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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
  • A01H6/1424—Chrysanthemum
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/30—Caryophyllaceae
  • A01H6/305—Dianthus carnations
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
• • C—CHEMISTRY; METALLURGY
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
  • C12Y114/13088—Flavonoid 3',5'-hydroxylase (1.14.13.88)

Definitions

• 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.
• 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).
• fiavonoid pathway 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,
• 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).
• CHS chalcone synthase
• 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.
• DHQ dihydroquercetin
• HMM dihydromyricetin
• 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.
• 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.
• 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.
• 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.
• the polypeptide is of petunia, verbena, delphinum, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg plant, lisianthus or campanula origin.
• 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.
• the gene construct is DNA it may be cDNA or genomic DNA.
• 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.
• 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.
• derivative is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally-occurring enzyme.
• 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.
• 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:
• transgenic plant exhibits one or more of the following properties:
• 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.
• 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.
• nucleic acid sequence in the plant may be constitutive, inducible or developmental.
• altered inflorescence means any alteration in flower colour relative to the naturally-occurring flower colour taking into account normal variations between flowerings.
• 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.
• 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.
• 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.
• 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.
• 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.
• Figure 5 is a diagrammatic representation of the binary expression vector pCGP653, contruction of which is described in Example 6.
• 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.
• 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.
• the Esche ⁇ chia coli strain used was:
• 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.
• Plasmid pCGP90 was constructed by cloning the cDNA insert from pCGP602 (International Patent Application PCT/AU92/00334; Publication Number WO
• the binary expression vector pCGP293 was derived from the Ti binary vector ⁇ CGN1559 (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 ⁇ /.,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.
• 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 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.
• 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 pCGP13 ⁇ 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 ⁇ CGP176 (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.
• 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).
• 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.
• the plasmid pCGPl458 was contructed using the 10 kb binary vector pBIN19 (Bevan,
• 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.
• Transformation of the Escherichia coli strain DH5 ⁇ -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 ⁇ g of plasmid DNA to 100 ⁇ 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 ⁇ g/mL kanamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the gentamycin-resistant transformants.
• 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 ⁇ -naphthalene acetic acid (NAA), 20 ⁇ M acetosyringone and 0.8% Difco Bacto Agar (pH 5.7).
• 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 sele ⁇ ion 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.
• 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 ⁇ E cool white fluorescent light) at 23 ⁇ 2. When plants were rooted and reached 4-6 cm tall they were acclimatised under mist.
• Plant tissues of the rose cukivar Royalty were transformed according to the method disclosed in PCT 91/04412, having publication number WO92/00371.
• 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.
• 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 ⁇ 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.
• 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.
• 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.
• 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).
• DNA was isolated from leaf tissue using an extra ⁇ ion buffer containing 4.5 M guanidinium thiocyanate, 50 mM EDTA pH 8.0, 25 mM sodium citrate pH 7.0,
• DNA was extra ⁇ ed by grinding tissue in the presence of liquid N2 in a mortar and pestle and adding 1ml of extra ⁇ ion buffer (0.14 M sorbitol, 0.22 M Tris-HCl [ ⁇ H8.0], 0.022 M EDTA, 0.8 M NaCl, 0.8% (w/v) CTAB, l%N-laurylsarcosine) heated at 65°C. Chloroform (200 ⁇ l) was added and the mixture incubated at 65°C for 15 min. Following centrifugation, the supernatant was phenol-chloroform extra ⁇ ed 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 30 ⁇ l TE buffer (pH 8.0).
• extra ⁇ ion buffer 0.14 M sorbitol, 0.22 M Tri
• the genomic DNA (10 ⁇ g) was digested for 16 hours with 60 units of EcoRI and ele ⁇ rophoresed 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.
• the suspension was filtered through Miracloth (Calbiochem) and centrifuged in a JA20 rotor for 10 minutes at 10,000 rpm. The supernatant was colle ⁇ ed and made to 0.2 g/ mL CsCl (w/v).
• RNA pellets were resuspended in TE/SDS (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% (w/v) SDS) and extra ⁇ ed 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 ele ⁇ rophoresed through 2.2 M formaldehyde/ 1.2% (w/v) agarose gels using running buffer containing 40 mM morpholinopropanesulphonic acid (pH
• 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.
• DNA fragments 50 to 100 ng were radioa ⁇ ively labelled with 50 ⁇ Ci of [ ⁇ - 32 P]- dCTP using an oligolabelling kit (Bresatec). Unincorporated [a- 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.
• anthocyanidin Analysis Prior to HPLC analysis the anthocyanin molecules present in petal extra ⁇ s 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 dete ⁇ or (MWD). Reversed phase chromatographic separations were performed on a Spherisorb S5 ODS2 cartridge column, 250 mm x 4 mm ID.
• anthocyanidin peaks were identified by reference to known standards.
• An alternative method for the analysis of anthocyanin molecules present in petal extra ⁇ s is to be found in Brugliera et al, 1994.
• HPLC analysis is condu ⁇ ed 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.
• Plant tissue was homogenised in a 10 times volume of ice-cold extra ⁇ ion 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-merca ⁇ toethanol 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 a ⁇ ivity.
• 3' ,5' -Hydroxylase Assay 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-merca ⁇ toethanol
• the assay rea ⁇ ion mixture typically contained 195 ⁇ L of plant extra ⁇ , 5 ⁇ L of 50 mM NADPH in assay buffer (100 mM potassium phosphate (pH8.0), 1 mM EDTA and 20 mM 2-mercaptoethanol), and 10
• the TLC plates were air-dried and the rea ⁇ ion produ ⁇ s localised by autoradiography and identified by comparison to non-radioa ⁇ ive naringenin, eriodi ⁇ yol, dihydroquercetin and dihydromyricetin standards which were run alongside the rea ⁇ ion produ ⁇ s and visualized under UV light.
• chimaeric genes contained in any one of the constru ⁇ s pCGP90, pCGP812, pCGP628, pCGP485, pCGP653, pCGP484 or pCGP1458 is introduced into plant varieties of rose, carnation and chrysanthemum using Agrobacterium-me ⁇ ia.te ⁇ 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 sele ⁇ ion and HPLC analysis is used to dete ⁇ 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 a ⁇ ivity 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 produ ⁇ ion of 3 ' ,5' -hydroxylase a ⁇ ivity.
• the plasmid pCGP485 was introduced into the carnation cukivar Website using Agrobacterium-me ⁇ iate ⁇ gene transfer, as described in Example 10. Integration of the constru ⁇ in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin sele ⁇ ion. HPLC analysis of the anthocyanin molecules present in petal extra ⁇ s is carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives.
• the plasmids pCGP485 and pCGP628 were introduced into the rose cukivar Royalty using Agrobacterium-mediate ⁇ gene transfer, as referred to in Example 11. Integration of the constru ⁇ in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin sele ⁇ ion. HPLC analysis of the anthocyanin molecules present in petal extra ⁇ s is again carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives.
• the plasmid pCGP1458 was introduced into the rose cukivar Kardinal using Agrobacterium-mediate ⁇ gene transfer, as described in Example 11. Integration of the constru ⁇ in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin sele ⁇ ion. HPLC analysis of the anthocyanin molecules present in petal extra ⁇ s is again carried out according to the procedure set out in Example 16, above, to show the presence of 3 ' ,5 ' -hydroxylated anthocyanin derivatives.
• the plasmids pCGP484, pCGP485 and pCGP628 were introduced into the chrysanthemum cukivar BlueRidge using Agrobacterium-me ⁇ iste ⁇ gene transfer, as described in Example 12. Integration of the constru ⁇ in the plant genome was confirmed by Southern analysis of plants obtained after kanamycin sele ⁇ ion. HPLC analysis of the anthocyanin molecules present in petal extra ⁇ s is again carried out according to the procedure set out in Example 16, above, to show the presence of 3' ,5' -hydroxylated anthocyanin derivatives.
• the expression of the introduced fiavonoid 3' ,5' -hydroxylase enzyme a ⁇ ivity in the transgenic plant is capable of having a marked effe ⁇ 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 affe ⁇ the individual outcome and the citing of specific colours should not be interpreted as defining the possible range.

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