CZ339899A3 - Plants with adjusted growth - Google Patents

Abstract

The lost formwork of the ceilings according to the invention is based on the fact that the ceilings according to the invention are loose consists of a concrete ceiling slab (1) on which they are | perpendicularly placed concrete ribs (2), with the soffit i the ceiling slab (1) is reinforced with reinforcing wires (3). Lost j the formwork of the ceilings is made of concrete made of fine sand fraction 0-5 mm and cement class 325 to 500. Thickness ceiling tiles (1), height and thickness of ribs (2) is directly dependent on the length of the ceiling ceiling (1), and the width »Ceiling ceiling tiles (1) are inversely dependent on length ceiling tiles (1). Lost formwork ceilings by 1 of the invention serves as a formwork for forming concrete ceilings, but not in itself a bearing part of the ceilings.

Description

Technical field

The invention describes the use of proteins or parts thereof that control cell division. Preferred are cell division directing proteins that bind to retinoblasome-like proteins. More preferred are cyclins, particularly D-type cyclins and genes encoding them, which are used to produce modified phenotypes, especially modified growth rate plants, or plants containing modified growth rate portions and / or modified relative size or altered plants architecture. The invention also relates to plant cells and plants expressing such DNA.

BACKGROUND OF THE INVENTION

All eukaryotic cells undergo the same series of steps in their division. The term "cell cycle" reflects the nature and universality of these steps. DNA (S) replication and cell division is normally temporarily separated by a gap (G1 and G2) in the G1-S-G2-M sequence in a eukaryotic cell cycle. This arrangement makes it possible to control critical DNA replication and mitosis processes. These emphasized cytological steps of the cell cycle are an ordered series of transiently organized molecular and cellular procedures that define the direction and order of the cycle. Cell cycle progression is likely to be regulated in all eukaryotes by the main control units operating at G1 to S and at the G2 to M phase interface. Passage through these control points requires activation of cyclin-dependent kinases (CDKs) whose catalytic activity and substrate specificity are determined specific regulatory subunits known as cyclins and interactions with other proteins that regulate the stage of complex phosphorylation (Atherton-Fessier et al., 1993, Semin. Call Biol. 4: 433-442; Solomon, 1993

Curr. Opin. Cell. Biol. 5: 180-186). In budding and dividing yeast, both phases G1 to S and G2 to M of transition are controlled by one CDK, encoded by the cdc2 + gene in Schizosaccharomyces pombe and in Saccharomyces cerevisiae CDC28. The association of p34 ^cdc2 (p34 ^cdc2 in budding yeast) with different cyclin partners differentiates two control points (Nasmyth 1993 Curr. Opin. Cell. Biol. 5: 166-179). A more complex situation occurs in mammalian cells. There are at least six related but different CDKs (encoded by the genes cdc2 / cdk1, cdk2, cdk3, cdkain4, cdk5 and cdk6) of different roles. Each CDK is associated with one or more cyclin-like partners (Fang and Newport 1991 Cell 66: 731-742; Meyerson et al. 1991 Cold Spring Harbor Symp. Quant. Biol. 56: 177-186; Meyerson et al. 1993 EMBO J. 11: 2909-2917; Xiong et al., 1992 Cell 71: 505-514; Tsai et al., 1993 and Development 119: 10291040; Van den Heuvel and Harlow, 1993 Science 262: 2050-2054;

Meyerson and Harlow, 1994 Mol. Cell. Biol. 14: 2077-2086). Type B cyclins are the major class involved in the G2 and M transition and associate with p34 ^cdc2 or its direct homologs (Nurse 1990 Nature 344: 503-508).

Cyclin B is one of two cyclins originally reported to accumulate in vertebrate eggs during interphase and is rapidly destroyed during mitosis (Evans et al., 1983 Cell 33: 389-396) and is a component of factor promoting aging in xenopus (Murray et al. 1989 Nature 339: 280-286).

Then, cyclin B homologues were identified in many eukaryotic species. Cyclin A is also widespread in multicellular organisms and its exact role is unknown, although the maximum incidence suggests some function in the S phase (Pines, 1993 Trends Biochem. Sci. 18: 195-197).

In S. cerevisiae yeast, phase transition is best described

G1 on S. Genetic studies define a point in the G1 phase called START. After passing through the START point, the cell enters • •·· · · · · <

S and undergo a complete cycle of division, resulting in two daughter cells again in the G1 phase (Hartwell, 1974, Bacteriol. Rev. 38: 164-198; Nasmyth 1993 Curr. Opin. Cell. Biol. 5: 166-179 ). The products of the three cyclin G1 genes in S. cerevisiae, called CLN1, CLN2, and CLN3, are in principle limiting START passage components (Richardson et al., 1989 Cell 59: 1127-113; Wittenberg et al., 1990 Cell 62: 225237; Tyers et. al., 1993, EMBO J. 12: 1955-1968). Transcription of CLN1 and CLN2 is activated in the G1 phase and the accumulation of protein products at a critical cutoff level through a positive feedback mechanism leads to activation of the p34 ^CDC28 kinase and passage through the START point (Dirick and Nasmyth, 1991 Nature 351: 754-757). G1 cyclins then degrade as a result of their FEST motifs to their primary sequences resulting from rapid protein degradation (Rogers et al., 1986 Science 234: 364-368; Lew et al., 1991 Cell 66: 11971206 (Reed, 1991, Trends Genet. 7: 95-99).

Yeast cycles of S. cerevisiae are at least partially in excess because yeast strains in which two of the three cyclin G1 genes are deleted and the third is under the control of a galactose-regulated promoter show a phenotype where galactose-dependent growth is phenotype. Such strains are used to identify Drosophila flies and human cDNA clones that rescue this cln-deficient phenotype on glucose plates when suppressing a single yeast CLN gene (Koff et al., 1991 Cell 66: 1217-1228; Lahue et al. , 1991 Genes Dev 5: 2166-2175, Leopold and O'Farrell, 1991 Cell 66: 1207-1216; Lew et al., 1991 Cell 66: 1197-1206, Xiong et al., 1991 Cell 65: 691-699 ). In this way, human cDNA encoding three new classes of cyclins C, D and E were determined. Although these cyclins exhibit only limited homology to the yeast CLN proteins, it has been shown to play an important role in understanding the control units that act in mammalian cells during G1 and G1. a restriction point at the boundary of the G1 and S phases (described in terms of the G1 and S phases). In Pardee, 1989 Science 246: 603-608, Matsushime et al., 1992 Cell 71: 323-334; Koff et al. 1992 Science 257:

1689-1694; Koff et al., 1993, Science 260: 536-539; Ando et al. 1993 Proc. Nati. Acad. Sci. USA 90: 8571-9575; Quelle et al. 1993 Genes Dev. 7: 1559-1571; Tsai et al., 1993b Oncogene 8:

1593-1602). Cyclin E may act as a rate limiting component at the G1 and S phase boundaries (Ohtsubo and Roberts, 1993 Science 259: 1908-1912; Wimmel et al., 1994 Oncogene 9: 995-997), while cyclin D levels dependence on serum growth factors (Matsushime et al. 1991 Cell 65: 701-713; Baldin et al., 1993 Genes Dev. 7: 812821; Sewing et al., 1993 J. Cell. Sci. 104: 545-555) that type D cyclins can form a bond between these signals and the continuation of the cell cycle.

An important factor involved in the regulation of cell cycle progression in mammals is the retinoblastoma susceptibility gene that encodes the retinoblastoma protein (Rb). Rb binds and inactivates the E2F family of transcription factors through the ability to inactivate most of its potential to control cell division in the G1 phase. E2F transcription factors are known to turn on the cyclin E and phase S genes and increase the amount of cyclin E and / or E2F, resulting in replication (Nevins, 1992 Science 258: 424-429; Johnson et al., 1993 Nature) 365: 349-352). The ability of Rb to inactivate E2F depends on its phosphorylation stage. During the G1 phase, Rb occurs in a phosphorylated state, but in the late G1 phase phosphorylation of Rb occurs by cyclin-dependent kinases, especially CDK4 complexed with its substantial regulatory subunit cyclin D (Pines, 1995Biochem. J. 308: 697- 711; Pines, 1995 Adv. Cancer Res. 66: 181-212) and CDK2 complexed with cyclin E (at the G1 / S phase interface) or cyclin A (in S phase). These multiple Rb phosphorylations cause release of E2F, which promotes transcription of the S phase genes.

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Plant cells have been used in the study of cell growth and division to define discrete phases of the eukaryotic cell cycle (Howard and Pele, 1953 Heredity 6 (suppl.): 216-273), but there is a lack of data regarding cell cycle control in plant systems. Plant cells that disrupted division in vivo due to quiescent or in vitro, causing nutrient deficiency, blocked baseline control points in G1 and G2 phases (van't Hof over Kovacs, 1972 In The Dynamics of Meristem Cell Populations, MW Miller and CC Keuhnert, eds (NY: Plenum pp 15-32; Gould et al., 1981 Protoplasm 106: 113; van't Hof, 1985 In the cell division eyel in plants, JA Bryant and D. Francis, eds (Cambridge: Cambridge University Press) pp. 1-13) This is broadly consistent with controls that function in other eukaryotic systems, although mature plant cells may be present either in G1 or G2 DNA content. (described by Evans and van 't Hof, 1974 Exp. and Development 7: 555-569; Gould et al., 1981 Protoplasma 106: 1-13), the population of G1 is predominant in general. cells that lack scarcity These cells block the G1 phase (Gould et al., 1981 Protoplasma 106: 1-13). There are strong analogies between the base control point in the G1 phase of the plant cell cycle, the START point in yeast, and the restriction point in mammalian cells.

Antibody or histone kinase HI assays have been used to identify the presence and localization of active CDC2-related kinases in plant cells (John et al. 1989 Plant Cell 1: 1185-1193; John et al., 1990 J. Cell Sci., 97: 627-630, John et al., 1991

Protoplasma 161: 70-74; Mineyuki et al., 1991 Protoplasma 162: 182-186; Chiatante et al., 1993 Plant Sci. 89: 13-21; Colasanti et al., 1993 Plant Cell 5: 1101-1111; John et al. 1993). From a large number of plant species including peas (Feiler and Jakobs, 1990 Proc. Natl.

alfalfa (described

Acad. Sci. USA 87: Hirt et al

Acad. Sci. USA 88: 61-69), Arabidopsis

5397-5401)

1991 Proc. Nati.

1636-1640; Hirt et al. 1993 Plant J. 4:

(described in Ferreira et al., 1991 Plant Cell 3: 531-540; Hirayama et al., 1991 1991 Gene 105: 159-165), soybean (Miao et al., 1993 Proc. Natl. Acad. Sci. USA 90: 943-947)

Antirrhinum (Fobert et al., 1994 EMBO

J. 13: 616-624) and maize (Colosanti et al., 1991 Proc. Natl. Acad. Sci. USA 88: 3377-3381) were isolated by hybridization with reduced stringency or insufficient polymerase chain reaction cDNA encoding functional homology of CDC2 kinases. Among other different plant species including carrots (Hata et al., 1991 EMBO J. 10:

2681-2688 'soybean (Hata et al., 1991)

Why. Nati. Acad cDNA sequences

EMBO J. 10: 2681-2688), Arabidopsis (Hemerly et al., 1992)

Why. Nati. Acad. Sci. USA 89: 3295-3299; Day and Reddy, 1994. Biochim. Biophys. Acta Gene Struct. Express. 1218: 115-118), alfalfa (Hirt et al., 1992 Plant Cell

4: 1531-1538), Antirrhinum (Fobert et al., EMBO J. 13: 616-624) and corn (Renaudin et al., 1994

Sci. USA 91: 7375-7379), a series of plant mitotic cyclins having A- or B-type characteristics or mixed A- and B-type features was isolated.

Soni et al. , 1995 Plant Cell 7: 85-103 identified a new yeast plant of three related cyclins in Arabidopsis by supplementing a yeast strain with insufficient levels of Gl cyclins. Individual members of this family show tissue-specific expression and are conservative in other plant species. They form a different group of plant clines and have been termed δ-type cyclins, indicating their similarity to mammalian type D cyclins. The sequence relationship between δ and D cyclins includes the N-terminal sequence of LxCxE. Leucine

preceded at position -1 or -2 by an amino acid and an acidic side chain (D, E). This motif was originally identified in certain viral oncoproteins and has been implicated in binding to retinoblastoma protein. By analogy with mammalian cyclin D, these plant homologs can mediate growth and phytohormonal signals in plant cell cycles. In this regard, cyclin 53 was rapidly induced by the growth cytokinin plant regulator upon re-stimulation of cells grown in suspension. Cyclin 52 was induced by a carbon source. In Reunadin et al. 1996 Plant Molecular Biology 32: 1003-1018 defines groups and nomenclature of plant cyclins and cyclins 5 are now called CycD cyclins.

Dahl et al., 1995 Plant Cell 7: 1847-1857 describes the identification of cyclin (cycMs4) in alfalfa. This cyclin is related to alfalfa cyclin 53.

Rb-like proteins have also been identified in plants. In Xie et al., 1996 EMBO J. 15. 4900-4908 and Grafi et al., 1996 Proc. Nati. Acad. Sci. USA 93: 8962-8967 describes the isolation and preliminary characterization of a corn derived Rb homolog.

Doerner et al., 1996 Nature 380: 520-523 describes the ectopic expression of type B cyclin (cyclAt originating from Arabidopsis) under the control of a promoter from the cdc2a gene in an Arabidopsis plant. The transgenic plants of cdc2a strongly expressing the transgene have a considerably increased root growth rate. Growth and development of lateral roots were accelerated by subsequent induction of indole acetic acid in transgenic plants relative to control plants.

Hemerly et al., 1995 EMBO J. 14: 3295-3299 describes a transgenic tobacco plant and Arapidopsis expressing a wild-type kinase or a dominant mutation thereof. The tenro enzyme is active in mitosis (CDC2a). The plants still over-produce wild-type CDC2a or a mutant form in which the CDC2a is a wild-type or mutant form in which the plants are &lt; Desc / Clms Page number 3 &gt; · ··· · ·· · «·· ·· ·· assumes that the accelerated cell cycle does not show a significant change in development. Mutant CDC2a that can block the cell cycle, expressed in Arabidopsis, has stopped cell division. Some tobacco plants still produce a later mutant kinase. These plants contained significantly fewer cells, but these cells were larger.

PCT patent WO 92/09685 discloses a method of controlling plant cell growth comprising modulating the amount of cell cycle protein in a plant for a time and under conditions suitable for controlling cell division. A preferred protein identified in the examples is p34 ^cdc2 kinase or similar proteins that function in mitosis.

WO 93/12239 describes plants with altered structure and other phenotypic effects, in particular the premature flowering period and a greater number of flowers. These plants were obtained by transformation with a genome that includes a yeast-derived cdc25 gene such as Schizosaccharomyces pombe.

WO 97/47647 describes the isolation and characterization of a plant DNA sequence that encodes a retinoblastoma protein, its use in controlling the growth of plant cells, and further describes plants and / or plant viruses as well as the use of vectors, plants or animals or animal cells treated with the plant retinoblastoma protein .

US patent 5,514,571 discloses the use of cyclin D1 as a negative regulator of mammalian cell proliferation. Overexpression of cyclin D1 blocks mammalian cell growth, while expression of blocking cyclin D1 promotes cell proliferation.

SUMMARY OF THE INVENTION

The invention describes a process for obtaining a plant with altered growth characteristics or altered architecture, in particular plants with reduced or increased growth rates, plants that previously bloom or plants that exhibit

an increased number of flowers in a single plant or larger organ size comprising the step of altering the amount or functional amount of a protein that controls cell division. This protein is capable of binding or phosphorylating an Rb-like protein. Preferably, a cell division controlling protein comprising a LxCxE binding motif or a related motif is preferred. Preferably, the motif is located in the N-terminal portion of the protein. Particularly preferred is cyclin type D.

Further disclosed is a method of obtaining a plant with altered growth characteristics or altered architecture comprising the step of altering the amount or functional amount of a cell division controlling protein. This method is performed by inserting a chimeric gene into the genome of plant cells that contain the following operably linked DNA fragments:

(a) a plant-expressible promoter region, in particular the CaMV35S promoter region,

(b) a transcribed region of DNA encoding an RNA or protein which, when expressed, either increases or decreases the amount or functional amount of the cell division controlling protein; and

c) 3'-terminal formation and a polyadenylation signal that is functional in plant cells.

In accordance with the invention, the transcribed DNA region encodes an antisense RNA, a ribozyme or a sense strand of RNA which, when expressed, decreases, inhibits or prevents expression of a cell division controlling protein, particularly endogenous type D cyclin.

Further, according to the invention, the transcribed DNA encodes a cell division controlling protein capable of binding to the pocket domain of an Rb-like protein. Preference is given to a cell division control protein comprising a LxCxE binding motif, more preferably type D cyclin, especially plant-derived cyclin D. Most preferred is a type D cyclin selected from the group of fcfcfc · fc · fcfc

Arabidopsis thaliana CYCD1, Arabidopsis thaliana CYCD2, Arabidopsis thaliana CYCD3, Nicotiana tabacum CYCD3; 1, Nicotiana tabacum CYCD3; 2, Helianthus tuberosus CYCD1; 1, Zea mays CYCD2 and Helianthus tuberosus.

Further, according to the invention, the transcribed RNA encodes a protein or peptide which, when expressed, increases said functional amount of a cell division controlling protein, in particular a protein or peptide selected from the group consisting of mutant type D cyclin, part D type cyclin, type D cyclin. showing a mutation in a cyclin box, a type D2 cyclin that exhibits the substitution of amino acid 185 or amino acid 155, a type D2 cyclin that exhibits a E185A or K155A mutation, a type D cyclin where the PEST sequences have been deleted, a type D cyclin where the LxCxE exchanged or deleted or type D cyclin, where the C-residue was removed from the LxCxE binding motif.

The invention further provides said chimeric genes.

The invention further provides plant cells, plants and their seeds comprising the chimeric genes of the invention and exhibiting changes in growth characteristics and / or changes in architecture.

The invention further provides the use of a cell division controlling protein capable of binding to and / or phosphorylating the pocket domain of an Rb-like protein, in particular a cell division controlling protein comprising a LxCxE binding motif in the N-terminal portion of the protein, particularly type D cyclins and genes encoding it , to control the growth characteristics or plant architecture. The cell division directing protein encodes a chimeric gene incorporated into the genome of the plant cells.

The term "plant architecture" means a general morphology defined by the relative size, position, and number of several parts of a plant (i.e., organs such as leaves, inflorescence, storage organs such as tubers, roots, stems, flowers or organ portions such as a crown calyx, anther, stigma, forehead, petiole, etc.).

• φ ····

The term "altering the architecture of a plant" means altering the morphology as a result of changing, for example, the number, size and position of organs or parts of organs. Obviously, a change in either one organ or part thereof, or several organs or parts thereof, will lead to a change in the plant architecture. This can be achieved by altering (i.e., enhancing or reducing) cell division activity in existing meristems and / or organ primordia, or by creating de novo merostems.

The term "co-suppression" refers to the process of transcription and / or post-transcriptional suppression of RNA accumulation in a sequence-specific manner resulting in suppression of the expression of homologous endogenous genes or transgenes. Suppression of endogenous gene expression can be achieved by introducing a transgene that contains a strong promoter operably linked to a DNA region, wherein the resulting transcribed RNA is a sense RNA comprising a nucleotide sequence having at least 75%, preferably at least 80%, preferably at least 85%, more is at least 90%, particularly at least 95%, of the coding or transcribed DNA sequence (anticoding) of the gene whose expression is suppressed. Preferably, the transcribed DNA region does not encode a functional protein. It is particularly preferred that the transcribed region does not encode a protein.

The term "plant-expressible promoter" means a promoter that is capable of directing transcription in a plant cell. It can be any promoter of plant origin, but also any promoter that does not originate from the plant and is capable of directing transcription in the plant cell. They may be, for example, viral or bacterial promoters, such as CaMV35S or T-DNA gene promoters.

The term &quot; gene expression &quot; means a process driven by regulatory regions, and in particular transcribes into RNA that is biologically active. That is, it is capable of interacting with another nucleic acid or protein or is able to translate into biologically active where the region of the DNA promoter is »φ φφφφ φ φ

A polypeptide or protein. The gene is said to encode RNA when the end product of gene expression is a biologically active RNA, such as an antisense RNA or a ribozyme. A gene is said to encode a protein when the end product of gene expression is a biologically active polypeptide or protein.

The term "gene" means a DNA fragment containing a DNA region ("transcribed DNA region") that is transcribed into an RNA molecule (eg, mRNA) in a cell under the control of appropriate regulatory regions. This is, for example, a plant-expressible promoter. Thus, a gene may contain several operably linked DNA fragments, such as a promoter, a 5 'leader sequence, a coding region, and a 3' region containing a polyadenylation site. An endogenous plant gene is a gene that is naturally found in plant species. A chimeric gene is any gene that is not normally found in plant species, or else it is any gene where the promoter is not linked to part or all of the transcribed DNA region or at least one regulatory region of the gene.

The invention is based on the unexpected finding that chimeric genes containing DNA encoding a protein controlling cell division binding to an Rb-like protein, in particular a plant-type D cyclin under the control of a plant-expressible promoter, can be stably incorporated into the plant cell genome without undesirable effects, and further enhanced expression of such a cell division controlling protein, particularly cyclin type D, in plants leads to specific changes in the growth rate and architecture of the resulting transformed plants.

The invention describes a change in the expression level or activity of a functional cell division controlling protein. Preferably, a stable change in the cells of the plant is preferred, wherein the architecture or growth rate is changed, or both features are transformed simultaneously in transformed plants and their offspring. It is preferred that the amount or functional amount of the cell division controlling proteins is controlled genetically by altering the expression of the genes.

9999 99 9 99 99 coding for these cell division controlling proteins. Increasing the amount and functional amount of the cell division controlling protein can be achieved, for example, by manipulating the copy number of the coding gene (s), altering the promoter region of the coding genes, or manipulating genes that directly or indirectly regulate the expression level of the cell division controlling protein. Alternatively, the amount of cell division controlling protein may be increased by stabilizing the mRNA encoding the cell division controlling protein or by stabilizing the cell division controlling protein, for example, by removing destructive motifs or so-called PEST sequences.

The functional amount and activity of the cell division controlling protein may be increased by reducing the amount of the cell division promoting antagonist or inhibitor by a method such as obtaining a cell with a protein such as an inactive cell division controlling protein similar to a protein whose functional amount is increased or a portion thereof. a cell division control that is still capable of binding an inhibitor or other regulatory protein, or still capable of binding to cyclin-dependent canines. The functional amount or activity of the cell division controlling protein can also be increased by altering or mutating the cell division controlling protein, thereby reducing or eliminating the binding of an antagonist or inhibitor of cell division related activity.

Reduction of the functional amount of cell division controlling protein can be achieved, for example, by reducing the amount of RNA encoding cell division controlling protein available in translation by a method such as antisense RNA, ribozyme treatment or co-suppression. Alternatively, the functional amount of the cell division controlling protein may be reduced by increasing the amount of the cell division promoting protein antagonist or inhibitor.

The term "cell division controlling protein" is a polypeptide or protein that is required to regulate the progression of the cell cycle of a eukaryotic cell. Preferably, the plant cell or

9 * · * • · · 9 1 9 9 9 9 · ·· ** 1 9 9 14 9 ···· · · · · « • 9 9 9 • 99,9191 9 9 9 9 9 9 9 9 9 99 91 protein that can cause input cells into the cell cycle or cause method cells cycle direct interaction

with a protein required to regulate cell cycle progression, or with a polypeptide or protein that is believed to have equivalent function but is not required for cell cycle regulation.

Suitable cell division controlling proteins are those capable of phosphorylating the Rb-like protein alone or in combination with other proteins. Preferably, a protein capable of phosphorylating an Rb-like protein in a plant cell at the G1 and S phase transition, or a protein capable of binding to the pocket domain of retinoblastoma-like proteins (Rb-like). Preferred are proteins having an LxCxE binding motif contained in an amino acid sequence or a related motif such as LxSxE or FxCxE (the binding motifs are represented by a one letter amino acid code, where x is any amino acid). Particularly preferred are cyclins that contain a LxCxE binding motif (and / or a related motif) in the non-terminal half of the protein. It is preferred that the motif be found in the first residues of 50 amino acids, particularly the first residues of 30 amino acids, such as type D cyclins, especially plant D cyclins. Preferred are cyclins from the group comprising Arabidopsis thaliana CYCD1, Arabidopsis thaliana CYCD2 Arabidopsis thaliana CYCD3; Nicotiana tabacum CYCD2; Nicotiana tabacum CYCD3; 1, Nicotiana tabacum CYCD3; 2, Helianthus tuberosus CYCD1; 1, Zea mays CYCD2 and Helianthus tuberosus CYCD3; 1 or cyclins with substantially similar protein sequences.

Said plant D cyclins were completely characterized by the amino acid sequence encoded by EMBL DNA # N ° X83369 from nucleotide position 104 to nucleotide 1108 for Arabidopsis thaliana CYCD1, EMBL # N ° X83370 from nucleotide position 195 to nucleotide 1346 for Arabidopsis thaliana CYCD2 , EMBL No. N ° X83371 from nucleotide position 266 to nucleotide 1396 for Arabidopsis thaliana CYCD3, nucleotide sequence SEQ ID NO: 1 from nucleotide position 182 to nucleotide 1243 for Nicotiana tabacum CYCD2 / 1, nucleotide sequence SEQ ID NO: 2 from nucleotide position 181 to nucleotide 1299 for Nicotiana tabacum CYCD3 / 1, with nucleotide sequence SEQ ID NO: 3 from nucleotide position 198 to nucleotide 1298 for Nicotiana tabacum CYCD3 / 2, nucleotide sequence SEQ ID NO: 4 from nucleotide position 165 to nucleotide 1109 in the case of Helianthus tuberosus CYCD1; 1, the nucleotide sequence of SEQ ID NO: 5 from nucleotide position 48 to nucleotide 1118 in the case of H elianthus tuberosus CYCD3 / 1 by the nucleotide sequence of SEQ ID NO: 21 from nucleotide position 316 to nucleotide 1389 of Zeam mays CYCD2.

It is contemplated that an increase, decrease, amount or functional amount or activity of these proteins controlling cell division in plant cells accelerates or delays, respectively, the G1 to S phase transition or increases or decreases the proportion of actively dividing cells based on their interaction with Rb-like proteins that affect the ability of the protein to inactivate certain transcription factors. Furthermore, it is considered that the expression of these cell division controlling proteins that interact with Rb-like proteins effectively allows cells to initiate the division process, whereas (over) expression of G2 / mitotic cyclins (such as type B cyclins or ccřc25 gene product) leads to faster G2 / cell cycle mitosis progress that has already begun.

The term "Rb-like proteins" is defined as proteins from the group consisting of human Rb-1 protein (Lee et al., 1987 Nature 329: 642-645; No. P06400), human P107 (Ewen et al., 1991 Cell 66: 1155-1164; No. L14812) and human p130 (Hannon et al., 1993 Genes Dev. 7: 2378-2391; No. A49370), RBF Drosiphila (Du et al., 1996 Genes and Development 10: 1206-). 1218; No. for the entry of DNA encoding the X96975 gene), mouse •

RB (Bernards et al., 1989 Proc. Natl. Acad. Sci. USA 86: 64746478; No. P13405), chicken RB (Boehmelt et al., 1994 Cell Growth Differ. 5: 221-230; No. X72218), Xenopus Rb (Destree et al.,

1992 Dev. Biol. 153: 141-149; A44879), ZmRb and Rb1 from Zae mays (Xie et al., 1996 EMBO J. 15: 4900-4908; Grafi et al., 1996 Proc. Natl. Acad. Sci. USA 93: 8962-8967; DNA entry number) coding genes: X98923; GenBank U52099) as well as proteins which simultaneously exhibit at least 25-30% amino acid sequence similarity to at least three members of the above group, and contain a conserved cysteine residue located at position 706 of human Rb-1 or at equivalent positions in other Rb-like proteins (Xie et al., 1996 EMBO J. 15. 4900-4908).

Rb-like proteins are members of a small family known as pocket proteins. This term was derived from a conserved two-part region called the pocket domain. It acts as a binding site for several growth-controlling proteins, such as the E2F family of transcription factors, type D cyclins, and viral oncoproteins. Subdomains A and B of the pocket region are more conserved compared to the rest of the protein (approximately 50 to 64% for subdomains A and B) and are separated by a non-conservative space bar. The pocket domains are between amino acids at positions 451 and 766 for human Rb, at positions 321 to 811 for human p107, at positions 438 to 962 for human p30, at positions 445 to 758 for mouse Rb, at positions 441 to 758 for chicken Rb, at positions 440 to 767 for Xenopus Rb, at positions 11 to 382 for ZmRb for corn, at positions 89 to 540 for Rbl of corn.

"Binding to the Rb-like protein or binding to the pocket region of the Rb-like protein can be analyzed by either an in vitro assay or one of the in vivo assays or a combination thereof.

In in vitro assays, binding is analyzed between the protein ···· »• · · · · · · •» • · · · · · • · · · · · · · · • ···· · · · · designated * ³⁵ S-methionone and glutathione transferase (GST) / pocket domain of an Rb-like protein, such as human Rb. Fusion with GST allows easy purification and fixation of the fusion protein to glutathione sepharose particles. The interaction between the test protein and the Rb-like protein is compared to the binding between the same protein and the GST fusion protein and the Rb-like protein with a conserved cysteine mutation at a position equivalent to systeine 706 in human Rb, such as human Rb C706F. Such an assay is described, for example, in Dowdy et al 1993 Cell 73: 499-511 and Ewen et al. 1993 Cell 73: 487-497. In another variation, the Rb-like protein and the Rb-binding protein were co-expressed in insect cells and the association was detected by gel filtration or co-immunoprecipitation (O'Reilly et al. 1992. Baculovirus expression vectors-A laboratory manual. Freeman and Co. New York) ).

The in vivo assay used to detect protein binding to the pocket domain of an Rb-like protein is a yeast two hybrid system (Fields and Song, 1989 Nature 340: 245-246). This analysis reveals the ability to reconstitute the functional activity of GAL4 from two separate fusion proteins containing the DNA binding domain (GAL4 ^BD ) and the GAL4 activation region ^{and d} ) fused to the pocket domain of an Rb-like protein and a test protein. Expression of plasmids containing chimeric genes encoding these fusion proteins is introduced into a yeast strain encoding suitable GAL4 inducible markers. For example, it is the HF7c strain (Feilloter et al., 1994 Nucl. Acids Res. 22: 1502-1503) containing Gal4-inducible markers HIS3 and LacZ or the Y190 strain (Harper et al., 1993 Cell 75: 805-816. Proteins that bind to the pocket domain of an Rb-like protein allow growth in the absence of histidine. An example of two hybrid assays demonstrating the interaction of a protein with an Rb-like protein is described in Durfee et al. 1993 Genes and Development 7: 555-569.

I

I · 4 · · · · · · · · · ·

It is preferred that control experiments such as separate introduction of expression plasmids into the same yeast strain or introduction of expression plasmids encoding fusion proteins comprising a DNA binding domain (GAL4) are included.

BDv activation region (GAL4 ^ad ) fused to the mutated pocket domain of an Rb-like protein. A mutation at C706 or an equivalent position is preferred.

Alternatively, an in vitro assay to determine binding between a protein and the pocket domain of an Rb-like protein involves the temporary expression of both proteins in plant cells. Protoplasts of the tobacco plant are preferred. Immunoprecipitation is also used using antibodies directed against one of the two proteins, where the co-precipitation of the other protein is determined.

"An Rb-like phosphorylating protein can be analyzed by an in vitro assay based on the use of ³² P-labeled adenosine triphosphate, monitoring the capacity of a protein (or a combination of proteins such as cyclins and cyclin-dependent kinase) to transfer labeled phosphate groups to the target protein. The term "cyclin" is defined as a regulatory protein comprising a regulatory region of about 100 amino acids known as a "cyclin box." The cyclin box is the binding site of cyclin-dependent kinases enabling it to exert its regulatory effect on the kinase activity of CDKs.

The cyclin box can be determined by comparing the amino acid sequence of a protein with a known amino acid sequence between Arabidopsis thaliana, Arabidopsis thaliana, Arabidopsis thaliana, cyclin boxes described in Renaudin et al., 1994 Proc. Nati. Acad. Sci. USA 91: 73757379; Renaudin et al., 1996 Plant Molecular Biology 32: 10031018; 1995 Plant Cell 7: 85-103; Hemerly et al.,

1992 Proc. Nati. Acad. Sci. USA 89:

cyclin boxing , as positions 81 to 186 CYCD1 positions 96 to 201 CYCD2 positions 86 to 191 CYCD3

3295-3299).

Acad.

Nati.

• · • ·

The amino acid sequence identified as a sequence comparison cyclin box should contain at least five conserved residues that are required for cyclin activity R (97), D (126), L (144), K (155), E (185) (labeled). the positions are from the CYCD sequence of Arabidopsis thaliana) (Soni et al., 1995 Plant Cell 7: 85-103; Renaudin et al., 1996 Plant Molecular Biology 32: 1003-1018).

Type D cyclins (cyclin D or CycD) are cyclins that are characterized by the presence of other characteristic sequences, such as the LxCxE motif or related motifs suitable for Rb-like proteins found in the N-terminal portion of the protein. They are preferably located between the N-terminus and the cyclin box, especially in the first 50 amino acids, preferably in the first 30 amino acids of the initiating methionine residue. Preferably, the leucine from the binding motif is preceded by an amino acid having an acid side chain (D, E) at the -1 or -2 position. Alternative binding motifs such as LxSxE or FxCxE can be found. Phelps et al 1992 J. Virol. 66: 2418-2427, it is reported that mutation of the LxCxE binding motif in human papillomavirus E7 to LxSxE does not affect the ability of the protein to bind to Rb-like proteins. Based on sequence homology of CycD1 (containing Arabidopsis thaliana CycD1 and Helianthus tuberosus CYCD1; 1), CysD2 (containing Arabidopsis thaliana CYCD2; Nicotiana tabacum CYCD2; 1, Zea mays CYCD2), CycD3 (containing Arabidopsis thaliana CYCD3, Nicotiana CYCD3, Nicotiana CYCD3; Nicotiana tabacum CYCD3 / 2, Helianthus tuberosus CYCD3; 1) Three groups of type D cyclins have been identified.

The nomenclature and sequence consensus for different types and groups of plant cyclins including type D cyclins is described in Renaudin et al., 1996 Plant Molecular Biology 32: 1003-1018) and can be used to classify novel cyclins based on their amino acid sequences.

• · · · · · · · · · · · · · · · · ·

Cell division directing proteins can be provided to cells directly, for example, by electroporation of protoplasts. in the presence of proteins controlling cell division or indirectly by transforming plant cells in plants with expressible chimeric genes that encode the test protein and are incorporated into the protoplast genome temporarily or stably.

In one embodiment of the invention, the amount or functional amount of a cell division controlling protein capable of phosphorylating an Rb-like protein or binding to the pocket domain of Rb-like proteins is increased by incorporating the chimeric gene into the genome of the plant cells to obtain a plant with altered growth rate or altered architecture. The gene contains the following operably linked DNA fragments:

(a) a plant-expressible promoter region, in particular the CaMV35S promoter region;

(b) a transcribed region of DNA encoding a protein which, when expressed, increases the amount or functional amount of the cell division controlling protein; and

c) 3'-terminal formation and polyadenylation signal functional in plant cells.

In a preferred embodiment of the invention, the expression level of cyclin D is increased by incorporation of a chimeric gene comprising a transcribed region of the DNA encoding cyclin D into the genome of the plant cell. This gene drives a plant-expressible promoter. Preferably, the transcribed DNA region comprises a nucleotide sequence selected from the group consisting of EMBL No. N ° X83369 nucleotide sequence from nucleotide position 104 to nucleotide 1108, EMBL No. N ° X83370 nucleotide sequence from nucleotide position 195 to nucleotide 1346, EMBL No. N nucleotide sequence. ° X83371 from nucleotide position 266 to nucleotide 1396, nucleotide sequence SEQ ID NO: 1 from nucleotide position 182 to nucleotide 1243, nucleotide sequence SEQ ID NO: 2 from nucleotide position 181 to nucleotide 1299, nucleotide sequence SEQ ID NO: 3 from nucleotide position 198 to nucleotide 1298, nucleotide • · ♦ 9

9 of SEQ ID NO: 4 from nucleotide position 165 to nucleotide 1109, nucleotide sequence of SEQ ID NO: 5 from nucleotide position 48 to nucleotide 1118, or nucleotide sequence of SEQ ID NO: 21 from nucleotide position 316 to nucleotide 1389 for Zeam mays CYCD2.

In a particularly preferred embodiment of the invention, the expression level of CycD2-type cyclin has been altered (i.e. enhanced) by the inclusion of the cycD2 chimeric gene in the plant cell genome. The chimeric gene contains a transcribed region of DNA encoding cyclin CysD2 driven by a plant expressible promoter. In this case, a constitutive promoter, especially the CaMV35S promoter, is preferred. Such a chimeric gene is the cycD2 gene of plasmid pCEC1. This change will change the morphology, architecture and growth characteristics of the transgenic plant, in particular to accelerate vegetative growth, in particular to change the growth rate of the transgenic plant.

The "attenuation or" enhancement of the measurable phenotypic trait is quantified as the difference between the average of the measurements associated with the description of that trait in different plants of one transgenic plant line and the average of the trait of this trait in type plants divided by the average of the trait of that trait. This difference is expressed as a percentage. Transgenic and control (wild-type plants) plants are cultured under conditions of nutrient delivery, light, humidity, appropriate temperature, and the like. Cultivation under standardized conditions is preferred. The preferred amount of amplification and attenuation is statistically significant, with a confidence level of 0.05 being preferred, in particular a confidence level of 0.01. It can be determined, for example, by one of the methods of analysis of variance (see, for example, Snedecor and Cochran, Statistical Methods The Iowa University Press, Ames, Iowa, U.S.A).

The enhancement of the vegetative growth of the transgenic plant is preferably monitored by measuring the increase in dry weight during wild growth in plants. The average dry weight increase is defined as the difference in dry weight average of transgenic and wild type plants, multiplied by 100 and divided by the average dry weight of wild type plants. A typical increase in dry weight, particularly in the initial growth period achieved by incorporation of the cycD2 chimeric genes of the invention is in the range of at least about 39% to 350%, and a range of 68% to 150% is particularly preferred.

It is understood that the increase in dry weight as a result of incorporation of the chimeric genes may vary depending on the plant species or the chimeric genes used. The invention emphasizes any substantial increase in dry weight in transgenic plants, especially if the dry weight is at least 1.4 to 4.5 times the dry weight of untransformed control plants, in particular at least 1.8 to 2.7 times the dry weight of untransformed control plants. In any case, the average dry weight of the transgenic plants differs significantly from the average dry weight of the untransformed plants.

Enhancement of the vegetative growth of the transgenic plant can also be determined by comparing the number of leaves visible on the transgenic plants and the wild-type control plants at a given time. It is expected that the difference in the number of leaves of transgenic plants in the middle of the growth period is at least 1.1 to 3 times, in particular 1.5 to 2 times the number of leaves in untransformed plants.

The vegetation growth enhancement of the transgenic plant can also be monitored by measuring the height of the stem (measured from soil level to the point of maximum growth) during the growth period. The average stem height increase is defined as the difference in the average stem height value of transgenic and wild-type plants, multiplied by 100 and divided by the average height of the wild-type plants. A typical increase in stem height by introducing the cycD2 chimeric genes of the invention is in the range of approximately 65% over time

0

early growth period, over 20 to 30% in the middle growth period to at least 10% in the flowering period, but may be higher, approximately 40-50% to 75% in the middle of the growth period and 15-20% at the end of the flowering stage.

It is understood that the stem height as a result of the introduction of the chimeric genes of the invention may vary depending on the plant species or the chimeric genes used. The invention emphasizes any substantial increase in stem height in transgenic plants, especially if the stem height is at least 1.1 to 3 times the stem height of untransformed control plants, in particular at least 1.5 to 2 times the stem height of untransformed control plants.

The difference in stem height between transgenic and control plants gradually decreases during growth, as the growth rate of the plants decreases during flowering. The final height of the transgenic plant may then be similar to the final height of the non-transgenic plants.

Transgenic plants that do not contain the cycD2 genes of the invention exhibit an increased growth rate compared to untransformed plants, resulting in a shortening of the time required to achieve a given dry weight or stem height. The term "growth rate" means an increase in the size of a plant or part of a plant per day, in particular an increase in stem height per day, and can be calculated as the difference between the size of a plant or plant part at the beginning and end of a period including a certain number of days. ) divided by the number of days. The increase in growth rate is preferably expressed with respect to the general definition of an increase in measurable phenotype, but can also be expressed as the ratio between the growth rate of transgenic plants versus the growth rate of untransformed control plants during the same period under the same conditions.

As described above, the increase in growth rate as a result of incorporation of chimeric genes, particularly the cycD2 chimeric genes of the invention, can vary depending on the plant species and used to emphasize any substantial transgenic plants, particularly, in the range of about 4 % and 85% 60%, especially from about 30% of the chimeric genes. The invention increases the growth rate if the growth rate is, especially from about 20% to 50%.

Acceleration of the vegetative growth of the transgenic plant can also be monitored by measuring the length and size of the largest leaf at different times during the growth period, while the leaves continue to grow. This measurable phenotype expresses the degree of increased maturity of the transgenic plants. The mean value of the largest leaf length increase (defined as rosdil between the average largest leaf length of transgenic and wild type plants, multiplied by 100 and divided by the average largest leaf length in wild type plants) obtained by introducing the chimeric genes of the invention is 7 to 31% (average value is about 17%), in the intermediate phase of growth this value is in the range of about 3 to 14% (average value is 7%).

Again, increasing the largest leaf size as a result of introducing the chimeric genes of the invention may germinate depending on the plant species or chimeric genes used, and the invention emphasizes any substantial increase in leaf growth or transgenic plant size.

The invention further provides a chimeric gene controlling cell division. In particular, the cycD2 chimeric genes, which can also be introduced into plants to increase the development of roots, in particular will increase the average value of the root length. In general, an increase in root development occurs concurrently with an increase in the vegetation portion above the ground (stem, leaves, flowers) and may range from about 40% to 70%. Again, this increase varies depending on the plant species and the chimeric genes used. The invention emphasizes any significant increase in root development, particularly a statistically significant increase.

·· ····

Further, the invention describes a chimeric cell division controlling gene, particularly the cycD2 chimeric genes, which can also be introduced into plants to increase size and also the number of flowers, particularly the number of fertilized flowers and the number of fertilized eggs in each flower. As a result of the increase in the number of fertilized flowers and the number of fertilized eggs in each flower (in general this leads to an increase in the number of seeds in the plant), the yield of seeds with each plant will increase. Obviously, the increased number of flowers and eggs in the flower as well as the increase in seed yield varies depending on the plant species transformed with the chimeric genes controlling the cell division of the invention or the chimeric genes used. A typical value of the increase in flower size as a result of the introduction of a chimeric gene comprising a DNA region encoding CysD2 under the control of the CaMV35S protmotor is in the range of at least about 4 to 30%, particularly at least 10 to 20%. A typical increase in the number is in the range of at least about 20 to 50%, in particular at least 24 to 45%, while the increase in the number of seeds in the plant (expressed in weight) is in the range of at least about 5 to 55%, especially at least 10 to 30%. about 25% is preferred.

In another embodiment of the invention, the chimeric gene controlling cell division, particularly the cycD2 chimeric genes, which can also be introduced into plants or their seeds, accelerates germination. It has been found that transgenic seeds containing the cycD2 chimeric genes of the invention can germinate at least 8. up to 16 hours faster than wild type control.

The aforementioned chimeric genes can also be introduced into plants to increase the average number of days required to achieve inflorescence development, effectively reducing the time the plants need to bloom. Transgenic plants containing the chimeric genes of the invention, as well as the maturity, especially the stage of inflorescence of a fierce but normal size flowering plant. Indeed, they achieve a shortening of the time it takes to reach the flowering stage can

Depending on the plant species or the chimeric genes used. Typically, transgenic plants carrying a chimeric gene comprising a DNA region encoding CycD2 under the control of the CaMV35S promoter exhibit a reduction in flow time of at least about 3% to 11-12%, particularly at least about 4% to 7%.

In another particularly preferred embodiment of the invention, a chimeric gene comprising a transcriptional DNA region encoding CycD3 is introduced into the plant under the control of a plant-expressible promoter, preferably a constitutive promoter, particularly a CaMV35S promoter, such as a chimeric gene comprising the nucleotide sequence of the cycD3 plants with altered morphological features or architecture, particularly altered specific plant parts or organs, particularly altered size and morphology of flowers with elongated and / or enlarged crown petals. Transgenic plants transformed with a chimeric gene containing a DNA region encoding CycD3 under the control of a plant-expressible promoter (and its progeny) exhibit an increased flower size of about 31% to 44%. However, these plants also bloom later in comparison to wild-type plants, which corresponds to an increase in flowering time of about 5 to 20%, especially about 8 to 16%.

In another embodiment of the invention, a functional amount of a cell division controlling protein capable of phosphorylating an Rb-like protein or binding to the pocket-like domain of Rb-like proteins, particularly cyclin type D, is increasing to obtain a plant with altered growth rate and architecture by incorporating the chimeric gene into the genome plant cells containing the following operably linked DNA fragments:

(a) the plant-expressible promoter region, in particular the CaMV35S promoter region,

b) a region of the transcribed DNA coding for a protein which, when expressed, increases the functional amount of the protein which, when expressed, increases the functional amount. A cell division controlling protein, preferably a portion of DNA encoding a mutant cell division controlling protein or a portion of a mutant cell division controlling protein. More preferably, a portion of the DNA encoding the mutant type D cyclin, particularly the type D cyclin, which exhibits a mutation in the cyclin box (particularly a substitution of amino acid 185 or amino acid 155 of type D cyclin, especially E185 or K155A) or type D cyclin in particular, where the C-terminus has been removed to remove FIST sequences, or type D cyclins, where the LxCxE binding motif has been altered or deleted, particularly where the C-residue has been deleted from the LxCxE binding motif, and

c) 3'-terminal formation and polyadenylation signal functional in plant cells.

Although not intended to be limited to mode of action, it is believed that mutant cell division control proteins exert their effects by masking inhibitors or anagonists of normal functional cell division control proteins.

It follows from this disclosure that chimeric genes comprising a transcribed DNA region encoding other type D cyclins, in particular CycD family cyclins derived from plants, can be used to obtain similar effects. These genes can be obtained from other plant species or varieties in a variety of ways, including hybridization, whereby available DNAs encoding CycD1, CycD2 or CycD3 are used as probes and the stringency of the hybridization conditions is reduced. Further, polymerase chain reaction-based methods using oligonucleotides based on the available type D cyclin nucleotide sequences may be used. Preferred are oligonucleotides having a nucleotide sequence encoding a consensus corresponding amino acid sequence, particularly oligonucleotides having a nucleotide sequence corresponding to sequences encoding conservative amino acid sequences in cycling box ··· «· ·

98 • 9 9 9

9 9 9

9 9 9

99 oligonucleotides of cyclins of each group of cyclins. These conserved amino acid sequences may be derived from available DNA encoding such amino acid sequences. A particularly preferred combination suitable for PCR amplification of plant D1 is an oligonucleotide selected from the group of oligonucleotides having SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and an oligonucleotide selected from the group of oligonucleotides having the DNA sequence shown in SEQ SEQ ID NO: 10 or SEQ ID NO: 11.

A particularly preferred combination of oligonucleotides suitable for PCR amplification of plant D2 type cyclins is an oligonucleotide selected from the group of oligonucleotides having the DNA sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 and an oligonucleotide selected from the group of oligonucleotides comprising the DNA sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15. A particularly preferred combination of nucleotides suitable for PCR amplification of plant cyclins of type D3 is an oligonucleotide selected from the group of oligonucleotides comprising the DNA sequence set forth in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 and an oligonucleotide selected. from the group of oligonucleotides having the DNA sequence shown in SEQ ID NO: 19 or SEQ ID NO. 20. The amplified DNA fragment was then used to screen the cDNA library and genomic library (under stringent conditions) to isolate full-length clones.

Alternatively, other genes encoding plant-derived cyclins can be obtained by methods such as functional complementation of yeast strains that are conditionally deficient in Gl-S cyclins as described in Soni et al., 1995 Plant Cell 7: 85-103 and Dahl et al., 1995 Plant Cell 7:

1847-1857 or by using a yeast two hybrid system (Fields and Song, 1989 Nature 340: 245-246) to isolate DNA sequences encoding cyclins binding to the pocket domain of Rb-like proteins as described above.

> · · · · ·

Further, some plants are known to contain more than one gene encoding type D cyclin of the same subgroup (for example, a tobacco plant comprises at least two genes of the CycD3 subgroup) and it is understood that these variants can be used in accordance with the invention.

Type D cyclins having an amino acid sequence substantially similar to the amino acid sequence described herein, such as mutant cyclins. however, they can be used to achieve the same effects. With respect to the amino acid sequence, the term "substantially similar" means when the two relevant sequences are aligned, the percent sequence identity, i.e. the number of positions with the same amino acid residues divided by the number of residues shorter of the two sequences, is greater than 80%, preferably greater than 90% . Alignment of the two amino acid sequences was performed using the Wilbur and Lipman algorithms (Wilbur and Lipman, 1983 Proc. Nat. Acad. Sci. USA 80: 726) using 20 amino acids, the word length being 2 amino acids and the gap bridging is 4. Computer analysis and interpretation of sequence data, including sequence alignment as described above, can be performed using Intelligenetics ™ Suite programs (Intelligenetics Inc, CA).

It will be appreciated that any DNA sequence encoding a cell division controlling protein, in particular type D cyclin, may be used in the construction of the cell division control genes of the invention, in particular DNA sequences which are wholly or partially synthesized artificially.

It is also evident that other plant-expressible promoters, especially constitutive promoters such as the Opine synthase promoter of Agrobacterium Ti or Ri-plasmids. In particular, the nopaline synthase promoter may be used to achieve the same effect. In the case of the existence of various forms of the CaMV35S promoter, it is clear to the person skilled in the art that the object of the invention can equally be achieved by the use of:

of these CaMV35S promoter alternatives as described in Hul and Howell, Virology, 86, 482 (1978).

The invention further provides plants with altered morphology or architecture limited to specific organs or tissues using a tissue-specific or organ-specific promoter to control expression of DNA that encodes a cell division-controlling protein, in particular cyclin type D. Such tissue or organ-specific promoters are known. and include, but are not limited to seed-specific promoters (eg, WO89 / 03887), original organ-specific promoters (An et al., 1996 The Plant Cell 8: 15-30), stem-specific promoters (Keller et. al., 1988 EMBO J. 7: 36253633), leaf specific promoters (Hudspeth et al., 1989 Plant Mol. Biol. 12: 579-589), mesophyll specific promoters (such as the light-inducible Rubisco promoter), promoters specific for roots (Keller et al., 1989 Genes

Devel. 3: 1643-1646, tuber-specific promoters (Keil et al., 1989 EMBO J. 8: 1323-1330), vascular tissue-specific promoters (Peleman et al., 1989 Gene 84: 359-369), promoters specific for meristem (such as the SHOOTMERISTEMLESS (STM) gene promoter) (Long et al., 1996 Nature 379: 66-69), a primordia-specific promoter (such as the CycD3a Anirrhinum gene promoter, Doonan et al., 1988 in " Plant Cell Division (Francis, Duditz and Inze, Eds., Portland Press, London) and the like.

In another embodiment of the invention, expression of a chimeric gene that encodes a cell division controlling protein can be controlled by applying a suitable chemical inducing agent, operably linking the DNA region encoding the cell division controlling protein to a promoter whose expression is induced by a chemical such as a gene promoter described in European application EP 0332104 or the gene promoter disclosed in WO 90/08826.

In another embodiment of the invention, expression of a chimeric gene that encodes a cell division directing protein can be controlled by use

site-specific recombinase and its corresponding cis-expressible plant-expressing sequences and cell division controlling protein

For example, an unrelated nucleotide sequence (preferably may contain a transcription and / or translation terminating signal) flanked by cis-acting sequences that is recognized by site-specific recombinase (eg, Iok or FRT sites) may be inserted between the promoter transcribed region. Alternatively, said expression can be controlled such that in plant cells containing said chimeric gene with a site-specific recombinase (eg Cre or FLP), the incorporated unrelated nucleotide sequence is eliminated by recombination, allowing expression of the chimeric gene controlling cell division.

It is contemplated that morphological changes due to overexpression in plants of cell division control proteins, in particular type D cyclins, caused by the introduction of a chimeric gene containing a DNA region encoding cell division control protein, particularly cyclin type D, expressed in plants may inactivate FEST sequences . FEST sequences are amino acids sequences that are rich in proline, glutamate or aspartate and serine or threonine, which hay between positively charged flanking sequences involved in the conversion of a protein containing such sequences (Tyers et al., 1992 EMBO

J. 11: 1773-1784; Cross, 1988. Mol. Cell. Biol. 8: 4675-4684; Wittenberg and Reed, 1988 Cell 54: 1061-1072; Salama et al. ,

Cell. Biol. 14: 7953-7966). Removal of these fists in yeast cyclins stabilizes them in vivo (Pines, 1995 Biochem. J. 308: 697-711).

PEST regions can be identified by computer analysis using software such as PESTFIND (Rogers et al., 1986 Science 234: 364-368; Rechsteiner,

1990 Seminars Cell Biol. 1: 433-440). DNA mutations encoding a protein controlling the division of cells with altered PEST sequences are driven by a promoter to enhance adaptation or

1994 Mol Sequences ·

··· · · · · ····

O / -) · ♦ ♦ ·······

JZ is well known in the art. The methods described in Sambrook et al., Molecular Cloning: A Laboratory, Manual, 2 ^nd . Ed. (1989), Cold Spring Harbor, New Yourk.

It is further contemplated that the quantitative effects of phenotypic changes may be altered, i.e., enhanced or attenuated, by expressing endogenous chimeric genes controlling cell division, particularly endogenous chimeric genes that encode CycD, as an alternative to using heterogeneous genes encoding similar proteins from other plants. It is preferred that heterogeneous genes, in particular heterogeneous genes encoding similar proteins, be used which exhibit less than about 65%, preferably less than 75%, preferably less than 65%, amino acid sequence identity with the endogenous cell division controlling protein.

The invention further discloses that plant morphology can be altered by down-regulating the expression of a functional cell division controlling protein, in particular cyclin type D. This can be achieved, for example, by using antisense DNA, ribozyme or co-suppression methods. A plant chimeric gene containing a region of DNA transcribed into RNA is introduced into plant cells, the production of which reduces, inhibits or prevents the expression of a cell division controlling protein, in particular cyclin type D. It prefers stable incorporation into the genome of plant cells.

In accordance with the invention, the DNA region of the chimeric gene encodes an antisense RNA that is complementary to at least a portion of the sense mRNA encoding a cell division controlling protein, particularly type D cyclin. Thus, the antisense RNA comprises a region that is complementary to the sense mRNA portion. It is preferred to form a continuation thereof of at least 50 bases. A length of 100 to 1,000 bases is particularly preferred. The antisense RNA may be complementary to any portion of the mRNA sequence: it may be complementary to a sequence adjacent to the 5 'end or to the dropping site, part or all of the leader region, the intron or exon region (or the bridging region).

intron or exon), a sense pre-mRNA, with a region that spans the non-coding or coding region, with all or part of the coding region comprising the 3'-end of the coding region and / or all or part of the 3'-end region or terminal RNA sequence Sequence similarity for the antisense RNA and the sense RNA complement encoding the cell division directing protein should be in the range of at least about 75 to 100%.

In another embodiment of the invention, the transcribed DNA region of the chimeric gene encodes a specific RNA enzyme or so-called ribozyme (described, for example, in WO 89/05852) that is capable of highly specific cleavage of sense mRNA that encodes a cell division controlling protein, particularly cyclin type D.

In another embodiment of the invention, the amount of functional cell division control protein, particularly type D cyclin, can be reduced by expressing a chimeric gene comprising a DNA region encoding a protein or polypeptide which, when expressed, reduces the amount of cell division control protein, particularly type D cyclin. or inhibits this protein to perform its function in cells. It is preferred that the chimeric gene encodes antibodies that bind to a cell division-controlling protein, particularly type D cyclin.

Reducing the amount or functional amount of a cell division controlling protein, in particular type D cyclin, in cells of a transgenic plant containing the chimeric genes of the invention results in a change in plant architecture. In particular, the height of the stem, the growth rate or the time to reach the flowering plant, in comparison with untransformed plants which are grown under the same conditions, are varied, or the time required for flowering can be increased. The effect obtained may vary depending on the plant species or chimeric genes used. The invention emphasizes any effect on architecture and / or rate of growth, in particular reducing stem height or slowing plant growth or prolonging the time required to develop a flower.

··· 9 9 · 9 · 9 · ······ 9 9 99 99 9

9 9 9 9 9 9 9 9 9 9 99 99

The growth rate retardation caused by the decrease in the amount of cell division controlling protein, preferably type D cyclin, especially CYCD2 type cyclin, is in the range of about 30% to 60%, especially in the range of about 35% to 50%.

The reduction in stem height caused by a decrease in the amount of cell division controlling protein, preferably cyclin type D, particularly CYCD2 type cyclin, is in the range of about 10% to 60%, especially in the range of about 30% to 50%. .

The prolongation of the time required to achieve inflorescence due to a decrease in the amount of cell division controlling protein is preferred, cyclin type D, particularly cyclin type D2 being in the range of about 10% to 40%, particularly in the range of about 15% to 38%.

The cell division chimeric gene may further comprise other regulatory or other function sequences, such as leader sequences [e.g. cab22L leader sequence from Petunia or omega leader sequence derived from TMV (Gallie et al., 1987 Nucl. Acids Res. 15: 3257-3273)],

3transcriptional termination signal and polyadenylation signal (e.g., octopine synthase gene (De Greve et al., 1982 J. Mol. Appl. Genet. 1. 499)), 3transcriptional termination signal and polyadenylation signal of the nopaline synthase gene (Depicker et al., 1982 J. Mol. Appl. Genet. 1: 561) or T-DNA gene 7 (Velten and Schell, 1985 Nucl. Acids Res. 13: 6998) and the like (Guerineau et al., 1991 Mol. Gen. Genet. 226 Proudfoot, 1991 Cell, 64: 671-674, Safacon et al., 1991 Genes Dev 5: 141-149, Mogen et al., 1990 Plant Cell, 2: 1261-1272, Munroe et al. , 1990 Gene 91: 151-158; Ballas et al., 1989 Nucl. Acids Res. 17: 7891-7903; Joshi et al., 1987 Nucl. Acids Res. 15: 9627-9639), translation initiation sequence in plants (1990). Joshi et al., 1987 Nucl. Acids Res. 15: 9627-9639), introns (Luehrsen and Walbot, 1991 Mol. Gen. Genet. 225: 81-93) and • 0 • 0 0 0 0 0 0 0 0 0 0 ·· · 0 0 0 0 0000 * 0 · · 0 0 0 »0 • 0 0 0> ··· 0 0 00 0000 0 0 00 similarly, operatively connected with the nucleotide sequence of the chimeric gene controlling cell division.

It is preferred that the recombinant DNA containing the chimeric gene controlling the cell division be accompanied by a chimeric marker gene. The marker chimer gene may comprise a DNA marker that is operably linked at its 5-terminus to a plant-expressible promoter. A constitutive promoter such as the CaMV 35S promoter or a light-inducible gene promoter encoding a small Rubisco subunit is preferred. At its 3'-end, the marker DNA is linked to the 3'-end of the transcription and polyadenylation signal. It is expected that the choice of the DNA marker is not critical and any suitable marker DNA may be used. For example, the marker DNA may encode a protein that provides the transformed plant cell with a distinctive color. For example, it is the Al gene (Meyer et al., 1987 Nature 330: 677), which confers herbicide resistance on transformed plant cells, or a bar gene encoding phosphinothricin resistance (described in EP 0,242,246) or can provide antibiotic resistance. Such a gene is the aac (6 ') gene encoding gentamycin resistance (WO94 / 01560).

Although it is clear that the invention is applicable to all plant species and varieties, it is particularly useful for altering the architecture or increasing the growth rate of plants that can be used commercially. It is expected that the enhancement of vegetation growth will be most pronounced in plants that do not undergo extensive breeding and selection due to rapid vegetation growth. The invention is particularly relevant to plants that are grown in a greenhouse, particularly to reduce the time required to reach the desired stage of development, such as the time required to achieve inflorescence, the formation of fruits or seeds. The invention is further relevant in accelerating the growth of trees, especially coniferous trees such as pine, poplar, Eucalyptus and the like. Another important application of the invention is φ · · φ · · · · · φ φ · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ Expansion of effective areas where plants can be cultivated. The time required to reach an economically important development stage is reduced. A particularly preferred plant where the invention may be advantageously applied is corn, oilseed rape, flaxseed, wheat, grass, alfalfa, legumes, tomatoes, lettuce, rice, barley, potatoes, tobacco, sugar beet, sunflowers and ornamental plants such as carnation, chrysanthemum, roses, tulips and the like.

Recombinant DNA containing a chimeric gene controlling cell division can be stably incorporated into the genome of the plant cell nucleus. The gene transfer can be effected by a vector which is an armless Ti-plasmid containing the chimeric gene of the invention and by Agrobacterium. This transformation can be accomplished using the procedure described, for example, in EP 0,116,718. When other plant transformation methods are used, such as direct gene transfer (described in EP 0,233,247), pollen-mediated transformation (described in EP 0,270,356; WO 85/01856 and US 4,684,611), virus-mediated plant RNA transformation (described in EP 0,067,553 and US 4,407,956), liposome-mediated transformation (see, for example, US 4,536,475), other types of vectors may be used.

Other methods, such as microprojectile bombardment as described by Fromm et al. , 1990 Bio / Technology 8: 833 and Gordon-Kamm et al. 1990 The Plant Cell 2: 603. Monocotyledon cells, such as major cereals, can be transformed using wounded and / or enzymatically degraded compact embryogenic tissue capable of forming compact embryogenic kallus or wounded and / or degraded immature embryos as described in WO 92 / 09696. The resulting transformed plant can then be suitably used to regenerate the transformed plant.

The transformed plant obtained can be used in a conventional breeding scheme to produce more transformed plants with

9 9 9 9 9 9 9 9 9 9 by the same characteristics or by incorporating a chimeric gene controlling cell division in other varieties of the same or related plant species. Seeds obtained from transformed plants contain a chimeric gene controlling the cell division of the invention, as a stable genome insert.

The following examples describe the construction of a cell division chimeric gene and the use of such a gene to modify the architecture and plant growth rate. Unless otherwise indicated in the examples, genetic engineering methods are performed according to standard protocols described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd . Ed. (1989), Cold Spring Harbor, New Yourk. Standard materials and methods of molecular processing of plants are described in Plant Molecular Biology Labfax (1993) RDD Croy; BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

List sequences: SEQ ID NO: 1: cDNA encoding CYCD2; Nicotiana tabacum SEQ ID NO: 2: cDNA encoding CYCD3; Nicotiana tabacum SEQ ID NO: 3: cDNA encoding CYCD3; 2 Nicotiana tabacum SEQ ID NO: 4: cDNA encoding CYCD1; 1 Helianthus tuberosus SEQ ID NO: 5: cDNA encoding CYCD3; Helianthus tuberosus SEQ ID NO: 6: T-DNA of pGSV5 SEQ ID NO: 7: PCR primer 1 SEQ ID NO: 8: PCR primer 2 SEQ ID NO: 9: PCR primer 3 SEQ ID NO: 10 PCR primer 4 SEQ ID NO: 11 PCR primer 5 SEQ ID NO: 12 PCR primer 6 SEQ ID NO: 13 PCR primer 7 SEQ ID NO: 14 PCR primer 8 SEQ ID NO: 15 Dec PCR primer 9 SEQ ID NO: 16 PCR primer 10

• flfl flfl flfl flflfl flflfl fl flfl flfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl flflfl ··· · * · ·

SEQ ID NO: 17: PCR primer 11 SEQ ID NO: 18: PCR primer 12 SEQ ID NO: 19: PCR primer 13 SEQ ID NO: 20: PCR primer 14 SEQ ID NO: 21: cDNA encoding CYCD2 Zea Mays Plasmid pCEC1 and pCRK9 are deposited within the institution

Belgian Coordinated Collections of Microoragnism (BCCM) Laboratory of Molecular Biology-Plasmidecollectie (LMBP)

Universiteite Gent

K.L.Ledeganckstraat 35

B-9000 Ghent, Belgium

The deposit was carried out on 11 March 1997 and is marked with the following numbers:

MC (1061) (pCEC1): BCCM / LMBP3657 DH5α (pCRK9): BCCM / LMBP3656

The plasmids pBlueScript-ZM18 were deposited in the institution

Belgian Coordinated Collections of Microorganisms (BCCM) Laboratory of Molecular Biology-Plasmidecollectie (LMBP)

Universiteit Gent

K. L. Ledeganckstraat

B-9000 Ghent, Belgium

The deposit was carried out on 19 March 1998 and is numbered

BCCM / LMBP 3866.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Construction of chimeric genes

1.1. Construction of the CaMV35S-AthcycD2 chimeric gene and inclusion in the T-DNA vector

The 1298 bp NcoI-SacI fragment containing the DNA encoding A.thaliana CYCD2 (having the nucleotide sequence EMBL # X83370, position 194-1332) was blunt-ended with Klenow polymerase and ligated into plasmid pART7 with linearized restriction enzyme SmaI (Gleave) (1992 Plant Mol Mol Biol 20: 1203-1207). This results in plasmid pCEC1. In this way, a chimeric gene flanked by NotI restriction sites was constructed, wherein the DNA encoding CYCD2 was operably linked to the CaMV35S promoter of CabbB-J1 isolate (Harpster et al., 1988 Mol. Gen. Genet. 212: 182-190) and the 3'ocs region ( MacDonald et al., 1991 Nucl. Acids Res. 19: 5575-5581). The chimeric gene was then inserted between the T-DNA border of the T-DNA vector containing the selective chimeric marker gene.

The cycE> 2 chimeric gene was excised from the plasmid pCEC1 using the NotI restriction enzyme and ligated into the plasmid pART27 (Gleave, 1992 Plant. Mol. Biol. 20: 1203-1207) linearized with the NotI restriction enzyme to produce pCEC5. Plasmid pART27 contains a selectable marker chimeric gene containing the following operably linked fragments: a nopaline synthase gene promoter encoding the neo region and the 3 'end of the nopaline synthase gene (An et al., 1988 Bunary vectors In: Gelvin SB, Schilperoort RA, Verma DPS (eds)) Plant Molecular Biology Manual pp. A3 / 1-A3 / 19 (Kluwer Academic Publishers, Dordrecht).

Alternatively, the cycD2 chimeric gene was excised from plasmid pCEC1 using a suitable restriction enzyme (eg, NotI) and inserted into the polylinker between the T-DNA border sequences of the pGSV5 vector, together with the chimeric selectable marker gene (pSSU-bar-3). De Almeida et al., 1989 Mol. Gen. Genet. 218: 78-86) to give pCEC5b.

Plasmid pGSV5 was derived from plasmid pGSC1700 (Cornelissen and Vandewiele, 1989 Nucl. Acids Res. 17: 833), but differs from later generated plasmids that do not contain the beta-lactamase gene and that its T-DNA is characterized by SEQ ID NO: 6.

1.2 Construction of the CaMV35S-AthcycD3 chimeric gene and inclusion in the T-DNA vector.

cDNA cDD3 was isolated as a 1335 bp BsII-DraI fragment containing blunt ends after Klenow polymerase treatment (exhibiting EMBL nucleotide sequence # X83371, nucleotide position 104-1439). This fragment was inserted into the SmaI restriction site of plasmid pUC18 to generate pRS14a. This clone carries the entire coding sequence of cycD3 with a translation initiation codon located immediately adjacent to the SmaI restriction site of plasmid pUC18 in such an orientation that the SacI restriction site of plasmid pUC18 is at the 5 'end of the cDNA cDD3 and the BamHI restriction site at the 3' end. The 1.35 kb SacI-BamHI fragment of pRS14a was isolated and ligated into the 26.6 kb SacI-BamHI fragment of pSLJ94 (Jones et al., 1992 Transgen. Research 1: 285-297) to give pCRK9. In this way, a chimeric gene was constructed in which the DNA region coding for cycD3 from A. thaliana was operably linked to the CaMV35S promoter and the 3'ocs region. In plasmid pCRK9, the chimeric gene is located between the edges of the T-DNA and is accompanied by a neo chimeric selection gene (Jones et al., 1992 Transgen. Research 1: 285-297).

Alternatively, the cycD3 chimeric gene is excised from plasmid pRS14a using appropriate restriction enzymes and inserted into the polylinker between the T-DNA flanking sequences of the pGSV5 vector together with a chimeric selectable marker gene (pSSU-har-3'ocs; DeAlmeida et al., 1989 Mol. Gen. Genet. 218: 78-86) to give plasmid pCRK9b.

Example 2: Transformation of a tobacco plant by T-DNA with vectors of Example 1 mediated by Agrobacterium

The T-DNA vectors pCEC5 and pCRK9 were introduced into Agrobacterium tumefaciens LBA4404 (Klapwijk et al., 1980 J. Bacteriol. 141: 128-136) by electroporation as described in fefe fefe fefe. Fe fe fe fe fe fe fe fe fe fe fe fe fe fe fe feef fe fe feef fefe feefefe in Walkerpeach and Velten (1995) In: Gelvin SB, Schilperoort RA, Verma DPS (eds) .) Plant Molecular Biology Manual pp. Kluwer Academie Publishers, Dordrecht). Transformants were selected using Spectinmycin and Tetracycline.

The T-DNA vectors pCEC5b and pCRK9b were introduced into A.tumefaciens C58ClRif ^{R by} triple crossing (Ditta et al., 1980 Proc. Natl. Acad. Sci. USA 77: 7347-7351).

The resulting Agrobacterium strains were used to transform Nicotiana tabacum variety Xanthi. In this case, the disk transformation of the leaves was applied as described in An et al., 1985 EMBO J. 4: 277-284.

Eight tobacco plants transformed with plasmid pCRK9 (designated as 1K9, 2K9, 3K9, 4K9, 8K9, 10K9, 17K9, 19K9 and 28K9) and 11 tobacco plants transformed with plasmid pCEC5 (designated C8 lines 1-3 and 5-12) were generated.

Plants transformed with pCRK9 T-DNA were examined for copy number of incorporated transgenes by Southern blotting, using the labeled pRS14a cDNA insert as probe. Each 2K9, 3K9 and 4K9 lines received one copy of the transgenes, while the 1K9 line contained three copies of the transgenes.

Plants transformed with pCEC5 T-DNA were analyzed for copy number of incorporated transgenes by Southern hybridization using BamHI digested DNA prepared from said plants and a 0.7 kb labeled NcoI-EcoRI fragment derived from a J22 cDNA (containing a portion of the cycD2 coding region; (Soni et al., 1995 Plant Cell 7: 85-103). Lines C8-2, C8-3, C8-5, C8-8, C81, C8-9. C8-10, C8-11, C8-12 showed all copies of transgenes, lines C8-7 had two copies, lines C8-6 had three copies, and lines C8-1 had four copies of transgenes.

TOs (primary transformants) self-fertilized to produce seeds (TI). Plants grown from T1 seeds were designated C8-T1-X, where X is the line number of the original transformant.

• 9 • 9 9 9 9 9 9 9 9 • 9 9 9 9 9

9999 9 9 9 9 99 99 9 • 999 9999

99999 99 99

Seeds from T1 plants were designated T2. Plants grown from such seeds were designated C8-T2-X, where X is again the line number of the original transformants. In the absence of populations, the plants were grown from T1 seeds.

Northern analysis confirmed transcription of the transgenes in at least the C8-1, C8-3, C8-7, 3K9, 4K9 and 8K9 lines.

Example 3: Phenotype analysis of transformed tobacco plants.

3.1. Tobacco plants containing the CaMV35S-AthCycD2 chimeric gene

Seeds from primary transformants (TO plants) were surface sterilized with 10% bleach for 15 minutes and then washed in sterile water. Surface sterilized seeds were germinated on GM medium containing kanamycin at a final concentration of 100 μ9 / πι1. Seeds on plates were stored over a period of 5 days at 4 ° C (vernalization) and then stored at 23 ^{° C} C in a growth chamber. All time data refers to the day of placement in the growth chamber. 18 days after transfer to the growth chamber (i.e. 23 days in total), kanamycin resistant seedlings were transferred to seed troughs containing soil and grown in a growth room at a photoperiod of 18 hours. After a further 10 days, these plants were transferred to 7 cm pots and after a further 15 days they were transferred to 20 cm pots where they remained for the remainder of the time of the expreiment. Plants in pots were incubated in a greenhouse. Daily photoperiod was supplemented by lighting for a total of 18 hours. The plants were placed in a greenhouse in a random array.

The measurement was started over the next two days (i.e. after 45 days or 27 days in soil; referred to as week 1) and repeated every week for seven weeks. The following number of plants were tested from each line: 22 plants from line C8-1, 7 plants from line C8-2, 22 plants from line C8-3, 8 plants from line C8-5, 6 plants from line C8-6, 22 plants from line C8-7, 5 plants «· ·············

From line C8-8, 6 plants from line C8-9, 4 plants from line C810, 6 plants from line C8-11, 5 plants from line C8-12, 34 plants from untransformed control (wild type).

The following parameters were analyzed: height of the plant from the soil surface to the highest point (shown in Table 1 as average height +/- standard deviation in cm), the length of the largest leaf at a defined time (shown in Table 2, expressed is the mean length +/- standard deviation in cm), the time (given in Table 3, as the mean time +/- standard deviation in days) when meristem inflorescence is visible to the naked eye (inflorescence of 0.25 cm and 1 cm), height at which meristem inflorescence is visible (shown in Table 3 as average length +/- standard deviation in cm), length of flower petals, width of root neck of flower petals (shown in Table 3 as average length and width + / standard deviation in mm), the total number of seed capsules per plant and the average seed yield per plant (by weight).

Transgenic plants showed an increased growth rate from the seedling stage, resulting in a higher average stem height value (Table 1). At week 3, all transgenic lineage populations are substantially higher than untransformed control plants (t-test, 95% probability rate), while lines C8-1, C8-2, C8-3, C8-5, C8-11 are substantially higher than untransformed controls at a confidence level of 99%. Increased growth rates lead to larger leaves at the indicated time, which corresponds to the period when leaf growth continues (Table 2) and to larger flowers, with the petals being on average longer than those of untransformed plants.

Transgenic plants also increased the number of flowers, as well as the number of fertilized flowers, resulting in more seed capsules and more seed yields (see Table 4). However, the number of seeds per capsule was u

99 99 ·· * 9 · 9 9 9 9

999999 · · 9 9 9 9

998 9 9 9 9

999 9 99 9999 99 99 transgenic plants higher than wild-type control plants. The yield of aberrant seeds in the C8-T1-6 line was due to an excessively high percentage of fallen off flowers.

It can be concluded that constitutive expression of the DNA encoding AthCycD2 leads to an increase in the number of seed capsules and the total seed yield per plant.

Root development was compared in wild-type and transgenic seedlings (see Table 4). The seeds were sterilized, plated on GM culture medium plates without selection, vernalized and stored in a vertical position in a growth room. Root length was measured 9 and 13 days after vernalization and the presence of lateral roots was recorded. Seeds of the C8T1-7 and C8-T2-2 (homozygous) lines were used. The C8-T1-7 line shows two inserts that segregate approximately 15: 1 on kanamycin plates. This line has grown to 35 seedlings, three of which represent the growth rate observed in wild-type seedlings. Data from these seedlings were recorded separately nine days after vernalization. A t-test was applied to determine the significance of the mean value differences and the significance level is given in the table. The ns symbol indicates an insignificant difference between samples. Thus, the increase in the vegetative growth of the apical parts seems to be in balance with the development of the roots.

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wonder. type 1 O 00 •"to 1 NJ C8-T1-11 C8-T1-10 C8-T1-9 and 1 -1 t what O "the _X 1 C8-T1-6 C8-T1-5 C8-T1-3 | C8-T1-2 C8-T1-1 Line rť Xk WHAT O WHAT OJ cn WHAT Xk 05 / 03 / 03 / he Xk Xk nj O WHAT <1 čn <33 what what <33 bu CD cn CL WHAT O O what cn O Xk OJ O 05 / (D 1+ 14 I4 14 H- H- H- 14 B · 14 14 14 CL tí -to NJ N) -to -to OJ -X ""to NJ * x NJ 3 o> čn OJ WHAT Xk Xk O ** 4 mX čn NJ Xk H 'l · ^-1 OJ cn O what Xk -X Xk cn 05 / what OJ cn rt •and NJ -AND cn 'C' O —0 what O -to CD O cn • * 4 03 / 03 / I- ¹ CL čn čn O čn Oj WHAT <1 OJ Xk NJ (D O WHAT O 00 NJ NJ cn * x WHAT CL 3 1+ 14 H- (4 14 (4 14 14 14 14 14 14 3 OJ cn OJ Xx OJ NJ Xk NJ _x Xk NJ OJ • H ^with M OJ WHAT O OJ Xk čn čn 03 / what NJ Xx OJ cn NJ 03 / NJ - * · 05 / - * · WHAT Xk -X 03 / rt cn 03 / Xk Xk Xk OJ Xk OJ Xk Xx Xx OJ CL -to NJ cn cn AND CD OJ Xk 03 / <o CD (D 03 / 03 / _x <1 05 / OJ -and. 05 / _x what WHAT CL 3 NJ Η * * 4 cn * 4 NJ NJ O CD Xk 05 / xX 3 H- 14 14 H- H- 14 H · 14 14 H- 14 H 'CO O -to cn CD 05 / NJ 05 / 05 / 03 / WHAT Xk 05 / 05 / O 05 / O WHAT _x Xk 05 / Č5 <í 05 / NJ - ^k OJ NJ O “4 NJ 05 / WHAT OJ 05 / cn cn * 4 ~ 4 05 / 05 / -4 cn * 4 05 / * 4 <33 cn cl (D CL 3 Xk what NJ cn NJ NJ Xk NJ O 05 / * 4 03 / O cn NJ OJ X. 05 / 05 / 05 / čn O O H * cn 03 / • * 4 O 05 / * 4 WHAT 14 Xx WHAT 14 I4 14 14 14 14 14 14 (4 14 “4 OJ cn -4 WHAT OJ -4 cn cn -x Xk what 03 / OJI čn. “4 what _x what OJ O ** 4 03 / WHAT NJ O NJ 03 / NJ OJ O Xk OJ WHAT • Jk - 'f-t 03 / O O O _to _x O «X WHAT cn OJ OJ 90 WHAT _x WHAT O O * 4 what cn Xx 03 / OJ • 4 - ^k Xk 05 / Xk Lx Xx CO cl 05 / O OJ ’* 4 ~ 4 to čn CD čn «X Xk NJ O CL 3 3 14 H- 14 cn H- O O OJ O 14 1+ 14 -and. «X 14 1+ 1+ H- H- -X _x O Xk - * WHAT NJ 03 / 03 / CD 05 / cn O O H 'Cn WHAT TO čn NJ NJ O OJ O Xb čn NJ O NJ - ^k Xk 03 / 03 / CD NJ 4 —X cn ft .and _jk _X _to _x _x CO H ¹ CL NJ Xk _X cn OJ _to Xk NJ Xk OJ Xk NJ —X O Xx NJ ”4 OJ what O OJ Xk OJ WHAT 03 / Xk kj What Xk čn cn čn what OJ (D O čn cn OJ NJ what O 03 / cn O UX> -3 H- H- what H- H · O H · H- 14 H- 1+ 14 3 _to. 14 NJ H- _to —X NJ —X NJ Η 'ΟΊ 00 NJ cn —0 O cn -to OJ ΙΌ * 4 Xx NJ -to. 05 / what OJ 05 / O * 4 03 / _x '* 4 03 / 00 05 / OJ what X OJ cn OJ W -4 cn O cn "to —- Ct- X » _X cn Xk 05 / cn 05 / Xx "-to OJ CO ^ <S cn cn —Λ. NJ CD čn O NJ 05 / WHAT WHAT Xk cn what cn * 4 05 / 03 / • * 4 O O CL (D what H · 03 / H- OJ 14 H- -4 (4 O 14 Xk 14 what O what 00 14 OJ 14 O O H- WHAT H- CL 3 '3 >. -4 "and NJ OJ H '-J WHAT O what * 4 03 / -to -to cn WHAT NJ WHAT - * Xk ’* 4 NJ NJ 05 / 03 / WHAT 05 / what -j. what COJ Xk 05 / WHAT * 4 05 / WHAT OJ 03 / cn OJ O

Table 1: Average length (cm) of transformed tobacco plants containing CaMV35S-AtcycD2

d H- < rt * < C8-T1-12 C8-T1-11 C8-T1-10 O 03 / 1 “4 1 what C8-T1-8 C8-T1-7 C8-T1-6 C8-T1-5 C8-T1-3 "O CD -H 1 ro IC8-T1-1 .AND I-— ¹ H- 0 H- (D —X _to _x _x _x _x X. __x (O to cn to ro «(« X cn to cn cn 03 / OJ cn 03 / cn AND to _X čn 03 / O what 03 / cn rt ~ 4 O OJ cn _x 03 / what 03 / 03 / cn to 0 cn O OJ O -4 - «4 03 / -'J ro cn OJ 0 d 1+ l + 1+ H- 1+ H- 1+ l + l + 1+ 1+ l + (1) 2 -to -to ro ro ro ro -X it -X -X what to _x 03 / O AND AND to 03 / to AND 03 / l · - ¹ to 03 / at. WHAT OJ ro —X O 03 / O 03 / to 03 / IO OJ OJ cn what what ro cn O IO 03 / ΓΟ ro ro »X ro ro ro ro ro ro cn -to O what O ro O - O OJ WHAT 03 / to 00 _X what cn O what čn what čn what what O OJ 03 / -to O O OJ 03 / cn O 0 £ L O OJ - ^ 4 O O OJ 03 / —X O CD (D 1+ l + H- 1+ 1+ l + 1+ l + l + 1+ 1+ l + ro -X -to ro ro _x _x -to -X OJ -to 10 AND to 03 / what O 00 to what AND AND Oh O 03 / -O O ro «X -4 what O to OJ cn 0 O cn ro cn O cn 4 -O to to ÍO ro ro ro ro ÍO ro ro ro ro ro ro 03 / cn 03 / 03 / -with WHAT what what what ~ 4 oj Lk 03 / čn OJ čn what what what OJ 03 / _to rt cn O OJ 03 / OJ he O ro M to to —X 'C' N3 O OJ OJ OJ OJ what ~ 4 cn «• X OJ to d 1+ l + H- 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 6 -X ro _x it ro -X -to ro OJ _to ro what 03 / AND 03 / X čn what AND 03 / O WHAT -X OJ 03 / O ro OJ _x cn 4 O 4 CD to what O 03 / ro cn 03 / 03 / to ro cn ro ro

Table 2: Average length (in cm) of the largest leaf of transformed tobacco plants containing CaMV35S-AtcycD2

· · • · φ · φ · φ ·

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Table 3A'-Flowering development (average height at 0.25 cm or 1 cm (in cm) of inflorescence, average time to inflorescence at 0.25 or 1 cm (in days after vernalization) in plants) of tobacco transformed with CaMV35SAthCycD2.

t

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what

Table 4A: Average number of seed capsules per plant, average seed weight of six capsules (g), average seed yield per plant (g) for tobacco plants transformed with CaMV35SAthCycD2.

line average, number of seeds, capsules diameter, seed weight 6 capsules (g) average seed yield / 1 grew (gi C8-T1-1 105.15 ± 14.96 1.085 + 0.174 19.015 C8-T1-2 127.29 ± 7.82 0.824 ± 0.137 17.481 C8-T1-3 110.46 ± 16.30 1.106 ± 0.179 20.361 C8-T1-5 97.86 ± 10.81 1.105 ± 0.178 18.023 C8-T1-6 78.60+ 12.97 1,078 ± 0.150 14.123 C8-T1-7 118.17 ± 15.64 1.131 ± 0.253 22.275 C8-T1-8 123.75 ± 4.78 1.123 ± 0.165 23.162 C8-T1-9 110.83 ± 20.91 1.090 ± 0.218 20.134 C8-T1-10 104.20 ± 10.99 1.122 ± 0.311 19.485 C8-T1-11 138.20 ± 8.35 1.116 ± 0.222 25.705 C8-T1-12 106.20 ± 12.62 1.134 ± 0.056 20.072 wonder. type 106.73 ± 16.47 0.938 ± 0.118 16.685

Table 4B: Comparison of root development in wild-type seedlings and transformed seedlings

line Number plant O. Ό lateral roots Average length roots (mm) surface significance ti 9 days after vernalization WT 23 36 15,326 ± 1,893 C8-T1-7 25 100 ALIGN! 26, 520 ± 1.971 0.001 3 33 14,000 ± 3,464 Ns C8-T2-2 28 100 ALIGN! 26,911 ± 2,064 0.001 13 days after vernalization WT 16 100 ALIGN! 28,188 ± 1,893 C8-T1-7 13 100 ALIGN! 53,846 ± 1,971 0.001 C8-T2-2 15 Dec 100 ALIGN! 51,267 ± 3,464 0.001

3.2. Tobacco plants containing the CaMV35S-AthCycD3 chimeric gene. Plants containing the CaMV35S-AthCycD3 chimeric genes were grown from T1 seeds and treated as described in 3.1. Measurements were started 49 days after germination. It was measured at an interval of approximately 7 days. For each line, the following number of plants were analyzed: 11 plants for line 1K9, 19 plants for line 3K9, 20 plants for line 4K9 and 18 plants for untransformed controls.

The following parameters were analyzed: the length and flow of the flower tube (in cm) and the time (days) when at least 75% of the plants reached at least the stage where the inflorescence was clearly evident, is shown in Table 5.

Table 5: Summary of measurements in tobacco plants containing the CaMV35S-AthCycD3 chimeric gene.

line Average flower tube length (cm) Average flower tube width (cm) Average time required to reach inflorescence of 1 cm (days) 1K9 5.66 ± 0.46 3.44 ± 0.27 100 ALIGN! 3K9 5.18 ± 0.37 3.20 + 0.35 100 ALIGN! 4K9 5.48 ± 0.38 2.90 ± 0.35 93 wt 3.96 ± 0.12 2.39 + 0.10 84

If the transgenic plant petal tube was on average longer than that of untransformed plants, then the transgenic plants had larger flowers and a longer period of time is required to reach the clear flowering stage.

Example 4: Isolation of cycD homolog genes from other plants

A cDNA library derived from exponentially growing cells of the BY-2 tobacco plant was constructed in a Lambda Express vector (Stratagene). Approximately 7.5 x 10 ⁵ library clones were plated and blots were made from each plate prepared using Hybond N ⁺ nylon membranes (Amersham Int.), Which were then fixed by baking at 80 ° C for two hours. The membranes were hybridized with heterogeneous cycD2 or cycD3 probes labeled with random oc- ³² PdCTP incorporation. The cycD3 probe contains the A. thaliana cycD3 fragment (a 405 bp HincII-KpnI fragment, exhibiting EMBL nucleotide sequence # X83371 from nucleotide position 557 to nucleotide 962). The cycD2 probe consists of a 1298 bp NcoI-SacI fragment from A. thaliana (showing the nucleotide sequence EMBL # X83370 from nucleotide position 194 to 1332). CycD3 hybridization was performed at 55 ° C and the membranes were washed twice for 10 minutes in 2xSSC / 0.1% SDS, followed by a 10 minute wash in 0.1 SSC / 0, 1% SDS. This is followed by autoradiography. CycD2 hybridization was performed at 48 ° C. The membranes were washed three times for rain for minutes in 2x SSC / 0.1% SDS. All washes were performed at room temperature. Isolated library clones were excised in vivo (according to the manufacturer's protocol) to form subclones in phagemid pBK-CMV (Stratagene) and DNA sequence was determined using standard methods. Sequence information was analyzed using GCG software (Genetic Computer Group) (1994). The cDNA sequences of cycD2; 1, cycD3; 1, and cycD3; 2 from the tobacco plant were represented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

Another cDNA library was prepared from polyadenylation RNA isolated from tubers, roots and leaves from Helianthus tuberosus. The cDNA was synthesized from the oligo (dT) primer and ligated into the lambda vector ZAPII into the EcoRI restriction site.

Approximately 1.25x10 ⁶ clones were plated. Replicates of plaque blots were prepared as described above and blots were hybridized using labeled probes. In addition, blots were probed using a cycD1 probe that contains an XbaI-Aval fragment of the 401 bp A. thaliana cycD1 gene (which has EMBL nucleotide sequence # X83369 from nucleotide position 312 to nucleotide position 713). Isolated clones were analyzed as described above. The sequences of cycD1; 1a of the cycD3; 1 genes originating from Helianthus tuberosus are represented in SEQ ID NO: 4 and SEQ ID NO: 5.

Another cDNA library was prepared from polyadenylation RNA isolated from Zea mays (Pa91xH99) xH99 sludge. The cDNA was synthesized from the oligo (dT) primer and ligated into the lambda vector ZAPII at the EcoRI restriction site. Approximately 1.25x10 6 clones were plated and plaque blot replicas as described above were prepared and hybridized using labeled probes as described above.

0 0 0 0 0 0 0 0 0 0 00 0 • 000 000 0000 900 00 00 00 00 00

Isolated clones were analyzed as described above. The cDNA sequence of cycD2 originating from Zea mays is provided in SEQ ID NO: 21.

Example 5: Construction of antisense chimeric genes and transformation of tobacco plants

A 1298 bp NcoI-SacI fragment containing DNA encoding A. thaliana-derived CYCD2 (having EMBL nucleotide sequence # X83370 from nucleotide position 194 to nucleotide position 1332) was treated with Klenow polymerase to form truncated blunt ends and ligated into a linearized pART7 vector restriction enzyme SmaI (Gleave, 1992 Plant.

Mol. Biol. 20: 1203-1207). A plasmid was selected where the DNA fragment was in such an orientation that the DNA encoding CYCD2 was introduced in a reverse fashion between the CaMV35S promoter of CabbB-J1 isolate (Harpster et al., 1988 Mol. Gen. Genet. 212: 182-190) and the 3 'region. ocs (MacDonald et al. 1991 Nucl. Acids. Res. 19: 5575-5581) such that, upon expression, antisense RNA is produced.

The chimeric anticoding gene was inserted between the T-DNA border sequence of the T-DNA vector, which also contains the selection marker chimeric gene. The cycD2 chimeric gene was excised from the plasmids pCEC2 using the NotI restriction enzyme and ligated into the NotI linearized plasmids (Gleave et al., 1992 Plant. Mol. Biol. 20: 1203-1207) to form pCEC6.

Tobacco plants were transformed with chimeric genes as described in Example 2.

Example 6: Transformant analysis

Plants transformed with the chimeric genes described in Example 5 were treated as described in Example 3.1 and the following number of plants were tested: 7 plants of line C9-2 and 6 plants of line C9-7.

The following parameters were analyzed: height of the plant from the soil surface to the highest point (Table 6;

Mean height ± standard deviation (in cm), length of the largest leaf at defined time (Table 7, given as mean length ± standard deviation (in cm)), time (Table 8, given as the mean time ± standard deviation (in days) at which meristem inflorescence is visible to the naked eye, the height at which meristem inflorescence is visible (Table 8, reported as mean length ± standard deviation (in cm)).

Transgenic plants exhibit a slowed growth rate that is evident from the seedling stage. The result is a smaller average stem height (Table 6). The increased growth rate leads to the formation of smaller leaves at a given time, which corresponds to the time at which plant growth continues (Table 7).

Table 6: Mean height (reported in cm) of transformed tobacco plants containing CaMV35S-anticoding cycD2.

line C9-2 C9-7 Untransformed control' Week 1 2.64 ± 1.22 2.75 ± 0.89 4.48 ± 1.63 Week 2 6.64 ± 1.68 6.07 ± 1.43 10.50 ± 3.33 Week 3 20.00 ± 3.74 17.21 ± 6.47 31.82 ± 7.89 Week 4 34.07 ± 6.13 28.50 ± 5.83 54.00 ± 7.89 Week 5 54.00 ± 8.87 45.14 ± 8.46 86.56 ± 10.91 Week 6 74.29 ± 9.97 61.29 ± 5.11 121.80 ± 18.28 Week 7 85.92 ± 12.03 71.50 ± 23.19 145.18 ± 19.44

Table 7: Difference in mean leaf length of transformed tobacco plants containing CaMV35Santicoding cycD2 and mean length values of largest leaves of untransformed tobacco plants (in cm).

9 • 9 9 9 9 9

999 99 9,999

9999999 9 99 99 99 9

9,999,999 * 99 9,999,999 99,99

line C9-2 C9-7 Untransformed control Week 1 -2.31 -4,30 0 Week 2 -3.26 -6, 44 0 Week 3 -4.35 -8.91 0

Table no.

inflorescence anticoding

8: Average flower size (mm), average height (cm), average time to development (days) for tobacco plants transformed with CaMV35ScycD2.

line Average inflorescence time (days) Average inflorescence height (cm) Average flower length (mm) C9-2 102 ^a 95 ON C9-7 89 ± 7.95 68.57 ± 9.62 38.31 Untransformed control 79 ± 2.39 111 ± 10.02 41.22

Only one plant developed inflorescence during the monitored time

Table 8b: The effect of anti-coding CycD2 expression on flower length in transgenic tobacco plants was analyzed in other lines (T1 generation) and statistically compared to wild-type plants using the t-test. The length of five flowers from each plant was measured and the average flower length of each transgenic line was calculated. The values of each independent transgenic line were compared to a wild-type plant using the ttest. The table shows a significance level that indicates whether the results of the comparison with the wild-type plant are statistically significant.

line Average flower length (mm) Significance

• · · β

C9-T1-1 41.05 ± 1.558 NS C9-T1-3 40.68 ± 1.574 NS C9-T1-7 38, 68 ± 1.991 NS C9-T1-10 39.78 ± 1.024 P <0.05 C9-T1-12 39.55 ± 1.568 P <0.05 Average value 40 ± 1.301 P <0.05 Wild type 41.22 ± 1.005

Example 7: Transformation of the oilseed T-DNA rapeseed plant described in Example 1 and similar vectors and analysis of the transformed plants

Hypocotyl explants of Brassica napus cultured and transformed essentially as described in De Block et al. 1989 Plant Physiol. 91: 694. The following method modifications were used:

hypocotyl explants were pre-cultured for one day on A2 culture medium (MS, 0.5 g / 1 Mes (pH 5.7), 1.2% glucose, 0.5% agarose, 1mg / 1 2.4- D, 0.25 mg / L of naphthaleneacetic acid (NAA) and 1 mg / L of 6-benzylaminopurine (BAP)).

-infectious A3 media Yippee MS, 0.5 mg / L +0.92 (pH5.7), 1.2% glucose, 0.1 mg / l NAA, C i, 75 mg / l BAP a 0.01 mg / l acid gibberelin (GA3). -selectional A5G media Yippee MS, 0.5 g / L +0.92 (pH5,7) , 1,2%

glucose, 40 mg / l adenine. SO ₄ , 0.5 g / l polyvinylpyrrolidone (PVP), 0.5% agarose, 0.1 mg / l NAA, 0.75 mg / l BAP, 0.01 mg / l GA3, 250 mg / l carbenicillin, 250 mg / l triacillin, 5 mg / l AgNO ₃ for three weeks. After this time, selection is continued on A5J medium (similar to A5G but containing 3% sucrose)

The regenerative medium A6 is MS, 0.5 g / l Mes (pH 5.7), 2% sucrose, 40 mg / l adenine. SO _4; 0.5 g / l PVP, 0.5% agarose,

0.0025 mg / l BAP and 250 mg / l triacillin.

-healthy shoots were transferred to rooting medium which was A9: half-concentration MS, 1.5% sucrose (pH5.8), 100 mg / l tracin, 0.6% agar in a 1 L container. MS is the abbreviation of Murashige and Skoog culture medium (Murashige and Skoog, 1962 Physiol. Plant. 15: 473).

Hypocotyl explants were infected with Agrobacterium tumefaciens strain C58ClRif ^R carrying a helper Tiplasmid, such as pGV4000, which was derived from plasmid pMP90 (Koncz and Schell 1986 Science 260: 536-539) obtained by incorporating a bacterial gene carrying a kanamycin-2-size fragment associated with a fragment of kanamycin 2 , 5 kb, which shows homology with the pGSV5 T-DNA vector, into the pMP90 vector, and the T-DNA vector obtained from the pGSV5 vector containing the chimeric genes described in Example 1 between the T-DNA flanking sequences and the marker chimeric gene (pCEC5b and pCRK9b).

The transgenic oilseed rape plants of the invention exhibit an accelerated vegetation program (higher growth rate), reduced time to reach the inflorescence stage, increased flowering and increased seed yield per plant.

Example 8: Transformation of maize plants with the vectors described in Example 1 and similar vectors and analysis of the transformed plants.

Corn plants were transformed with the vectors described in Example 1 of WO 92/09696. The transgenic maize plants containing the chimeric genes of the invention exhibit an accelerated vegetation program (increased growth rate), reduced time to reach the inflorescence stage, increased number of flowers and increased seed yield per plant.

Example 9: Transformation of Tomato Plants with Vectors of Example 1 and Similar Vectors and Analysis of Transformed Plants.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ···· · ·· · · · · · · · · · ·

Tomato plants were transformed with the vectors described in Example 1 and De Block et al. 1987 Embo J. 6: 2513-2518. The transgenic tomato plants containing the chimeric genes of the invention exhibit an accelerated vegetation program (increased growth rate, reduced time to reach the inflorescence stage, increased number of flowers and increased yield of fruit per plant).

Example 10: Transformation of Lettuce Plants with Vectors Described in Example 1 and Similar Vectors and Analysis of Transformed Plants.

Lettuce plants were transformed with the vectors of Example 1. The vectors are described in Micheimore et al.

1987 Plant Cell Rep. 6: 439-442. Transgenic lettuce plants containing the chimeric genes of the invention exhibit an accelerated vegetation program (increased growth rate), a reduced time to reach the inflorescence stage, an increased number of flowers, and an increased seed yield per plant.

Example 11: Further analysis of the progeny line of transgenic tobacco plants transformed with the CaMV35SAthCycD2 constructs of Example 3 in a segregation and non-segregation population

The progeny population (segregating or non-segregating) of plants from two lines of transgenic tobacco plants transformed with the CaMV35SAthCycD2 constructs (line 2 and line 5 of Example 3) were analyzed for inflorescence time and increase in vegetative growth by measuring the average stem height or average dry weight of the plants.

Segregation of the transgenes was monitored by assessing their resistance to kanamycin. In the case of the segregation population, 32 plants were analyzed, while in the case of non-segregation populations, 12 plants were analyzed. Non-transformed populations also included 12 plants.

The following populations were used:

Β · Β ΒΒ ΒΒ ΒΒ β Β β Β β β β β β β β β β β β β β β β β β β β β β β β β β β β β

Β Β β * Β «ΒΒΒ

Β «· Β · ···· Β · Β *

Segregation population:

Line 2:

C8-T1-2 (T1 seeds from primary transformant C8-2; segregates 3: 1 for T-DNA)

C8-T2-2 (T2 seeds from self-fertilized C8-T1-2 # 3 plants that are homozygous, seed segregates 3: 1 for T-DNA)

C8-T2-2 (T2 seeds from crossing a C8-T1-2 # 3 plant with a wild-type plant using wild-type plant pollen as parent pollen. This seed segregates 1: 1 for T-DNA and all plants containing T-DNA are hemizygotic)

Line5

C8-T1-5 (T1 seeds from the primary transformant C8-5; segregates

3: 1 for T-DNA) C8-T2-5 (T2 seeds from self-fertilized plant C8-T1-5 # 304, which are hemizygous, Non-segregating population the seed segregates 3: 1 for T-DNA) Line 2 C8-T2-2 (T2 seeds from self-fertilized plant C8-T1-2 # 302,

which are homozygous for T-DNA)

Line 5

AND

C8-T2-2 (T2 seeds from C8-T1-5 # 121 crossed with wild-type plants using wild-type pollen of the parental plant. All T-DNA containing # 121 plants are hemizygous and all T2 seeds are hemizygous for T -DNA) «· 99 99 9999

9 9 9999

99999 · 9 9 9 9 9 99 9

9 9 9 9 9

9 9 9 9 9 9 99 99

C8-T2-2 (seeds Τ2 from self-fertilized plants C8-T1-5 # 121 All plants # 121 are hemizygous and T-DNA and all T2 seeds are hemizygous for T-DNA)

The effect of overexpression of CycD2 on the length of time required to initiate inflorescence in transgenic tobacco plants was measured, and the values were statistically compared to those of the same parameters measured in a wild-type control population using a non-parametric t-test. transgenic populations are not the same. The time required by each plant to develop inflorescence of 0.5 cm was recorded and the average number of days after vernalization was calculated. Values for each transgenic population were compared to that for the wild-type population using the t-test. Data obtained for segregation lines were separated from data obtained for kanamycin resistant and kanamycin-sensitive populations. Data obtained from the kanamycin-resistant population were also reported separately for the homozygous kanamycin-resistant sub-population (no longer segregating) and for the hemizygous kanamycin-resistant sub-population (further segregating 3: 1). These data are shown in Table 9. Table 10 shows the average stem height values of the transgenic non-segregation lines at a different time after vernilization compared to the wild-type population (statistically evaluated). A significance level of less than 0.05 is considered a highly significant difference between the average height of each transgenic line and the mean height of the controls. NS means an insignificant difference between populations. In addition, the seedlings biomass from said non-segregating populations were compared to wild-type seedlings during early vegetation growth. The seedlings were harvested on the marked days after vernalization and weighed by weighing before drying at 70 ° C for two days. The average dry weight was calculated. · 9 · · · · «·« «« «« «« «« «« «« «« «« «« «« « · · · · 9 9 9 99 9 9 9 9 9 9 seedlings and standard deviation and results are shown in Table 11.

Table 9: Effect of CycD2 overexpression on the length of time required to initiate inflorescence development in transgenic tobacco plants

Population Average flowering time 0.5 cm (days) Standard deviation Rate significance Non-segregation lines WT 72,125 2,258 - C8-T2-2 (302) 63.62 3,863 0.001 s.opi.) C8-T1-5 (121 67.78 2,438 0, 02 samopl.) C8-T1-5 (120xWT) 64.18 1,991 0.001 Diameter 65.19 2,258 0.001 Segregation lines C8-T1-2 KanR 59, 04 2,973 0.001 Hemizyg. 59, 32 3,110 0.001 Homozyg. 58.50 2,507 0.001 Citl. to kanamycin 73.00 5,994 NS C8-T2-2 pl 3xWT Kan R 59.44 2,756 0.001 Citl. to kanamycin 69.10 2,846 NS C8-T2-2 pl 3 59.20 2,141 0, 001 s.opi., KanR Hemizyg. 59.25 1,653 0.001 Homozyg. 59.38 3,021 0.001 Citl. to kanamycin 73.50 5,431 NS C8-T1-5 KanR 62.67 3,367 0.001 Citl.na kanamyc. 71.50 5,782 NS C8-T2-5 pl 304 s.opi. Hemizygot. 64.50 3,030 0.001 Homozygot. 65.83 2,483 0,002 Citl. to kanamycin 74.50 7,764 NS

fe · · · · · · · ·

rate significance 3_ C8-T2-5 (120 x WT) rate significance - o f -> co ω i * - * H ω cn i • cn 0 I.E H rate significance C8-T2-2 (302 p.opi.) wonder. type population 0.001 ! 4.37 + 0.378 O b O 4.25 ± 0.507 0.002 3.55 + 0.451 1.48 + 0.238 ω CL P H ' 0.001 8.14 + 1.914 0.001 6.02 i 1.662 0.001 6.02 + 1.662 3.50 + 0.831 37 days i_I 0.001 14.76 + 2.550 0.001 11.11 ± 3.823 1ΌΌ 11.11 ± 3.823 7.59 + 1.932 4 days !AND 0.001 25.18 + 3,314 0.001 19.35 +, 6.528 0.05 19.35 ± 6.528 14.59 + 3.816 cn CL P 0.001 39.91 ± 4.898 O b O 32.81 + 9.181 0.05 32.81 ± 9.181 25.81 ± 6.030 4 9 days O O O 75.04 ± 6.258! 0.001 71.82 + 7,604 0.05 67.50 + 12,281 58.24 + 8.423 Cn Ol CL P H ' 0.01 117.83 + | 9.808 0.01 119.86 + 10.675 0.01 120.12 + 12,829 107.06 + 8,306 63 days AND 0.05 146.54 ± 13.422 O b O 152.82 + 11,297 0.01 151.46 + 15,253 136.75 ± 7,105 O CL P H ' ns 160.71 ± 15.183 0.001 170.73 ± 11,130 0.05 165.23 + 14,696 153.94 + 7.430 77 days ns 174.1 + 14,963 O b 192.18 ± 7.846 ns 177.23 ± 13,935 177.71 ± 13.129 finite height

Table 10: Statistical comparison of stem height of transgenic tobacco plants containing CaMV35SAthCycD2 with wild type controls.

cn

wonder. type C8-T2-5 (120 x WT) C8-T2-5 (121 p.opi.) C8-T2-2 (302 pp.) population 1.2 ± 0.510 5.48 ± 1.130 4.30 ± 1.623 3.75 ± 1.462 17 days 13.16 ± 3.09 39.51 ± 10.13 29.05 + 10.50 22.11 + 6.59 23 days 29.88 + 14.89 79.81 ± 20.36 49.14 ± 8.51 53.19 + 9.97 28 days 135 ± 60.72 476 ± 120.2 351 ± 67.6 337 + 58.3 34 days 382 ± 90.3 946 ± 154 547 + 24.9 530 ± 60.0 38 days

• 9 9 9 9 9 9

4 9 9 4

9999994 9 99 99 99 9

9 9 4 9 9 4

999 9 99 9999 9

h st ° ω <

κ o

o c

tr o

α t?

o ct o

St σ

ΗΟ flj ω

· <

x ω

N <

q, α> '

Γ +

Hi

SH ts ω

Ό ω

ti ts

I— ¹

Ηts

H <D

Φ

Ό

Hi

St ° (D <

Hi ts tr o

G.

ts o

r + fu

Hσ

HÍU ω

X x

o ts

X

Hi

O

H- ¹

Table 11: Dry weight values (mg) obtained from non-segregating populations of transgenic seedlings that overexpress CycD2 and wild-type seedlings (WT) at various times after vernalization. In all cases, the t-test indicates that there is a very substantial difference between t »9 · ·» 9 9 «99 99 • 9 9 9 9 * 9 ·

9 9 9 9 ·

9 · · · 9 9 9 9 9

9 9 9 9 9

9999 98 99

Sequence Protocol (2) Information about SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1284 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA per mRNA (iii) HYPOTHETICAL: no

(iv) POSITIVE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (ix)

(A) Name / Key: (B) Position: 182 ..1243 (D) Other Information:

/ note = "cDNA encoding cyclin CYCD2; 1 (xi) Description of sequence: SEQ ID NO: 1:

CAAATTTTTC TCCCTTCTAT AGTCTCTTTC CTGTTCTCTT AAAAATCCTT AAAAATTTAT 60 TTTTTTTAAC AATCTCATGT AAATGGGATT AAATTTTGTA AAAATATAAG A.TTTTGATAA 120 AGGGGGTTTA ATTATAACAT AGTAAATTAA GATTTTTTTT TTGCTTTGCT AGTTTGCTTT 180 AATGGCAGCT GATAACATTT ATGATTTTGT AGCCTCAAAT CTTTTATGTA CAGAAACAAA 240 AAGTCTTTGT TTTGATGATG TTGATTCTTT GACTATAAGT CAACAGAACA TTGAAACTAA 300

··

GAGTAAAGAC TTGAGCTTTA ACAATGGTAT TAGATCAGAG CCATTGATTG ATTTGCCAAG 360 TTTAAGTGAA GAATGCTTGA GTTTTATGGT GCAAAGGGAA ATGGAGTTTT TGCCTAAAGA 420 TGATTATGTC GAGAGATTGA GAAGTGGAGA TTTGGATTTG AGTGTGAGAA AAGAGGGTCT 480 TGATTGGATT TTGAAGGCTC ATATGCACTA TGGATTTGGA GAGCTGAGTT TTTGTTTGTC 540 GATAAATTAC TTGGATCGAT TTCTATCTCT GTATGAATTG CCAAGAAGTA AAACTTGGAC 600 AGTGCAATTG TTAGCTGTGG CCTGTCTATC ACTTGCAGCC AAAATGGAAG AAATTAATGT 660 TCCTTTGACT GTTGATTTAC AGGTAGGGGA TCCCAAATTT GTATTTGAAG GCAAAACTAT 720 ACAAAGAATG GAACTTTTGG TATTAAGCAC ATTGAAGTGG AGAATGCAAG CTTATACACC 780 TTACACATTC ATAGATTATT TTATGAGAAA GATGAATGGT GATCAAATCC CATCTCGGCC 840 GTTGATTTCT GGATCAATGC AACTGATATT AAGCATAATA AGAAGTATTG ATTTCTTGGA 900 ATTCAGGTCT TCTGAAATTG CAGCATCAGT GGCAATGTCT GTTTCAGGGG AAATACAAGC 960 AAAAGACATT GATAAGGCAA TGCCTTGCTT CTTCATACAC TTAGACAAGG GTAGAGTGCA 1020 GAAGTGTGTT GAACTGATTC AAGATTTGAC AACTGCTACT ATTACTACTG CTGCTGCTGC 1080 CTCATTAGTA CCTCAAAGTC CTATTGGAGT GTTGGAAGCA GGAGCATGCT TGAGCTACAA 1140 AAGTGGTGAT GAoAGAACAG TTGGATCATG TACAACTTCT TCACATACTA AAAGGAGAAA 1200 ACTTGACACA ' TCATCTTTAG AGCATGGGAC TTCAGAAAAG TTGTGAATCT GAATTTTCCC 1260 TTTTTAAAAA AAAAAAAAAA AAAA 1284

(2) SEQ ID NO: 2 Information:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1679 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA per mRNA (iii) HYPOTHETICAL: no

(iv) POSITIVE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (ix)

(A) Name / Key: (B) Position: 181 ..1299 ·· ·· ··· (D) Other Information:

/ note = cDNA encoding cyclin CYCD3; 1 (xi) Description of sequence: SEQ ID NO: 2:

AAACGAGTCT CTGTGTACTC CTCCTCCTAT AGCTTTTCTC TCTTCTTCTC TTCACACCTC 60

CCACAACACA CAATCAGACA AAATAGAGAG GAAAATGAGT ATGGTGAAAA AGCTTTGTTT 120

TGTATAATGA GAAAAAGAGA TTTATATACA TCTCTTCTTC TACTTCCTTC TTACTAGAAG 180

ATGGCAATAG AACACAATGA GCAACAAGAA CTATCTCAAT CTTTTCTTTT AGATGCTCTT 240

TACTGTGAAG AAGAAGAAGA AAAATGGGGA GATTTAGTAG ATGATGAGAC TATTATTACA 300

CCACTCTCTT CAGAAGTAAC AACAACAACA ACAACAACAA CAAAGCCTAA TTCTTTATTA 360

CCTTTGCTTT TGTTGGAACA AGATTTATTT TGGGAAGATG AAGAGCTTCT TTCACTTTTC 420

TCTAAAGAAA AAGAAACCCA TTGTTGGTTT AACAGTTTTC AAGATGACTC TTTACTCTGT 480

TCTGCCCGTG TTGATTCTGT GGAATGGATT TTAAAAGTGA ATGGTTATTA TGGTTTCTCT 540

GCTTTGACTG CCGTTTTAGC CATAAATTAC TTTGACAGGT TTCTGACTAG TCTTCATTAT 600

CAGAAAGATA AACCTTGGAT GATTCAACTT GCTGCTGTTA CTTGTCTTTC TT7AGCTGCT 660

AAAGTTGAAG AAACTCAAGT TCCTCTTCTT TTAGATTTTC AAGTGGAGGA TGCTAAATAT 720

GTGTTTGAGG CAAAAACTAT TCAAAGAATG GAGCTTTTAG TGTTGTCTTC ACTAAAATGG 780

AGGATGAATC CAGTGACCCC ACTTTCATTT CTTGATCATA TTATAAGGAG GCTTGGGCTA 840

AGAAATAATA TT.CACTGGGA ATTTCTTAGA AGATGTGAAA ATCTCCTCCT CTCTATTATG 900

GCTGATTGTA GATTCGTACG TTATATGCCG TCTGTATTGG CCACTGCAAT TATGCTTCAC 960

GTTATTCATC AAGTTGAGCC TTGTAATTCT GTTGACTACC AAAATCAACT TCTTGGGGTT 1020

CTCAAAATTA ACAAGGAGAA AGTGAATAAT TGCTTTGAAC TCATATCAGA AGTGTGTTCT 1080

AAGCCCATTT CACACAAACG CAAATATGAG AATCCTAGTC ATAGCCCAAG TGGTGTAATT 1140

GATCCAATTT ACAGTTCAGA AAGTTCAAAT GATTCATGGG ATTTGGAGTC AACATCTTCA 1200

TATTTTCCTG TTTTCAAGAA AAGCAGAGTA CAAGAACAGC AAATGAAATT GGCATCTTCA 1260

ATTAGCAGAG TTTTTGTGGA AGCTGTTGGT AGTCCTCATT AAAATCAATC ACCTGATTTA 1320

TCTCTTTTCT TTCTTATTAC CAACTATGGT GGTAATAATA TTTATTGATA TTCAGAAGTA 1380

TTTACCTTTA ATGTCATTTT CAAAAATTAC ATGAAAATGG AAAAAAAGAA AAGAAGAGCT 1440

TAGCTGGTGG TTGCAGTTGG CAGAGAAGAG GACTGGCTTT TTTTTGCAGG AGTGTAGTCT 1500

ACTACTACTG GAAAGCAGAG ATAGAGAGAG GAGAAAAGAC AGAAAATCTG CACTATTTGT 1560

TTTTTCTCTA TTCATATCAA TTCTCTCTTA GGTCCTTTTC ATGCATGCAT ACTTTTGATG 1620

GACATATTTT ATATATTTAC TATAATCATA AATTCTTGAA TAAAAAAAAA AAAAAAAAA 1679 (2) •

9 9 to '

Information about SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1431 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA per mRNA (iii) HYPOTHETICAL: no (iv) POSITIVE: no (iv) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (ix)

(A) Name / Key: (B) Position: 1981..1298 (D) Other Information:

/ note = cDNA encoding cyclin CYCD3; 2 (xi) Description of sequence: SEQ ID NO: 3:

CACCTTTACT CTCTTCTCCT TTTTGGCTCT TCCCATTCTC TCCTTCTCTT TCTTTATTTT 60 CTGTCCTGTA GAGAGAGAGA GAAAGTATAA GCAAAGCAGC AGATATGTTA CTGGGTCCAA 120 GATTGAGTTT TGGCTTACCT TGAAGATAAT GAGTAGAGCC TGCATTGTCT TCTTCCGTCA 180 AGAAGAAGAA GAAGAAGATG GTTTTCCCTT TAGATACTCA GCTCCTAAAT CCAATCTTTG 240 ATGTCCTTTA CTGTGAGGAA GATCGATTCT TGGACGATGA TGA7TTAGGA GAATGGTCTA 300 GTACTTTAGA ACAAGTAGGA AATAATGTGA AAAAGACTCT ACCTTTATTA GAATGTGACA 360 TGTTTTGGGA AGATGACCAG CTTGTCACTC TTTTAACTAA GGAAAAAGAG TCTCATTTGG 420 GTTTTGATTG TTTAATCTCA GATGGAGATG GGTTTTTAGT GGAGGTTAGA AAAGAGGCAT 480 TGGATTGGAT GTTGAGAGTC ATTGCTCACT ATGGTTTCAC TGCTATGACT GCTGTTTTAG 540 CTGTGAATTA TTTTGATAGG TTTGTATCTG GACTCTGCTT TCAGAAAGAT AAGCCTTGGA 600 TGAGTCAACT TGCTGCTGTG GCTTGTCTTT CTATTGCTGC TAAAGTGGAA GAGACCCAAG 660 TCCCCCTTCT CTTAGACCTC CAAGTGGCTG ATTCAAGATT TGTGTTTGAG GCAAAGACTA 720 TTCAGAGAAT GGAACTCTTG GTGCTCTCCA CTCTTAAGTG GAAAATGAAT CCAGTGACAC 780 CACTATCTTT CATTGATCAT ATCATGAGGA GATTTGGATT CATGACCAAT CTACATTTGG 840 ATTTTCTTAG GAGATGTGAA CGCCTCATTC TTGGTATTAT CACTGATTCT AGGCTCTTGC 900 ATTATCCTCC atctgttatt GCAACTGCAG TAGTGTATTT CGTGATCAAT GAGATTGAGC 960

· T t 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 · «0 ···· ·· ··

CTTGCAATGC AATGGAATAC CAGAATCAGC TCATGACTGT TCTTAAAGTC AAACAGGATA 1020 , GTTTTGAAGA ATGCCATGAT CTTATTCTAG AGCTAATGGG CACTTCTGGC TACAATATCT 1080 GCCAAAGCCT CAAGCGCAAA CATCAATCTG TACCTGGCAG TCCAAGTGGA GTTATCGATG 1140 CATATTTTAG TTGCGACAGC TCTAATGATT CGTGGTCGGT AGCATCTTCA ATTTCATCGT 1200 CACCAGAACC TCAGTATAAG AGGATCAAAA CTCAGGATCA GACAATGACA CTGGCTCCAC 1260 TGAGTTCTGT TTCTGTCGTT GTGGGCAGTA GTCCTCGTTG ATCAGTATCT CATTCTCTAG 1320 ATTATCTAGT ATTACGGCTA TGGTTACTAT ATGATCTCTC TTTTTTGGTA TGTTCTCTTA 1380 AACTGCAGTT GCACAATGCT CTGATGTTCC ATTAAAAAAA AAAAAAAAAA AND 1431

(2) SEQ ID NO: 4 information:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1788 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA per mRNA (iii) HYPOTHETICAL: no (iv) POSITIVE: no (iv) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Helianthus tuberosus (ix)

- (AND) Name / Key: - (B) Position: 165 ..1109 (D) Other information:

/ note = cDNA encoding cyclin CysD1; 1 Sequence description: SEQ ID NO: 4:

(xi)

ΦΦ • Φ φ φ φ

φ φ

φφφφ φ

φ φ

φ φ

• φ · · • · ΦΦΦ • • • • • • • •

ΦΦ ·· φ φ φ φ φ φ φ φ

ΦΦ

ΦΦ

GACAACAATG ACTTCTACTC ACTATTCACT ACTTACTAAT CACTGCAACT TCTCCGGCCA 60 CTTTTCACCT CAAACCGCCG GAACTCCGCC GCTCCGGTCG ACGGTGAATC ACTGAATCTT 120 AGCAATTATG TTCACAACAG TATGAACAAT CAACACCGGT CATCATGTCA ATCTCGTGCT 180 CTGACTGCTT CTCCGACTTA . CTCTGCTGCG AGGACTCCGG CATATTATCC GGCGACGACC 240 GGCCGGAGTG CTCCTATGAT TTCGAATATT CCGGCGACTT TGATGATTCG ATCGCGGAGT 300 TTATAGAACA GGAGAGAAAG TTCGTTCCAG GAATCGATTA CGTCGAGCGA TTTCAATCGC 360 AAGTTCTCGA TGCTTCTGCT AGAGAAGAAT CGGTTGCCTG GATCCTTAAG GTGCAACGGT 420 TTTACGGATT TCAGCCGTTG ACGGCGTACC TCTCCGTTAA CTATCTGGAT CGTTTCATCT 480 ATTGCCGTGG CTTCCCGGTG GCAAATGGGT GGCCCTTGCA ACTCTTATCT GTAGCATGCT 540 TGTCTTTAGC TGCTAAAATG GAGGAAACCC TTATTCCTTC TATTCTTGAT CTCCAGGTTG 600 AAGGTGCAAA ATATATTTTC GAGCCGAAAA CAA.TCCGAAG AATGGAGTTT CTTGTGCTTA 660 GTGTTTTGGA TTGGAGACTA AGATCCGTTA CACCGTTTAG CTTTATCGGC TTCTTTTCGC 720 ACAAAATCGA TCCATCTGGA ATG7ATACGG GTTTCCTTAT CTCAAGGGCA ACACAAATTA 780 TCCTCTCAAA TATTCAAGAA GCTAGTTTAC TTGAGTATTG GCCATCATGT ATTGCTGCTG 840 CAACAATACT TTGTGCAGCA AGTGATCTTT CTAAATTCTC ACTTATCAAT GCTGATCATG 900 CTGAATCATG GTGTGATGGC CTTAGCAAAG AGAAGATCAC AAAATGTTAC AGACTTGTAC 960 AATCTCCAAA GATATTGCCG GTACATGTTC GAGTCATGAC GGCTCGAGTG AGTACTGAGT 1020 CAGGTGACTC ATCGTCGTCG TCTTCTTCGC CATCGCCTTA CAAAAAGAGG AAACTAAATA 1080 ACTACTCATG GATAGAGGAG GACAAAAGAT GAAAATAAGG AGACAAAATA ΑΑΤΑΑΑΤΑΑΑ 1140 TCCGGATTCC TCTCTATATT TTTTAAAGGA ATCAACAAAT ΑΤΑΤΑΤΑΑΑΑ AAAAAAAATG 1200 GAGTCAGGAA AAGCAACGAA AGCCGCCGGA GGAAGAAAAG GCGCCGGAGC GAGGAAGAAG 1260 TCCGTCACAA AGTCCGTCAA AGCCGGTCTC CAGTTCCCCG TCGGAAGAAT CGCTAGGTTT 1320 CTAAAAAAAG GCCGATACGC TCAACGTACC GGATCCGGAG CTCCGATCTA CCTTGCTGCT 1380 GTTCTAGAAT ACCTTGCTGC TGAGGTTTTG GAGTTGGCGG GAAATGCAGC GAGAGATAAC 1440 AAGAAGACAA GGATAAACCC TAGGCACTTG CTATTGGCTG TTAGGAACGA TGAGGAATTG 1500 GGGAAATTGC TTGCTGGTGT TACTATTGCT AGTGGAGGTG TGTTGCCCAA TATCAATCCG 1560 GTTCTTTTGC CCAAGAAGTC TTCTTCTTCT TCTGCTGCTG AGAAGACCCC CAAATCTAAA 1620 AAGTCGCCTA AAAAGGCTGC TTAGATAGAT GTTTCTGGTT ATAGTTGGTT AGATTAAGTT 1680 GAAGCAAAAC AGTCTCTTTT GTTCAATTAG TCGTCTGGCA ATGTAACTAT TTTGGTCGTC 1740

TTCAAAATGT TAATTGGATA CTATCTTCTT ΤΑΑΑΑΑΑΑΑΑ ΑΑΑΑΑΑΑΑ

1788

(2) SEQ ID NO: 5 information:

* ···· · «· · · ··· · ······· · · · ······

(and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 1414 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: double-stranded (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA per mRNA

(iii) HYPOTHETICAL: no (iv) POSITIVE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Helainthus tuberosus (ix)

(AND) Name / Key: - (B) Position: 48 ..1118 (D) Other information: / note = cDNA encoding cyclin CYCD3;

(xi) Sequence description: SEQ ID NO: 5:

TTGAACCTTC ATTTCTTTTC TTTTCTTCTT TCTAATCACC AACCCCAATG GCCATTTTAT 60

CACCATATTC ATCTTCTTTC TTAGACACAC TCTTTTGCAA TGAACAACAA GATCATGAAT 120

ATCATGAATA TGAGTATGAA GATGAATTTA CACAAACCAC CCTCACAGAT TCATCTGATC ISO

TCCATCTTCC CCCCCTGGAC CAACTAGATT TGTCATGGGA ACATGAAGAG CTTGTGTCCT 240

TGTTCACAAA AGAACAAGAG CAGCAAAAAC AAACCCCTTG TACTCTCTCT TTTGGCAAAA 300

CTAGTCCCTC AGTTTTTGCT GCTCGTAAAG AGGCTGTAGA TTGGATCCTT AAGGTCAAAA 360

GTTGTTATGG ATTCACACCT CTTACAGCCA TTTTAGCCAT CAATTATCTT GATAGGTTTC 420

TTTCTAGCCT CCATTTTCAA GAAGATAAAC CTTGGATGAT TCAACTTGTT GCTGTTAGTT 480

GTCTCTCTTT AGCTGCTAAA GTTGAAGAAA CTCAAGTGCC ACTCTTACTA GATCTTCAAG 540

TAGAGGACAC TAAGTACTTG TTTGAGGGTA AAAACATACA AAAAATGGAG CTTTTGGTGA 600

TGTCAACTTT GAAATGGAGG ATGAACCCAG TGACACCAAT CTCATTTCTT GATCACATTG 660

TAAGAAGGCT TGGATTAACT GATCATGTTC ATTGGGATTT TTTCAAGAAA TGTGAAGCTA 720

TGATCCTTTG TTTAGTTTCA GATTCAAGAT TCGTGTGTTA TAAACCATCC GTGTTGGCCA 780

CAGCTACAAT GCTTCACGTT GTAGATGAAA TTGATCCTCC CAATTGTATT GACTACAAAA 840

GTCAACTTCT GGATCTTCTC AAAACCACTA AGGACGACAT AAACGAGTGT TACGAGCTCA 900

TTGTCGAGCT AGCT7ACGAT CATCACAACA AACGAAAACA TGATGCAAAC GAGACAACAA 960

CCAATCCGGT TAGTCCAGCT GGCGTGATCG ATTTCACTTG TGATGAAAGT TCAAATGAGT 1020 • 9 · · · · ·

71 • · · • · · 9 9999 9 9 99 9 9 • · · • · 9 9 9 9 9 9 9 CATGGGAACT TAATGCTCAT CATTTCCGCG AGCCTTCATT CAAGAAAACA AGAATGGATT 1080 CAACAATTCG GGTTCGGGTT TGGTTCACTT ATAAGCTTTA ATCGAGGGTA GTTGTAAACA 1140 TGTAATCCGC ATGCACGCTA TTAATCCTAC GGTCCACTAC TACATATAAT CGGCCTATAA 1200 AATTATAGGT TAAGATGACC AGTCGTAGGC GTCGAGATGT CCTTATGGTT GGTCAATTTC 1260 TCTATGGTTT TAGGTCGTTT TTAATGTGAG ataaattaaa TTCGGTATGT TAAGTCTTTA 1320 TCAAGGAATG GACGTTATAT TTATTGTTTG ATATTGAGAA TTAAATTCCA TGGGAAAAAA 1380 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 1414

(2) Information about SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 17 pairs of cravings (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide PCR

for (xi) Sequence description: SEQ ID NO: 7:

AND

GCMTGGATYC

TYAAGGT (2) Information about SEQ ID NO: 8:

and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 17 pairs of cravings (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid

(A) DESCRIPTION: /desc.= "oligonucleotide primer 2 for PCR (xi) Description of sequence: SEQ ID NO: 8:

TGCTTGTCWT TAGCTGC 17 (2) Information about SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: /desc.= "oligonucleotide primer 3 for PCR (xi) Sequence description: SEQ ID NO: 9:

AAGAATGGAR YTTCTTGT 18 (2) Information about SEQ ID NO: 10:

(and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 19 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) PCR DESCRIPTION: /desc.= "oligonucleotide primer

(xi) Sequence description: SEQ ID NO: 10:

ARAGNATYCY KGCWGCAGC (2) Information about SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

• flfl · · · · · · · · · · · · · · ·

flfl flflfl · • flfl • · flfl (AND) LENGTH: 19 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide primer PCR (xi) Sequence Description: SEQ ID NO: 11: CCRTCACACC AWGNYTCAG (2) Information on SEQ ID NO: 12: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 16 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide primer PCR

(xi) Sequence description: SEQ ID NO: 12:

pro pro

TGGWGATTTG GATTTG (2) Information about SEQ ID NO: 13:

(and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 17 pairs of cravings (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide primer PCR (xi) Sequence Description: SEQ ID NO: 13:

for

ATNAANTACT TGGATCG • (2) Information about SEQ ID NO: 14:

(and)

SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 19 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide PCR (xi) Sequence Description: SEQ ID NO: 14: AGCTTGCANT CTCCANTTC (2) Information on SEQ ID NO: 15: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 17 pairs of cravings (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide PCR

(xi)

Sequence Description: SEQ ID NO: 15:

for

TAGAAGNCC TGAANTC

SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 pairs of baffles (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid • 4 · · · 4 ♦

4 · · · · ·

4444 ·· · 4 · 4 9 4

4 44 44 4 4 9 (A) DESCRIPTION: /desc.= "oligonucleotide primer 10 for PCR (xi) Description of sequence: SEQ ID NO: 16:

GANTGGATNY TNAARGT 17 (2) Information about SEQ ID NO: 17:

(and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 17 pairs of cravings (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D). TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide for PCR

(xi) Sequence description: SEQ ID NO: 17:

AAGABAARCC WTGGATG 17 (2) SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: /desc.= "oligonucleotide primer 12 for PCR (xi) Sequence description: SEQ ID NO: 18:

GTKGAAGARA

CTCAAGTBCC (2) Information about SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

• · • ·

 76: ··: * · • · · · • · · · • · · • · · · · · • · · • · · • · (AND) LENGTH: 24 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: another nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide primer 13 for PCR (xi) Sequence Description: SEQ ID NO: 19: TGGNGTNACW GGNTKCATYY 24 (2) Information about SEQ ID NO: 20 (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 20 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: single chain (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: other nucleic acid (AND) DESCRIPTION: /desc.= "oligonucleotide primer PR14 for PCR (xi) Sequence Description: SEQ ID NO: 20: GCWGNNGCNA NNNCAGANGG 20 May (1) Information about SEQ ID NO: 21 (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 1846 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: double-stranded (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA per mRNA

(iii) HYPOTHETICAL: no (iv) POSITIVE: no (v) ORIGINAL SOURCE:

77 • · · · · · · · · · · · · BBB · · • ······ · • · · · · «·· · ·· ·· • ·· • · · B · · « • · · Β B « Β Β B · (A) ORGANISM: Zea mays (ix) FEATURES: (A) Name / Key: - (B) Position: 316 ..1389 (D) Other information: / Note = CDNA encoding cyclin

CYCD2 (xi) Description of sequence: SEQ ID NO: 21:

L 1 λ CTAGCCGGCG TCGTCCTCCC CCTCTCHCGC TCCTCTGTCC TCCCCTCTCC 60 ACTTGAGAAG AACACAATTA GGAAAAAAAG GCAAAAAACA TTTACCTTTT TTCTATCTGT 120 ATATTATCTG aataaatcaa GAGGAGGAAG AGGGGAGGGA GCGAGGGAGG GGGAGGAGTA 180 GCAAATCCAG ACTCCATAGC AACCAGCTCG CGAGAAGGGG AAAAGGGGGA GGAAGAGCTT 240 CGCTTGTGTA TTGATTGCTC GCTGCTCCAG TCCCTGCATT CGTGCCGTTT TTGGCAAGTA 300 GGTGGCGTGG CAAGCATGGT GCCGGGCTAT GACTGCGCCG CCTCCGTGCT GCTGTGCGCG 360 GAGGAGAACG CTGCTATTCT CGGCCTGGAC GACGATGGGG AGGAGTCCTC CTGGGCGGGC 420 GCCGC7ACGC CGCCACGTGA CACCGTCGCC GCCGCCGCCG CCACCGGGGT CGCCGTCGAT 480 GGGATTTTGA CGGAGTTCCC CTTGCTCTCG GATGACTGCG TTGCGACGCT CGTGGAGAAG 540 GAGGTGGAGC ACATGCCCGC GGAGGGGTAC CTCCAGAAGC TGCAGCGACG GCATGGGGAC 600 CTGGATTTGG CCGCCGTCAG GAAGGACGCC ATCGATTGGA TTTGGAAGGT CATTGAGCAT 660 TACAATTTCG CACCGTTGAC TGCCGTTTTG TCTGTGAACT ACCTCGATAG ATTCCTCTCC 720 ACGTATGAGT TCCCTGAAGG CAGAGCTTGG ATGACTCAGC TCTTGGCAGT GGCTTGCTTG 780 TCTTTGGCTT CGAAAATCGA AGAGACTTTT GTGCCACTCC CCTTGGATTT GCAGGTAGCG 840 GAGGCAAAGT TTGTTTTTGA GGGAAGGACC ATAAAAAGGA TGGAGCTTCT GGTGCTAAGC 900 ACCTTAAAGT GGAGGATGCA TGCTGTTACT GCTTGCTCAT TTGTTGAATA CTTTCTTCAT 960 AAATTGAGTG ATCATGGTGC ACCCTCCTTG CTTGCACGCT CTCGCTCTTC GGACCTTGTC 1020 TTGAGGACCG CTAAAGGTGC TGAATTCGTG GTATTCAGAC CCTCCGAGAT TGCTGCCAGT 1080 GTTGCACTTG CTGCTATCGG CGAATGCAGG AGTTCTGTAA TTGAGAGAGC TGCTAGTAGC 1140

9

78 • · · · · • ···· · · · • · · · ·· · · ·· 9 9 · 9 9 TGCAAATATT TGGACAAGGA GAGGGTTTTA AGATGCCATG AAATGATTCA AGAGAAGATT 1200 ACTGCGGGAA GCATTGTCCT AAAGTCTGCT GGATCATCAA TCTCCTCTGT GCCACAAAGC 1260 CCAATAGGTG TCCTGGACGC TGCAGCCTGT CTGAGTCAAC AAAGCGATGA CGCTACTGTC 1320 gggtctcCtg CAGTATGTTA CCATAGTTCT TCCACAAGCA GAGAA G GATCACTAGA 1380 CGTCTACTCT AATTGTGGTA CGCTTCAGGT GTGCTCCTCA CCGCTCTAGG AGTTTTTGAT 1440 TGGTTCAAAC ATCTTAAATT TAGTTTGGCC GCTGGAGGAT TATGGTTTAG TCAAGTAGTT 1500 GCTGAATGGA CAACAAAACA CGCACACTAC TTGGTCCATA AAGACAAGAA AATAACTGGC 1560 AGCGTCCCGC GAGCCAGCGC TGCAATCCAG TTCATGCAAG ACCCTAGAGT CCAGGGGGGG 1620 TGCTGGTGTA GGTAGAGAGG GAACAAGGCA TTCACATACG CCGTAGAGAT GAGAGAGCCT 1680 CTCGTATGTT TTGTACTTTT GCTCCTTCAG TTTGCAATGA ACTATATAAA CAAGGATTGC 1740 CTTGGGGCAG TGAACATTTG TCGGATGAAA AGAATCAAAA AGGATGGGGG TGGGCAGAGG 1800 AATAGAACAA TTTGATATAT TTCCATAAAC TAAAAAAAAA AAAAAA 1846

Claims (19)

PATENT CLAIMS 2. 3. 4. 5. and) (b) C) 6. 2. 3. 4. 5. and) (b) C) 6. A method of obtaining a plant having altered growth characteristics or altered architecture, comprising the step of adjusting the amount and functional amount of a plant cell division control protein, wherein the cell division control protein is capable of binding or phosphorylating an Rb-like protein. The method of claim 1, wherein said cell division controlling protein comprises in its Nterminal portion a binding motif of an Rb-like protein. The method of claim 2, wherein said Rb-like protein binding motif is LxCxE. The method of claim 3, wherein said cell division control protein is type D cyclin. The method of any one of claims 1 to 4, wherein said amount or functional amount of said cell division control protein is adjusted such that: a chimeric gene comprising the following operably linked DNA fragments is expressed in said cells of said plant: a plant-expressible promoter region, a transcribed region of DNA encoding an RNA or protein which, when expressed, either increases or decreases said amount or a functional amount of said cell division control and 3'-end formation and polyadenylation signal functional in plant cells. The method of claim 5, wherein said transcribed DNA region encodes an antisense RNA, ribozyme, or sense strand of RNA that, when expressed, reduces, inhibits, or prevents expression of a cell division controlling protein. 7. The method of claim 6, wherein said transcribed DNA region encodes an antisense RNA which, when expressed, reduces, inhibits, or prevents expression of endogenous Type D cyclin. The method of claim 5, wherein said transcribed DNA region encodes a cell division controlling protein capable of binding or phosphorylating an Rb-like protein. The method of claim 5, wherein said transcribed DNA region encodes a cell division controlling protein comprising a binding motif of an Rb-like protein. The method of claim 9, wherein said binding motif is LxCxE. The method of claim 5, wherein said transcribed DNA region encodes a type D cyclin. 12. The method of claim 11 wherein said type D cyclin is from plants. The method of claim 12, wherein said type D cyclin is selected from the group consisting of Arabidopsis thaliana CYCD1, Arabidopsis thalina CYCD2, Arabidopsis thaliana CYCD3, Nicotiana tabacum CYCD3; 1, Nicotiana tabacum CYCD2; 1, Nicotiana tabacum CYCD3; 2, Helianthus tuberosus CYCD1; 1, Zea mays CYCD2 and Helianthus tuberosus CYCD3. The method of claim 12, wherein said transcribed DNA region comprises a nucleotide sequence selected from the group consisting of EMBL nucleotide sequence X83369 from nucleotide position 104 to nucleotide position 1108, EMBL nucleotide sequence X83370 from nucleotide position 195 to nucleotide position 1346, nucleotide sequence EMBL # X83371 from nucleotide position ·· * · 266 to nucleotide position 1396, nucleotide sequence SEQ ID NO: 1 from nucleotide position 182 to nucleotide position 1243, nucleotide sequence SEQ ID NO: 2 from nucleotide position 181 to nucleotide position 1299, nucleotide sequence SEQ ID NO: 3 from nucleotide position 198 to nucleotide position 1298, nucleotide sequence SEQ ID NO: 4 from nucleotide position 165 to nucleotide position 1109, nucleotide sequence SEQ ID NO: 5 from nucleotide position 48 to nucleotide position 1118, or nucleotide sequence of SEQ ID NO: 21 from nucleotide position 316 to nucleotide position 1389. 15. The method of claim 5, wherein said transcribed DNA region encodes a protein or peptide that, when expressed, increases said functional amount of said cell division controlling protein. 16. The method of claim 15, wherein said transcribed DNA region encodes a protein or peptide selected from the group consisting of a mutant type D cyclin, a portion of type D cyclins, a type D cyclin having a mutation in a cyclin box, type D2 cyclin, having a substitution of amino acid 185 or amino acid 155, a cyclin of type D2 which exhibits a mutation of E185A or K155A, a cyclin of type D where the PEST sequence has been deleted, a cyclin of type D where the LxCxE binding motif has been changed or deleted C-residue of the LxCxE binding motif. The method of any one of claims 5 to 16, wherein said plant expressible region of the plant is a CaMV35S promoter region. 18. The method of claim 6 or 7, wherein said modified growth characteristic is a reduced growth rate. 19. The method of claims 8 to 17 wherein said modified growth characteristic is an increased growth rate. * · Method according to claims 8 to 17, characterized in that said modified growth characteristic is faster germination. The method of claims 8 to 17, wherein said modified growth characteristic is a reduction in flowering time. Method according to claims 8 to 17, characterized in that said modified structure comprises an increased number of flowers per plant or an increased number of seeds per plant or an increased number of fruits per plant. A chimeric gene as described in any one of claims 5 to 17. A plant cell comprising the chimeric genes of claim 23. A plant essentially comprising the plant cells of claim 24. The plant of claim 25 that grows in a greenhouse. The plant of claim 23, selected from the group consisting of pine, poplar, Eucalyptus tree, alfalfa, legumes, grasses, maize, oilseed rape, flax seeds, wheat, brassica vegetables, tomatoes, lettuce, rice, barley, potatoes, tobacco, sugar beet, sunflower, carnation, chrysanthemum, rose or tulip. Plant seed according to any one of claims 25 to 27, characterized in that it comprises the chimeric genes according to claim 23. Isolated DNA fragment marked u j ici with E by wherein said nucleotide sequence comprises a nucleotide sequence SEQ ID NO: 1 from position nucleotide 182-1243. Isolated DNA fragment marked u j ici with E by wherein said nucleotide sequence comprises a nucleotide sequence SEQ ID NO: 2 from position nucleotide 181-1299. Isolated DNA fragment marked u j ici with E by wherein said nucleotide sequence comprises a nucleotide sequence SEQ ID NO: 3 from position nucleotides 198-1298. —F »» »» - »2 2. Λ. Λ · · · · · · »» »· · · 32. An isolated DNA fragment comprising the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 165 to 1109. 33. An isolated DNA fragment comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide position 48-1118. 34. An isolated DNA fragment comprising the nucleotide sequence of SEQ ID NO: 21 from nucleotide position 316 to 1389. 35. Use of a cell division controlling protein capable of binding the pocket domain of an Rb-like protein or capable of phosphorylating an Rb protein, altering the growth characteristics or plant structure. The use of claim 35, wherein the cell division control protein comprises in its N-terminal portion a LxCxE binding motif. The use of claim 36, wherein the cell division controlling protein is type D cyclin. The use of claim 37, wherein the cell division controlling protein is type D cyclin selected from the group consisting of Arabidopsis thaliana CYCD1, Arabidopsis thalina CYCD2, Arabisopsis thaliana CYCD3, Nicotiana tabacum CYCD3; 1, Nicotiana tabacum CYCD2; 1, Nicotiana tabacum CYCD3; Helianthus tuberosus CYCD1; 1, Zea mays CYCD2 and Helianthus tuberosus CYCD3. The use of any one of claims 34 to 37, wherein the cell division controlling protein is encoded by a chimeric gene incorporated in the genome of plant cells. 40. Use of DNA that encodes a cell division controlling protein capable of binding or phosphorylating an Rb-like protein, altering the growth characteristics or plant structure. The use of claim 40, wherein the cell division controlling protein is cyclin type D. »· ·· 0 0 0 · 0 · · · 0 0 0 · 00 ·· ·· The use of claim 41, wherein said DNA comprises a nucleotide sequence selected from the group consisting of EMBL nucleotide sequence X83369 from nucleotide position 104 to nucleotide position 1108, EMBL nucleotide sequence X83370 from nucleotide position 195 to nucleotide 1346, EMBL nucleotide sequence No. X83371 from nucleotide position 266 to nucleotide position 1396, nucleotide sequence SEQ ID NO: 1 from nucleotide position 182 to nucleotide position 1243, nucleotide sequence SEQ ID NO: 2 from nucleotide position 181 to nucleotide position 1299, nucleotide sequence SEQ ID NO: 3 from nucleotide position 198 to nucleotide position 1298, nucleotide sequence SEQ ID NO: 4 from nucleotide position 165 to nucleotide position 1109, nucleotide sequence SEQ ID NO: 5 from nucleotide position 48 to nucleotide 1118, or nucleotide sequence SEQ ID NO: 21 of nucleotide 316 to nucleotide position 1389.