WO2000052168A1 - Method of selecting transformed cells and tissues - Google Patents

Abstract

The present invention provides a method of detecting or identifying transformed or transfected plant cells, tissues or organs that are hormone-dependent, and reagents therefor.

Description

METHOD OF SELECTING TRANSFORMED CELLS AND TISSUES

FIELD OF THE INVENTION

The present invention relates generally to a method of selecting cells and tissues that carry foreign genetic material, in particular foreign DNA, as a complement to the genome of the tissue or cell. More particularly, the present invention relates to a novel use for cell cycle control proteins in detecting and/or identifying transformed or transfected plant cells, tissues or organs, such as, for example, plant cells, tissue or organs for which the processes of endoreplication and/or endoreduplication and/or cell division and/or cell expansion and/or growth and/or viability and/or regeneration into whole plants, are hormone-dependent. The present invention also provides gene constructs for use in the inventive method. The invention clearly extends to those plants, and any plant parts, propagules, cells, tissues or organs prepared according to the method described herein.

GENERAL

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

BACKGROUND TO THE INVENTION

5 Transgenic plants provide the means for modifying many plant phenotypes, including those phenotypes related to aspects of plant growth, development, biochemistry and physiology, such as, for example, to increase yield, and/or to increase abiotic stress resistance. Because the frequency of transformation events in a transformation protocol is generally low, a means is required to select or identify those transformed 0 cells from the non-transformed cells, which are normally present in far more greater numbers.

Accordingly, known techniques for plant transformation utilise the expression of a selectable marker gene, to identify or detect transformed cells, and to distinguish any 5 transformed cells from the untransformed cells, and to select those transformed cells, and preferably, to facilitate the enhanced growth, regeneration, and development of the transformed cells compared to non-transformed cells. In such protocols, the selectable marker gene, in addition to the gene-of-interest which confers a desired trait on the plant, is transferred to the plant, by any one of a number of transformation 0 methods.

Selectable markers used in modern plant biotechnology are generally dominant genes, which encode proteins conferring resistance to one or more cytotoxic compounds, such as, for example, antibiotics or herbicides (Yoder and Goldsbrough, 25 1994). Cells which are resistant to the cytotoxin are selected in the presence of the compound. However, cytotoxins such as antibiotics and herbicides often have adverse effects on both untransformed cells and transformed cells, including the slowing or inhibition of callus formation and plantlet regeneration.

30 Additionally, such compounds produce adverse environmental consequences, making their use undesirable. Accordingly, it is possible that, in future, regulatory authorities will require transformed plant material to be free of selectable marker genes that encode resistance proteins having cytotoxin specificities. Moreover, in light of the adverse environmental consequences associated with using such selectable marker genes, there is likely to be reduced demand in the market for plants carrying such resistance genes.

Development and environmental adaptation are highly regulated processes in plants. These processes are not cell-autonomous but rather involve extensive communication between different parts of the plant. Amongst the most important mobile signals involved in this long-distance communication are plant hormones such as auxins, cytokinins, abscisic acid, gibberellins, and ethylene. Other signals, so far not defined as plant hormones, include salicyclic acid, jasmonic acid and brassinosteroids.

There are plethora of data showing that the external application of plant hormones has profound effects on development, metabolism and environmental fitness. For example, the external application of 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. Additionally, exogenous cytokinin application following anthesis in cereals enhances grain set and yield and the phase of nuclear and cell division in the developing endosperm of cereal grains is accompanied by a peak of cytokinin concentration, suggesting a role for cytokinins in grain development in cereals (Herzog, 1980; Morgan et al., 1983). 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).

In transformation protocols, both cytokinin and auxin are required to be present in approximately equal amounts in plant media, for successful callus formation from differentiated plant tissues, such as leaf discs, to occur. The requirement for cytokinin can be overcome by expressing genes encoding cytokinin biosynthetic enzymes. Examples of such enzymes are isopentenyltransferse (ipt gene; WO93/07272, US5496732, US5689042) and the Agrobacterium rhizogenes rolC gene product (EP- A-334383). However, the ectopic expression of such genes in plant tissue is known to produce adverse pleiotropic effects, in particular the so-called 'shooty phenotype'.

To address the problem of shooty phenotype, Ebinuma et al. (1997) embedded the ipt selectable marker gene in a transposable genetic element (i.e. a transposon), such that it could be removed by activation of transposon excision following the processes of selection and regeneration, thereby eliminating expression of the ipt gene in the regenerated plantlets.

In an alternative approach, Kakimoto et al. (1996) used the Arabidopsis thaliana CKI1 gene as a selectable marker gene. The Ckl1 gene is involved in the cytokinin signal transduction pathway, and can be expressed in a tightly-regulated manner to avoid pleiotropic effects. The use of the CKI1 gene as selectable marker is also documented in W099/38988.

Those skilled in the art are aware that it is often desirable to introduce a number of different genes into plants, each expressing a desired polypeptide to confer a novel trait. However, it is not advantageous to introduce these genes in the form of a single gene construct, because of the difficulty associated with producing large gene constructs, which often rearrange and are unstable. Additionally, large gene constructs have a tendency to be unstable in the plant genome, or incorporated at low frequency into the plant genome. Accordingly, such traits are generally introduced into the plant genome by multiple transformation events, a process known as "stacking", wherein each transformation event requires selection. Whilst the Ckl1 gene is perhaps one example of a selectable marker gene that overcomes the problems associated with the use of cytotoxic compounds to select transformed plant cells, tissues, organs, and plantlets, a wide range of selectable marker genes capable of overcoming this problem is required for use in the art, to facilitate the stacking of new genes in the genome of a plant.

SUMMARY OF THE INVENTION In work leading up to the present invention, the present inventors sought to develop novel selectable markers for selecting transformed plant cells, tissues, organs or whole plants, without using cytotoxic compounds in the selection process, and which avoided the pleiotropy associated with the prior art.

The inventors discovered that the cytokinin-mediated and/or gibberellin-mediated induction of mitosis (M-phase) in plants can be achieved by ectopically expressing a cell cycle control protein (CDK) therein, such as, for example, the fission yeast Cdc25 phosphoprotein phosphatase, or other CDK that mimics the bioactivity of Cdc25. In this regard, the inventors have discovered that the ectopic expression of the yeast Cdc25 phosphoprotein phosphatase, or other CDKs, in plants and/or plant cells can override the inhibition of DNA replication (S phase) under growth limiting conditions. Without being bound by any theory or mode of action, it is likely that the ectopic expression of a CDK in a plant cell, in particular Cdc25, or a CDK protein that mimics Cdc25, releases the inhibition of mitosis and/or DNA replication that would otherwise be imposed by endogenous Cdc2 activity in the cell, thereby allowing cells to enter mitosis and/or DNA replication.

CDKs are intracellular proteins, 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.

The present inventors have developed gene constructs that provide for the controlled expression of CDKs, in particular the fission yeast Cdc25 protein, and other CDK proteins that mimic the bioactivity of endogenous Cdc25, in particular cells, tissues and organs of plants. Accordingly, one aspect of the invention provides a gene construct or vector comprising a nucleic acid molecule which consists of a selectable marker gene selected from the group consisting of:

(i) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter sequence that is operable in a plant ;

(ii) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element; and

(iii) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a constitutive promoter sequence that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element.

Preferably, the gene construct of the present invention further comprises the nucleotide sequence(s) of one of more gene(s)-of-interest which are intended to be introduced to the plant to confer a desired phenotype thereon. The gene construct of the invention further comprises nucleotide sequences required for maintenance, and/or replication, in a prokaryotic cell, and for expression and/or integration in a plant cell, as will be known to those skilled in the art.

Surprisingly, the gene constructs developed by the inventors are useful in selecting transformed plant cells in the absence of exogenous cytokinin or gibberellin. Based upon the role of cell cycle control proteins in producing cytokinin-mediated and/or gibberellin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics in plants, as elucidated by the present inventors, the gene construct of the invention may be applied to the selection of any cell, tissue, organ or whole organism that expresses and/or exhibits cytokinin-mediated and/or gibberellin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics, from a background of cells, tissues, organs or whole organisms that do not exhibit such characteristics. By way of exemplification, the formation of transformed calli from tobacco leaf discs on medium lacking exogenous cytokinin, is provided in Example 6. The transformed calli selected using the inventive method described herein are regenerated into whole transgenic plants.

Accordingly, a second aspect of the present invention provides a method of detecting or identifying a transformed or transfected plant cell, tissue, organ, or plantlet that is hormone-dependent, comprising expressing a cell cycle control protein in said plant cell, tissue or organ is under the operable control of a plant-expressible regulatable promoter sequence, for a time and under conditions sufficient for the normally hormone-mediated cell division and/or hormone-mediated tissue differentiation to occur in the absence of the hormone.

The present invention is preferably applicable to the transformation of gibberellin- dependent and/or cytokinin-dependent plant cells, tissues, and organs.

Moreover, expression of the cell cycle control protein is preferably achieved by introducing the gene construct of the invention, or the selectable marker gene perse into a plant cell. Accordingly, in a further preferred embodiment, the present invention provides a method for selecting transgenic cells, tissues or organs comprising:

(i) introducing to a plant cell, tissue or organ a gene construct or vector comprising a nucleotide sequence that encodes a cell cycle control protein operably in connection with a regulatable promoter sequence; and (ii) expressing said cell cycle control protein in one or more of said cells, tissues or organs of the plant for a time and under conditions sufficient for cell division and/or cell differentiation to occur in the absence of exogenous cytokinin and/or gibberellin.

This aspect of the invention clearly encompasses further introducing nucleotide sequence(s) of one of more gene(s)-of-interest which are intended to be introduced to the plant to confer a desired phenotype thereon. Such nucleotide sequences may be introduced to the plant cell on the same gene construct as the selectable marker gene of the invention, or alternatively, on a separate nucleic acid molecule wherein it is preferably introduced simultaneously to the plant cell with the gene construct comprising the selectable marker gene ofthe invention, such as by co-transfection, co- transformation, co-electroporation, or as a co-precipitate on gold or other microprojectile particles, etc.

More preferably, this aspect of the invention further provides for the regeneration of organised and differentiated tissues from the transformed cells expressing the cell cycle control protein, including plantlets and other plant parts, and whole plants.

A third aspect of the invention provides plant cells, tissues, organs and plant parts, propagules and progeny plants that have been selected using the inventive process described herein or alternatively or in addition, which comprise the gene constructs of the invention. Such plants may be substantially free of the selectable marker gene, particularly if produced using a selectable marker gene integrated into an excisable genetic element, and subsequent excision of the excisable genetic element.

BRIEF DESCRIPTION OF THE DRAWINGS

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. Total RNA was extracted from tobacco cells either in the absence of added dexamethasone (lane 0), or after 12 h induction with 0.01 μM, or 0.10 μM, or 1.00 μM, or 10.00 μM dexamethasone and then loaded onto agarose gels (60 μg 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 0.01 μM dexamethasone (Lane 2), or 0.10 μM dexamethasone (Lane 3), or 1.00 μM dexamethasone (Lane 4), or 10.00 μM dexamethasone (Lane 5) and then loaded onto SDS/polyacrylamide gels (50 μg aliquots total soluble protein per lane), transferred to membrane support and probed with antibody specific for the Cdc25-specific probe. p67^Cdc25 was detected by western blot of 50 μg aliquots of total soluble p67 ^Cdc2protein.

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 μM dexamethasone. Data were also obtained for both transformed cells (O) and for control non-transformed cells (Δ) grown under identical culture conditions.

Figure 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 μM dexamethasone (-D), or alternatively, that have been induced with 0.1 μM 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 (Δ) 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 μM 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 10⁶) 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 μM dexamethasone or cytokinin. Data show cell number for both transgenic cells induced using dexamethasone (D) or cytokinin (Δ), and for non-transgenic cells induced using dexamethasone (O).

Figure 2-4 is a graphical representation showing the change in activities of Cdc25 phosphatase (Δ) 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^suc1 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 μM NAA (panels c,d) or with 0.56 μM BAP (panels e,f) or 5.4 μM NAA plus 0.56 μM 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 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. Both cultures were sampled after 6 days of batch culture, when cell cycle progress has arrested at a G1/S control point in non-transgenic cells (2N peak in A) but has been driven through this point in a majority of the transgenic cells resulting in accumulation mostly in G2 phase (4n peak in B) and in some cases an additional traverse of S phase has been induced without intervening mitosis, resulting in endoreduplication of the genome and generation of 8n nuclei (seen in B).

Figure 6 is a photographic representation of leaf discs treated with Agrobacterium containing a binary plant transformation vector carrying the Cdc25 coding sequence under the operable control of a promoter containing the glucocorticoid response elements (GREs). Agrobac.er/^'um-treated leaf discs are incubated on a medium containing B5 salts supplemented with 250 mg/1 ammonium nitrate, 20 g/1 glucose, 0.5 g/1 MES, 40 mg/1 adenine, 0.8% agar, 0.1 mg/l IAA, 500 mg/l cefotaxime and 50 mg/l kanamycine (pH 5.7). When incubated on this medium that contains auxin but lacks cytokinin, in the presence of 0.1 to 1μM dexamethasone, only cells that are expressing the introduced Cdc25 gene survive and proliferate (compare Figure 6A, no dexamethasone added and Figure 6B, dexamethasone added, circles indicate calli) whereas untransformed cells die. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the invention provides a gene construct or vector comprising a nucleic acid molecule which consists of a selectable marker gene selected from the group consisting of: (i) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter sequence that is operable in a plant ; (ii) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element; and (iii) a selectable marker gene comprising a nucleotide sequence that encodes a ceil cycle control protein placed operably in connection with a constitutive promoter sequence that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element.

As used herein, the term "gene construct" refers to any nucleic acid molecule that comprises one or more chimeric genes suitable for introducing into a plant cell, tissue, organ, or plant part, including a plantlet, and preferably which is capable of being integrated into the genome of a plant.

As used herein, the word "vector" shall be taken to refer to a linear or circular DNA sequence which includes a gene construct as hereinbefore defined, and which includes any additional nucleotide sequences to facilitate replication in a host cell and/or integration and/or maintenance of said gene construct or a part thereof in the host cell genome.

Preferred vectors include plasmids, cosmids, plant viral vectors, and the like. More preferably, the vector consists of a plasmid or cosmid containing T-DNA to facilitate integration of DNA into the plant genome, such as, for example, binary transformation vectors, super-binary transformation vectors, co-integrate transformation vectors, Ri- derived transformation vectors, suitable for use in any known method of transforming plant cells, and tissues.

The term "vector" also includes any recombinant virus particle or cell, in particular a bacterial cell or plant cell, which comprises the gene construct of the invention. For example, a plant virus, such as a gemini virus, amongst others, may be engineered to express the cell cycle protein, or alternatively, a gene construct may be introduced into Agrobacterium tumefaciens or Agrobacterium rhizogenes, for subsequent transfer to a plant cell using art-recognised techniques for plant cell transformation. Accordingly, it is within the scope of the invention to include such embodiments.

The term "cell cycle" as used herein 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. However, the cell cycle also includes modified cycles such as endomitosis, acytokinesis, polyploidy, polyteny, endopolyploidisation and endoreduplication or endoreplication.

The term "cell cycle interacting protein", "cell cycle protein", or "cell cycle control protein" as denoted herein 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, homologues, alleles or precursors (eg preproproteins or preproteins) thereof.

Cell cycle control proteins and their role in regulating the cell cycle of eukaryotic organisms are reviewed in detail by John (1981) and the contributing papers therein (Norbury and Nurse 1992; Nurse 1990; Ormond and Francis 1993) and the contributing papers therein (Doerner et al. 1996; Elledge 1996; Francis and Halford 1995; Francis et al. 1998; Hirt er a/. 1991 ; Mironov er a/. 1999) which are incorporated herein by way of reference.

The term "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. 1989; Murray and Kirschner 1989; Renaudin et al. 1996; Soni et al. 1995; Sorrell et al. 1999; Swenson et al. 1986); cyclin dependent kinase inhibitor (CKI) proteins such as ICK1 (Wang et al. 1997), FL39, FL66, FL67 (PCT/EP98/05895), Sid , Far1 , Rum1 , p21 , p27, p57, p16, p15, p18, p19 (Elledge 1996; Pines 1995), 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; Russeell and Nurse 1987b) homologues of Cdc2 such as Cdc2MsB (Hirt et al. 1993) CdcMs kinase (Bbgre et al. 1997) Cdc2 T14Y15 phosphatases such as Cdc25 protein phosphatase or p80Cdc25 (Bell et al. 1993; Elledge 1996; Kumaghi and Dunphy 1991; Russell and Nurse 1986) and Pyp3 (Elledge 1996) Cdc2 protein kinase or p34Cdc2 (Colasanti et al. 1991 ; Feiler and Jacobs 1990; Hirt et al. 1991 ; John et al. 1989; Lee and Nurse 1987; Nurse and Bissett 1981 ; Ormrod and Francis 1993) Cdc2a protein kinase (Hemerly et al. 1993) Cdc2 T14Y15 kinases such as weel or p107wee1 (Elledge 1996; Russell and Nurse 1986; Russell and Nurse 1987a; Russell and Nurse 1987a; Sun et al. 1999) mik1 (Lundgren et al. 1991) and myt1 (Elledge 1996); Cdc2 T161 kinases such as Cak and Civ (Elledge 1996); Cdc2 T161 phosphatases such as Kap1 (Elledge 1996); Cdc28 protein kinase or p34Cdc28 (Nasmyth 1993; Reed et al. 1985) p40MO15 (Fequet et al. 1993; Poon et al. 1993) chkl kinase (Zeng et al. 1998) cdsl kinase (Zeng et al. 1998) growth associated H1 kinase (Labbe etal. 1989; Lake and Salzman 1972; Langan 1978; Zeng ef al. 1998) MAP kinases described by (Binarova et al. 1998; Bδgre et al. 1999; Calderini et al. 1998; Wilson et al. 1999).

Other 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. Other 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.

For the present purpose, the term "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. The term "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, CDC16, and plant-derived homologues thereof (Cohen-Fix and Koshland 1997; Hochstrasser 1998; Krek 1998; Lisztwan et al. 1998) and Plesse et al. in (Francis er al. 1998)).

A nucleotide sequence that encodes a cell cycle control protein will be understood to refer to any nucleotide sequence, or more preferably a structural gene or mutant thereof which is expressed in a plant cell to produce functional cell cycle control protein, wherein said protein 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 endoreduplication.

For the present purpose, the term "a nucleotide sequence that encodes a cell cycle control protein" shall be taken to be synomymous with the term "cell cycle control gene", to mean 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, Weel , Nim1 , MAP kinases, etc. Preferably, such a nucleotide sequence will exert such regulatory control at the post-translation level, via interactions involving the polypeptide product expressed therefrom.

More specifically, a "cell cycle control gene" means a gene which is involved in the control of entry of the cell into the S-phase and progression through the S phase, such as, for example, cyclin dependent kinases (CDK), cycline 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.

Cell cycle control genes further 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.

For the present purpose, the term "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 an animal mitogen or plant hormone (auxin, cytokinin, ethylene, gibberellic acid, abscisic acid and brassinosteroid).

Preferably, the gene construct of the invention comprises a cell cycle control gene which encodes a cell cycle control protein selected from the group consisting of: (i) a Cdc25 protein or a homologue, analogue or derivative thereof; (ii) a Cdc25 substrate protein that mimics the bioactivity of a Cdc25 protein or a homologue, analogue or derivative thereof; and (ii) a modified Cdc25 substrate protein that mimics the bioactivity of a Cdc25 protein, or a homologue, analogue or derivative thereof.

In a particularly preferred embodiment, the gene construct of the invention comprises a cell cycle control gene which encodes a Cdc25 protein or a homologue, analogue, or derivative thereof. In all cells, the switch that raises activity of Cdc2 at entry into mitosis is the Cdc25-catalysed removal of phosphate from threonine-14 and/or tyrosine-15 in Cdc2. Prior to the onset of mitosis the activity of the Cdc2 protein involved in this process is inactivated by wee-1 and/or mik-1 mediated phosphorylation of threonine-14 and/or tyrosine-15 in Cdc2. 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, animal cells contain several CDKs and several Cdc25 proteins. 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. 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.

"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.

To produce such homologues of a cell cycle control protein such as Cdc25, 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 α-helical structures or β-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. Preferably, 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. For example, 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.

Because the inventive method described herein involves modifying the G1/S and/or the G2/M phase transition of plant cells by ectopically expressing a cell cycle control protein therein, such as a Cdc25 protein, Cdc2 protein or cyclin protein, and, in particular a Cdc25 protein, and this expression alters the balance between Cdc25 dephosphorylation and wee-1/mik-1 phosphorylation of their respective substrates, the inventive method can be performed equally, using a Cdc25 substrate protein and/or modified Cdc25 substrate protein. The invention clearly encompasses gene constructs for the expression of such molecules. Accordingly, particularly 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, and which mimic the bioactivity of Cdc25.

In the present context, the term "Cdc25 substrate", or similar term, shall be taken to refer to any protein that is regulated directly or indirectly by Cdc25, and more particularly, any protein that is dephosphorylated by Cdc25, including, but not limited to cyclin-dependent kinase (CDKs). The CDK may be an A-type or a B-type CDK, preferably it is an A-type (or PSTAIRE-type CDK) and more preferably the CDK is Cdc2a, the key enzyme driving entry into S-phase and/or into mitosis (M-phase).

The inventors have also discovered that expression of a substrate of Cdc25, in particular a Cdc2 protein, in plants or plant cells can override the arrest of DNA replication (S phase) under growth-limiting conditions. Similarly, cyclin B also cooperates with Cdc25 to override the DNA synthesis checkpoint in plant cells. As a consequence, the ectopic expression of Cdc25, cyclin B or Cdc2 proteins in plant cells can partially sustain endoreplication.

As used herein, unless specifically stated otherwise, or the context requires otherwise, reference to "a Cdc2 protein" shall be taken to include a reference to a Cdc2a protein, and, in particular, a reference to a Cdc2a polypeptide of plant origin, and including the A. thaliana Cdc2a polypeptide. Similarly, reference herein to "a modified Cdc2 protein" shall be taken to include a reference to a modified Cdc2a protein, and, in particular, a reference to a modified Cdc2a polypeptide of plant origin, and including the A. thaliana Cdc2aAtA14Y15 polypeptide.

Without being bound by any theory or mode of action, the substitution or deletion of the phosphorylation sites of a protein that is an a substrate for a Cdc25 phosphoprotein phosphatase mimics the effect of a constitutive phosphatase activity, such as the effect of Cdc25 protein phosphatase ( p80 ^Cdc25) activity. Moreover, the substitution or deletion of the phosphorylation sites of a Cdc25 substrate (eg Cdc2a) further mimics the effect of down-regulated kinase activity, such as a down-regulation ofthe wee-1 kinase and/or the mik-1 kinase. Those skilled in the art will know that the wee-1 kinase and/or the mik-1 kinase adds the inhibitory phosphate on threonine-14 and/or tyrosine 15. Thus phosphorylated protein will not be produced at high steady- state concentrations in either the absence of phosphorylation or when phosphatases are expressed at raised levels or when kinase(s) is (are) expressed at lowered levels. Accordingly, the Cdc25-induced effects described herein can also be obtained by the regulated expression of a modified substrate of Cdc25.

Similar effects are also observed for cyclin protein expression, in particular mitogenic cyclin expression, such as, for example, cyclin B, and more particularly, CycMs2 cyclin expression, and, as a consequence, the present invention clearly extends to the use of nucleotide sequences encoding cyclin proteins in the construction of the selectable marker genes referred to herein.

The term "modified Cdc25 substrate" refers to a homologue, analogue or derivative of a Cdc25 substrate protein 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. For example, substitution of threonine and tyrosine at positions 14 and 15 of cyclin- dependent kinases (CDKs) for alanine and phenylalanine, respectively, can produce one or more cytokinin-like effects in the plant, similar to those observed following constitutive Cdc25 expression in the plant or alternatively to those observed following down-regulation of wee-1 and/or mik-1.

The term "modified Cdc25 substrate" also refers to a homologue, analogue or derivative of a substrate of Cdc25 that mimics the effect of Cdc25 to result in earlier switching from quiescence to active cycling and/or early entry of cells into S-phase and/or into mitosis (M-phase).

Accordingly, a modified Cdc25 substrate encompasses any Cdc2 protein, including Cdc2a, or a homologue, analogue or derivative thereof which is non- phosphorylateable, or dephosphorylated, such that the modification removes the inhibitory effect of the phosphates on Cdc2 (or Cdc2a) and/or removes inhibition of Cdc2 (or Cdc2a) resulting from phosphorylation by wee-1 and/or mik-1 , or otherwise increases Cdc2 (or Cdc2a) activity.

Preferably, a non-phosphorylatable form of Cdc2a is free of the phosphate at the tyrosine at position 15 (i.e. tyrosine-15 or Y15) and optionally (though not necessarily) free of the phosphate at threonine at position 14 (i.e. threonine-14 or T14).

A particularly preferred modified substrate of Cdc25 is a modified Cdc2a wherein both threonine-14 and tyrosine-15 have been substituted with alanine and phenylalanine, respectively, to produce Cdc2aA14F15.

The present inventors have shown that the ectopic expression of Cdc25, or a modified Cdc25 substrate which consists ofthe Cdc2aA14F15 polypeptide, or a cyclin B protein, produces growth effects and/or one or more cytokinin-like effects in plants, especially increased meristem activity and outgrowth of lateral buds, leading to increased branching of plants. Without being bound by any theory or mode of action, these effects are presumably the result of the ectopically-expressed protein being both active and expressed at a high level.

"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 ^Cdc25 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. Alternatively or in addition, 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.

Other examples of 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. Additionally, homopolymers or heteropolymers comprising one or more copies ofthe 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.

In a particularly preferred embodiment of the invention, the nucleotide sequence encoding a cell cycle control protein consists of a structural gene sequence encoding the fission yeast Cdc25 phosphoprotein phosphatase, or a biologically-active homologue, analogue or derivative thereof as described hereinabove, in particular a Cdc25 substrate or modified Cdc25 substrate. The present invention clearly contemplates the use of nucleotide sequences encoding plant-derived cell cycle control proteins which mimic the bioactivity of the fission yeast Cdc25 protein, and, in particular, a nucleotide sequence encoding a Cdc2aA14F15 protein.

The gene construct of the invention is preferably designed to facilitate expression of a functional cell cycle control protein for the selection of transformed plant cells. To effect expression of the cell cycle control protein in a plant cell, tissue or organ, either the protein may be introduced directly to said cell, such as by microinjection means or altematively, an isolated nucleic acid molecule encoding said protein may be introduced into the cell, tissue or organ in an expressible format.

By "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 intracellular or extracellular signal, such as an environment 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, auxin, cytokinin, glucocorticoid, etc), hormone analogue (iodoacetic acid (IAA), 2,4-D, etc), metal (zinc, copper, iron, etc) or dexamethasone, amongst others. As will be known to those skilled in the art, 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".

Preferably, 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, syntectic 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-operable promoter sequence.

Reference herein to a "promoter" is to be taken in its broadest context and 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 (ie., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.

The term "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.

The term "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. For example, copper-responsive, glucocorticoid-responsive ordexamethasone-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.

In the context of the present invention, the promoter is a plant-operable promoter sequence. By "plant-operable" 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.

The terms "plant-operable promoter sequence" and "promoter sequence operable in a plant" or similar term shall be taken to be equivalent to the term "plant-expressible promoter sequence".

In the present context, a "regulatable promoter sequence" is a promoter that is capable of conferring expression on a structural gene sequence, in particular a cell cycle protein-encoding nucleotide sequence, in a particular cell, tissue, or organ or group or cells, tissues or organs of a plant, optionally under specific conditions, however is generally not expressed throughout the plant under all conditions. Accordingly, 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.

Preferably, the regulatable promoter sequence is selected from the group consisting of: cell-specific promoter sequences; tissue-specific promoter sequences; organ- specific promoter sequences; cell cycle gene specific promoter sequences; tissue- specific inducible promoter sequences; environmentally-inducible promoter sequences; chemically-inducible promoter sequences; wound-inducible promoter sequences; hormone-inducible promoter sequences; and pathogen-inducible promoter sequences.

The term "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.

Similarly, the term "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.

Similarly, the term "organ-specific" shall be taken to indicate that expression is predominantly in a particularly plant organ albeit not necessarily exclusively in that plant organ.

Similarly, the term "cell cycle specific" or similar shall be taken to indicate that expression is predominantly under control of the cell cycle, or capable of being cyclic such that it occurs in one or more phases of the cell cycle, albeit not necessarily in consecutive phases of the cell cycle, or in cycling cells. ln an alternative embodiment, the promoter is a constitutive promoter sequence, subject to the proviso that said promoter sequence is integrated into an excisable genetic element.

As will be known to those skilled in the art, a "constitutive promoter sequence" is a promoter sequence that confers expression predominantly throughout the plant, albeit not necessarily in every cell, tissue or organ under all conditions, a strong constitutive promoter is one which confers a high level of ectopic expression on a structural gene to which it is operably connected, predominantly throughout the plant, albeit not necessarily in every cell, tissue or organ under all conditions.

Those skilled in the art will readily be capable of selecting appropriate promoter sequences for use in regulating appropriate expression of the cell cycle control protein from publicly-available or readily-available sources, without undue experimentation, such as, for example, any one or more of the promoters listed in Table 1.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.

TABLE 1 EXEMPLARY PLANT-OPERABLE PROMOTERS FOR USE IN PERFORMING THE PRESENT INVENTION

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Placing a nucleic acid 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.

In the construction of 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 (ie., 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. Similarly, 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 (ie., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.

Preferred tissue-specific inducible promoter sequences include the light-inducible rbcs- 1A or rbcs-3A promoter sequences, anoxia-inducible maize Adh1 gene promoter sequences (Howard et al., 1987; Walker et al., 1987), hypoxia-inducible maize Adh1 gene promoter sequences (Howard et al. 1987; Walker et al., 1987), and the temperature-inducible heat shock promoter sequences. Such environmentally- inducible promoters are reviewed in detail by Kuhlemeier ef al. 1987.

Preferred chemically-inducible promoter sequences include the 3-β- indoylacrylic acid- inducible Tip promoter; IPTG-inducible lac promoter; phosphate-inducible promoter; L-arabinose-inducible araS 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 ef al. 1996); and any one or more 5 of the chemically-inducible promoters described by Gatz ef al. (1996), amongst others.

Preferred wound-inducible or pathogen-inducible promoter sequences include the phenylalanine ammonia lyase (PAL) gene promoter (Ebel ef al. 1984), chalcone synthase gene promoter (Ebel ef al., 1984) or the potato wound-inducible promoter 0 (Cleveland et al, 1987), amongst others.

Preferred hormone-inducible promoter sequences include the abscisic acid-inducible wheat 7S globulin gene promoter and the wheat Em gene promoter (Marcotte et al., 1988); an auxin-responsive gene promoter; ora gibberellin-inducible promoter such 15 as the Amy32b gene promoter (Lanahan ef al., 1992), amongst others.

Preferred constitutive plant-operable promoter sequences include the CaMV 35S promoter sequence, CaMV 19S promoter sequence, the octopine synthase (OCS) promoter sequence, or nopaline synthase (NOS) promoter sequence (Ebert ef al, 20 1987), amongst others.

In the case of constitutive promoters or promoters that induce expression throughout the entire plant, these may be modified by the addition of nucleotide sequences derived from one or more of the tissue-specific promoters listed in Table 1 , or 25 alternatively, nucleotide sequences derived from one or more of the above-mentioned tissue-specific inducible promoters, to confer tissue-specificity thereon. For example, the CaMV 35S promoter may be modified by the addition of maize Adh1 promoter sequence, to confer anaerobically-regulated root-specific expression thereon, as described previously (Ellis ef al., 1987). Such modifications can be achieved by routine experimentation by those skilled in the art and are clearly encompassed by the present invention.

Preferably, the selectable marker gene further includes a transcription termination sequence which is operable in a plant (i.e. a "terminator"), placed operably in connection with the nucleotide sequence encoding the cell cycle control protein, and, preferably, placed downstream, or at the 3'-terminus, of said nucleotide sequence.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signal 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.

Examples of 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 lea mays zein gene terminator sequence, the RBC-1A gene terminator, and the RBC-3A gene terminator sequences, amongst others.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for sue in performing the invention. Such sequences may readily be used without any undue experimentation.

Those skilled in the art will be well aware of the requirements for expressing foreign genes in plants and, as a consequence, will readily be capable of producing the gene construct of the invention, and expressing same in plant cells, tissues, organs, or whole plants, from the teaching provided herein.

In particular embodiments of the invention, it is desirable to remove the selectable marker gene of the invention following selection of transfected or transformed cells expressing said gene, such as, for example, to avoid any undesirable side-effects arising from ectopic expression of the cell cycle control protein in specific cells, tissues or organs of the regenerated plant, or progeny plants derived therefrom. This may be achieved by including one or more excisable genetic elements adjacent to, or in proximity to, the 5'- and/or 3'- ends of the selectable marker gene.

As used herein, the term "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. By "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. In performing the present invention, 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. Preferably, 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 frt nucleotide sequences.

Known site-specific recombination systems, for example the crellox system (Dale and Ow, 1991 ; Russell et al, 1992) and the flp/frf system (Lloyd and Davis, 1994; Lyznik ef al, 1995) which comprise a loci for DNA recombination flanking a selected gene, specifically lox or frt genetic sequences, combination with a recombinase, ere or tip, which specifically contacts said loci, producing site-specific recombination and deletion of the selected gene. In particular, 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.

Furthermore, International Patent Application No. PCT/US92/05640 (The United States of America as represented by the Secretary of Agriculture, USA) filed 6 July, 1992 discloses a method of excising and segregating selectable marker genes in higher plants using site-specific recombination systems such as the cre//ox or flp/fr systems wherein plant cells are first transformed with a recombinant vector which contains a plant-expressible selectable marker gene operably linked to loci for DNA recombination and the selectable marker gene is subsequently excised from transformed plants by further transforming the plant cells with a second recombinant vector which contains a plant-expressible, site-specific recombinase gene or, alternatively, by cross- pollinating the first-mentioned transformed plant with a second transformed plant which expresses a recombinant site-specific recombinase. As a consequence, the selectable marker gene contained in the first-mentioned transformed plant is excised. According to PCT/US92/05640, the recombinant site-specific recombinase gene is also linked to a selectable marker gene which must be removed to produce a plant which is free of selectable marker transgenes. This approach, therefore, requires at least one generation of conventional plant breeding to remove the second selectable marker gene.

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. Preferably, a site-specific recombinase will induce excision of intervening DNA located between two such recombination loci. The terms "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.

Preferred recombinase genes according to the present invention include the ere gene (Abremski ef a/., 1983) and flp gene (Golic et al., 1989; O'Gorman et al., 1991 ). In a particularly preferred embodiment of the present invention, the recombinase gene is the ere gene or a homologue, analogue or derivative thereof which is capable of encoding a functional site-specific recombinase.

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. In a particularly preferred embodiment of the present invention, 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 selectable marker gene of the invention. As will be known to those skilled in the art, 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.

For example, 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. In one embodiment of the current invention, 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.

Alternatively, a substantially purified recombinase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment. Typically, the site-specific recombinase coding region will be operably linked to regulatory sequences enabling expression of the site-specific recombinase in the eukaryotic cell. In a preferred embodiment of the present invention, the site-specific recombinase sequences is operably linked to an inducible promoter.

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 (Dale ef al. 1990), and A. thaliana (Osborne ef al. 1995).

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.

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.

In a most particularly preferred embodiment, however, 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 ef 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.

As will be known to those skilled in the art, for recombination mediated by a transposon to occur, a pair of DNA sequences comprising inverted repeat transposon border sequences, flanking the excisable genetic element sequence, and a specific transposase enzyme, are required. The 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 Spm system and the Mu system. All of these systems are operable in Zea mays, and at least the Ac/Ds and the Spm system function in other plants (Fedoroff et al. 1993, Schlappi ef al. 1993, Van Sluys et al. 1987).

Preferred transposon sequences for use in the gene constructs ofthe 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.

As with the use of site-specific recombinase systems, 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 the selectable marker gene of the invention. 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.

For example, 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. Alternatively, a substantially purified transposase protein could be introduced directly into the eukaryotic cell, eg., by micro-injection or particle bombardment. Typically, the transposase coding region will be operably linked to regulatory sequences enabling expression of the transposase in the eukaryotic cell. In a preferred embodiment of the present invention, the transposase-encoding sequence is operably linked to an inducible promoter.

In the present context, transposon border sequences are organized as inverted repeats flanking the excisable genetic element. As 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 selectable marker.

Likewise, 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. Alternatively, said site-specific recombinase gene or transposase gene are included in the same or in a different excisable genetic element as the selectable marker gene.

Preferably, the gene construct further comprises one or more nucleotide sequences comprising gene(s)-of-interest, which are intended to be introduced into the plant cell, tissue, organ, plantlet, or whole plant, to confer one or more genotypes or phenotypes thereon. However, wherein the gene construct utilises an excisable genetic element, this is positioned such that recombination mediated by the excisable genetic element does not result in a recombination event leading to excision of the gene(s)-of-interest. For example, the excisable genetic element flanking the selectable marker gene can be positioned at some distance from the gene(s)-of-interest.

Although intended for the transformation of a eukaryotic organism and/or the expression of genes contained therein, the gene constructs of the present invention may need to be propagated in a prokaryotic organism such as the bacteria Esche chia coli or Agrobacterium tumefaciens. Accordingly, the gene constructs described herein may further comprise genetic sequences corresponding to a bacterial origin of replication and/or a second, bacteria-operable selectable marker gene, such as, for example an antibiotic-resistance gene, suitable for the maintenance and replication of said gene construct in a prokaryotic organism. Such sequences are well-known in the art. Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the gene construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.

Preferred originals of replication include, but are not limited to, the f1-oή and co/E1 origins of replication.

Suitable bacteria-operable selectable marker genes include the ampicillin resistance (Amp^r), tetracycline resistance gene (Tc^r), bacterial kanamycin resistance gene (Kan^r), neomycin phosphotransferase gene (πpfll), hygromycin resistance gene, β- glucuronidase (GUS) gene, and chloramphenicol acetyltransferase (CAT) gene, amongst others.

In a further embodiment of the present invention, the gene construct of the present invention is also suitable for integration into the genome of a cell in which it is expressed. Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or gene construct into the genome of a host cell, certain additional genetic sequences may be required. For example, the successful integration of DNA into the genome of a plant cell mediated by Agrobacterium tumefaciens requires the presence of one or more left and/or right T-DNA border regions flanking the genetic sequence to be integrated. Accordingly, the gene construct of the invention may optionally further comprise additional genetic sequences as required for its integration into the genome of a eukaryotic cell, in particular a plant cell.

Wherein the gene construct of the invention is intended for use in plants, it is particularly preferred that it be further modified for use in >4grofjacfer/wm-mediated transformation of plants by the inclusion of one or more left and/or right T-DNA border sequences. To facilitate Agrobacferium-mediated transformation, the selectable marker gene, with or without flanking excisable genetic element sequences, and, where applicable, the gene(s)-of-interest, are usually placed between the left and/or right T- DNA border sequences, if more than one of said sequences is present.

Surprisingly, the gene constructs developed by the inventors are useful in selecting transformed plant cells in the absence of exogenous cytokinin or gibberellin. Based upon the role of cell cycle control proteins in producing cytokinin-mediated and/or gibberellin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics in plants, as elucidated by the present inventors, the gene construct of the invention may be applied to the selection of any cell, tissue, organ or whole organism that expresses and/or exhibits cytokinin-mediated and/or gibberellin-mediated morphological characteristics and/or biochemical characteristics and/or physiological characteristics, from a background of cells, tissues, organs or whole organisms that do not exhibit such characteristics. By way of exemplification, the formation of transformed calli from tobacco leaf discs on medium lacking exogenous cytokinin, is provided in Example 6. The transformed calli selected using the inventive method described herein are regenerated into whole transgenic plants.

Accordingly, a second aspect of the present invention provides a method of detecting or identifying a transformed or transfected plant cell, tissue, organ, or plantlet that is hormone-dependent, comprising expressing a cell cycle control protein in said plant cell, tissue or organ is under the operable control of a plant-operable promoter sequence, for a time and under conditions sufficient for the normally hormone- mediated cell division and/or hormone-mediated tissue differentiation to occur in the absence of the hormone.

According to this embodiment ofthe invention, because the cells in which the invention is performed are hormone-dependent and selection is carried out in the absence of those hormones which are required for hormone-mediated cell division and/or hormone-mediated tissue differentiation to occur, only those cells which contain and express the cell cycle control protein will divide and differentiate into tissues, organs, plantlets, or whole plants.

As used herein, the term "hormone-dependent" means any cell, tissue or organ that requires the exogenous application of a cytokinin or gibberellin to facilitate or produce cell division and/or cell proliferation in primary culture in vitro.

Those skilled in the art may be aware that plant cells in culture require cytokinin as well as auxin for cell proliferation. In a minority of tissues, such as the shoot internode meristem of the Graminae, a hormone other than cytokinin, in particular gibberellin, appears to be required for cell proliferation.

The present inventors have confirmed the cytokinin requirement, for tissues other than the shoot internode meristem of the Graminae. In particular, the inventors have observed that whilst auxin alone is able to stimulate the enlargement cells derived from excised tobacco pith tissue, cytokinin is also required for cell division to occur. Only in the presence of both auxin and cytokinin do tobacco cells proliferate and form callus, as measured by the incoφoration of bromodeoxyuridine (BrdU) into replicating DNA.

With regard to the dependence of cells upon gibberellin, Sauter ef al. (1995) showed that within 4 hours of gibberellin application to rice stems, a synchronous decline occurs in the number of cells that are in G2 phase, and cells move into mitosis, indicating that the G2-arrested cells had been stimulated. However, the application of gibberellins to plant tissues to promote cell division is also accompanied undesirable pleiotropic side-effects, in particular the induction of flowering, stem elongation, seed germination, fruit and seed development.

The inability of genetically unmodified plant cells derived directly from the plant to divide without added cytokinin or gibberellin in addition to auxin forms the basis for the positive selection method of the present invention, which depends upon the unexpected ability of cell cycle control protein expression to replace the requirement for these hormones. In this embodiment of the invention, plant cells that ectopically- express a cell cycle control protein, in particular a Cdc25 protein, or a Cdc25 substrate or modified Cdc25 substrate that mimics the bioactivity of Cdc25, under the control of a plant-operable promoter sequence, proliferate without added cytokinin or, in the case of certain tissues such as the shoot meristem internode, without added gibberellin, thereby providing a strong positive selection for transformed or transfected cells on medium lacking cytokinin or gibberellin, as the case may be. This positive selection for transgenic cells is particularly advantageous for plant breeding by gene transfer, wherein the cell cycle control gene operably in connection with a plant-expressible promoter is introduced into the plant cell at the same time as the gene(s)-of-interest.

Accordingly, it is particularly preferred that the hormone-dependent cell, tissue, or organ is a gibberellin-dependent or cytokinin-dependent cell, tissue, or organ. Preferably, the gibberellin-dependent plant tissue is the meristem shoot internode or intercalary meristem ofthe youngest stem internode derived from a monocotyledonous plant species, in particular the Graminae.

As used herein, the term "gibberellin-dependent" shall be taken to refer to a naturally- occurring plant cell, tissue or organ that at least requires the application of exogenous gibberellin to promote cell division and/or proliferation in vitro.

As used herein, the term "cytokinin-dependent" shall be taken to refer to a naturally- occurring plant cell, tissue or organ that at least requires the application of exogenous cytokinin to promote cell division and/or proliferation in vitro.

In a preferred embodiment, expression of the cell cycle control protein is achieved by expressing nucleic acid encoding the cell cycle control protein, preferably by introducing a gene construct as described herein into a plant cell by standard transfection or transformation procedures and incubating said cell under conditions sufficient for expression of the cell cycle control protein encoded by the selectable marker gene to occur.

The selectable marker gene, or a gene construct comprising the same, may be introduced into a cell using any known method for the transfection or transformation of said cell. Wherein 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.

As stated supra, the present inventive method includes a situation wherein nucleotide sequence(s) comprising one of more gene(s)-of-interest which are introduced to the plant, and optionally, expressed therein to confer a desired phenotype thereon. The requirements for expression of the gene(s)-of-interest in the plant cell are the same as for the expression of other structural genes in plants (e.g., a suitable plant-operable promoter sequence, transcriptional termination sequence, appropriate codon usage, etc). As will be known to those skilled in the art, such gene(s)-of-interest may be introduced to the plant cell on the same gene construct as the selectable marker gene of the invention. Alternatively, gene(s)-of-interest may be introduced to the plant cell on a separate nucleic acid molecule, wherein it is preferably introduced simultaneously to the plant cell with the gene construct comprising the selectable marker gene of the invention, such as by co-transfection, co-transformation, co-electroporation, or as a co- precipitate on gold or other microprojectile particles, etc.

As will be known to those skilled in the art, if the gene(s)-of-interest are introduced to the plant on a separate gene construct to the gene construct of the invention, then that gene construct may comprise its own nucleotide sequences required for maintenance and/or replication in prokaryotic cells, and for expression and/or integration in the plant cell. In cases of co-transfection/co-transformation, cells are initially selected by virtue of the expression of the selectable marker gene of the invention, and a further selection is performed to confirm and identify those cells carrying both gene constructs, such as, for example by nucleic acid detection methods, immunological methods, or, if the gene-of-interest is expressed to produce a visible phenotype, by the observation of that phenotype. The present invention encompasses all such embodiments.

By "transfect" is meant that the gene construct or vector or an active fragment thereof comprising the cell cycle control gene operably under the control of the promoter sequence is introduced into said cell without integration into the cell's genome.

By "transform" is meant that the gene construct or vector or an active fragment thereof comprising the cell cycle control gene operably under the control of the promoter sequence is stably integrated into the genome of the cell.

Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI₂ and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens ef al, 1982; Paszkowski ef al., 1984), PEG-mediated uptake to protoplasts (Armstrong ef al. 1990) microparticle bombardment, electroporation (Fromm ef al, 1985), microinjection of DNA (Crossway ef al. 1986), microparticle bombardment of tissue explants 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 of Agrobacterium to the plant tissue as described essentially by An ef. al. (1985), Herrera-Estrella ef al. (1983a, 1983b, 1985).

For microparticle bombardment of cells, 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 ef a/., (US Patent No. 5,122,466) and Sanford and Wolf (US Patent No. 4,945,050). When using ballistic transformation procedures, the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 μm 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, whether by organogenesis or embryogenesis, 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. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (eg., apical meristem, axillary buds, and root meristems), and induced meristem tissue (eg., cotyledon meristem and hypocotyl meristem).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

The regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, 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 (eg., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (eg., in plants, a transformed root stock grafted to an untransformed scion). ln accordance with the inventive method, selection and/or regeneration is performed in the absence of exogenous hormone in the culture medium. For example, in the case of cytokinin-dependent cells and tissues, these processes are performed in the absence of exogenous cytokinin. Additionally, gibberellin-dependent cells and tissues will be cultured in the absence of exogenous gibberellin. It is particularly preferred for the hormone-free media to be used during callus-induction and/or during the regeneration of organised plant tissues from transformed selected cells or transformed selected calli.

Preferably, 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. In a particularly preferred embodiment, plants are transformed by an in planta method using Agrobacterium tumefaciens such as that described by Bechtold ef a/., (1993) or Clough ef 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. Those skilled in the art will be aware that the selection of tissue for use in such a procedure may vary, however it is preferable generally to use plant material at the zygote formation stage for in planta transformation procedures.

Without being bound by any theory or mode of action, the inventors have discovered that cytokinin modifies the phosphorylation of a Cdc25 substrate, in particular a Cdc2 protein and, as a consequence, the transformation and/or regeneration of plants in the absence of cytokinin facilitates the recovery of plants which express said Cdc25 substrate and functional homologues, analogues and derivatives thereof at a sufficiently high level to show significant differences from wild-type non-transformed plants. Accordingly, the transformation of plants using gene constructs comprising nucleotide sequences encoding Cdc25, substrates or modified substrates of Cdc25, in particular a Cdc2 protein, such as, but not limited to, Cdc2a or modified Cdc2a, and, in particular Cdc2aA14F15, placed operably under the control of strong, constitutive promoter sequences, are particularly preferred for the purposes of selecting transformed plant cells and tissues in accordance with the present invention.

Wherein the selectable marker gene is integrated into an excisable genetic element, it is particularly preferred that the excisable genetic element is induced to excise, together with the integrated nucleotide sequence encoding the cell cycle control protein, following the selection process, such that the selected cells and/or differentiated cells, and/or the regenerated plant tissues, plantlets or whole plants, are no longer capable of expressing the cell cycle control protein. In this way, the transformed plants cells and tissues express the introduced gene(s)-of-interest, but do not exhibit the hormone-mediated characteristics conferred by the introduced cell cycle control protein.

Accordingly, a third aspect of the invention provides plant cells, tissues, organs and plant parts, propagules and progeny plants that have been selected using the inventive process described herein or alternatively or in addition, which comprise the gene constructs of the invention. Such plants may be substantially free of the selectable marker gene, particularly if produced using a selectable marker gene integrated into an excisable genetic element, and subsequent excision of the excisable genetic element.

The positive selection method of the present invention is applicable to any plant that is amendable to transformation using known procedures, such as, for example, any monocotyiedonous 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.,Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.

Accordingly, 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.

Those plants and plant parts that have been produced according to the inventive method are identified by standard nucleic acid hybridisation and/or amplification techniques to detect the presence of the selectable marker gene or a gene construct comprising same. Alternatively, in the case of transformed plant cells, tissues, and plants wherein the selectable marker gene has bene excised, it is possible to detect a footprint in the genome of the plant which has been left following the excision event, using such techniques.

As used herein, the term "footprint" shall be taken to refer to any derivative of a selectable marker gene or gene construct described herein which is produced by excision, deletion or other removal of the selectable marker gene from the genome of a cell transformed previously with said gene construct.

A footprint generally comprises at least a single copy of the recombination loci or transposon used to promote excision. However, a footprint may comprise additional sequences derived from the gene construct, for example nucleotide sequences derived from the left border sequence, right border sequence, origin of replication, recombinase-encoding or transposase-encoding sequence if used, or other vector- derived nucleotide sequences. Accordingly, a footprint is identifiable according to the nucleotide sequence of the recombination locus or transposon of the gene construct used, such as, for example, a sequence of nucleotides corresponding or complementary to a lox site or frt site.

Art-recognised methods are used to identify the plants produced according to the inventive method, such as, for example, the standard nucleic acid hybridisation and/or amplification methods described by Ausubel et al. (1992) and/or McPherson ef al. (1991 ) and/or Sambrook ef al. . (1989), which are incorporated herein by way of reference.

The present invention is further described with reference to the following non-limiting Examples and to the drawings.

EXAMPLE 1

CELL CULTURE, PROTEIN AND ENZYME METHODS

Cell culture Suspension cultured cells of Nicotiana plumbaginifolia 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.

Antibodies

Polyclonal antibodies were raised in rabbits using the carboxy terminal amino acid sequence of the tobacco cdc2 protein, designated as cdc2a, as an immunogen. 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.

Assay of cdc2 and Cdc25 activities

Both cdc2 and Cdc25 enzyme activities were extracted from tobacco cells, by grinding the cells in liquid nitrogen. For cdc2 extraction, NDE buffer containing 25Mm HEPES (pH 7.2) with protease and phosphatase inhibitors was used. For Cdc25 extraction, PDE buffer, containing 25mM MOPS (pH 7.2), 100mM NaCl, 10mM DTT, 5jM EDTA, 1 mM EGTA, 1% NP-40, 50mM NaF, 0.5mM PMSF, 3μg ml^'1 leupeptin, and 20μg ml^"1 aprotinin, was used.

Immunoprecipitates of cdc2 and Cdc25 were obtained by reaction with 25 I 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 I 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 2μM spermidine.

To measure cdc2 activity, the phosphorylation of HI histone was followed. To measure Cdc25 activity, assays were conducted in two stages. First, Cdc25 immunoprecipitates from 500μg total soluble plant protein were incubated for 30 min at 30 °C in Cdc25 assay buffer with 0.25μg tyrosine phosphorylated cdc2 substrate that had been purified with p13^sυc1 -beads from 500μg protein arrested Cdc25-22 mutant fission yeast. The phosphatase reaction was stopped by removing the complexed Cdc25/cdc2 by sedimentation. In the second stage of the Cdc25 assay, 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 5mg of extracted plant protein was used as starting material, and the NDE buffer was modified to include 2.5mM sodium vanadate and 1mM phosphotyrosine, and the immune complexes were washed with HDW buffer supplemented with 1 mM with sodium vanadate.

Western blots of cell-derived protein were probed with anti-phosphotyrosine mouse monoclonal (PY99, Santa Cruz Biotechnology, S.C, USA), followed by [¹²⁵l]-labelled second antibody, and the signal obtained was detected by Phosphorimage analysis.

Northern blots RNA was extracted from cells ground in liquid nitrogen, into 2 volumes of 10 mM Tris/HCI (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 % EDTA, 1% (w/v) SDS and 2 volumes of phenol:chloroform:iso-amylalcohol 25:24:1 at 4c and fractioned. RNA was electrophoresed on agarose gels, transferred to membrane and probed with the 65- bp BglU-Xbal fragment of the Cdc25 gene, using standard procedures. EXAMPLE 2

Expression of yeast Cdc25 makes cell division in plant cells independent of cytokinin

The effect of ectopic expression of yeast Cdc25 in plants 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 enzymatically 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. When cytokinin stimulates entry into mitosis, dephosphorylafion of cdc2 is one of the events that occur, but it was uncertain whether the hormone might have several effects in the cell cycle.

To test this possibility we therefore arranged the inducible expression of the fission yeast Cdc25 gene tobacco under the control of a dexamethasone-inducible promoter. Only if the sole essential action of cytokinin is to cause dephosphorylafion and activation of cdc2 kinase can the ectopic expression of the Cdc25 gene substitute for presence cytokinin at mitosis.

We now report that the sole essential action of 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. The GRE-Cdc25, together with the constitutively- expressed NOS promoter-GR construct, were inserted into the vector pBin19, which contains pnos;nptil 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.

At the high concentration of 10μM dexamethasone, cells commonly arrested at prophase in mitotic catastrophe but lower inducer concentrations allowed colony formation and generated cell lines in which inducible expression of Cdc25 was detected by Western blot analysis using antibody against glutathione-S-transferase (GST)-Cdc25 fusion protein.

Inducible cell lines contained yeast Cdc25 DNA (detected by Southern blots, now shown) and in 0.01-1 OμM 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 0.01-1 OμM induced division (Figure 1 -3) and a sharp optimum concentration of 0.1 μM dexamethasone was observed in independent clones, consistent with requirement for 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.

Induced synthesis of Cdc25 in cells at the cytokinin control point in late G2 resulted in appearance of Cdc25 activity, which was detected by its activation of yeast cdc2 HI histone kinase that was provided a substrate in low activity form, phosphorylated on tyrosine 15 and amenable to activation by Cdc25 (Figure 2-1 to Figure 2-7). The induced Cdc25 phosphatase activity peaked at 6 h and provides an explanation for the increase in cdc2 kinase activity, which increased while Cdc25 was active (Figure 2-2). Specific recovery of cdc2a and Cdc25 was indicated by precompetifion with cdc2a peptide antigen and by preimmune anti-Cdc25 serum or anti-Cdc25 antibody precompeted with inactive GST-Cdc25 (Figure 2-1.

To test whether the effectiveness of ectopically expressed Cdc25 derived from the operation of mechanisms present in normal mitosis, transgenic cells induced with dexamethasone were monitored for Cdc25 phosphatase and cdc2 kinase activity

(Figure 2-2; Figure 2-3) in parallel with cells induced with cytokinin (Figure 2-3; Figure

2-4). Both showed increase in Cdc25 activity and then cdc2a kinase activity leading to division. A control over Cdc25 activity at post-translational level is indicated by the absence of a higher Cdc25 catalytic activity when yeast enzyme was expressed in addition to the endogenous Cdc25 (Figure 2-2; Figure 2-4). This suggests that the additional yeast enzyme comes under homeostatic controls that are conserved between yeasts and plants. Post-translational control of Cdc25 activity is known to be complex, tolerant to different levels of the protein, and to involve activating phosphorylations and ubiquitin-directed proteolysis.

The temporal correlation of induced Cdc25 phosphatase activity with increase in cdc2 kinase activity (Figure 2-2; Figure 2-4) suggested that the phosphatase is responsible. We tested this by investigating whether Cdc25 enzyme could activate cdc2 from cells in prophase and whether the extent of activation by Cdc25 declined when activation had already occurred in vivo. Data presented in Figure 2-5 show that excess bacterially-synthesised CST-Cdc25 could activate plant cdc2 enzyme that was extracted in prophase between 3 hours and 12 hours, and that the extent of activation declined in proportion with activation that had previously occurred. These data are consistent with the increase in Cdc25 phosphatase driving prophase progression by dephosphorylating cdc2. After 12 hours, cdc2 activity declined during anaphase and the enzyme then became unresponsive to GST-Cdc25, consistent with the anaphase decline in activity being due to proteolysis of cyclin, as observed for cyclin 1 b in maize mitosis.

The low level of Cdc25 activity in cells that are arrested by limiting cytokinin, as at time zero (ie., 0 hours) in Figure 2, indicates that down-regulation of Cdc25 activity is part of the cytokinin control mechanism and that induced Cdc25 therefore provides a biologically relevant signal. 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. Thus, unexpectedly 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. EXAMPLE 3

Evidence for the presence of Cdc25 protein in plant cells

The ability of 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 an immunoprecipitafion 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 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, unambigously 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).

We also assayed phosphotyrosine in the cdc2a kinase that increased in activity when the hormonal block was released. As shown in Figure 2-7, levels of tyrosine phosphate in cdc2a declined after induction of Cdc25, as the catalytic activity of cdc2a increased (Figure 2-2), indicating that a decline in phosphotyrosine caused by induction of Cdc25 in transgenic cells stimulates entry of cells into mitosis.

To further test the evidence for Cdc25 presence in genetically unmodified plant cells, we tested for immunological cross-reactivity between plant Cdc25 and authentic fission yeast Cdc25. In western blot analyses, antibodies against fission yeast Cdc25 detected a protein of 67 kDa in a tobacco cell fraction obtained using the mitotic protein p13^suc1 as an affinity ligand to purify cell cycle proteins (Figure 3, lane 1 ). Moreover, the binding of antibody to this 67 kDa tobacco protein was eliminated by pre-competition with authentic yeast Cdc25 protein (Figure 3, lane 2), suggesting that the yeast and plant Cdc25 protein share protein epitopes, such as primary amino acid sequences, secondary, or tertiary structures. The size of the 67 kDa tobacco protein correlates with the known size of other Cdc25 molecules.

EXAMPLE 4

Expression of 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 40ml 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). When inserted into the genome of the transgenic plant cells induction could be obtained by the presence of dexa methasone. The GRE-cdc25, together with the constitutively- expressed NOS promoter-GR construct, were inserted into the vector pBin19, which contains pnos ptil for kanamycin resistance, and was introduced into cells of N. plumbaginifolia by electroportion into protoplasts. Clones resistant to kanamycin were selected and their arrest properties investigated. Non transgenic cells (A) and transgenic cells (B) were cultured in identical medium containing 0.1 micromolar dexamethasone (see Figure 5). After 6 days of growth, when cell cycle progression was arrested in the non-transgenic cells, cells were harvested by centrifugation at gav = 1643 for 3 min in a swing out head. Cells were washed once with washing buffer (0.3 M Mannitol, 0.12 M NaCl, 3.0 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 5.8) then resuspended in two volumes of digestion solution (washing buffer supplemented with cell wall digesting enzymes; with 2%(w/v) cellulysin (Calbiochem), 2% driselase (Fluka), 2% macerozyme (Calbiochem) and 4% hemicellulase (Sigma). Wall digestion was carried out at 37°C with shaking at 120-125 rpm and was monitored by microscopy for 90 min until all cells were converted into spherical protoplasts. The protoplasts were collected by centrifugation at 500rpm (gav=42) for 3 minutes and washed once with washing buffer. Release of the nuclei was done by adding 2 ml of Galbraith buffer (45 mM Mg CI2, 30 mM sodium citrate, 20 mM 3-(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. Nuclei were stained with propidium iodide (PI) by incubation in PI solution of 20 microgram/ml in 1 mM ethylenediaminetetracetic 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. Both cultures were sampled after 6 days of batch culture, when cell cycle progress has arrested at a G1/S control point in non-transgenic cells (2N peak in A) but has been driven through this point in a majority of the transgenic cells resulting in accumulation mostly in G2 phase (4n peak in B) and in some cases an additional traverse of S 5 phase has been induced without intervening mitosis, resulting in endoreduplication of the genome and generation of 8n nuclei (seen in B).

EXAMPLE 5

Cdc25 as a selectable marker gene in transformed plants 0 In the present example of the application of the present invention, discs excised from tobacco leaves are treated with Agrobacterium tumefaciens containing the binary vector pBIN19 comprising within the T-DNA borders the following gene constructs:

(1 ) the nptll gene placed operably under control of the pNOS promoter and upstream of the NOS terminator; 5 (2) the fission yeast Cdc25 gene placed operably under the control of a modified plant promoter that contains rat glucocorticoid response elements (GREs), which are responsive to rat glucocorticoid receptor protein (GR) in the presence of dexamethasone (Schena et al., 1991 ), downstream flanked by a CaMV35S terminator; and 0 (3) a NOS promoter-GR gene-NOS terminator construct that expresses GR constitutively in plant cells.

The binary vector is introduced into plant cells, essentially according to De Block ef a/., (1993). Agrobacfer/um-treated leaf discs are incubated on a medium containing B5 salts supplemented with 250 mg/l ammonium nitrate, 20 g/l glucose, 0.5 g/l MES, 25 40 mg/i adenine, 0.8% agar, 0.1 mg/l IAA, 500 mg/l cefotaxime and 50 mg/l kanamycine (pH 5.7). When incubated on this medium that contains auxin but lacks cytokinin, in the presence of 0.1 to 1 μM dexamethasone, only cells that are expressing the introduced Cdc25 gene survive and proliferate (compare Figure 6A, no dexamethasone added and Figure 6B, dexamethasone added) whereas untransformed cells die. The calli resulting from sustained rapid proliferation of the transformed cells (see Figure 6B) are transferred after 3 to 5 weeks to the same medium without dexamethasone and IAA, but with 1 mg/l BA to allow shoot formation.

When the transformed plant tissue is incubated on agar medium that contains auxin and 1 M dexamethasone, but lacking cytokinin, only cells containing the binary vector in a format such that the expression of Cdc25 occurs therein are capable of proliferating and form transgenic callus. Finally, to allow root formation, shooting calli are transferred to medium without auxin, or alternatively or in addition, with the root- promoting hormone indole butyric acid.

An advantage of the inventive method is that, whilst cytokinin expressed from the ipt gene or exogenously supplied from the medium allows the proliferation of neighbouring non-transgenic cells, the ectopic expression of Cdc25 in transformed is highly localised and, as a consequence, highly-specific. Accordingly, the present invention overcomes the need to carry out extensive genetic crossing to eliminate progeny that do not contain the desired gene, in order to establish lines able to transmit the new character to future generations according to standard Mendelian inheritance.

Moreover, in the present inventive method, the plantlet relies entirely on normal endogenous hormone production during the later regeneration stages and develops into a normal plant. The present method generates plants no longer expressing Cdc25, completely normal in growth, and having gained the new beneficial trait conferred by gene transfer. In contrast, when cytokinin is expressed from the ipt gene or exogenously supplied from the medium, the continuing raised cytokinin synthesis alters development and growth of plantlets. REFERENCES

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Claims

WE CLAIM:

1. A method of detecting or identifying transformed or transfected plant cells, tissues or organs that are hormone-dependent, comprising expressing a nucleotide sequence encoding a cell cycle control protein in said plant cell, tissue or organ operably under the control of a plant-operable promoter sequence, for a time and under conditions sufficient for hormone-mediated cell division and/or hormone-mediated cell differentiation to occur in the absence of exogenous hormone in the growth medium, and selecting and/or detecting the proliferating cells.

2. The method according to claim 1 , further comprising introducing a nucleic acid molecule to the plant cell which comprises the nucleotide sequence of a gene- of-interest.

3. The method according to claim 2, wherein the nucleotide sequence of the gene- of-interest is introduced on the nucleic acid molecule as the nucleotide sequence encoding the cell cycle control protein.

4. The method according to claim 2, wherein the nucleotide sequence of the gene- of-interest is introduced to the plant cell, tissue, or organ on a different nucleic acid molecule to the nucleotide sequence encoding the cell cycle control protein.

5. The method according to claim 1 wherein the hormone is cytokinin, and the hormone-mediated cell division is cytokinin-mediated cell division, and the hormone-mediated tissue differentiation is cytokinin-mediated tissue differentiation.

6. The method according to claim 1 , wherein the hormone is gibberellin, and the hormone-mediated cell division is gibberellin-mediated cell division, and the hormone-mediated tissue differentiation.

7. The method according to claim 1 , wherein the cell cycle control protein is a Cdc25 protein or a bioactive homologue, analogue or derivative thereof.

8. The method according to claim 7, wherein the Cdc25 protein is the fission yeast Cdc25 protein.

9. The method according to claim 7 wherein the analogue of a Cdc25 protein comprises a Cdc25 substrate or a modified Cdc25 substrate which mimics the bioactivity of said Cdc25 protein.

10. The method according to claim 9, wherein the modified Cdc25 substrate is a non-phosphorylateable Cdc2 protein.

11. The method according to claim 10, wherein the modified Cdc25 substrate comprises a threonine residue at amino acid position-14 and/or a tyrosine residue at amino acid position-15.

12. The method according to claim 11 , wherein the modified Cdc25 substrate is a Cdc2aA14F15 protein.

13. The method according to claim 7 wherein the analogue of a Cdc25 protein comprises a cyclin protein.

14. The method according to claim 1 , wherein the nucleotide sequence encoding the cell cycle control protein is placed integrated into an excisable genetic element or positioned between at least two excisable genetic elements to facilitate the excision of said nucleotide sequence following selection/detection, and wherein said excision is mediated by a protein which acts on said excisable genetic element(s).

15. The method according to claim 14, wherein the excisable genetic element comprises a transposon and wherein the protein which acts on said excisable genetic element(s) comprises a transposase.

16. The method according to claim 14, wherein the excisable genetic element comprises a recombination locus and wherein the protein which acts on the excisable genetic element(s) comprises a site-specific recombinase protein.

17. The method according to claim 1 , wherein the promoter sequence is selected from the group consisting of: cell-specific promoter sequences; tissue-specific promoter sequences; organ-specific promoter sequences; cell cycle gene specific promoter sequences; tissue-specific inducible promoter sequences; environmentally-inducible promoter sequences; chemically-inducible promoter sequences; wound-inducible promoter sequences; hormone-inducible promoter sequences; and pathogen-inducible promoter sequences.

18. The method according to claim 17, wherein the promoter sequence is a chemically-inducible promoter sequence.

19. The method according to claim 18, wherein the promoter sequence is inducible by the exogenous application of dexamethasone.

20. The method according to claim 1 , wherein the promoter sequence is a strong constitutive promoter sequence that is operable in a plant.

21. The method according to claim 20 wherein the promoter sequence is the CaMV 35S promoter sequence.

22. The method according to claim 1 , wherein cells expressing the cell cycle control protein are cultured in the presence of one or more auxins.

23. A method of detecting or identifying transformed or transfected plant cells, tissues, or organs that are cytokinin-dependent, comprising expressing a nucleotide sequence encoding a Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a dexamethasone-inducible promoter in the presence of one or more auxins for a time and under conditions sufficient for cytokinin-mediated cell division and/or cytokinin-mediated tissue differentiation to occur in the absence of exogenous cytokinin in the growth medium, and selecting and/or detecting proliferating cells.

24. A method of detecting or identifying transformed or transfected plant cells, tissues or organs that are gibberellin-dependent, comprising expressing a nucleotide sequence encoding a Cdc25 protein or a homologue, analogue or derivative thereof operably under the control of a dexamethasone-inducible promoter in the presence of one or more auxins for a time and under conditions sufficient for gibberellin-mediated cell division and/or gibberellin-mediated tissue differentiation to occur in the absence of exogenous gibberellin in the growth medium, and selecting and/or detecting proliferating cells.

25. The method according to claim 23 or 24, wherein the plant is a monocotyledonous plant.

26. The method according to claim 25, wherein the plant belongs to the Graminae.

27. The method according to claim 24, wherein the plant tissue is shoot internode meristem tissue or intercalary meristem tissue or the callus a cell derived from said tissue.

28. The method according to claim 1 , wherein the nucleotide sequence encoding the cell cycle control protein is expressed by a process comprising introducing a gene construct that comprises said nucleotide sequence operably in connection with the plant-operable promoter sequence into a plant cell and culturing said plant cell under conditions sufficient for transcription and translation to occur.

29. The method according to claim 28 wherein culturing of the plant cell under conditions sufficient for transcription and translation to occur includes incubation of said cell for a time and under conditions sufficient for callus formation and/or organogenesis and/or embryogenesis to occur in the absence of exogenous cytokinin and/or gibberellins.

30. The method according to claim 29 wherein the organogenesis or embryogenesis includes regeneration of the plant cell into organised tissues or a whole plant.

31. A transformed plant produced by the method according to claim 30.

32. A gene construct or vector comprising a nucleic acid molecule which consists of a selectable marker gene selected from the group consisting of:

(i) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter sequence that is operable in a plant ;

(ii) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a regulatable promoter that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element; and

(iii) a selectable marker gene comprising a nucleotide sequence that encodes a cell cycle control protein placed operably in connection with a constitutive promoter sequence that is operable in a plant, wherein said selectable marker gene is integrated into an excisable genetic element.

33. The gene construct or vector according to claim 32, further comprising a nucleotide sequence which encodes a gene-of-interest.

34. The gene construct or vector according to claim 32, wherein the cell cycle control protein is a Cdc25 protein or a bioactive homologue, analogue or derivative thereof.

35. The gene construct or vector according to claim 34, wherein the Cdc25 protein is the fission yeast Cdc25 protein.

36. The gene construct or vector according to claim 34, wherein the analogue of a Cdc25 protein comprises a Cdc25 substrate or a modified Cdc25 substrate which mimics the bioactivity of said Cdc25 protein.

37. The gene construct or vector according to claim 36, wherein the modified Cdc25 substrate is a non-phosphorylateable Cdc2 protein.

38. The gene construct or vector according to claim 37, wherein the modified Cdc25 substrate comprises a threonine residue at amino acid position-14 and/or a tyrosine residue at amino acid position-15.

39. The gene construct or vector according to claim 38, wherein the modified Cdc25 substrate is a Cdc2aA14F15 protein.

40. The gene construct or vector according to claim 34, wherein the analogue of a Cdc25 protein comprises a cyclin protein.

41. The gene construct or vector according to claim 32, wherein the excisable genetic element comprises a transposon.

42. The gene construct or vector according to claim 32, wherein the excisable genetic element comprises a recombination locus.

43. The gene construct or vector according to claim 32, wherein the promoter sequence is selected from the group consisting of: cell-specific promoter sequences; tissue-specific promoter sequences; organ-specific promoter sequences; cell cycle gene specific promoter sequences; tissue-specific inducible promoter sequences; environmentally-inducible promoter sequences; chemically-inducible promoter sequences; wound-inducible promoter sequences; hormone-inducible promoter sequences; and pathogen-inducible promoter sequences.

44. The gene construct or vector according to claim 43, wherein the promoter sequence is a chemically-inducible promoter sequence.

45. The gene construct or vector according to claim 44, wherein the promoter sequence is inducible by the exogenous application of dexamethasone.

46. The gene construct or vector according to claim 32, wherein the promoter sequence is a strong constitutive promoter sequence that is operable in a plant.

47. The gene construct or vector according to claim 46, wherein the promoter sequence is the CaMV 35S promoter sequence.

48. A gene construct or vector comprising a nucleic acid molecule which consists of a selectable marker gene comprising a nucleotide sequence encoding a Cdc25 protein placed operably under the control of a dexamethasone-inducible promoter sequence that is operable in a plant.

49. The gene construct or vector according to claim 48, wherein the Cdc25 protein is the fission yeast Cdc25 protein.

50. The gene construct or vector according to claim 48, wherein the selectable marker gene is placed within T-DNA border sequences to facilitate integration of said gene into the genome of a plant.

51. The gene construct or vector according to claim 48, wherein the selectable marker gene further comprises a terminator sequence that is operable in a plant.

52. The gene construct or vector according to claim 51 , wherein the terminator sequence is the CaMV 35S terminator sequence.

53. The gene construct or vector according to claim 48, further comprising a bacterial selectable marker gene.

54. The gene construct or vector according to claim 53, wherein the bacterial selectable marker gene confer resistance on a bacterial cell to kanamycin or an equivalent antibiotic compound thereto.

55. The gene construct or vector according to claim 48, wherein the dexamethasone-inducible promoter sequence comprises one or more GRE sequences which are responsive to a glucocorticoid receptor protein in the presence of dexamethasone.

56. The gene construct or vector according to claim 55, further comprising a gene construct which encodes the glucocorticoid receptor protein placed operably under control of a plant-operable promoter sequence and positioned within said gene construct or viral vector such that the glucocorticoid receptor protein is capable of being expressed in the transformed plant cell.

57. A method of detecting or identifying transformed or transfected plant cells, tissues or organs that are hormone-dependent, comprising expressing a nucleotide sequence encoding a cell cycle control protein in said plant cell, tissue or organ operably under the control of a plant-operable promoter sequence, for a time and under conditions sufficient for hormone-mediated cell division and/or hormone-mediated cell differentiation to occur in the absence of exogenous hormone in the growth medium, and selecting and/or detecting the proliferating cells, and wherein the nucleotide sequence encoding the cell cycle control protein is contained within the gene construct or viral vector according to claim 32.

58. A method of detecting or identifying transformed or transfected plant cells, tissues or organs that are hormone-dependent, comprising expressing a nucleotide sequence encoding a Cdc25 protein in said plant cell, tissue or organ operably under the control of a plant-operable promoter sequence, for a time and under conditions sufficient for hormone-mediated cell division and/or hormone-mediated cell differentiation to occur in the absence of exogenous hormone in the growth medium, and selecting and/or detecting the proliferating cells, and wherein the nucleotide sequence encoding the Cdc25 protein is contained within the gene construct or viral vector according to claim 32.

59. A plant cell, tissue, organ, plant part, propagule or whole plant comprising the gene construct or vector according to claim 32 or a footprint thereof.

60. A plant cell, tissue, organ, plant part, propagule or whole plant comprising the gene construct or vector according to claim 48.