AU2002210243A1

AU2002210243A1 - Manipulation of flowering and plant architecture

Info

Publication number: AU2002210243A1
Application number: AU2002210243A
Authority: AU
Inventors: Michael Emmerling; Eng Kok Ong; Timothy Ivor Sawbridge; German Spangenberg
Original assignee: Agriculture Victoria Services Pty Ltd; AgResearch Ltd
Current assignee: Agriculture Victoria Services Pty Ltd; AgResearch Ltd
Priority date: 2000-10-19
Filing date: 2001-10-17
Publication date: 2002-07-04
Anticipated expiration: 2021-10-17

Description

MANIPULATION OF FLOWERING AND PLANT ARCHITECTURE

The present invention relates to nucleic acids or nucleic acid fragments encoding amino acid sequences for proteins involved in the control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development in plants, and the use thereof for the modification of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development in plants.

Most plants have several growth phases. Following seed embryo germination, the plant apical meristem goes through a vegetative phase generating leaf primordia with axillary meristems. The axillary meristems will generate side branches or will rest dormant until apical dominance is removed. Upon receiving appropriate signals, the apical meristem switches to reproductive development (flowering). The switch is controlled by various physiological signals and genetic pathways that will coordinate flowering. The apical meristem switched from vegetative to reproductive phase will produce an inflorescence. Inflorescences are the flower-bearing parts of the plant. Flowers are plant structures that support gametophyte development. Inflorescences are of two basic types, determinate (where apical meristems grow indefinitely generating floral meristems from their periphery) and indeterminate (where apical meristem is transformed into a floral meristem terminating apical growth and with subsequent growth taking place from axillary meristems). Floral meristems produce a defined number of floral organ primordia in concentric whorls, which develop into sepals, petals, stamens or carpels (in the dicot flower); and into palea, lodicule, stamens and carpels (in the grass flower).

A series of homeotic genes that specify floral meristem identity and determine the fate of floral organ primordia have been identified. These genes interact to specify the basic floral organs of the dicot flower. These interactions can be summarized through the 'ABC" model of floral organ identity, where the first (outer) whorl organs (sepals) are specified by A-function genes, the second whorl organs (petals) are specified by a combination of A- and B-function activities, third whorl organs (stamens) by a combination of B- and C-function, and fourth whorl (carpels) by a C-function activity.

Most of these genes belong to a family of transcription factors characterized by the presence of a conserved DNA-binding domain, named the MADS-box. These genes (MADS-box genes or MADS) thus encode plant MADS-box proteins and are involved in flower and embryo development. Identified MADS-box genes in the model crucifer Arabidopsis thaliana include the meristem identity A-function genes APETALA1 (AP1) and CAULIFLOWER (CAL); the organ identity B-function genes APETALA3 (AP3) and PISTILLATA (PI); and the organ identity C-function gene AGAMOUS (AG).

The gene CENTRORADIALIS (CEN) of snapdragon (Antirrhinum) is required to maintain an indeterminate shoot identity.

The APETALA2 (AP2) gene of Arabidopsis thaliana has a role in the specification of floral meristem identity and floral organ identity. AP2 is required for A-function organ identity. Mutations of AP2 lead to homeotic conversion of floral organs, for example conversion of sepals to leaves or to carpels, conversion of petals to stamens, absence of petals.

Other plant genes involved in pattern formation and having a putative role in vegetative and floral development have a conserved DNA-binding domain, named the Homeo-box. These genes (Homeo-box genes or HB genes) thus encode plant homeodomain proteins that may function as transcription factors in controlling downstream target genes. Mutations in HB genes may result in abnormal leaf and flower development.

It would be useful however if other genes involved in specifying floral meristem identity, involved in the fate of floral development could be identified. In this regard the inventors have identified the following new genes: LpMADS, LpCEN, LpAP2, and LpHB. It would be desirable to have methods of manipulating plant life cycles and growth phases eg. the transition from the vegetative to the reproductive stage, flowering processes, flowering and plant architecture and inflorescence and flower development in plants, including grass species such as ryegrasses (Loliυm species) and fescues (Festuca species), thereby facilitating the production of, for example, pasture and turf grasses and pasture legumes with enhanced or shortened or modified life cycles, enhanced or reduced or otherwise modified inflorescence and flower development, inhibited flowering (eg. non-flowering), modified flowering architecture (eg. indeterminate and determinate), earlier or delayed flowering, enhanced or modified number of leaves, enhanced or reduced or otherwise modified number of reproductive shoots, enhanced persistence and improved herbage quality, enhanced seed and leaf yield, altered growth and development, leading to improved seed production, improved biomass production, improved pasture production, improved pasture quality, improved animal production and reduced environmental pollution (e.g. reduced pollen allergens, reduced nitrogenous waste).

Perennial ryegrass (Lolium perenne L.) is a key pasture grass in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.

Clovers (Trifolium species) such as white clover (T. repens), red clover (T. pratense) and subterranean clover (T. subterraneum), and lucerne (M. sativa) and medics (Medicago species) are fructan-devoid, starch-accumulating key pasture legumes in temperate climates throughout the world.

While nucleic acid sequences encoding some of the enzymes involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development have been isolated for certain species of plants, there remains a need for materials useful in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development, in a wide range of plants, particularly in forage and turf grasses and legumes including ryegrasses and fescues, and for methods for their use. It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

In one aspect, the present invention provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for the following proteins from a ryegrass (Lolium) or fescue (Festuca) species, or functionally active fragments or variants thereof: MADS-box proteins (MADS),

CENTRORADIALIS (CEN), APETALA2 (AP2), and Homeo-box proteins (HB).

The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to MADS, CEN, AP2 and HB. Such proteins are referred to herein as MADS-like, CEN-like, AP2-like, and HB-like, respectively.

The individual or simultaneous enhancement or down-regulation or otherwise manipulation of MADS-box gene activities in plants may alter flower and embryo and seed development, may for example enhance or inhibit embryo differentiation and growth, may alter flower organ identity through conversion of one floral organ in another, may lead to absence of individual floral organs, may lead to male and/or female sterility, may increase the number of specific floral organs, may enhance or inhibit and otherwise alter flowering, may enhance or delay and otherwise alter flowering in time, and may increase or otherwise alter the number of leaves made before flowering.

The enhancement or otherwise manipulation of CEN activity in plants may alter the control of phase change, may promote vegetative growth indefinitely, may delay or otherwise alter flowering in time, and may increase or otherwise alter the number of leaves made before flowering.

The down-regulation or otherwise manipulation of AP2 activity in plants may alter flower organ identity through conversion of one floral organ in another, may lead to absence of individual floral organs, may increase the number of specific floral organs, and may alter flowering architecture. The enhancement or ectopic expression or otherwise manipulation of HB activity in plants may alter the control of phase change, may promote or reduce vegetative growth, may delay or otherwise alter flowering, may alter floral organ identity, and may alter plant architecture e.g. enhanced branching, increased bushiness.

Manipulation of flowering and plant architecture has significant consequences for a wide range of applications in plant production. For example, it has applications in inducing male sterility for hybrid seed production, in changing flower architecture for enhancing value of ornamentals, in delaying flowering in forage grasses thus stopping the formation of the less digestible stems and increasing herbage quality, in altering flowering time allowing early maturing crops, in delaying vegetative phase and thus increasing biomass production, in increasing branching and thus leading to enhanced bushiness in fruit trees, in altering plant size and leading to shorter plant stature, in blocking flowering and reducing the release of allergenic pollen, etc.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne).

The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.

The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment. Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term "consensus contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid fragments) may be assembled into a single contiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a MADS or MADS-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1 , 3, 4, 6, 7, 9, 10, 12, 14, 15, 17, 19, 21 , 23, 25, 27, 29, 54, 59, 63, 68, 72, 76, 81 , 86 and 90 hereto (Sequence ID Nos: 1 , 3 to 11 , 12, 14 to 18, 19, 21 to 23, 24, 26, 28 to 30, 31 , 33, 35, 37, 39, 41 , 43, 45 to 49, 90, 92, 94, 96, 98, 100, 102, 104 and 106, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CEN or CEN-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 30, 32 and 49 hereto (Sequence ID Nos: 50, 52 to 54 and 88, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an

AP2 or AP2-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 33, 35, 36, and 38 hereto (Sequence ID Nos: 55, 57 to 68, 69 and 71 to 74, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an HB or HB-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 39, 41 , 43, 45, and 47 hereto (Sequence ID Nos: 75, 77, 79, 81 and 83 to 87, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

By "functionally active" in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) encodes a polypeptide capable of modifying control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development, in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned nucleotide sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides. The nucleic acids or nucleic acid fragments encoding at least a portion of several proteins involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development have been isolated and identified. The nucleic acids or nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction, ligase chain reaction).

For example, genes encoding other proteins involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or

RNA. For example, polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acids or nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments may be isolated (Ohara et al. (1989,) Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217; the entire disclosures of which are incorporated herein by reference). Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs.

In a second aspect of the present invention there is provided a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of MADS and MADS-like, CEN and CEN-like, AP2 and AP2-like, HB and HB-like proteins; and functionally active fragments and variants thereof.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated MADS or MADS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8, 11 , 13, 16, 18, 20, 22, 24, 26, 28, 55, 60, 64, 69, 73, 77, 82, 87 and 91 hereto (Sequence ID Nos: 2, 13, 20, 25, 27, 32, 34, 36, 38, 40, 42, 44, 91 , 93, 95, 97, 99, 101 , 103, 105 and 107, respectively); and functionally active fragments and variants thereof. In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CEN or CEN-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures

31 and 50 hereto (Sequence ID Nos: 51 and 89, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated AP2 or AP2-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures

34 and 37 hereto (Sequence ID Nos: 56 and 70, respectively); and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated HB or HB-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 40,

42, 44 and 46 hereto (Sequence ID Nos: 76, 78, 80 and 82, respectively); and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides it is meant that the fragment or variant has one or more of the biological properties for the proteins MADS, MADS-like, CEN, CEN-like, AP2, AP2-like, HB and HB-like, respectively. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned amino acid sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids. In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group. Variations in genotype are important in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNP's), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.

Applicants have identified a number of SNP's of the nucleic acids and nucleic acid fragments of the present invention. These are indicated (marked with grey on the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. See for example, Figures 3, 6, 9, 14, 29, 35, 38 and 47 (Sequence ID Nos: 3 to 11 , 14 to 18, 21 to 23, 28 to 30, 45 to 49, 57 to 68, 71 to 74, and 83 to 87, respectively).

Accordingly, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to the present invention, or complements or sequences antisense thereto, and functionally active fragments and variants thereof.

In a still further aspect of the present invention there is provided a method of isolating a nucleic acid or nucleic acid fragment of the present invention including a single nucleotide polymorphism (SNP), said method including sequencing nucleic acid fragments from a nucleic acid library.

The nucleic acid library may be of any suitable type and is preferably a cDNA library.

The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.

The sequencing may be performed by techniques known to those skilled in the art.

In a still further aspect of the present invention, there is provided use of nucleic acids or nucleic acid fragments of the present invention including SNP's, and/or nucleotide sequence information thereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment according to the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, e.g. tagging QTLs for herbage quality traits, flowering intensity, flowering time, number of tillers, leafiness, bushiness, seasonal growth pattern, herbage yield, flower architecture, plant stature. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.

In a still further aspect of the present invention there is provided a construct including a nucleic acid or nucleic acid fragment according to the present invention.

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases may also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

In a still further aspect of the present invention there is provided a vector including a nucleic acid or nucleic acid fragment according to the present invention.

The term "vector" as used herein includes both cloning and expression vectors. Vectors are often recombinant molecules including nucleic acid molecules from several sources. In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid and terminator being operatively linked.

By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non- chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable, or integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter. A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransf erase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet) and gymnosperms. In a preferred embodiment, the vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), even more preferably perennial ryegrass, including forage- and turf-type cultivars. In a preferred embodiment, the vectors may be used to transform dicotyledons, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago sativa).

Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

Cells incorporating the vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a construct or a vector of the present invention. The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass, including both forage- and turf-type cultivars. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a dicotyledon, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago sativa).

The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.

The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

In a further aspect of the present invention there is provided a method of modifying the control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or flower and/or inflorescence development in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or vector according to the present invention.

By "an effective amount" it is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference. Using the methods and materials of the present invention, plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or inflorescence and/or flower development may be increased, decreased or otherwise modified relative to an untransformed control plant. For example, the number of leaves produced before flowering, the number of floral organs, the number of branches, the plant stature, the number of phytomers, the number of inflorescences and/or flowers, may be increased, decreased or otherwise modified. They may be increased or otherwise modified, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased or otherwise modified, for example, by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures

Figure 1 shows the consensus contig nucleotide sequence of LpMADSa (Sequence ID No: 1).

Figure 2 shows the deduced amino acid sequence of LpMADSa (Sequence

ID No: 2).

Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSa (Sequence ID Nos: 3 to 11).

Figure 4 shows the consensus contig nucleotide sequence of LpMADSb

(Sequence ID No: 12). Figure 5 shows the deduced amino acid sequence of LpMADSb (Sequence ID No: 13).

Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSb (Sequence ID Nos: 14 to 18). .

Figure 7 shows the consensus contig nucleotide sequence of LpMADSc (Sequence ID No: 19).

Figure 8 shows the deduced amino acid sequence of LpMADSc (Sequence ID No: 20).

Figure 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSc (Sequence ID Nos: 21 to 23).

Figure 10 shows the nucleotide sequence of LpMADSd (Sequence ID No: 24).

Figure 11 shows the deduced amino acid sequence of LpMADSd

(Sequence ID No: 25).

Figure 12 shows the consensus contig nucleotide sequence of LpMADSe (Sequence ID No: 26).

Figure 13 shows the deduced amino acid sequence of LpMADSe (Sequence ID No: 27).

Figure 14 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSe (Sequence ID Nos: 28 to 30).

Figure 15 shows the nucleotide sequence of LpMADSf (Sequence ID No: 31). Figure 16 shows the deduced amino acid sequence of LpMADSf (Sequence ID No: 32).

Figure 17 shows the nucleotide sequence of LpMADSg (Sequence ID No: 33).

Figure 18 shows the deduced amino acid sequence of LpMADSg

(Sequence ID No: 34).

Figure 19 shows the nucleotide sequence of LpMADSh (Sequence ID No: 35).

Figure 20 shows the deduced amino acid sequence of LpMADSh (Sequence ID No: 36).

Figure 21 shows the nucleotide sequence of LpMADSi (Sequence ID No: 37).

Figure 22 shows the deduced amino acid sequence of LpMADSi (Sequence ID No: 38).

Figure 23 shows the nucleotide sequence of LpMADSj (Sequence ID No:

39).

Figure 24 shows the deduced amino acid sequence of LpMADSj (Sequence ID No: 40).

Figure 25 shows the nucleotide sequence of LpMADSk (Sequence ID No: 41).

Figure 26 shows the deduced amino acid sequence of LpMADSk (Sequence ID No: 42).

Figure 27 shows the consensus contig nucleotide sequence of LpMADSi (Sequence ID No: 43). Figure 28 shows the deduced amino acid sequence of LpMADSi (Sequence ID No: 44).

Figure 29 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSi (Sequence ID Nos: 45 to 49).

Figure 30 shows the consensus contig nucleotide sequence of LpCENa (Sequence ID No: 50).

Figure 31 shows the deduced amino acid sequence of LpCENa (Sequence ID No: 51).

Figure 32 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCENa (Sequence ID Nos: 52 to 54).

Figure 33 shows the consensus contig nucleotide sequence of LpAP2a (Sequence ID No: 55).

Figure 34 shows the deduced amino acid sequence of LpAP2a (Sequence

ID No: 56).

Figure 35 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpAP2a (Sequence ID Nos: 57 to 68).

Figure 36 shows the consensus contig nucleotide sequence of LpAP2b

(Sequence ID No: 69).

Figure 37 shows the deduced amino acid sequence of LpAP2b (Sequence ID No: 70). Figure 38 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpAP2b (Sequence ID Nos: 71 to 74).

Figure 39 shows the nucleotide sequence of LpHBa (Sequence ID No: 75).

Figure 40 shows the deduced amino acid sequence of LpHBa (Sequence

ID No: 76).

Figure 41 shows the nucleotide sequence of LpHBb (Sequence ID No: 77).

Figure 42 shows the deduced amino acid sequence of LpHBb (Sequence ID No: 78).

Figure 43 shows the nucleotide sequence of LpHBc (Sequence ID No: 79).

Figure 44 shows the deduced amino acid sequence of LpHBc (Sequence ID No: 80).

Figure 45 shows the consensus contig nucleotide sequence of LpHBd (Sequence ID No: 81 ).

Figure 46 shows the deduced amino acid sequence of LpHBd (Sequence

ID No: 82).

Figure 47 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpHBd (Sequence ID Nos: 83 to 87).

Figure 48 shows a plasmid map of the cDNA encoding perennial ryegrass

LpCen.

Figure 49 shows the nucleotide sequence of perennial ryegrass LpCen cDNA (Sequence ID No: 88). Figure 50 shows the deduced amino acid sequence of perennial ryegrass LpCen cDNA (Sequence ID No: 89).

Figure 51 shows plasmid maps of sense and antisense constructs of LpCen in pDH51 transformation vector.

Figure 52 shows screening by Southern hybridisation for RFLPs using

LpCen as a probe.

Figure 53 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADSi .

Figure 54 shows the nucleotide sequence of perennial ryegrass LpMADSi cDNA (Sequence ID No: 90).

Figure 55 shows the deduced amino acid sequence of perennial ryegrass LpMADSi cDNA (Sequence ID No: 91).

Figure 56 shows plasmid maps of sense and antisense constructs of LpMADSi in pDH51 transformation vector.

Figure 57 shows screening by Southern hybridisation for RFLPs using

LpMADSi as a probe.

Figure 58 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADSi b.

Figure 59 shows the nucleotide sequence of perennial ryegrass LpMADSi b cDNA (Sequence ID No: 92).

Figure 60 shows the deduced amino acid sequence of perennial ryegrass LpMADSi b cDNA (Sequence ID No: 93).

Figure 61 shows plasmid maps of sense and antisense constructs of LpMADSi b in pDH51 transformation vector. Figure 62 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-1.

Figure 63 shows the nucleotide sequence of perennial ryegrass LpMADS2-

1 cDNA (Sequence ID No: 94).

Figure 64 shows the deduced amino acid sequence of perennial ryegrass

LpMADS2-1 cDNA (Sequence ID No: 95).

Figure 65 shows plasmid maps of sense and antisense constructs of LpMADS2-1 in pDH51 transformation vector.

Figure 66 shows screening by Southern hybridisation for RFLPs using LpMADS2-1 as a probe.

Figure 67 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-2.

Figure 68 shows the nucleotide sequence of perennial ryegrass LpMADS2-

2 cDNA (Sequence ID No: 96).

Figure 69 shows the deduced amino acid sequence of perennial ryegrass

LpMADS2-2 cDNA (Sequence ID No: 97).

Figure 70 shows plasmid maps of sense and antisense constructs of LpMADS2-2 in pDH51 transformation vector.

Figure 71 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-3.

Figure 72 shows the nucleotide sequence of perennial ryegrass LpMADS2-

3 cDNA (Sequence ID No: 98).

Figure 73 shows the deduced amino acid sequence of perennial ryegrass LpMADS2-3 cDNA (Sequence ID No: 99). Figure 74 shows plasmid maps of sense and antisense constructs of LpMADS2-3 in pDH51 transformation vector.

Figure 75 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS3.

Figure 76 shows the nucleotide sequence of perennial ryegrass LpMADS3 cDNA (Sequence ID No: 100).

Figure 77 shows the deduced amino acid sequence of perennial ryegrass LpMADS3 cDNA (Sequence ID No: 101).

Figure 78 shows plasmid maps of sense and antisense constructs of LpMADS3 in pDH51 transformation vector.

Figure 79 shows screening by Southern hybridisation for RFLPs using LpMADS3 as a probe.

Figure 80 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS4.

Figure 81 shows the nucleotide sequence of perennial ryegrass LpMADS4 cDNA (Sequence ID No: 102).

Figure 82 shows the deduced amino acid sequence of perennial ryegrass LpMADS4 cDNA (Sequence ID No: 103).

Figure 83 shows plasmid maps of sense and antisense constructs of LpMADS4 in pDH51 transformation vector.

Figure 84 shows screening by Southern hybridisation for RFLPs using LpMADS4 as a probe.

Figure 85 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS4-2. Figure 86 shows the nucleotide sequence of perennial ryegrass LpMADS4- 2 cDNA (Sequence ID No: 104).

Figure 87 shows the deduced amino acid sequence of perennial ryegrass LpMADS4-2 cDNA (Sequence ID No: 105).

Figure 88 shows plasmid maps of sense and antisense constructs of

LpMADS4-2 in pDH51 transformation vector.

Figure 89 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADSδ.

Figure 90 shows the nucleotide sequence of perennial ryegrass LpMADSδ cDNA (Sequence ID No: 106).

Figure 91 shows the deduced amino acid sequence of perennial ryegrass LpMADSδ cDNA (Sequence ID No: 107).

Figure 92 shows plasmid maps of sense and antisense constructs of LpMADSδ in pDHδl transformation vector.

Figure 93 shows screening by Southern hybridisation for RFLPs using

LpMADSδ as a probe.

Figure 94 shows the regeneration of transgenic tobacco plants from direct gene transfer to protoplasts of chimeric ryegrass genes involved in flowering and plant development.

Figure 9δ shows a subgrid of a microarray for the expression profiling of perennial ryegrass flowering and plant development genes. Red represents up- regulated expression, green represents down-regulated expression and yellow represents no change in expression. For example, an overlay of microarray images probed with leaf blade tissues (red) and root tissues (green). Expression level is relatively expressed as up-regulated in leaf blade (red), down-regulated in leaf blade (green) and no change in expression (yellow). Figure 96 shows a genetic linkage map of perennial ryegrass NA6 showing map location of ryegrass genes involved in flowering and plant development.

EXAMPLE 1

Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for MADS, Cen, AP2, and HB proteins from perennial ryegrass (Lolium perenne)

cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass (Lolium perenne) were prepared. The characteristics of the libraries are described in Table 1.

TABLE 1

cDNA libraries from perennial ryegrass (Lolium perenne)

The cDNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101 , USA), tailed and size fractionated, according to the protocol provided by Clontech. The cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA, USA). The Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).

Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the δ insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate 0 partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2

DNA sequence analyses δ The cDNA clones encoding MADS, CEN, AP2 and HB proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 216:403-410) searches. The cDNA sequences obtained were analysed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTN algorithm provided by 0 the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI. δ The cDNA sequences obtained and identified were then used to identify additional identical and/or overlapping cDNA sequences generated using the BLASTN algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence alignment. The 0 consensus contig sequence was then used as a query for a search against the SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.

EXAMPLE 3

Identification and full-length sequencing of perennial ryegrass MADS and δ Cen cDNAs encoding flowering and plant development proteins.

To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of perennial ryegrass cDNAs encoding flowering and plant development proteins was identified and fully sequenced.

0 Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches. Full-length cDNAs were identified by alignment of the query and hit sequences using Sequencher (Gene Codes Corp., AnnArbor, Ml 48108, USA). The original plasmid was then used to transform chemically competent XL-1 cells (prepared in- δ house, CaCI₂ protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the 0 best initial sequencing result.

Sequencing was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases the sequencing could be done from both δ' and 3' end. The sequences of the oligonucleotide primers are shown in Table 2. In some instances, however, an δ extended poly-A tail necessitated the sequencing of the cDNA to be completed from the δ' end. TABLE 2 List of primers used for sequencing of the full-length cDNAs

Contigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRI cut site both at the δ' and 3' end.

Plasmid maps and the full cDNA sequences of perennial ryegrass Cen, MADS1 , MADSI b, MADS2-1 , MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADSδ were obtained (Figures 48, 49, δ3, δ4, δ8, δ9, 62, 63, 67, 68, 71 , 72, 75, 76, 80, 81 , 85, 86, 89 and 90).

EXAMPLE 4

Development of transformation vectors containing chimeric genes with Cen, MADS1, MADSIb, MADS2-1 , MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 cDNA sequences from perennial ryegrass

To alter the expression of flowering and plant development proteins Cen, MADS1 , MADSI b, MADS2-1 , MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADSδ, through antisense and/or sense suppression technology and for over-expression of these key enzymes in transgenic plants, a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRI and Xbal for directional and non-directional cloning into the target vector.

TABLE 3 List of primers used to PCR-amplify the open reading frames

After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pDHδl , a pUC18-based transformation vector containing a CaMV 3δS expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass Cen, MADS1 , MADSI b, MADS2-1 , MADS2-2, δ MADS2-3, MADS3, MADS4, MADS4-2 and MADSδ in sense and antisense orientations under the control of the CaMV 3δS promoter were generated (Figures δ1 , 56, 61 , 65, 70, 74, 78, 83, 88 and 92).

EXAMPLE 5

Production of transgenic tobacco plants carrying chimeric Cen, MADS1 , 0 MADSI b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 genes from perennial ryegrass

A set of transgenic tobacco plants carrying chimeric Cen, MADS1 , MADSI b, MADS2-1 , MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 cDNA genes from perennial ryegrass were produced.

δ pDHδl -based transformation vectors with LpCen, LpMADSi , LpMADSi b,

LpMADS2-1 , LpMADS2-2, LpMADS2-3, LpMADS3, LpMADS4, LpMADS4-2 and LpMADSδ cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 3δS promoter were generated.

0 Direct gene transfer experiments to tobacco protoplasts were performed using these transformation vectors.

The production of transgenic tobacco plants carrying the perennial ryegrass Cen, MADS1 , MADSI b, MADS2-1 , MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADSδ cDNAs under the control of the constitutive CaMV 3δS δ promoter is described here in detail. Isolation of mesophyll protoplasts from tobacco shoot cultures

2-4 fully expanded leaves of a 6 week-old shoot culture were placed under sterile conditions (work in laminar flow hood, use sterilized forceps, scalpel and blades) in a 9 cm plastic culture dish containing 12 ml enzyme solution [1.0% (w/v) δ cellulase "Onozuka" R10 and 1.0% (w/v) Macerozyme^® R10]. The leaves were wetted thoroughly with enzyme solution and the mid-ribs removed. The leaf halves were cut into small pieces and incubated overnight (14-18 h) at 2δ°C in the dark without shaking

The protoplasts were released by gently pipetting up and down, and the 0 suspension poured through a 100 μm stainless steel mesh sieve on a 100 ml glass beaker. The protoplast suspension was mixed gently, distributed into two 14 ml sterile plastic centrifuge tubes and carefully overlayed with 1 ml Wδ solution.

After centrifugation for δ min. at 70g (Clements Orbital δOO bench centrifuge, swing-out rotor, 400 rpm), the protoplasts were collected from the interphase and δ transferred to one new 14 ml centrifuge tube. 10 ml Wδ solution were added, the protoplasts resuspended by gentle tilting the capped tube and pelleted as before.

The protoplasts were resuspended in δ-10 ml Wδ solution and the yield determined by counting a 1 :10 dilution in a haemocytometer.

Direct gene transfer to protoplasts using polyethylene glycol 0 The protoplasts were pelleted [70g (Clements Orbital δOO bench centrifuge,

400 rpm) for δ min.] and resuspended in transformation buffer to a density of 1.6 x 10⁶ protoplasts/ml. Care should be taken to carry over as little as possible Wδ solution into the transformation mix. 300 μl samples of the protoplast suspension (ca. δ x 10⁵ protoplasts) were aliquotted in 14 ml sterile plastic centrifuge tubes, 30 δ μ\ of transforming DNA were added. After carefully mixing, 300 μ\ of PEG solution were added and mixed again by careful shaking. The transformation mix was incubated for 16 min. at room temperature with occasional shaking. 10 ml Wδ solution were gradually added, the protoplasts pelleted [70g (Clements Orbital 600 bench centrifuge, 400 rpm) for δ min.] and the supernatant removed. The 0 protoplasts were resuspended in O.δ ml K3 medium and ready for cultivation 36

Culture of protoplasts, selection of transformed lines and regeneration of transgenic tobacco plants

Approximately δ x 10⁵ protoplasts were placed in a 6 cm petri dish. 4.δ ml of a pre-warmed (melted and kept in a water bath at 40-4δ°C) 1 :1 mix of K3:H δ medium containing 0.6% SeaPlaque™ agarose were added and, after gentle mixing, allowed to set.

After 20-30 min the dishes were sealed with Parafilm^® and the protoplasts were cultured for 24 h in darkness at 24°C, followed by 6-8 days in continuous dim light (6 /vmol m^"2 s^"1, Osram L36 W/21 Lumilux white tubes), where first and 0 multiple cell divisions occur. The agarose containing the dividing protoplasts was cut into quadrants and placed in 20 ml of A medium in a 260 ml plastic culture vessel. The corresponding selection agent was added to the final concentration of 60 mg/l kanamycin sulphate (for npt2 expression) or 2δ mg/l hygromycin B (for hph expression) or 20 mg/l phosphinotricin (for bar expression). Samples were δ incubated on a rotary shaker with 80 rpm and 1.26 cm throw at 24°C in continuous dim light.

Resistant colonies were first seen 3-4 weeks after protoplast plating, and after a total time of 6-8 weeks protoplast-derived resistant colonies (when 2-3 mm in diameter) were transferred onto MS morpho medium solidified with 0.6% (w/v) 0 agarose in 12-well plates and kept for the following 1-2 weeks at 24°C in continuous dim light (δ vmol m^"2 s^' Osram L36 W/21 Lumilux white tubes), where calli proliferated, reached a size of 8-10 mm, differentiated shoots that were rooted on MS hormone free medium leading to the recovery of transgenic tobacco plants (Table 4 and Figure 94). TABLE 4 Production of transgenic tobacco calli carrying chimeric ryegrass genes (in sense and antisense orientaion) involved in the regulation of flowering and plant development

EXAMPLE 6

Genetic mapping of perennial ryegrass genes involved in flowering and plant development

The cDNAs representing genes involved in flowering and plant δ development were amplified by PCR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped in the Fi (first generation) population, NA₆ x AUβ- This population was made by crossing an individual (NA₆) from a North African ecotype with an individual (AU₆) from the cultivar Aurora, 0 which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1 x CTAB method of Fulton et al. (1996).

Probes were screened for their ability to detect polymorphism using the DNA (10 μg) of both parents and δ F-i progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindi 11. Hybridisations were carried out using the method of δ Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 62, 67, 66, 79, 84, and 93).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1 :1 ratio 0 were mapped using MAPMAKER 3.0 (Lander et al. 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of δ.O and ordered within each linkage group using a LOD threshold of 2.0.

Loci representing genes involved in flowering and plant development 6 mapped to the linkage groups as indicated in Table δ and in Figure 96. These gene locations can now be used as candidate genes for quantitative trait loci for flowering and plant development. TABLE 5

Map locations of ryegrass genes encoding proteins involved in flowering and plant development across two genetic linkage maps of perennial ryegrass (NA6 and AU6)

Linkage group

Probe Polymorphic Mapped with Locus NA6 AU6

LpAP2b Hind III LpAP2b.1 1

LpAP2b.2 1 1

LpCENa Y Dra \ LpCENa δ

LpHBa Y EcoR V LpHBa.1 6

LpHBa.2 7

LpHBb Y EcoR I LpHBb δ δ

LpHBd Y Hind III LpHBd.1 6 6

LpHBd.2 6

LpMADSi /L Y EcoR V LpMADSi 3 pMADSIa

LpMADS3 Y EcoR V LpMADS3 7 δ

LpMADS4-1 Y EcoR I LpMADS4-1 7

EXAMPLE 7

Expression profiling of cDNAs encoding proteins involved in flowering and plant development using microarray technology cDNAs encoding proteins involved in flowering and plant development were PCR amplified and purified. The amplified products were spotted three times on each amino-silane coated glass slide (CMT-GAPS, Corning, USA) using a microarrayer MicroGrid (BioRobotics, UK). Spotting solution was also spotted in every subgrid of the microarray as negative and background controls. The duplicates were placed about 800 micron apart to prevent competitive hybridisation. Table 6 gives details on the tissues used to extract total RNA.

TABLE 6

List of hybridization probes used in expression profiling of perennial ryegrass genes encoding proteins involved in flowering and plant development using microarrays

Fluorescence labelled probes were synthesis by reversed transcribing RNA and incorporating Cyanine 3 or δ labelled dCTP. The probes were hybridised onto microarrays. In each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the microarrays were washed and scanned using a confocal laser scanner (ScanArray 3000, Packard, USA). The images obtained were quantified and analysed using Imagene 4.1 and GeneSight 2.1 (BioDiscovery, USA). Data were judged as not present (-), low expression (+), medium expression (++), high expression (+++) and highly expression (++++) (Table 7).

i

TABLE 7 Results of expression profiling of ryegrass genes encoding proteins involved in flowering and plant development

TABLE 7 (cont.)

REFERENCES

Feinberg, A. P., Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.

Frohman et al. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad Sci. USA 85:8998

Gish and States (1993) Identification of protein coding regions by database similarity serach. Nature Genetics 3:266-272

Lander, E.S., Green P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations. Genomics 1 : 174-181.

Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L, Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain. Science 243:217-220

Ohara, O., Dorit, R.L., Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA 86:6673-5677

Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press

Sharp, P.J., Kreis, M., Shewry, P.R., Gale, M.D. (1988). Location of α-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75: 286-290. Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

It will also be understood that the term "comprises" (or its grammatical variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

Documents cited in this specification are for reference purposes only and their inclusion is not an acknowledgement that they form part of the common general knowledge in the relevant art.

Claims

46

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a protein selected from the group consisting of MADS-box proteins (MADS), CENTRORADIALIS (CEN), APETALA2 (AP2), and Homeo-box δ proteins (HB) from a ryegrass (Lolium) or fescue (Festuca) species, or a functionally active fragment or variant thereof.

2. A nucleic acid or nucleic acid fragment according to Claim 1 , wherein said ryegrass or fescue species is perennial ryegrass (Lolium perenne).

3. A nucleic acid or nucleic acid fragment according to Claim 1 , 0 encoding a MADS or MADS-like protein and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1 , 3, 4, 6, 7, 9, 10, 12, 14, 15, 17, 19, 21 , 23, 25, 27, 29, 54, 69, 63, 68, 72, 76, 81 , 86 and 90 hereto (Sequence ID Nos: 1 , 3 to 11 , 12, 14 to 18, 19, 21 to 23, 24, 26, 28 to 30, 31 , 33, 36, 37, 39, 41 , 43, 46 to 49, 90, 92, 94, 96, 98, 100, 102, 104 and 106, 6 respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

4. A nucleic acid or nucleic acid fragment according to Claim 1 , encoding a CEN or CEN-like protein and including a nucleotide sequence selected 0 from the group consisting of (a) sequences shown in Figures 30, 32 and 49 hereto (Sequence ID Nos: 60, 62 to 64 and 88, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

5 5. A nucleic acid or nucleic acid fragment according to Claim 1 , encoding an AP2 or AP2-like protein and including nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 33, 36, 36, and 38 hereto (Sequence ID Nos: δδ, 67 to 68, 69 and 71 to 74, respectively) ; (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

6. A nucleic acid or nucleic acid fragment according to Claim 1 , encoding an HB or HB-like protein and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 39, 41 , 43, 46, and 47 hereto (Sequence ID Nos: 76, 77, 79, 81 and 83 to 87, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

7. A construct including a nucleic acid or nucleic acid fragment according to Claim 1.

8. A vector including a nucleic acid or nucleic acid fragment according to Claim 1.

9. A vector according to Claim 8, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked.

10. A plant cell, plant, plant seed or other plant part, including a construct according to claim 7 or a vector according to Claim 8.

11. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 10.

12. A method of modifying plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or flower and/or inflorescence development in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to

Claim 1 , a construct according to claim 7 and/or a vector according to Claim 8.

13. Use of a nucleic acid or nucleic acid fragment according to Claim 1 , and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

14. A substantially purified or isolated polypeptide from a ryegrass δ (Lolium) or fescue (Festuca) species, selected from the group consisting of the proteins MADS and MADS-like, CEN and CEN-like, AP2 and AP2-like, HB and HB-like proteins; and functionally active fragments and variants thereof.

16. A polypeptide according to Claim 14, wherein said ryegrass is perennial ryegrass (Lolium perenne).

0 16. A polypeptide according to Claim 14, wherein said polypeptide is

MADS or MADS-like and includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, δ, 8, 11 , 13, 16, 18, 20, 22, 24, 26, 28, δδ, 60, 64, 69, 73, 77, 82, 87 and 91 hereto (Sequence ID Nos: 2, 13, 20, 25, 27, 32, 34, 36, 38, 40, 42, 44, 91 , 93, 95, 97, 99, 101 , 103, 105 and 107, 6 respectively); and functionally active fragments and variants thereof.

17. A polypeptide according to Claim 14, wherein said polypeptide is CEN or CEN-like includes an amino acid sequence selected from the group consisting of sequences shown in Figures 31 and 60 hereto (Sequence ID Nos: 61 and 89, respectively); and functionally active fragments and variants thereof.

0 18. A polypeptide according to Claim 14, wherein said polypeptide is

AP2 or AP2-like and includes an amino acid sequence selected from the group consisting of sequences shown in Figures 34 and 37 hereto (Sequence ID Nos: 66 and 70, respectively); and functionally active fragments and variants thereof.

19. A polypeptide according to Claim 17, wherein said polypeptide is HB or HB-like and includes an amino acid sequence selected from the group consisting of sequences shown in Figures 40, 42, 44 and 46 hereto (Sequence ID

Nos: 76, 78, 80 and 82, respectively); and functionally active fragments and variants thereof.