NZ266401A

NZ266401A - Transgenic flowering plants modified to enable expression of flavonoid 3'5' hydroxylase

Info

Publication number: NZ266401A
Application number: NZ266401A
Authority: NZ
Inventors: Timothy Albert Holton; Yoshikazu Tanaka
Original assignee: Int Flower Dev Pty Ltd
Priority date: 1993-05-20
Filing date: 1994-05-20
Publication date: 1996-09-25
Also published as: SG45175A1; KR100337755B1; CA2163220A1; PL177743B1; EP0703982A4; PL311691A1; CN1127015A; JPH08511683A; EP0703982A1; WO1994028140A1

Description

<div class="application article clearfix" id="description"> New Zealand No. 266401 International No. PCT/AU94/00265 Priority Dota<2): Compete Specification Filed: Class: Publication Dirt®: : p.O. Journal No: i NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Transgenic flowering plants Name, address and nationality of applicant(s) as in international application form: INTERNATIONAL FLOWER DEVELOPMENTS PTY. LTD., of 16 Gipps Street, Collingwood, Victoria 3066, Australia - A VicJoriQ CorpOrc^ion- \. IR 94/28140 PCT/AU94/00265 266401 TRANSGENIC FLOWERING PLANTS The present invention relates generally to transgenic flowering plants. More particularly, 5 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. The flower industry strives to develop new and different varieties of flowering plants, 10 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 15 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. 20 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 flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, retuuidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The 25 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 flavonoid pigments (hereinafter referred to as the 30 "flavonoid 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 94/28140 PCT/AU94/00265 -2 - of three molecules of malonyl-CoA with one molecule of /7-coumaroyl-CoA. This reaction is cata'ysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2\4,4',6'-tetrahydroxychalcone, is normally rapidly isonierized to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). Naringenin is 5 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), 10 respectively. Two key enzymes involved in this pathway are flavonoid 3'-hydroxylase and flavonoid 3',5'-hyrtioxylase. The flavonoid 3'-hydroxylase acts on DHK to produce DHQ and on naringenin to produce eriodictyol. The flavonoid 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 15 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 20 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'-25 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 30 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 O Q:\OPER\EJH\266401.172 - 20/6/56 -3 - and/or its derivatives relative to non-transgenic plants of the same species. It has been determined in accordance with the present invention that genetic constructs which comprise a promoter from a gene encoding a flavonoid pathway enzyme operably linked to a flavonoid 3',5'-hydroxylase are capable of directing expression of high levels of delpkinin-derived 5 anthocyanins. 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 selected from rose, carnation and chrysanthemum or progeny thereof or cut-flowers therefrom wherein said 10 plant carries a genetic construct comprising a promoter from a gene encoding an enzyme of the flavonoid pathway operably linked to a gene encoding a flavonoid 3',5'-hydroxylase wherein said transgenic plant produces higher levels of anthocyanins derived from delphinidin relative to non-transgenic plants of the same species. 15 Preferably, the flavonoid 3',5'-hydroxylase is of petunia, verbena, delphinum, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg plant, lisianthus or campanula origin. Most preferably, the flavonoid 3',5'-hydroxylase is of petunia origin. 20 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 25 which is capable of being integrated into a plant genome to produce the transgenic plant of the present invention. The chimaeric gene construct carries a plant promoter from a gene encoding an enzyme of the flavonoid pathway. A preferred promoter is from the gene encoding chalcone synthase (CHS) and is N-Z. PATgjTOFFICEj -7 AUG 1996 RT;:- 2 6 fi k n 1PCT,AU 94/00265 Bonprtwaiw tw U t y I RECEIVED 0 4 JUL 1995 4- referred to herein as the "CHS promoter". The CHS promoter is particularly preferred since it directs tfie high level expression of genetic sequences operably linked down stream thereof. The most preferred binary vectors are pCGP484, pCGP485, pCGP653 5 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 10 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 15 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 transgenic plant exhibits one or more of the following properties: (i) production of 3',5'-hydroxylase-specific mRNA; 20 (ii) production of 3',5'-hydroxylase protein; (iii) production of delphinidin and/or its derivatives; and/or (iv) altered infloresence. 25 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-30 transgenic endogenous levels; and/or (iv) altered infloresence. N&W iiJtAi-ANO 1 5 OCT 1995 PATENT OFFICE AMENDED SHEET IPEA/AU WO 94/28140 - 5 - PCT/AU94/00265 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 5 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 0^ 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-10 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 15 DHK and/or other suitable substrates into anthocyanin 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 20 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 25 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 30 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 WO 94/28140 PCT /AU94/00265 - 6 - 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 5 introducing into said plant a gene construct containing a nucleic acid sequence encoding a flavonoid 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. 10 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 15 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 20 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 25 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. 30 The present invention is further described by reference to the following non-limiting Figures and Examples. WO 94/28140 - 7 - PCT / AU94/00265 In the Figures: Figures 1(A) and (B) are schematic representations of the biosynthesis pathway for the flavonoid pigments. Enzymes involved in the first part of the pathway have been indicated as follows: PAL = Phenylalanine ammonia-lyase; C4H = Cinnamate 4-5 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 10 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 binaiy expression vector pCGP812, contruction of which is described in Example 3. Gent = the gentamycin resistance gene; 15 LB = left border; RB = right border; nptll = the expression cassette for neomycin phosphotransferase II; GUS = the /^-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, 20 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. 25 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. 30 WO 94/28140 - 8 - PCT / ATJ94/00265 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 5 neomycin phosphotransferase H. 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 10 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 pCGPl458, 15 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. 20 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 25 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-FLT? fragment, 30 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 WO 94/28140 PCT/AU94/00265 - 9- Xbal. The arrow indicates the 2.3kb product expected in transformed plants. EXAMPLE 1 Materials 5 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}- 3 naringenin (5.7 Ci/mmole) and[ HJ-dihydroquercetin (12.4 Ci/mmole) were obtained from Amersham. All enzymes were obtained from commercial sources and used 10 according to the manufacturer's recommendations. The Escherichia coli strain used was: DH5a supE44. AflacZYA-ArgF)U169. <f>80lacZAM!5. hsdR17 (r^-, mj^+), recAl. endAl. gvrA96. thi-1. relAl. deoR. (Hanahan, 1983 and BRL, 15 1986). The disarmed Agrobacterium tu.mefaciens 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. 20 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. 25 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. WO 94/28140 PCT / AU94/00265 - 10 - EXAMPLE 2 Construction of pCGP 90 Plasmid pCGP90 was constructed by cloning the cDNA insert from pCGP602 5 (International Patent Application PCT/AU92/00334; Publication Number WO 93/01290) in a sense orientation behind the Mac promoter (Comai et al1990) of PCGP293. The binary expression vector pCGP293 was derived from the Ti binary vector 10 pCGN1559 (McBride and Summerfelt, 1990). Plasmid pCGN1559 was digested with Kpnl and the overhanging 3' ends were removed with T4 DNA polymerase according to standard protocols (Sambrook et aL, 1989). The vector was then further digested with Xbal and the resulting 5' overhang was repaired using the Klenow fragment of DNA polymerase I. The vector was then re-ligated to give pCGP67. A 1.97 kb PstI 15 fragment containing the Mac promoter, mas terminator and various cloning sites (Comai et aL, 1990) was isolated from pCGP40 and inserted into the PstI site of pCGP67 to give pCGP293. Plasmid pCGP40 was constructed by removing the GUS gene 0efferson et aL, 1987) 20 as a BamHI-SacI fragment from pCGN7334 and replac ag 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 etaL, 1990). 25 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. 30 94/28140 PCT/AU94/00265 - 11 - EXAMPLE 3 Construction of pCGP 812 The binary expression vector pCGP812 was derived from the Ti binary vector 5 pCGN1558 (McBride and Summerfelt, 1990). A 5.2 kb Xhol fragment containing the chimaeric mas-35S-GUS-ocs gene was isolated from pKIWl 101 (Jannsen 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 HindUI/Kpnl digestion and sub-cloned into the Hindm/Kpnl sites of pCGN1558 to give pCGP83. 10 Plasmid pCGP83 was restricted with Kpnl and the overhanging 3' ends were removed with T4 DNA polymerase according to standard protocols (Sambrook et <2/.,1989). A Smal-BamHl adaptor (Pharmacia) was then ligated to the flushed Kpnl sites to give BamHI "sticky" ends. A 3.8 kb BglH fragment containing the chimaeric Mac-Hfl-mas 15 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 BamHJ-Kpnl fragment containing the above-mentioned Hfl cDNA insert from pCGP602 with BamHI-Kpnl 20 ends of pCGP40. EXAMPLE 4 Construction of pCGP 485 The binary vector pCGP485 was derived from the Ti binary vector pCGNl547 25 (McBride and Summerfelt, 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 30 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 94/28140 PCT / AU94/00265 - 12 - kb Smal/Xhol fragment from pCGPl3A 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. 5 EXAMPLE 5 Construction of pCGP 628 Plasmid pCGP176 (International Patent Application PCT/AU92/00334; Publication Number WO 93/01290) was digested with EcoRI and Spel. The digested DNA was 10 filled in with Klenow fragment according to standard protocols (Sambrook etaL,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. 15 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 20 protocols (Sambrook et al,1989). It was then digested with HindUI. 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/HindlH fragment,, This plasmid product was designated pCGP672. 25 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 30 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 WO 94/28140 PCT / AU94/00265 - 13 - 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). 5 CHSA-782 5' GTTTTCCAAATCTTGACGTG 3' CHSA+34 5' ACGTGACAAGTGTAAGTATC 3' 10 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-.W/2-PLTP) in the opposite orientation. 15 EXAMPLE 8 Construction of pCGP 1458 The plasmid pCGP1458 was contracted using the 10 kb binary vector pBIN19 (Bevan, 1984) as the backbone. Plasmid pBIN19 was digested with EcoRI and the resulting 5' 20 overhang was filled in using Klenow fragment, according to standard protocols (Sambrook et al,1989). 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 polymerase and this fragment was then ligated into the filled in EcoRI site of the plasmid pBINl9. 25 EXAMPLE 9 Transformation of E. coli and A. tumcfactens Transformation of the Escherichia coli strain DH5a-cells with one or other of the vectors pCGP812, pCGP90, pCGP485, pCGP628, pCGP653, pCGP484 or pCGPl458 30 was performed according to standard procedures (Sambrook et al., 1989) or Inoue et al., (1990). WO 94/28140 PCT/AU94/00265 - 14 - The plasmid pCGP812, pCGP90, pCGP485, pCGP628, pCGP653, pCGP484 or pCGP1458 was introduced into the appropriate Agrobacterium tumefaciens strain by-adding 5 pg of plasmid DNA to 100 fjL of competent Agrobacterium tumefaciens cells prepared by inoculating a 50 mL MG/L (Garfinkel and Nester, 1980) culture and 5 growing for 16 h with shaking at 28. The cells were then pelleted and resvispended in 0.5 mL of 85% (v/v) 100 iaM CaCl2/15% (v/v) glycerol. The Jyt^K-Agrobacterium. mixture was frozen by incubation in liquid N£ 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 10 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 //g/mL kanamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the 15 gentamycin-resistant transformants. EXAMPLE 10 Transformation of Dianthus caryophyllus a. Plant Material 20 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 25 the dissecting microscope before co-cultivation. b. Co-cultivation of Agrobacterium and Dianthus Tissue Agrobacterium tumefaciens strain AGLO (Lazo et aL, 1991), containing any one of the binary vectors pCGP90, pCGP812, pCGP485 or pCGP653, was maintained at 4 on 30 MG/L (Garfinkel and Nester, 1980) agar plates with 100 mg/L gentamycin. A single 8 colony was grown overnight in liquid MG/L broth and diluted to 5 x 10 cells/mL next day before inoculation. Dianthus tissue was co-cultivated with Agrobacterium WO 94/28140 PCT/AU94/00265 - 15 - on Murashige and Skoog's (1962) medium (MS) supplemented with 3% sucrose (w/v), 5 mg/L a-naphthalene acetic acid (NAA), 20//M acetosyringone and 0.8% Difco Bacto Agar (pH 5.7). 5 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 10 axillary shoots from stem explants. After 6-8 weeks on selection 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 15 were transferred to MS medium supplemented with 3% sucrose, 500 mg/L ticarcillin and 0.4% {w/v) Gelrite Gellan Gum (Schweizerhall) for root induaion. All cultures were maintained under a 16 hour photoperiod (120 fJE 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 20 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 fjE mercury halide light). EXAMPLE 11 25 Transformation of Rosa hybrida 1. Rosa hybrida cv Royalty Plant tissues of the rose cultivar Royalty were transformed according to the method disclosed in PCT 91/04412, having publication number W092/00371. 30 2. Rosa hybrida cv Kardinal a. Plant Material Kardinal shoots were obtained from Van Wyk and Son Flower Supply, Victoria, 94/28140 PCT / AU94/00265 -16- 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 5 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 (Janssen and Gardner 1989) and AGL0, 10 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 8 grown overnight in liquid MG/L broth. A final concentration of 5 x 10 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 : 15 8317/pCGP812). A longitudinal cut was made through the shoot tip and an aliquot of 2 fA 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 AGLO, containing the binary vector pCGP1458, was 20 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. 25 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 30 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 WO 94/28140 PCT/AU94/00265 -17- 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 [iE 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 5 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 fiE mercury halide light). After 1-2 weeks potted plants were moved to glasshouse (Day/Night temperature : 25-28/14) and grown to flowering. 10 EXAMPLE 12 Transformation of Chrysanthemum morifolium a. Plant Material Chrysanthemum morifolium (cv. Blue Ridge, Pennine Chorus) cuttings were obtained 15 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. 20 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, pCGP485 or pCGP628, was grown on MG/L agar plates containing 50 mg/L rifampicin and 10 mg/L gentamycin. A single 25 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 100-200/xl aliquot of each frozen Agrobacterium was grown overnight in liquid MG/L containing 50 mg/L rifampicin and 10 mg/L gentamycin. A final concentration of 5 x 10^ cells/mL was prepared the next day by dilution in 30 liquid MS containing 3% (w/v) sucrose. Stem sections 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. WO 94/28140 PCT/AU94/00265 - 18 - c. Recovery of Transgenic Chrysanthemum Plants After co-cultivation, the stem sertions were transferred to seleaion medium. After 3-4 weeks, regenerating explants were transferred to fresh medium. Adventitious shoots which survived the kanamycin seleaion were isolated and transferred to MS medium 5 containing kanamycin and cefotaxime for shoot elongation and root induaion. All cultures were maintained under a 16 hour photoperiod (80 fiE cool white fluorescent light) at 23 ± 2°C. Leaf samples were colleaed 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 10 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 fiE mercury halide light). After 2 weeks potted plants were moved to glasshouse (Day/Night temperature : 25-28°C/14°C) and grown to flowering. 15 EXAMPLE 13 Southern Analysis a. Isolation of Genomic DNA from Dtanthus DNA was isolated from tissue essentially as described by Dellaporta et al., (1983). The 20 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 extraaion buffer containing 4.5 M 25 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 N2 following which extraaion buffer was added (5 mL/g of tissue) and the solution mixed on a rotating wheel for 16 h. The mixture. /as then phenol: chloroform: isoamylalcohol (50:49:1) extrarted twice and the genomic DNA 30 precipitated by adding three volumes of ethanol and centrifuging for 15 min at 10,000 rpm. WO 94/28140 PCT / AU94/00265 - 19 - c. Isolation of Genomic DNA from Rosa DNA was extracted by grinding tissue in the presence of liquid N2 in a mortar and pestle and adding 1ml of extraaion buffer (0.14 M sorbitol, 0.22 M Tris-HCl [pH8.0], 5 0.022 M EDTA, 0.8 M NaCl, 0.8% (w/v) CTAB, l%N-laurylsarcosine) heated at 65°C. Chloroform (200//1) was added and the mixture incubated at 65°C for 15 min. Following centrifugation, the supernatant was phenol-chloroform extracted 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 10 70% ethanol. The pellet was vacuum-dried and resuspended in 30//1 TE buffer (pH 8.0). d. Southern Blots The genomic DNA (10 /tg) was digested for 16 hours with 60 units of EcoRI and 15 electrophoresed 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. 20 Southern analysis of putative transgenic Dianthus, Rosa and Chrysanthemum plants obtained after selection on kanamycin confirmed the integration of the appropriate chimaeric gene into the genome. 25 EXAMPLE 14 Northern Analysis a. Dianthus and Chrysanthemum RNA Total RNA was isolated from tissue that had been frozen in liquid N£ and ground to a fine powder using a mortar and pestle. An extraaion buffer of 4 M guanidinium 30 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 WO 94/28140 PCT/AU94/00265 - 20- centrifuged in a JA20 rotor for 10 minutes at 10,000 rpm. The supernatant was collected 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 5 were resuspended in TE/SDS (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% (w/v) SDS) and extracted 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. 10 RNA samples were electrophoresed 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 32 Hybond-N filters (Amersham) as described by the manufacturer and probed with P- 8 6 labelled cDNA fragment (10 cpm//ig, 2 x 10 cpm/mL). Prehybridization (1 h at 15 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 /ig/mL) was added with the ^^-labelled probe for the hybridization step. Filters were washed in 2 x SSC/ 1% (w/v) SDS at 65°C for 1 to 2 hours and then 0.2 20 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. 25 b. Rosa RNA Total RNA was extracted from petals (buds and of flowers 5 days post-harvest) according to the method of Manning, 1991. 0 WO 94/28140 - 21 - PCT/AU94/00265 EXAMPLE 15 ^P-Labelling of DNA Probes 32 DNA fragments (50 to 100 ng) were radioaaively labelled with 50 /*Ci of [or- P}- 32 5 dCTP using an oligolabelling kit (Bresatec). Unincorporated [a- Pj-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column. EXAMPLE 16 Anthocyanidin Analysis 10 Prior to HPLC analysis the anthocyanin molecules present in petal extracts 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 detector 15 (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 extracted from petal segments (ca. 50 mg) with 5 ml of 20 methanol containing 1% (v/v) of aqueous 6M hydrochloric acid. Extracts were diluted with water (1:9) and Altered (Millex HV, 0.45/i) prior to injection into the HPLC system. b. Hydrolysis of anthocyanins 25 Crude methanolic extracts (100 /tL) obtained in a. above were evaporated to dryness in Pierce Reacti-Vials using a stream of dry nitrogen at room temperature. The residues were dissolved in 200/tL 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, 0.45/i) prior to HPLC analysis. 30 WO 94/28140 PCT / AU94/00265 - 22 - c. Chromatography Separation of flower pigments was effected via gradient elution using the following system: 5 Solvent A: (triethylamine: conc. H3PO4 : H2O) (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 10 Detection: 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 extracts is to be found in Brugliera et al., 1994. 15 HPLC analysis is conducted 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 20 pCGP653 in transgenic carnation flowers are shown in Table 1. WO 94/28140 PCT/AU94/00265 -23 -TABLE 1 HPLC Analysis of pCGP90, pCGP485 and pCGP653 Transgenic Flowers Sample % Delphinidin % Pelargonidin %Cvanidm NON-TRANSGENIC CARNATION: Cultivar: Red Sim 0 85.3 0.8 TRANSGENIC CARNATION: Red Sim + pCGP90 0) Acc «* 1933 1.9 82.7 nd** (ii) Acc #2011 3.7 76.9 nd Red Sim + pCGP485 (1) Acc # 3654B 13.0 75.1 2.3 Red Sim + pCGP653 0 Acc # 3660/2 18.1 71.4 3.2 (ii) Acc #3655 35.6 49.1 7.5 * Acc it — plant accession number ** nd -» not detected 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 extraaion 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-mercaptoethanol 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'-hydroxylasc activity. 94/28140 PCT/AU94/00265 -24 - 3' ,5' -Hydroxylase Assay 3',5'-Hydroxylase enzyme activity was measured using a modified version of the method described by Stotz and Forkmann (1982). The assay reaction mixture typically contained 195 fiL of plant extract, 5fiL of 50 mM NADPH in assay buffer (100 mM potassium phosphate (pH8.0), 1 mM EDTA and 20 mM 2-mercaptoethanol), and 10"* 14 dpm [ C] naringenin in a final volume of 200 /zL. Following incubation at 23 overnight, the reaction mixture was extracted twice with 0.5 mL of ethylacetate. The ethyl acetate phase was dried under vacuum and then resuspended in 10 fiL of ethyl acetate. The radio-labelled flavonoid 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 reaction products localised by autoradiography and identified by comparison to non-radioactive naringenin, eriodictyol, dihydroquercetin and dihydromyricetin standards which were run alongside the reaction products and visualized under UV light. EXAMPLE 18 Transformation of various cultivars The chimaeric genes contained in any one of the constructs pCGP90, pCGP812, pCGP628, pCGP485, pCGP653, pCGP484 or pCGPl458 is introduced into plant varieties of rose, carnation and chrysanthemum using Agrobacterium-medisted 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 selection and HPLC analysis is used to detect 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 activity 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 production of 3',5'-hydroxylase activity. 94/28140 PCT/AU94/00265 - 25 - EXAMPLE 19 Carnation cv. Crowley Sim + pCGP 90 The plasmid pCGP90 was introduced into the carnation cultivar Crowley Sim using Agrobacterium-mediated gene transfer, as described in Example 10. Integration of the construct in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin seleaion. Nine plants were examined for the presence of the nptll and Hfl genes and for the produaion of delphinidin. Eight of the nine plants analyzed were positive for both nptll and Hfl but HPLC analysis was unable to detea any evidence of delphinidin produaion 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 cultivar Laguna using Agrobacterium-medizted gene transfer, as described in Example 10. Integration of the construa in the plant genome was confirmed by Southern analysis of pijuits obtained after kanamycin seleaion. HPLC analysis of the anthocyanin molecules present in petal extraas 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 94/28140 PCT / AU94/00265 -26- exogenous DNA sequence, ie: the Hfl cDNA sequence, introduced via transformation with the binary vector pCGP485. EXAMPLE 21 Rose cv. Royalty + pCGP 485/pCGP 628 The plasmids pCGP485 and pCGP628 were introduced into the rose cultivar Royalty using Agrobacterium-mcdiatcd gene transfer, as referred to in Example 11. Integration of the construct in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin selection. HPLC analysis of the anthocyanin molecules present in petal extracts 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 vectors pCGP485 or pCGP628. EXAMPLE 22 Rose cv. Kardinal + pCGP 1458 The plasmid pCGPl458 was introduced into the rose cultivar Kardinal using Agrobacterium-medizted gene transfer, as described in Example 11. Integration of the construct in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin selection. HPLC analysis of the anthocyanin molecules present in petal extracts 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 vector pCGP1458. EXAMPLE 23 Chrysanthemum cv. BlueRidge + pCGP 484/pCGP 485/pCGP 628 The plasmids pCGP484, pCGP485 and pCGP628 were introduced into the chrysanthemum cultivar BlueRidge using Agrobacterium-mediated gene transfer, as described in Example 12. Integration of the construct in the plant genome was 94/28140 PCT/AU94/00265 - 27 - confirmed by Southern analysis of plants obtained after kanamycin selection. HPLC analysis of the anthocyanin molecules present in petal extracts 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 vectors pCGP484, pCGP485 or pCGP628. EXAMPLE 24 Altered Inflorescence The expression of the introduced flavonoid 3' ,5' -hydroxylase enzyme activity in the transgenic plant is capable of having a marked effect 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 affect 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 construct pCGP653 described above, an obvious bluing effect on the petals was observed. The normally-orange-red colour of Red Sim carnation cultivars (corresponding approximately tc 45A/B of the Royal Horticultural Society's Colour Chart) had changed to a blue/purple hue. WO 94/28140 PCT/AU94/00265 - 28 - 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 collectively, and any and all combinations of any two or more of said steps or features. 94/28140 PCT / AU94/00265 - 29 - REFERENCES: rr»> Bethesda Research Laboratories. BRL pUC host: E. coli DH5a competent cells. Bethesda Res. Lab. Focus 8(2): 9, 1986. Bevan, M. Nucleic Acids Res. 12: 8711-8721, 1984. Brugliera, F., Holton, T.A., Stevenson, T.W., Farcy, E., Lu, C-Y. and Cornish, E.C. Plant J. 5(1): 81-92, 1994. Comai, L., Moran, P. and Maslyar, D., Plant Molecular Biology 15: 373-381, 1990. Dellaporta, S.J., Wood, J. and Hick, J.B. Plant Mol Biol. Rep. 1: 19-21, 1983. Ebel, J. and Hahlbrock, K. In The Flavonoids: Advances in Research Since 1980. Harborne, J.B. (Ed.), Academic Press, New York, USA, 641-679, 1988. Forkmann, G. Plant Breeding 106: 1-26, 1991. Garfinkel, D.J. and Nester, E.W. J. Bacterial 144: 732-743, 1980. Hahlbrock, EL and Grisebach, H. Annu. Rev. Plant PbysioL 30: 105-130, 1979. Hanahan, D. J. Mol Biol. 166: 557, 1983. Holton, T.A. PhD Thesis, University of Melbourne, Australia, 1992. Holton, T.A. and Graham, M.W. Nucleic Acids Res. 19:1156, 1991. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholz, D., Rogers, S.G. and Fraley, R.T. Science 227: 1229-1231, 1985. PCT/AU94/00265 -30 - Inoue, H., Nojima, H. and Okayama, H.- Gene 96: 23-28, 1990. Jannsen, B-J.J. and Gardner, R.C. Plant Mol. Biol. 14: 61-72, 1989. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. EMBO J. 6(13): 3901-3907, 1987. Kandreck, K.A. and Black, N.D. Growing media for ornamental plants and turf. p317, NSW University Press, Kensington, Australia, 1984. Koes R.F. Genes involved in flavonoid biosynthesis in Petunia, hybrida: The chalcone synthase multigene family. PhD Thesis, Vrije Universiteit, Amsterdam, The Netherlands, 1988. Lazo, G.R., Pascal, A.S. and Ludwig, R.A. Bio/technology 9: 963-967, 1991. McDonnell, R.E., Clarke R.D., Smith, L.A. and Hinchee, M.A. Plant Mol. BiolRep. 4: 380-386, 1987 Manning, K. AnaL Biochem. 195: 45-50, 1991. Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory Press, USA, 1989. Schram, A.W., Jonsson, L.M.V. and Bennink, G.J.H. "Biochemistry of flavonoid synthesis in Petunia hybrida." In: Petunia Sink, K.C. (Ed.), Springer-Verlag, Berlin, Geiix*any, pp 68-75,1984. Sommer, H. and Saedler, H. Mol Gen. Genet. 202: 429434, 1986. Stafford, HA. Flavonoid Metabolism. CRC Press, Inc. Boca Raton, Florida, USA, 1990. WO 94/28140 PCT/AU94/00265 - 31 - Stotz, G. and Forkmann, G. Z Naturforsch 37c: 19-23, 1982. Vercruysse, S.A.R., Delcour, J.A. and Dondeyne, P. /. Chromatography 324: 495-497, 1985. "Wiering, H. and De Vlaming, P. "Inheritance and Biochemistry of Pigments." In: Petunia Sink, K.C. (Ed.), Springer-Verlag, Berlin, Germany, pp 49-65, 1984. Q:\OPER\EJHN266401.172 * 20/6/96 9 2 66 4 03) -32- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS:

1. A transgenic plant selected from rose, carnation and chrysanthemum or progeny thereof or cut-flowers therefrom wherein said plant carries a genetic construct comprising a promoter from a gene encoding an enzyme of the flavonoid pathway operably linked to a gene encoding a flavonoid 3',5'-hydroxylase wherein said transgenic plant produces higher levels of anthocyanins derived from delphinidin relative to non-transgenic plants of the same species.

2. A transgenic plant according to claim 1 wherein the flavonoid 3',5'-hydroxylase 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 flavonoid 3',5'-hydroxylase is of petunia origin.

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

5. A transgenic plant according to any one of claims 1 to 4 wherein the promoter is from the gene encoding chalcone synthase (CHS).

6. A transgenic plant according to claim 5 wherein the genetic construct is contained in a plasmid selected from pCGP484, pCGP485, pCGP653 and pCGP1458.

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

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

9. A transgenic plant according to claim 3 or 4 or 5 or 6 wherein said plant is a chrysanthemum. N.Z. PATENT OFFICE - 7 AUG 1996 RECEIVED 950703,q:V>{Xj\ejh,00265.cta^33 26 6 401 r^ceiveIj {9 9u? -33 -

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

14. A method for producing a transgenic plant selected from rose, carnation and chrysanthemum, said method comprising introducing into said plant a gene construct comprising a promoter from a gene encoding an enzyme of the flavonoid pathway operably linked to a nucleic acid sequence encoding a flavonoid 3',5'-hydroxylase characterised 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 respective species.

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

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

17. A method according to claim 15 wherein the flavonoid 3',5'-hydroxylase is of lisianthus origin.

18. A method according to any one of claims 14 to 17 wherein the promoter is from the gene encoding chalcone synthase (CHS).

19. A method according to claim 15 wherein the genetic construct is contained in a plasmid selected from pCGP484, pCGP485, pCGP653 and pCGP1458. AMENDED SHEEi PEA/AM N6W ZEALAfrfO 1 5 OCT 1995 PATENT OFFICE 0 C Z> f f\ A PCT/AU 94/00265 9S07ll.q:\operyjh,00265.dm^4 £ 0 U Q | RECElVED 1 1 JUL 1995 - 34-

20. A method according to claim 16 or 17 or 18 or 19 wherein said plant is a rose.

21. A method according to claim 16 or 17 or 18 or 19 wherein said plant is a carnation.

22. A method according to claim 16 or 17 or 18 or 19 wherein said plant is a chrysanthemum.

23. A method according to claim 14 wherein said transgenic plant exhibits altered - inflorescence.

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

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

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

27. A binary vector comprising a gene construct which is capable of being integrated into a plan* genome to produce the transgenic plant according to claim 1.

28. A binary vector according to claim 27 wherein the gene construct is a chimaeric gene construct

29. A binary vector according to claim 28 wherein the promoter is the CHS gene promoter. M8VV .: j j i 1 5 OCT M5 Fwwr Cvnoi: AMENDED SHEET IPEA/AU ,266 4 0 - 35 -

30. A transgenic plant substantially as herein described with reference to the accompanying examples.

31. A method for producing a transgenic plant substantially as herein described with reference to the accompanying examples.

32. A binary vector substantially as herein described with reference to the accompanying examples.

33. A transgenic plant as claimed in claim 30 and substantially as herein described with reference to the examples.

34. A method for producing a transgenic plant as claimed in claim 14 and substantially as herein described with reference to the accompanying drawings.

35. A binary vector as claimed in claim 27 and substantially as herein described with reference to the accompanying examples. N.Z. PATENT OFFICE '-J AUG 1996 RECEIVED </div>