WO1994028140A1

WO1994028140A1 - Transgenic flowering plants

Info

Publication number: WO1994028140A1
Application number: PCT/AU1994/000265
Authority: WO
Inventors: Timothy Albert Holton; Yoshikazu Tanaka
Original assignee: International Flower Developments Pty. Ltd.
Priority date: 1993-05-20
Filing date: 1994-05-20
Publication date: 1994-12-08
Also published as: JPH08511683A; KR100337755B1; EP0703982A4; CN1127015A; CA2163220A1; NZ266401A; SG45175A1; PL311691A1; PL177743B1; EP0703982A1

Abstract

The present invention relates generally to transgenic flowering plants. More particularly, the present invention is directed to transgenic rose, carnation and chrysanthemum plants genetically modified to enable expression of flavonoid 3',5'-hydroxylase thereby permitting the manipulation of intermediates in the flavonoid pathway.

Description

TRANSGENIC FLOWERING PLANTS

The present invention relates generally to transgenic flowering plants. More particularly, the present invention is directed to transgenic rose, carnation and chrysanthemum plants genetically modified to enable expression of fiavonoid 3',5'-hydroxylase thereby permitting the manipulation of intermediates in the fiavonoid pathway.

The flower industry strives to develop new and different varieties of flowering plants, with improved characteristics ranging from disease and pathogen resistance to altered inflorescence. Although classical breeding techniques have been used with some success, this approach has been limited by the constraints of a particular species' gene pool. It is rare, for example, for a single species to have a full spectrum of coloured varieties. Accordingly, substantial effort has been directed towards attempting to generate transgenic plants exhibiting the desired characteristics. The development of blue varieties of the major cutflower species rose, carnation and chrysanthemum, for example, would offer a significant opportunity in both the cutflower and ornamental markets.

Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the only pigment in yellow or orange flowers. The fiavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation, methylation and vacuolar pH (Forkmann, 1991).

The biosynthetic pathway for the fiavonoid pigments (hereinafter referred to as the "fiavonoid pathway") is well established and is shown in Figure 1 (Ebel and Hahlbrock,

1988; Hahlbrock and Grisebach, 1979; Wiering and de Vlaming, 1984; Schram et al,

1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of _/7-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2',4,4',6'-tetrahydroxychalcone, is normally rapidly isomerized to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3 -hydroxylase (F3H) to produce dihydrokaempferol (DHK).

The B-ring of dihydrokaempferol can be hydroxylated at either the 3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. Two key enzymes involved in this pathway are fiavonoid 3 '-hydroxylase and fiavonoid 3',5'-hydroxylase. The fiavonoid 3 '-hydroxylase acts on DHK to produce DHQ and on naringenin to produce eriodictyol. The fiavonoid 3',5'-hydroxylase (hereinafter referred to as 3',5'-hydroxylase) is a broad spectrum enzyme catalyzing hydroxylation of naringenin and DHK in the 3' and 5' positions and of eriodictyol and DHQ in the 5' position (Stotz and Forkmann, 1982), in both instances producing pentahydroxyflavanone and DHM, respectively. The pattern of hydroxylation of the B- ring of anthocyanins plays a key role in determining petal colour.

Because of the aforesaid gene pool constraints, many of the major cutflower species lack the 3',5'-hydroxylase and consequently cannot display the range of colours that would otherwise be possible. This is particularly the case for roses, carnations and chrysanthemums, which constitute a major proportion of the world-wide cutflower market.

There is a need, therefore, to modify plants and in particular roses, carnations and chrysanthemums, to generate transgenic plants which are capable of producing the 3 ',5'- hydroxylase thereby providing a means of modulating DHK metabolism, as well as the metabolism of other substrates such as DHQ, naringenin and eriodictyol. Such modulation influences the hydroxylation pattern of the anthocyanins and allows the production of anthocyanins derived from delphinidin, thereby modifying petal colour and allowing a single species to express a broader spectrum of flower colours. There is a particular need to generate transgenic plants which produce high levels of anthocyanins derived from delphinidin. In accordance with the present invention, gene constructs are generated and used to make transgenic plants which express high levels of delphinidin and/or its derivatives relative to non-transgenic plants of the same species. The production of these high levels of delphinidin and related molecules is particularly useful in developing a range of plants exhibiting altered inflorescence properties.

Accordingly, one aspect of the present invention contemplates a transgenic plant or its progeny selected from rose, carnation and chrysanthemum wherein said plant produces a polypeptide having fiavonoid 3',5'-hydroxylase activity and produces higher levels of anthocyanins derived from delphinidin relative to non-transgenic plants of the same respective species.

More particularly, the present invention is directed to a transgenic plant or its progeny selected from rose, carnation and chrysanthemum wherein said plant expresses a polypeptide having fiavonoid 3',5'-hydroxylase activity and produces higher levels of delphinidin and/or derivatives of delphinidin relative to non-transgenic plants of the same respective species.

Preferably, the polypeptide is of petunia, verbena, delphinum, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg plant, lisianthus or campanula origin.

Preferably, the peptide is fiavonoid 3',5'-hydroxylase and most preferably a petunia 3',5'- hydroxylase.

The gene construct of the present invention comprises a nucleic acid molecule encoding a sequence encoding 3',5'-hydroxylase and where necessary comprises additional genetic sequences such as promoter and terminator sequences which allow expression of the molecule in the transgenic plant. When the gene construct is DNA it may be cDNA or genomic DNA. Preferably, the DNA is in the form of a binary vector comprising a chimaeric gene construct which is capable of being integrated into a plant genome to produce the transgenic plant of the present invention. The chimaeric gene construct may have a plant promoter such as CHS or the 3',5'-hydroxylase gene sequence may be modified such as to enhance expression and lead to increased levels of delphinidin and/or its derivatives. The CHS promoter is particularly convenient since it is a plant promoter in the fiavonoid pathway and directs the high level expression of genetic sequences down stream of the promoter. The most preferred binary vectors are pCGP484, pCGP485, pCGP628, pCGP653 and pCGP1458.

By "nucleic acid molecule" as used herein is meant any contiguous series of nucleotide bases specifying a sequence of amino acids in 3',5'-hydroxylase. The nucleic acid may encode the full length enzyme or a functional derivative thereof. By "derivative" is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally-occurring enzyme. In this regard, the nucleic acid includes the naturally- occurring nucleotide sequence encoding 3',5'-hydroxylase or may contain single or multiple nucleotide substitutions, deletions and/or additions to said naturally-occurring sequence. The terms "analogues" and "derivatives" also extend to any functional chemical equivalent of the 3',5'-hydroxylase, the only requirement of the said nucleic acid molecule being that when used to produce a transgenic plant in accordance with the present invention said transgemc plant exhibits one or more of the following properties:

(i) production of 3',5'-hydroxylase-specifιc mRNA; (ii) production of 3',5'-hydroxylase protein; (iii) production of delphinidin and/or its derivatives; and/or (iv) altered infloresence.

More particularly, said transgenic plant exhibits one or more of the following properties:

(i) increased levels of 3',5'-hydroxylase-specific mRNA above non-transgenic endogenous levels;

(ii) increased production of 3',5'-hydroxylase protein;

(iii) elevated levels of production of delphinidin and/or its derivatives above non- transgenic endogenous levels; and/or (iv) altered infloresence. The nucleic acid molecules used herein may exist alone or in combination with a vector molecule and preferably an expression-vector. Such vector molecules replicate and/or express in eukaryotic and/or prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule may additionally contain a sequence useful in facilitating said integration and/or a promoter sequence capable of directing expression of the nucleic acid molecule in a plant cell. The nucleic acid molecule and promoter may be introduced into the cell by any number of means such as by electroporation, micro-projectile bombardment or Agrobacterium- mediated transfer.

In accordance with the present invention, a nucleic acid molecule encoding 3 ',5'- hydroxylase may be introduced into and expressed in a transgenic plant selected from the list consisting of rose, carnation and chrysanthemum thereby providing a means to convert DHK and/or other suitable substrates into anthocya in derivatives of anthocyanidins such as petunidin, malvidin and especially delphinidin. The production of these anthocyanins may contribute to the production of a variety of shades of blue colour or blue-like colour or may otherwise modify flower colour by diverting anthocyanin production away from pelargonidin, cyanidin and peonidin and their derivatives and towards delphinidin and its derivatives. Expression of the nucleic acid sequence in the plant may be constitutive, inducible or developmental. The expression "altered inflorescence" means any alteration in flower colour relative to the naturally-occurring flower colour taking into account normal variations between flowerings. Preferably, the altered inflorescence includes production of various shades of blue, purple or pink colouration different to those in the non-transgenic plant.

The present invention also contemplates a method for producing a transgenic flowering plant exhibiting elevated levels of production of delphinidin and/or its derivatives above non-transgenic endogenous levels, said method comprising introducing into a cell of a plant selected from the list consisting of rose, carnation and chrysanthemum, a nucleic acid molecule encoding a sequence encoding 3',5'-hydroxylase under conditions permitting the eventual expression of said nucleic acid molecule, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid molecule into the 3',5'-hydroxylase enzyme. The present invention is also directed to a method for producing a transgenic plant selected from rose, carnation and chrysanthemum, said method comprising introducing into said plant a gene construct containing a nucleic acid sequence encoding a fiavonoid 3',5'-hydroxylase characterised in that said transgenic plant produces higher levels of anthocyanin derived from delphinidin relative to non-transgenic plants of the same respective species.

In a preferred embodiment, the transgenic flowering plant exhibits altered inflorescence properties coincident with elevated levels of delphinidin production, and the altered inflorescence includes the production of blue flowers or other bluish shades depending on the physiological conditions of the recipient plant. In certain plant species it may be preferable to select a "high pH line", such being defined as a variety having a higher than average petal vacuolar pH. The origin of the recombinant 3',5'-hydroxylase or its mutants and derivatives may include, petunia, verbena, delphinium, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, lisianthus, campanula or eggplant.

Consequently, the present invention extends to a transgenic rose, carnation or chrysanthemum plant containing all or part of a nucleic acid molecule representing 3', 5'- hydroxylase and/or any homologues or related forms thereof and in particular those transgenic plants which exhibit elevated 3',5'-hydroxylase-specific mRNA and/or elevated production of delphinidin derivatives and/or altered inflorescence properties. The transgenic plants, therefore, contain a stably-introduced nucleic acid molecule comprising a nucleotide sequence encoding the 3 ',5 '-hydroxylase enzyme. The invention also extends to progeny from such transgenic plants and also to reproduction material therefor (e.g. seeds). Such seeds, especially if coloured, will be useful inter alia as proprietary tags for plants.

The present invention is further described by reference to the following non-limiting Figures and Examples. In the Figures:

Figures 1(A) and (B) are schematic representations of the biosynthesis pathway for the fiavonoid pigments. Enzymes involved in the first part of the pathway have been indicated as follows: PAL = Phenylalanine ammonia-lyase; C4H = Cinnamate 4- hydroxylase; 4CL = 4-coumarate: CoA ligase; CHS = Chalcone synthase; CHI = Chalcone flavanone isomerase; F3H = Flavanone 3 -hydroxylase; DFR = Dihydroflavonol-4-reductase; UFGT = UDP-glucose: flavonoid-3-O-glucosyltransferase. The later steps correspond to conversions that can occur in P. hybrida flowers and include: 1 = addition of a rhamnose sugar to the glucosyl residue of cyanidin-3-glucoside and delphinidin-3-glucoside; 2 = acylation and 5-O-glucosylation; 3 = 3' methylation; 4 = 5' methylation; 5 = 3'5' methylation.

Figure 2 is a diagrammatic representation of the binary expression vector pCGP812, contruction of which is described in Example 3. Gent = the gentamycin resistance gene; LB = left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II; GUS = the yS-glucuronidase coding region. Chimaeric gene insert is as indicated, and described in Example 3. Restriction enzyme sites are marked.

Figure 3 is a diagrammatic representation of the binary expression vector pCGP485, contruction of which is described in Example 4. Gent = the gentamycin resistance gene; LB = left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II. Chimaeric gene insert is as indicated, and described in Example 4. Restriction enzyme sites are marked.

Figure 4 is a diagrammatic representation of the binary expression vector pCGP628, contruction of which is described in Example 5. Gent = the gentamycin resistance gene; LB = left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II. Chimaeric gene insert is as indicated, and described in Example 5. Restriction enzyme sites are marked. Figure 5 is a diagrammatic representation of the binary expression vector pCGP653, contruction of which is described in Example 6. Gent = the gentamycin resistance gene; LB = left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II. Chimaeric gene insert is as indicated, and described in Example 6. Restriction enzyme sites are marked.

Figure 6 is a diagrammatic representation of the binary expression vector pCGP484, contruction of which is described in Example 7. Gent = the expression cassette for the gentamycin resistance gene; LB = left border; RB = right border; nptll = neomycin phosphotransferase II. Chimaeric gene insert is as indicated, and described in Example 7. Restriction enzyme sites are marked.

Figure 7 is a diagrammatic representation of the binary expression vector pCGP1458, contruction of which is described in Example 8. nptl = the neomycin phosphotransferase I resistance gene; ; LB «= left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II. Chimaeric gene insert is as indicated, and described in Example 8. Restriction enzyme sites are marked.

Figure 8 shows a photograph of an autoradiographic representation of a Southern hybridization of Royalty callus tissue transformed with pCGP628. Genomic DNA was digested with EcoRI and probed with the 720bp EcoRV internal fragment of Hfl cDNA. Negative controls (N) are Royalty callus tissue transformed with pCGP 293. The postive control (H) contains lOpg of the Hfl fragment. The arrows indicate the 2kb EcoRI fragment expected in transformed plants.

Figure 9 shows a photograph of an autoradiographic representation of a Southern hybridization of Chrysanthemum cv. Blue Ridge plants, transformed with pCGP484. Genomic DNA was digested with Xbal, which releases a 2.3kb Hfl-PLTP fragment, and probed with a 1.8kb FspI/BspHI fragment released from pCGP602, containing the Hfl cDNA. Negative control (N) is genomic DNA isolated from non-transformed Blue Ridge plants. The postive control (P) is plasmid DNA of pCGP485 digested with Xbal. The arrow indicates the 2.3kb product expected in transformed plants.

EXAMPLE 1

Materials Eriodictyol and dihydroquercetin were obtained from Carl Roth KG and naringenin was obtained from Sigma. Dihydromyricetin was chemically synthesized from

3 myricetin (Extra Synthese, France) by the method of Vercruysse et al. (1985). [ H]- naringenin (5.7 Ci/mmole) and [ 3 H]-di .hydroquercetin (12.4 Ci/mmole) were obtained from Amersham. All enzymes were obtained from commercial sources and used according to the manufacturer' s recommendations.

The Escheήchia coli strain used was:

DH5σ supE44, ΔQacZYA-Ar F)U169, tf>80lacZΔM15, hsdR17 (rj^-, ^+), recAl, endAl, gyrA96, thi-1. relAl, deoR. (Hanahan, 1983 and BRL, 1986).

The disarmed Agrobacterium tumefaciens strains AGLO (Lazo et al, 1991) and LBA4404 (Hoekema et al, 1983) were obtained from Dr R Ludwig, Department of Biology, University of California, Santa Cruz, USA and Calgene, Inc. CA, USA, respectively.

The armed Agrobacterium tumefaciens strain ICMP 8317 was obtained from Dr Richard Gardner, Centre for Gene Technology, Department of Cellular and Molecular Biology, University of Auckland, New Zealand.

The cloning vector pBluescript was obtained from Stratagene.

Plants were grown in specialised growth rooms with a 14 hr day length at a light intensity of 10,000 lux minimum and a temperature of 22 to 26. EXAMPLE 2

Construction of pCGP 90

Plasmid pCGP90 was constructed by cloning the cDNA insert from pCGP602 (International Patent Application PCT/AU92/00334; Publication Number WO

93/01290) in a sense orientation behind the Mac promoter (Comai et al, 1990) of pCGP293.

The binary expression vector pCGP293 was derived from the Ti binary vector ρCGN1559 (McBride and Summerfek, 1990). Plasmid pCGN1559 was digested with Kpnl and the overhanging 3' ends were removed with T4 DNA poiymerase according to standard protocols (Sambrook et Λ/.,1989). The vector was then further digested with Xbal and the resulting 5' overhang was repaired using the Klenow fragment of DNA poiymerase I. The vector was then re-ligated to give pCGP67. A 1.97 kb PstI fragment containing the Mac promoter, mas terminator and various cloning sites (Comai et al, 1990) was isolated from pCGP40 and inserted into the Pstl site of pCGP67 to give pCGP293.

Plasmid pCGP40 was constructed by removing the GUS gene (Jefferson et al, 1987) as a BamHI-SacI fragment from pCGN7334 and replacing it with the BamHl-SacI fragment from pBluescribe M13 that includes the multicloning site. Plasmid pCGN7334 (obtained from Calgene, Inc. CA, USA), was constructed by inserting the fragment containing the chimaeric Mac-GUS-mas gene into the Xhol site of pCGN7329 (Comai et al, 1990).

The BamHI-Kpnl fragment containing the above-mentioned cDNA insert was then isolated from pCGP602 and ligated with a BamHI/Kpnl fragment of pCGP293. Correct insertion of the insert in pCGP90 was established by restriction analysis of DNA isolated from gentamycin resistant transformants. EXAMPLE 3

Construction of pCGP 812

The binary expression vector pCGP812 was derived from the Ti binary vector pCGN1558 (McBride and Summerfek, 1990). A 5.2 kb Xhol fragment containing the chimaeric mas-35S-GUS-ocs gene was isolated from pKIWI 101 Qannsen and Gardner, 1989) and sub-cloned into the Xhol site of pBluescript KS to give pCGP82. The 5.2 kb fragment was then re-isolated by Hindlll/Kpnl digestion and sub-cloned into the Hindlll/Kpnl sites of pCGN1558 to give pCGP83.

Plasmid pCGP83 was restricted with Kpnl and the overhanging 3' ends were removed with T4 DNA poiymerase according to standard protocols (Sambrook et al.,19S9). A Smal-BamHI adaptor (Pharmacia) was then ligated to the flushed Kpnl sites to give BamHI "sticky" ends. A 3.8 kb Bglll fragment containing the chimaeric Mzc-Hfl-mas gene from pCGP807 (described below) was ligated with the BamHI "sticky" ends of pCGP83 to yield pCGP812 (Figure 2).

The plasmid pCGP807 was constructed by ligating the 1.8 kb BamHt-Kpnl fragment containing the above-mentioned Hfl cDNA insert from pCGP602 with BamHI-Kpnl ends of pCGP40.

EXAMPLE 4 Construction of pCGP 485

The binary vector pCGP485 was derived from the Ti binary vector pCGN1547 (McBride and Summerfek, 1990). A chimaeric gene was constructed consisting of (i) the promoter sequence from a CHS gene of snapdragon; (ii) the coding region of the above-mentioned cDNA insert from pCGP602 from petunia, and (iii) a petunia phospholipid transferase protein (PLTP) terminator sequence. The CHS promoter consists of a 1.2 kb gene fragment 5' of the site of translation initiation (Sommer and Saedler, 1986). The petunia cDNA insert consists of a 1.6 kb Bcll/Fspl fragment from the cDNA clone of pCGP602 (International Patent Application PCT/AU92/00334; Publication Number WO 93/01290). The PLTP terminator sequence consists of a 0.7 kb Smal/Xhol fragment from pCGP13Δ Bam (Holton, 1992), which includes a 150 bp untranslated region of the transcribed region of the PLTP gene. The chimaeric CHS/cDNA insert/PLTP gene was cloned into the PstI site of pCGN1547 to create pCGP485.

EXAMPLE S Construction of pCGP 628 Plasmid ρCGP176 (International Patent Application PCT/AU92/00334; Publication Number WO 93/01290) was digested with EcoRI and Sgel. The digested DNA was filled in with Klenow fragment according to standard protocols (Sambrook et -*/.,1989), and self-ligated. The plasmid thereby obtained was designated pCGP627. An Xbal/Kpnl digest of pCGP627 yielded a 1.8 kb fragment which was ligated with a 14.5 kb fragment obtained by Xbal/Kpnl digestion of pCGP293. The plasmid thus created was designated pCGP628.

EXAMPLE 6 Construction of pCGP 653 Plasmid pCGP293 (described above in Example 2) was digested with Xbal and the resulting 5' overhang was filled in using Klenow fragment according to standard protocols (Sambrook et -*/.,1989). It was then digested with Hind lI. During this procedure, the Mac promoter (Comai et al, 1990) was deleted. A 0.8 kb petunia CHS- A promoter from pCGP669 (described below) was ligated into this backbone as a blunt-ended EcoRI/Hindlll fragment. This plasmid product was designated pCGP672.

An Xbal/Asp718 digestion of pCGP807 (described in Example 3, above) yielded a 1.8 kb fragment containing the Hfl cDNA, which was ligated with a 16.2 kb Xbal/Asp718 fragment from pCGP672. The plasmid thus created was designated pCGP653.

A promoter fragment of the CHS-A gene was amplified by PCR, using the oligonucleotides CHSA-782 and CHSA+34 as primers (see sequences below) and

Petunia hybrida V30 genomic DNA as template. The PCR product was cloned into ddT-tailed pBluescript (Holton and Graham, 1991) and the orientation of the gene fragment verified by restriction enzyme mapping. The plasmid thus created was designated pCGP669. The oligonucleotide primers were designed to the published sequence of the petunia CHS-A promoter (Koes, 1988).

CHSA-782

5' GTTTTCCAAATCTTGACGTG 3'

CHSA+34

5' ACGTGACAAGTGTAAGTATC 3'

EXAMPLE 7

Construction of pCGP 484

Construction of pCGP484 was identical to that for pCGP485, outlined above in Example 4, except that pCGP484 contains the 3.5 kb PstI fragment (containing the chimaeric gene CHS-Hfl-VLTP) in the opposite orientation.

EXAMPLE 8

Construction of pCGP 1458

The plasmid pCGPl458 was contructed using the 10 kb binary vector pBIN19 (Bevan,

1984) as the backbone. Plasmid pBIN19 was digested with EcoRI and the resulting 5' overhang was filled in using Klenow fragment, according to standard protocols (Sambrook et al, 19S9). Plasmid pCGP485 was digested with PstI to remove the chimaeric CHS/cDNA insert/PLTP gene as a 3.5 kb fragment. The 3' overhang resulting from PstI digestion was removed with T4 DNA poiymerase and this fragment was then ligated into the filled in EcoRI site of the plasmid pBIN19.

EXAMPLE 9

Transformation of E. coli and A. tumefaciens

Transformation of the Escherichia coli strain DH5σ-cells with one or other of the vectors pCGP812, pCGP90, pCGP485, pCGP628, pCGP653, pCGP484 or pCGPl458 was performed according to standard procedures (Sambrook et al, 1989) or Inoue et al, (1990). The plasmid pCGP812, pCGP90, pCGP485, pCGP628, pCGP653, pCGP484 or pCGP1458 was introduced into the appropriate Agrobacterium tumefaciens strain by adding 5 μg of plasmid DNA to 100 μ of competent Agrobacterium tumefaciens cells prepared by inoculating a 50 mL MG/L (Garfinkel and Nester, 1980) culture and growing for 16 h with shaking at 28. The cells were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM CaCl2/15% (v/v) glycerol. The ONA-Agrobacterium mixture was frozen by incubation in liquid N2 for 2 min and then allowed to thaw by incubation at 37 for 5 min. The DNA/bacterial mixture was then placed on ice for a further 10 min. The cells were then mixed with 1 mL of MG/L media and incubated with shaking for 16 h at 28. Cells of A. tumefaciens carrying either pCGP812, pCGP90, pCGP485, pCGP628, pCGP653 or pCGP484 were selected on MG/L agar plates containing 100 /g/mL gentamycin. Cells of A. tumefaciens carrying pCGP1458 were selected on MG/L agar plates containing 100 μg/mL kanamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the gentamycin-resistant transformants.

EXAMPLE 10 Transformation of Dianthus caryophyllus a. Plant Material Dianthus caryophyllus, (cv. Crowley Sim, Red Sim, Laguna) cuttings were obtained from Van Wyk and Son Flower Supply, Victoria, Australia. The outer leaves were removed and the cuttings were sterilized briefly in 70% (v/v) ethanol followed by 1.25% (w/v) sodium hypochlorite (with Tween 20) for 6 minutes and rinsed three times with sterile water. All the visible leaves and axillary buds were removed under the dissecting microscope before co-cultivation.

b. Co-cultivation of Agrobacterium and Dianthus Tissue

Agrobacterium tumefaciens strain AGL0 (Lazo et al, 1991), containing any one of the binary vectors pCGP90, pCGP812, pCGP485 or pCGP653, was maintained at 4 on MG/L (Garfinkel and Nester, 1980) agar plates with 100 mg/L gentamycin. A single colony was grown overnight in liquid MG/L broth and diluted to 5 x 10 8 cells/mL next day before inoculation. Dianthus tissue was co-cultivated with Agrobacterium on Murashige and Skoog' s (1962) medium (MS) supplemented with 3% sucrose (w/v), 5 mg/L σ-naphthalene acetic acid (NAA), 20 μM acetosyringone and 0.8% Difco Bacto Agar (pH 5.7).

c. Recovery of Transgenic Dianthus Plants

Co-cultivated tissue was transferred to MS medium supplemented with 1 mg/L benzylaminopurine (BAP), 0.1 mg/L NAA, 150 mg/L kanamycin, 500 mg/L ticarcillin and 0.8% Difco Bacto Agar (selection medium). After three weeks, explants were transferred to fresh selection medium and care was taken at this stage to remove axillary shoots from stem explants. After 6-8 weeks on seleαion medium healthy adventitious shoots were transferred to hormone free MS medium containing 3% sucrose, 150 mg/L kanamycin, 500 mg/L ticarcillin, 0.8% Difco Bacto Agar. At this stage GUS histochemical assay (Jefferson, 1987) and/or NPT II dot-blot assay (McDonnell et al, 1987) was used to identify transgenic shoots. Transgenic shoots were transferred to MS medium supplemented with 3% sucrose, 500 mg/L ticarcillin and 0.4% (w/v) Gelrite Gellan Gum (Schweizerhall) for root induction. All cultures were maintained under a 16 hour photoperiod (120 μE cool white fluorescent light) at 23 ± 2. When plants were rooted and reached 4-6 cm tall they were acclimatised under mist. A mix containing a high ratio of perlite (75% or greater) soaked in hydroponic mix (Kandreck and Black, 1984) was used for acclimation, which typically lasts 4-5 weeks. Plants were acclimatised at 23°C under a 14 hour photoperiod (200 /E mercury halide light).

EXAMPLE 11 Transformation of Rosa hybrida

1. Rosa hybrida cv Royalty

Plant tissues of the rose cukivar Royalty were transformed according to the method disclosed in PCT 91/04412, having publication number WO92/00371.

2. Rosa hybrida cv Kardinal a. Plant Material

Kardinal shoots were obtained from Van Wyk and Son Flower Supply, Victoria, Australia. Leaves were removed and the remaining shoots (5-6 cm) were sterilized in 1.25 % (w/v) sodium hypochlorite (with Tween 20) for 5 minutes followed by three rinses with sterile water. Isolated shoot tips were soaked in sterile water for 1 hour and precultured for 2 days on MS medium containing 3% sucrose, 0.1 mg/L BAP, 0.1 mg/L kinetin, 0.2 mg/L Gibberellic acid, 0.5% (w/v) polyvinyl pyrrolidone and 0.25% Gelrite Gellan Gum, before co-cultivation.

b. Co-cultivation of Agrobacterium and Rosa shoot Tissue

Agrobacterium tumefaciens strains ICMP 8317 anssen and Gardner 1989) and AGL0, containing the binary vector pCGP812, was maintained at 4°C on MG/L agar plates with 100 mg/L gentamycin. A single colony from each Agrobacterium strain was grown overnight in liquid MG/L broth. A final concentration of 5 x 10 8 cells/mL was prepared the next day by dilution in liquid MG/L. Before inoculation, the two

Agrobacterium cultures were mixed in a ratio of 10:1 (AGL0/pCGP812 : 8317/pCGP812). A longitudinal cut was made through the shoot tip and an aliquot of 2 μ\ of the mixed Agrobacterium cultures was placed as a drop on the shoot tip.

The shoot tips were co-cultivated for 5 days on the same medium used for preculture.

Agrobacterium tumefaciens strain AGL0, containing the binary vector pCGPl458, was maintained at 4°C on MG/L agar plates with 100 mg/L kanamycin. A single colony from each Agrobacterium strain was grown overnight in liquid MG/L broth. A final

8 concentration of 5 x 10 cells/mL was prepared the next day by dilution in liquid

MG/L.

c. Recovery of Transgenic Rosa Plants

After co-cultivation, the shoot tips were transferred to selection medium. Shoot tips were transferred to fresh selection medium every 3-4 weeks. Galls observed on the shoot tips were excised when they reached 6-8 mm in diameter. Isolated galls were transferred to MS medium containing 3% sucrose, 25 mg/L kanamycin, 250 mg/L cefotaxime and 0.25% Gelrite Gellan Gum for shoot formation. Shoots regenerated from gall tissue were isolated and transferred to selection medium. GUS histochemical assay and callus assay were used to identify transgenic shoots. Transgenic shoots were transferred to MS medium containing 3% sucrose, 200 mg/L cefotaxime and 0.25% Gelrite Gellan Gum for root induction. All cultures were maintained under 16 hour photoperiod (60 μE cool white fluorescent light) at 23 ± 2. When the root system was well developed and the shoot reached 5-7 cm in length the transgenic rose plants were transferred to autoclaved Debco 514110/2 potting mix in 8 cm tubes. After 2-3 weeks plants were replanted into 15 cm pots using the same potting mix and maintained at 23 under a 14 hour photoperiod (300 μE mercury halide light). After 1-2 weeks potted plants were moved to glasshouse (Da /Night temperature : 25-28/14) and grown to flowering.

EXAMPLE 12 Transformation of Chrysanthemum morifolium a. Plant Material

Chrysanthemum morifolium (cv. Blue Ridge, Pennine Chorus) cuttings were obtained from F & I Baguley Flower and Plant Growers, Victoria, Australia. Leaves were removed from the cuttings, which were then sterilized briefly in 70% (v/v) ethanol followed by 1.25% (w/v) sodium hypochlorite (with Tween 20) for 3 minutes and rinsed three times with sterile water. Internodal stem sections were used for co- cultivation.

b. Co-cultivation of Agrobacterium and Chrysanthemum Tissue Agrobacterium tumefaciens strain LBA4404 (Hoekema et al, 1983), containing any one of the binary vectors pCGP90, pCGP484, ρCGP485 or pCGP628, was grown on MG/L agar plates containing 50 mg/L rifampicin and 10 mg/L gentamycin. A single colony from each Agrobacterium was grown overnight in the same liquid medium. These liquid cultures were made 10% with glycerol and 1 mL aliquots transferred to the freezer (-80). A 100-200μl aliquot of each frozen Agrobacterium was grown overnight in liquid MG/L containing 50 mg/L rifampicin and 10 mg/L gentamycin.

8 A final concentration of 5 x 10 cells/mL was prepared the next day by dilution in liquid MS containing 3% (w/v) sucrose. Stem seαions were co-cultivated, with

Agrobacterium containing any one of LBA4404/pCGP90, LBA4404/pCGP484,

LBA4404/pCGP485 or LBA4404/pCGP628, on co-cultivation medium for 4 days. c. Recovery of Transgenic Chrysanthemum Plants

After co-cultivation, the stem seαions were transferred to seleαion medium. After 3-4 weeks, regenerating explants were transferred to fresh medium. Adventitious shoots which survived the kanamycin seleαion were isolated and transferred to MS medium containing kanamycin and cefotaxime for shoot elongation and root induαion. All cultures were maintained under a 16 hour photoperiod (80 μE cool white fluorescent light) at 23 ± 2°C. Leaf samples were colleαed from plants which rooted on kanamycin and Southern blot analysis was used to identify transgenic plants. When transgenic chrysanthemum plants reached 4-5 cm in length they were transferred to autoclaved Debco 51410/2 potting mix in 8 cm tubes. After 2 weeks plants were replanted into 15 cm pots using the same potting mix and maintained at 23°C under a 14 hour photoperiod (300 μE mercury halide light). After 2 weeks potted plants were moved to glasshouse (Day/Night temperature : 25-28°C/14°C) and grown to flowering.

EXAMPLE 13 Southern Analysis a. Isolation of Genomic DNA from Dianthus

DNA was isolated from tissue essentially as described by Dellaporta et al, (1983). The DNA preparations were further purified by CsCl buoyant density centrifugation (Sambrook et al, 1989).

b. Isolation of Genomic DNA from Chrysanthemum

DNA was isolated from leaf tissue using an extraαion buffer containing 4.5 M guanidinium thiocyanate, 50 mM EDTA pH 8.0, 25 mM sodium citrate pH 7.0,

0.1 M 2-mercaptoethanol, 2% (v/v) lauryl sarcosine. The plant tissue was ground to a fine powder in liquid N following which extraαion buffer was added (5 mL/g of tissue) and the solution mixed on a rotating wheel for 16 h. The mixture was then phenol: chloroform: isoamylalcohol (50:49:1) extraαed twice and the genomic DNA precipitated by adding three volumes of ethanol and centrifuging for 15 min at 10,000 rpm. c. Isolation of Genomic DNA from Rosa

DNA was extraαed by grinding tissue in the presence of liquid N2 in a mortar and pestle and adding 1ml of extraαion buffer (0.14 M sorbitol, 0.22 M Tris-HCl [ρH8.0], 0.022 M EDTA, 0.8 M NaCl, 0.8% (w/v) CTAB, l%N-laurylsarcosine) heated at 65°C. Chloroform (200μl) was added and the mixture incubated at 65°C for 15 min. Following centrifugation, the supernatant was phenol-chloroform extraαed and then added to an equal volume of isopropanol, inverting to mix. This mixture was centrifuged and the pellet washed with 95% ethanol, re-centrifuged and washed with 70% ethanol. The pellet was vacuum-dried and resuspended in 30μl TE buffer (pH 8.0).

d. Southern Blots

The genomic DNA (10 μg) was digested for 16 hours with 60 units of EcoRI and eleαrophoresed through a 0.7% (w/v) agarose gel in a running buffer of TAE (40 mM Tris-acetate, 50 mM EDTA). The DNA was then denatured in denaturing solution (1.5 M NaCl/0.5 M NaOH) for 1 to 1.5 hours, neutralized in 0.5 M Tris-HCl (pH 7.5)/ 1.5 M NaCl for 2 to 3 hours and the DNA was then transferred to a Hybond N (Amersham) filter in 20 x SSC.

Southern analysis of putative transgenic Dianthus, Rosa and Chrysanthemum plants obtained after seleαion on kanamycin confirmed the integration of the appropriate chimaeric gene into the genome.

EXAMPLE 14

Northern Analysis a. Dianthus and Chrysanthemum RNA

Total RNA was isolated from tissue that had been frozen in liquid N2 and ground to a fine powder using a mortar and pestle. An extraαion buffer of 4 M guanidinium isothiocyanate, 50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.1% (v/v) Sarkosyl, was added to the tissue and the mixture was homogenized for 1 minute using a polytron at maximum speed. The suspension was filtered through Miracloth (Calbiochem) and centrifuged in a JA20 rotor for 10 minutes at 10,000 rpm. The supernatant was colleαed and made to 0.2 g/ mL CsCl (w/v). Samples were then layered over a 10 mL cushion of 5.7 M CsCl, 50 mM EDTA (pH 7.0) in 38.5 mL Quick-seal centrifuge tubes (Beckman) and centrifuged at 42,000 rpm for 12-16 hours at 23 in a Ti-70 rotor. Pellets were resuspended in TE/SDS (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% (w/v) SDS) and extraαed with phenol:chloroform:isoamyl alcohol (25:24:1) saturated in 10 mM EDTA (pH 7.5). Following ethanol precipitation the RNA pellets were resuspended in TE/SDS.

RNA samples were eleαrophoresed through 2.2 M formaldehyde/ 1.2% (w/v) agarose gels using running buffer containing 40 mM morpholinopropanesulphonic acid (pH

7.0), 5 mM sodium acetate, 0.1 mM EDTA (pH 8.0). The RNA was transferred to Hybond-N fikers (Amersham) as described by the manufaαurer and probed with 32 P-

8 6 labelled cDNA fragment (10 cpm/μg, 2 x 10 cpm/mL). Prehybridization (1 h at 42°C) and hybridization (16 h at 42°C) was carried out in 50% (v/v) formamide, 1 M

NaCl, 1% (w/v) SDS, 10% (w/v) dextran sulphate. Degraded salmon sperm DNA (100

32 μg/mL) was added with the P-labelled probe for the hybridization step.

Fikers were washed in 2 x SSC/ 1% (w/v) SDS at 65°C for 1 to 2 hours and then 0.2 x SSC/ 1% (w/v) SDS at 65°C for 0.5 to 1 hour. Filters were exposed to Kodak XAR film with an intensifying screen at -70 for 48 hours.

Northern analysis of Dianthus cv. Red Sim transformed with plasmid pCGP90 indicated that eight of thirteen plants were positive.

b. Rosa RNA

Total RNA was extraαed from petals (buds and of flowers 5 days post-harvest) according to the method of Manning, 1991. EXAMPLE 15

³²P-Labelling of DNA Probes DNA fragments (50 to 100 ng) were radioaαively labelled with 50 μCi of [σ- 32 P]- dCTP using an oligolabelling kit (Bresatec). Unincorporated [a- 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

EXAMPLE 16

Anthocyanidin Analysis Prior to HPLC analysis the anthocyanin molecules present in petal extraαs were acid hydrolysed to remove glycosyl moieties from the anthocyanidin core. The hydroxylation pattern on the B ring of the anthocyanin pigments was determined by HPLC analysis of the anthocyanidin core molecule. The HPLC system used in this analysis was a Hewlett-Packard 1050 equipped with a multiwavelength deteαor (MWD). Reversed phase chromatographic separations were performed on a Spherisorb S5 ODS2 cartridge column, 250 mm x 4 mm ID.

a. Extraction of anthocyanins and flavonoids

Flower pigments were extraαed from petal segments (ca. 50 mg) with 5 ml of methanol containing 1% (v/v) of aqueous 6M hydrochloric acid. Extraαs were diluted with water (1:9) and filtered (Millex HV, 0.45μ) prior to injeαion into the HPLC system.

b. Hydrolysis of anthocyanins Crude methanolic extraαs (100 μL) obtained in a. above were evaporated to dryness in Pierce Reaαi- Vials using a stream of dry nitrogen at room temperature. The residues were dissolved in 200μL 2M HCl, vials were capped and then heated at 100°C for 30 minutes. Hydrolysis mixtures were diluted with water (1:9) and filtered (Millex HV, 0.45μ) prior to HPLC analysis. c. Chromatography

Separation of flower pigments was effeαed via gradient elution using the following system: Solvent A: (triethylamine: cone. H3PO4 : H₂0) (3:2.5:1000)

Solvent B: acetonitrile

Gradient Conditions: 5% B to 40% B over 20 minutes Flow Rate: 1 ml/min Temperature: 35°C Deteαion: MWD with simultaneous data acquisition at 280, 350 and 546nm.

The anthocyanidin peaks were identified by reference to known standards. An alternative method for the analysis of anthocyanin molecules present in petal extraαs is to be found in Brugliera et al, 1994.

HPLC analysis is conduαed to determine the presence of delphinidin, pelargonidin and cyanidin pigments in samples of carnation, chrysanthemum and rose tissues having been transformed with one or other of the plasmids pCGP90, pCGP485, pCGP484, pCGP628, pCGP653 or pCGP1458. Representative data of pCGP90, pCGP485 and pCGP653 in transgenic carnation flowers are shown in Table 1.

TABLE 1

HPLC Analysis of ρCGP90, ρCGP485 and pCGP653 Transgenic Flowers

Sample % D 'elphinidin % Pe largonidin % Cvanidin

NON-TRANSGENIC CARNATION:

Cultivar: Red Sim 0 85.3 0.8

TRANSGENIC CARNATION:

Red Sim + pCGP90

(i) Ace #* 1933 1.9 82.7 nd**

(ii) Ace # 2011 3.7 76.9 nd

Red Sim + pCGP485

(i) Ace # 3654B 13.0 75.1 2.3

Red Sim + pCGP653

(i) Ace # 3660/2 18.1 71.4 3.2

(ii) Ace # 3655 35.6 49.1 7.5

* Ace # = plant accession number ** nd = not deteαed

EXAMPLE 17 Preparation of Plant Extracts for Assay of 3' ,5' -Hydroxylase Activity

Plant tissue was homogenised in a 10 times volume of ice-cold extraαion buffer (100 mM potassium phosphate (pH 7.5), 1 mM EDTA, 0.25 M sucrose, 0.25 M mannitol, 0.1% (w/v) BSA, 0.1 mg/mL PMSF, 20 mM 2-mercaρtoethanol and 10 mg/mL polyclar AT). The homogenate was centrifuged at 13,000 rpm in a JA20 rotor (Beckman) for 10 min at 4°C and an aliquot of the supernatant assayed for 3' , 5' - hydroxylase aαivity. 3' ,5' -Hydroxylase Assay

3 ' ,5 ' -Hydroxylase enzyme aαivity was measured using a modified version of the method described by Stotz and Forkmann (1982). The assay reaαion mixture typically contained 195 μL of plant extraα, 5μL of 50 mM NADPH in assay buffer (100 mM potassium phosphate (pH8.0), 1 mM EDTA and 20 mM 2-mercaptoethanol), and 10

1 Λ dpm [ C] naringenin in a final volume of 200 μL. Following incubation at 23 overnight, the reaαion mixture was extraαed twice with 0.5 mL of ethylacetate. The ethyl acetate phase was dried under vacuum and then resuspended in 10 μL of ethyl acetate. The radio-labelled fiavonoid molecules were then separated on cellulose thin layer plates (Merck Art 5577, Germany) using a chloroform: acetic acid: water (10:9:1, v/v) solvent system. At the completion of the chromatography, the TLC plates were air-dried and the reaαion produαs localised by autoradiography and identified by comparison to non-radioaαive naringenin, eriodiαyol, dihydroquercetin and dihydromyricetin standards which were run alongside the reaαion produαs and visualized under UV light.

EXAMPLE 18

Transformation of various cultivars The chimaeric genes contained in any one of the construαs pCGP90, pCGP812, pCGP628, pCGP485, pCGP653, pCGP484 or pCGP1458 is introduced into plant varieties of rose, carnation and chrysanthemum using Agrobacterium-meάia.teά gene transfer, as described in Examples 10, 11 and 12. Integration of the appropriate chimaeric gene into the plant genome is confirmed by Southern analysis of plants obtained after kanamycin seleαion and HPLC analysis is used to deteα the presence of anthocyanins as described in Example 16, above.

Plants successfully rendered transgenic and which are able to express the transgene in accordance with the present invention, have significant levels of 3 ' ,5 '-hydroxylase enzyme aαivity in addition to 3' ,5' -hydroxylated anthocyanins (seen in Example 16), compared with non-transgenic controls which do not contain the gene necessary for the produαion of 3 ' ,5' -hydroxylase aαivity. EXAMPLE 19

Carnation cv. Crowley Sim + pCGP 90

The plasmid pCGP90 was introduced into the carnation cukivar Crowley Sim using Agrobacterium-meάi^→sxeά gene transfer, as described in Example 10. Integration of the construα in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleαion. Nine plants were examined for the presence of the nptll and Hfl genes and for the produαion of delphinidin. Eight of the nine plants analyzed were positive for both nptll and Hfl but HPLC analysis was unable to deteα any evidence of delphinidin produαion by these plants (see Table 2; "Kan" = kanamycin).

Table 2

# Acc# Kan Hfl Delphinidin

1 1930A + +

2 1942B + +

3 2008B

4 2217A + +

5 2217B + +

6 2338A + +

7 2338B + +

8 2338C + +

9 2338D + +

EXAMPLE 20 Carnation cv. Laguna + pCGP 485

The plasmid pCGP485 was introduced into the carnation cukivar Laguna using Agrobacterium-meάiateά gene transfer, as described in Example 10. Integration of the construα in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleαion. HPLC analysis of the anthocyanin molecules present in petal extraαs is carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives. These 3' , 5' - hydroxylated anthocyanins are only produced as a result of the expression of the exogenous DNA sequence, ie: the Hfl cDNA sequence, introduced via transformation with the binary veαor pCGP485.

EXAMPLE 21 Rose cv. Royalty + pCGP 485/ρCGP 628

The plasmids pCGP485 and pCGP628 were introduced into the rose cukivar Royalty using Agrobacterium-mediateά gene transfer, as referred to in Example 11. Integration of the construα in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleαion. HPLC analysis of the anthocyanin molecules present in petal extraαs is again carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives. These 3 ' ,5 ' -hydroxylated anthocyanins are only produced as a result of the expression of the exogenous DNA sequence, ie: the Hfl cDNA sequence, introduced via transformation with either of the binary veαors pCGP485 or pCGP628.

EXAMPLE 22 Rose cv. Kardinal + pCGP 1458

The plasmid pCGP1458 was introduced into the rose cukivar Kardinal using Agrobacterium-mediateά gene transfer, as described in Example 11. Integration of the construα in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleαion. HPLC analysis of the anthocyanin molecules present in petal extraαs is again carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives. These 3' ,5' -hydroxylated anthocyanins are only produced as a result of the expression of the exogenous DNA sequence, ie: the Hfl cDNA sequence, introduced via transformation with the binary veαor pCGP1458.

EXAMPLE 23 Chrysanthemum cv. BlueRidge + pCGP 484/ρCGP 485/ρCGP 628

The plasmids pCGP484, pCGP485 and pCGP628 were introduced into the chrysanthemum cukivar BlueRidge using Agrobacterium-meάisteά gene transfer, as described in Example 12. Integration of the construα in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleαion. HPLC analysis of the anthocyanin molecules present in petal extraαs is again carried out according to the procedure set out in Example 16, above, to show the presence of 3' ,5' -hydroxylated anthocyanin derivatives. These 3' ,5' -hydroxylated anthocyanins are only produced as a result of the expression of the exogenous DNA sequence, ie: the Hfl cDNA sequence, introduced via transformation with any one of the binary veαors pCGP484, pCGP485 or _PCGP628.

EXAMPLE 24

Altered Inflorescence

The expression of the introduced fiavonoid 3' ,5' -hydroxylase enzyme aαivity in the transgenic plant is capable of having a marked effeα on flower colour. Floral tissues in transgenic plants may change from the pale pinks and reds of the non-transgenic control plants to colours ranging from a darker pink maroon to a blue/purple colour. The colours may also be described in terms of numbers from the Royal Horticultural Society' s Colour Chart. In general, the changes can be described as moving the colour from the pale-to-mid pink hues of 60C/D - 65C/D, to the darker bluer/purpler hues represented by many, but not all, of the colour squares between 70 and 85. It should be remembered that other biochemical and physiological conditions will affeα the individual outcome and the citing of specific colours should not be interpreted as defining the possible range.

In the case of the transgenic carnation flower, Accession Number 3655, produced using the plasmid construα pCGP653 described above, an obvious bluing effeα on the petals was observed. The normally-orange-red colour of Red Sim carnation cukivars (corresponding approximately to 45A/B of the Royal Horticultural Society' s Colour Chart) had changed to a blue/purple hue. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or colleαively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. A transgenic plant or its progeny seleαed from rose, carnation and chrysanthemum wherein said plant produces a polypeptide having flavonoid 3' ,5' -hydroxylase aαivity and produces higher levels of anthocyanins derived from delphinidin relative to non- transgenic plants of the same respeαive species.

2. A transgenic plant according to claim 1 wherein the polypeptide is of petunia, verbena, delphinium, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg plant, lisianthus or campanula origin.

3. A transgenic plant according to claim 2 wherein the polypeptide is a 3' , 5' hydroxylase of petunia origin.

4. A transgenic plant according to claim 2 wherein the polypeptide is a 3 ' ,5' hydroxylase of lisianthus origin.

5. A transgenic plant according to claim 3 wherein the polypeptide is a 3' , 5' - hydroxylase encoded by a genetic sequence contained in a plasmid seleαed from pCGP484, pCGP485, pCGP628, pCGP653 and pCGP1458.

6. A transgenic plant according to claim 3 or 4 or 5 wherein said plant is a rose.

7. A transgenic plant according to claim 3 or 4 or 5 wherein said plant is a carnation.

8. A transgenic plant according to claim 3 or 4 or 5 wherein said plant is a chrysanthemum.

9. A transgenic plant according to claim 1 exhibiting altered inflorescence.

10. A transgenic plant according to claim 6 exhibiting altered inflorescence.

11. A transgenic plant according to claim 7 exhibiting altered inflorescence.

12. A transgenic plant according to claim 8 exhibiting altered inflorescence.

13. A method for producing a transgenic plant seleαed from rose, carnation and chrysanthemum, said method comprising introducing into said plant a gene construα containing a nucleic acid sequence encoding a flavonoid 3' ,5' -hydroxylase charaαerised in that said transgenic plant produces higher levels of an anthocyanidin derivative of anthocyanins derived from delphinidin relative to non-transgenic plants of the same respeαive species.

14. A method according to claim 13 wherein the 3 ' ,5 ' -hydroxylase is of petunia, verbena, delphinium, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg plant, lisianthus or campanula origin.

15. A method according to claim 14 wherein the 3 ' ,5 ' -hydroxylase is of petunia origin.

16. A method according to claim 14 wherein the 3 ' ,5 ' -hydroxylase is of lisianthus origin.

17. A method according to claim 15 wherein the 3' ,5' -hydroxylase is encoded by a genetic sequence contained in a plasmid seleαed from pCGP484, pCGP485, pCGP628, pCGP653 and pCGPl458.

18. A method according to claim 15 or 16 or 17 wherein said plant is a rose.

19. A method according to claim 15 or 16 or 17 wherein said plant is a carnation.

20. A method according to claim 15 or 16 or 17 wherein said plant is a chrysanthemum.

21. A method according to claim 13 wherein said transgenic plant exhibits altered inflorescence.

22. A method according to claim 18 wherein said transgenic plant exhibits altered inflorescence.

23. A method according to claim 19 wherein said transgenic plant exhibits altered inflorescence.

24. A method according to claim 20 wherein said transgenic plant exhibits altered inflorescence.

25. A binary veαor comprising a gene construα which is capable of being integrated into a plant genome to produce the transgenic plant according to claim 1.

26. A binary veαor according to claim 25 wherein the gene construα is a chimaeric gene construα.

27. A binary veαor according to claim 25 wherein the gene construα comprises a plant promoter.

28. A binary veαor according to claim 27 seleαed from pCGP484, pCGP485, pCGP653 and pCGP1458.

29. A binary veαor according to claim 25 or 26 wherein the veαor is pCGP628.