiguously of plant origin, that is both present following cytokinin stimulation and capable of activating plant Cdc2 ( Figure 2-6, compare lanes 2 and 5). Moreover, the plant Cdc25 activity is slightly more effective than the heterologous yeast Cdc25 in activating plant Cdc2 in the tobacco cells tested ( Figure 2-6, compare lanes 5 and 6).
  • the fission yeast Cdc25 coding sequence is cloned between the promoter of a class I patatin gene ( Liu ef al., 1991) and the transcription termination signals of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens.
  • the B repeat region and the distal region of the A repeat of the patatin promoter is used, without the proximal region of the A repeat.
  • the proximal region of the A repeat of the patatin promoter confers sucrose-responsiveness in various tissues, which is not a desirable characteristic for our purposes (Grierson et al, 1994).
  • This construct is placed in a binary vector, mobilized to Agrobacterium tumefaciens, and the introduced into potato plants.
  • the Cdc25 protein is expressed under the control of the Class I patatin promoter when the first stolon starts to tuberize, consistent with the expression pattern for the patatin gene (Liu ef a/., 1991). At this stage, expression is associated with both internal and external phloem. After tuber induction has occurred, promoter activity is found both in tuberized stolons and in non-tuberized stolons. Expression then expands to the entire storage parenchyma, cortex and pith, but remains absent from the periderm.
  • Class II patatin promoters are expressed in the periderm and as such are complementary to the Class I promoters (K ⁇ ster-T ⁇ pfer et al.,; Liu ef al, 1991 ; Nap et al, 1992), it is beneficial to have Cdc25 expression driven by both Class I and Class II promoters within the same plant. Because the Class I patatin promoter is not expressed before the first stolon initiates tuberization, no effects of Class I patatin- Cdc25 transgenes is seen on tuber initiation.
  • the Class I patatin promoter drives Cdc25 expression very early after tuber initiation onwards, allowing a maximal impact of Cdc25 activity on organ formation and, as a consequence, on tuber size.
  • the fission yeast Cdc25 coding sequence is cloned between the promoter of the soybean SAUR gene (Li ef al, 1992 ) and the transcription termination signals of the nopaline synthase (NOS) gene oi Agrobacterium tumefaciens.
  • the SAUR promoter is inducible by auxins.
  • This chimeric gene construct is introduced between the T-DNA borders of the binary vector pBI121 or similar vector and mobilised into Agrobacterium tumefaciens. Poplar is transformed by transformation using standard procedures.
  • Transgenic poplar trees containing this construct show increased lignin content, correlated with an increased stem diameter and the higher ratio of vascular tissue to pith and cortex cells.
  • a similar phenotype in poplar is produced when the Cdc25 expression is driven by the rolB promoter oi Agrobacterium rhizogenes (Nilsson et al, 1997), that is expressed in cambial cells (i.e. the dividing cells of the vascular tissue).
  • the fission yeast Cdc25 coding sequence is placed operably in connection with the endosperm-specific Itr1 promoter from barley, or a synthetic promoter containing the endosperm box (GCN motif) of the barley Hor2 gene (Vicente-Carbajosa et al, 1998).
  • the Cdc25 structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene.
  • Cereals, in particular rice, maize, wheat and barley, are transformed using standard procedures, in particular microprojectile bombardment or transformation systems, with the gene constructs.
  • the grain size and starch storage capacity of the endosperm of the seeds of transformed plants is increased relative to otherwise isogenic non-transformed plants.
  • Cdc25 under the control of meristem-specific promoters reduces apical dominance in A. thaliana and B. napus plants
  • the fission yeast Cdc25 coding sequence is placed operably in connection with the shoot meristem-specific LEAFY promoter (Weigel ef al, 1992), or the KNOTTED-like Arabidopsis thaliana knatl promoter (Accession number AJ 131822), or the KNOTTED-like Malus domestica kn1 promoter ( Accession No. Z71981 ), or the A. thaliana CLAVATA1 promoter (Accession number AF049870).
  • the Cdc25 structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene.
  • NOS nopaline synthase
  • Transformed plants exhibit cytokinin-like effects at the level of the shoot (and flower) meristem, resulting in reduced apical dominance.
  • Cdc25 under the control of the cab-6 or ubi7 promoters reduces leaf necrosis and chlorosis in lettuce plants
  • the fission yeast Cdc25 coding sequence is placed operably in connection with the leaf-specific cab-6 gene promoter derived from Pinus (Yamamoto ef al, 1994) or senescence-specific ubi7 gene promoter (Garbarino ef al, 1995).
  • the Cdc25 structural gene is placed upstream of the transcription termination signals of the Agrobacterium tumefaciens nopaline synthase (NOS) gene. Lettuce is transformed as described by Bechtold ef al, 1993.
  • Leaf deterioration (chlorosis and necrosis) in lettuce for example as a consequence of post-harvest storage, is delayed in transformed lettuce plants compared to non- transformed control plants.
  • yeast Cdc25 under growth limiting conditions overrides the block in DNA replication.
  • Methods Cells of N. plumbaginifolia were maintained in CSV medium containing 9 millimolar 2,4-D and 0.23 millimolar kinetin and were diluted every seven days by transferring 5ml culture into 50 ml of fresh medium.
  • Cells in which the full length Cdc25 gene could be inducibly expressed were created by joining the fission yeast Cdc25 gene to a modified plant promoter that contained rat glucocorticoid response elements (GREs), which are responsive to rat glucocorticoid receptor protein (GR).
  • GREs rat glucocorticoid response elements
  • GR rat glucocorticoid receptor protein
  • the GRE-Cdc25 together with the constitutively-expressed NOS promoter-GR construct, were inserted into the vector pBin19, which contains pnos:nptll for kanamycin resistance, and was introduced into cells of N. plumbaginifolia by electroporation into protoplasts. Clones resistant to kanamycin were selected and their arrest properties investigated.
  • the protoplasts were collected by centrifugation at 5000 rpm (gav-42) for 3 min and washed once with washing buffer. Release of the nuclei was done by adding 2 ml of Galbraith buffer (45 mM Mg CI2, 30mM sodium citrate, 20mM 2-(N-Morpholino) propanesulfonic acid (MOPS), 1% Triton X-100, pH 7.0. Nuclear release was monitored by microscopy and was complete by 5 min, then debris was discarded by filtering through a 30 micrometer-pore Nylon filter twice.
  • Galbraith buffer 45 mM Mg CI2, 30mM sodium citrate, 20mM 2-(N-Morpholino) propanesulfonic acid (MOPS), 1% Triton X-100, pH 7.0.
  • Nuclei were stained with propidium iodide (PI) by incubation in PI solution of 20 microgram/ml in 1 mM ethylenediaminetetraacetic acid (EDTA) in a 25°C waterbath for 15 min. Then the nuclei were fixed by adding paraformaldehyde to a final concentration of 2% and incubated in a 25°C waterbath for 15 min. Nuclear DNA content was examined by flow cytometry in a Becton Dickinson FACScan with filter wavelength for FL-2 of 585 nm. Data-analysis was done with WinMDI 2.7 written by Joseph Trotter, The Scripps Research Institute, La Jolla, California 92037.
  • Figure 5 indicates the frequency profile of nuclei with 2n, 4n and 8n amounts of DNA isolated from cultures in which cell division is arresting, in (A) nontransgenic cells, (B) transgenic cells in which the Cdc25 gene is joined to a glucocorticoid regulated promoter and was induced by the presence of 0.1 micromolar dexamethasone.
  • PCNA rice proliferating cell nuclear antigen

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Abstract

L'invention concerne un procédé de modification, dans des plantes, d'une ou plusieurs de leurs propriétés ou caractéristiques morphologiques, biochimiques ou physiologiques, induites par des cytokinines, telles qu'au moins une réponse adaptative à l'environnement et/ou un processus de développement, ce procédé consistant à faire s'exprimer, dans la plante, une protéine de régulation du cycle cellulaire, notamment la phosphoprotéine phosphatase Cdc25, sous la commande d'une séquence promoteur pouvant être régulée.
PCT/AU2000/000134 1999-02-26 2000-02-25 Procede de modification de la morphologie, biochimie ou physiologie de plantes, a l'aide de cdc25 WO2000052171A1 (fr)

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WO2001023594A2 (fr) * 1999-09-27 2001-04-05 Pioneer Hi-Bred International, Inc. Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
WO2006100112A1 (fr) * 2005-03-25 2006-09-28 Cropdesign N.V. Plantes a rendement accru et procede de fabrication de celles-ci
WO2007141189A2 (fr) 2006-06-08 2007-12-13 Basf Plant Science Gmbh végétaux aux caractéristiques de croissance améliorées et procédé d'obtention
CN102243150A (zh) * 2010-05-14 2011-11-16 河北省农林科学院昌黎果树研究所 一种适于流式细胞分析的果树成熟叶片细胞核提取方法
US8420890B2 (en) 2006-03-28 2013-04-16 Cornell Research Foundation, Inc. Use of NAP gene to manipulate leaf senescence in plants
CN103940646A (zh) * 2014-03-26 2014-07-23 浙江省农业科学院花卉研究开发中心 一种红掌细胞核悬液制备方法
CN103940656A (zh) * 2014-03-26 2014-07-23 浙江省农业科学院花卉研究开发中心 一种松萝凤梨细胞核悬液制备方法
US9226515B2 (en) 2004-02-03 2016-01-05 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
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WO2001023594A2 (fr) * 1999-09-27 2001-04-05 Pioneer Hi-Bred International, Inc. Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
WO2001023594A3 (fr) * 1999-09-27 2001-12-06 Pioneer Hi Bred Int Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire
US10154679B2 (en) 2004-02-03 2018-12-18 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
US9226515B2 (en) 2004-02-03 2016-01-05 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
WO2006100112A1 (fr) * 2005-03-25 2006-09-28 Cropdesign N.V. Plantes a rendement accru et procede de fabrication de celles-ci
US8420890B2 (en) 2006-03-28 2013-04-16 Cornell Research Foundation, Inc. Use of NAP gene to manipulate leaf senescence in plants
EP2436761A1 (fr) 2006-06-08 2012-04-04 BASF Plant Science GmbH Installations dotées de caractéristiques de croissance améliorées et procédé de fabrication de celles-ci
US8273952B2 (en) 2006-06-08 2012-09-25 Basf Plant Science Gmbh Plants having improved growth characteristics and method for making the same
EP2436760A1 (fr) 2006-06-08 2012-04-04 BASF Plant Science GmbH Installations dotées de caractéristiques de croissance améliorées et procédé de fabrication de celles-ci
WO2007141189A2 (fr) 2006-06-08 2007-12-13 Basf Plant Science Gmbh végétaux aux caractéristiques de croissance améliorées et procédé d'obtention
CN102243150A (zh) * 2010-05-14 2011-11-16 河北省农林科学院昌黎果树研究所 一种适于流式细胞分析的果树成熟叶片细胞核提取方法
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CN103940656A (zh) * 2014-03-26 2014-07-23 浙江省农业科学院花卉研究开发中心 一种松萝凤梨细胞核悬液制备方法
CN114853859A (zh) * 2022-04-29 2022-08-05 安徽农业大学 茶树水通道蛋白基因CsAQP95及其应用

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