USPP21892P3 - Dianthus plant named ‘FLORIAMETRINE’ - Google Patents

Dianthus plant named ‘FLORIAMETRINE’ Download PDF

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USPP21892P3
USPP21892P3 US12/291,865 US29186508V USPP21892P3 US PP21892 P3 USPP21892 P3 US PP21892P3 US 29186508 V US29186508 V US 29186508V US PP21892 P3 USPP21892 P3 US PP21892P3
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floriametrine
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dianthus
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Filippa Brugliera
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International Flower Developments Pty Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13

Definitions

  • the present invention relates generally to the field of genetic modification of plants. More particularly, the present invention is directed to genetically-modified carnation plants expressing unique color phenotypes in selected parts of the plants.
  • the flower or ornamental plant industry strives to develop new and different varieties of flowers and/or plants.
  • An effective way to create such novel varieties is through the manipulation of flower color.
  • Classical breeding techniques have been used with some success to produce a wide range of colors for almost all of the commercial varieties of flowers and/or plants available today. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have the full spectrum of colored varieties.
  • novel colored varieties of plants or plant parts such as flowers, foliage and stems would offer a significant opportunity in both the cut flower and ornamental markets.
  • desired (including novel) colored varieties of carnation is of particular interest. This includes not only different colored flowers but also anthers and styles.
  • flavonoids are the most common and contribute a range of colors from yellow to red to blue.
  • the flavonoid molecules that make the major contribution to flower color are the anthocyanins, which are glycosylated derivatives of cyanidin and its methylated derivative peonidin, delphinidin and its methylated derivatives petunidin and malvidin and pelargonidin.
  • Anthocyanins are localized in the vacuole of the epidermal cells of petals or the vacuole of the sub epidermal cells of leaves.
  • the flavonoid pigments are secondary metabolites of the phenylpropanoid pathway.
  • the biosynthetic pathway for the flavonoid pigments is well established, (Holton and Cornish, Plant Cell 7:1071-1083, 1995; Mol et al., Trends Plant Sci. 3:212-217, 1998; Winkel-Shirley, Plant Physiol. 126:485-493, 2001a; and Winkel-Shirley, Plant Physiol.
  • the enzymes are phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL).
  • PAL phenylalanine ammonia-lyase
  • C4H cinnamate 4-hydroxylase
  • 4CL 4-coumarate: CoA ligase
  • the first committed step in the pathway involves the condensation of three molecules of malonyl-CoA (provided by the action of acetyl CoA carboxylase (ACC) on acetyl CoA and CO 2 ) with one molecule of p-coumaroyl-CoA. This reaction is catalyzed by the enzyme chalcone synthase (CHS).
  • CHS chalcone synthase
  • the product of this reaction is normally rapidly isomerized by the enzyme chalcone flavanone isomerase (CHI) to produce naringenin. Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
  • CHI chalcone flavanone isomerase
  • F3H flavanone 3-hydroxylase
  • the pattern of hydroxylation of the B-ring of DHK plays a key role in determining petal color.
  • the B-ring can be hydroxylated at either the 3′, or both the 3′ and 5′ positions, to produce dihydroquercetin (DHQ) or dihydromyricetin (DHM), respectively.
  • DHQ dihydroquercetin
  • HMM dihydromyricetin
  • Two key enzymes involved in this part of the pathway are flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′-hydroxylase (F3′5′H), both members of the cytochrome P450 class of enzymes.
  • the production of colored anthocyanins from the dihydroflavonols involves dihydroflavonol-4-reductase (DFR) leading to the production of the leucoanthocyanidins.
  • DFR dihydroflavonol-4-reductase
  • the leucoanthocyanidins are subsequently converted to the anthocyanidins, pelargonidin, cyanidin and delphinidin.
  • These flavonoid molecules are unstable under normal physiological conditions and glycosylation at the 3-position, through the action of glycosyltransferases, stabilizes the anthocyanidin molecule thus allowing accumulation of the anthocyanins.
  • DFR The substrate specificity shown by DFR can regulate the anthocyanins that a plant accumulates.
  • Petunia and cymbidium DFRs do not reduce DHK and thus they do not accumulate pelargonidin-based pigments (Forkmann and Ruhnau, Z Naturforsch C. 42c, 1146-1148, 1987, Johnson et al., Plant Journal, 19, 81-85, 1999).
  • Many important floricultural species including iris, delphinium, cyclamen, gentian, cymbidium are presumed not to accumulate pelargonidin due to the substrate specificity of their endogenous DFRs (Tanaka and Brugliera, 2006, supra).
  • the DFR enzyme is capable of metabolizing two dihydroflavonols to leucoanthocyanidins which are ultimately converted through to anthocyanins pigments that are responsible for flower color.
  • DHK is converted to leucopelargonidin, the precursor to pelargonidin-based pigments, giving rise to apricot to brick-red colored carnations.
  • DHQ is converted to leucocyanidin, the precursor to cyanidin-based pigments, producing pink to red carnations.
  • Carnation DFR is also capable of converting DHM to leucodelphinidin (Forkmann and Ruhnau, 1987 supra), the precursor to delphinidin-based pigments.
  • naturally occurring carnation lines do not contain a F3′5′H enzyme and therefore do not synthesize DHM.
  • Nucleotide sequences encoding F3′5′Hs have been cloned (see International Patent Application No. PCT/AU92/00334 incorporated herein by reference and Holton et al., Nature, 366:276-279, 1993 and International Patent Application No. PCT/AU03/01111 incorporated herein by reference). These sequences were efficient in modulating 3′, 5′ hydroxylation of flavonoids in petunia (see International Patent Application No. PCT/AU92/00334 and Holton et al., 1993 supra), tobacco (see International Patent Application No. PCT/AU92/00334), carnations (see International Patent Application No. PCT/AU96/00296 incorporated herein by reference) and roses (see International Patent Application No. PCT/AU03/01111).
  • Carnations are one of the most extensively grown cut flowers in the world.
  • Standard carnations are intended for cultivation under conditions in which a single large flower is required per stem. Side shoots and buds are removed (a process called disbudding) to increase the size of the terminal flower. Sprays and/or miniatures are intended for cultivation to give a large number of smaller flowers per stem. Only the central flower is removed, allowing the laterals to form a ‘fan’ of flowers.
  • Spray carnation varieties are popular in the floral trade, as the multiple flower buds on a single stem are well suited to various types of flower arrangements and provide bulk to bouquets used in the mass market segment of the industry.
  • Standard and spray cultivars dominate the carnation cut-flower industry, with approximately equal numbers sold of each type in the USA.
  • spray-type varieties account for 70% of carnation flowers sold by volume, whilst in Europe spray-type carnations account for approximately 50% of carnation flowers traded through out the Dutch auctions.
  • the Dutch auction trade is a good indication of consumption across Europe.
  • Kortina Chanel line of carnations Dianthus caryophyllus cv. Kortina Chanel.
  • the variety has excellent growing characteristics and a moderate to good resistance to fungal pathogens such as Fusarium.
  • purple/blue spray carnations were not available.
  • ‘FLORIAMETRINE’ The following traits represent the characteristics of the new Dianthus cultivar ‘FLORIAMETRINE’. These traits distinguish this cultivar from other commercial varieties. ‘FLORIAMETRINE’ may exhibit phenotypic differences with variations in environmental, climatic and cultural conditions, without any variance in genotype.
  • FIG. 1 is a photographic representation of the flower. Colors may appear different from the actual colors due to light reflection but are as accurate as possible by conventional photography.
  • FIG. 3 is a photographic representation of a high resolution scan of a Southern blot autoradiograph showing 10 ⁇ g of EcoRI-treated genomic DNA from the transgenic carnation line 19907, in comparison to 10 ⁇ g of EcoRI-treated genomic DNA from the carnation lines Kortina Chanel, Vega and Purple Spectro, hybridized with the NtALS probe.
  • FIG. 4 is a photographic representation of the ‘Kortina Chanel’ control on the left and the cultivar ‘FLORIAMETRINE’ on the right.
  • the present invention relates to a new and distinct cultivar of carnation that is grown for use as a flowering plant for pots and containers.
  • the new cultivar is known botanically as Dianthus caryophyllus and is referred to hereinafter by the cultivar name ‘FLORIAMETRINE’.
  • ‘FLORIAMETRINE’ is a complex transgenic plant comprising genetic sequences encoding at least two F3′5′H molecules and at least one DFR.
  • the vector pCGP2442 used to transform meristematic cells contains a chimeric AmCHS 5′: Salivia F3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene and the 35S 5′: SuRB selectable marker gene cassette of the plasmid pWTT2132.
  • the new variety originated in vitro by Agrobacterium tumefaciens -mediated transformation of meristematic cells of the Kortina Chanel (unpatented) carnation with the pCGP2442 vector at Florigene Pty Ltd., in Bundoora, Victoria, Australia. Cuttings of Dianthus caryophyllus cv. Kortina Chanel were obtained from Van Wyk and Son Flower Supply, Victoria or Propagation Australia, Queensland, Australia.
  • Transgenic plants containing the chimeric AmCHS 5′: SaliviaF3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, and a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene were successfully generated from the cells.
  • the plants also contains genes for acetolactate synthase resistance (SuRB) transformation selection markers.
  • the transformation and regeneration process is described in International Patent Application No. PCT/US92/02612; International Patent Application No. PCT/AU96/00296; and Lu et al., Bio/Technology 9: 864-868, 1991, the contents of each of which are incorporated by reference.
  • the primary focus of the carnation generation program was to produce new cultivars of carnations which exhibited a selected and desired purple/violet color in the spray background.
  • the term ‘FLORIAMETRINE’ was selected because of its pronounced production of delphinidin or delphinidin-based molecules pigments.
  • the new variety was selected from a group of 74 transgenic lines of which only three produced flowers with a significant shift in color into the violet, purple/violet range.
  • ‘FLORIAMETRINE’ is essentially similar to the parent in the morphological aspects of the flower, but can be further distinguished from the parent throughout the accumulation of pigment in the filaments and anthers of the flower. This is a new phenotype of the transgenic line. Some styles and anthers of ‘FLORIAMETRINE’ also have a shift in color to light purple, whereas the styles and anthers from flowers of the parent line were a cream-white color.
  • the new variety was originally selected in vitro as a regenerated shoot from a ‘Kortina Chanel’ carnation meristematic cell that had been transfected with Agrobacterium tumefaciens AGL0 (Lazo et al., Bio/technology 9:963-967, 1991) carrying the plasmid pCGP2442.
  • Stem surface Glabrous and glaucous Glabrous and glaucous Stem color 137B 137B Branching Little branching from Little branching from the the axils of lower leaves axils of lower leaves
  • Node color 192D 192D Node dimensions 6 mm diameter and 6 mm diameter and 3 mm 3 mm in length in length.
  • the DFR genomic fragments used in this application were isolated from petunia.
  • the petunia DFR enzyme is only capable of using DHQ and DHM as a substrate, but not DHK (Holton and Cornish, 1995 supra). This ensures that most or all of the anthocyanidin produced is delphinidin.
  • the F3′5′H coding sequences in the chimeric genes used in the new construct were from pansy (carnANS 5′: BP F3′5′H #18: carnANS 3′ in pCGP2205) and salvia (AmCHS 5′: Salvia F3′5′H #47: petD8 3′ in pCGP2122) as these represent the two expression cassettes that were the most efficient in producing the highest levels of delphinidin in the Kortina Chanel spray carnation.
  • the transformation vector pCGP2442 ( FIG. 2 ) contains a chimeric AmCHS: Salvia F3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene and the 35S 5′: SuRB selectable marker gene cassette of the plasmid pWTT2132 (see International Patent Application No. PCT/AU03/01111 incorporated herein by reference).
  • the disarmed Agrobacterium tumefaciens strain used was AGL0 (Lazo et al., 1991 supra).
  • Plasmid DNA was introduced into the Agrobacterium tumefaciens strain AGL0 by adding 5 ⁇ g of plasmid DNA to 100 ⁇ L of competent AGL0 cells prepared by inoculating a 50 mL LB culture (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 1989) and incubation for 16 hrs with shaking at 28° C. The cells were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM CaCl 2 /15% (v/v) glycerol.
  • the DNA- Agrobacterium mixture was frozen by incubation in liquid N 2 for 2 minutes and then allowed to thaw by incubation at 37° C. for 5 minutes. The DNA/bacterial mix was then placed on ice for a further 10 minutes. The cells were then mixed with 1 mL of LB (Sambrook et al., 1989 supra) media and incubated with shaking for 16 hrs at 28° C. Cells of A. tumefaciens carrying the plasmid were selected on LB agar plates containing appropriate antibiotics such as 50 ⁇ g/mL tetracycline or 100 ⁇ g/mL gentamycin. The confirmation of the plasmid in A. tumefaciens was done by restriction endonuclease mapping of DNA isolated from the antibiotic-resistant transformants.
  • Genomic DNA was isolated from leaf tissues as described by Dellaporta et al., Molecular Biology Reporter 1(14):19-21, 1983.
  • the genomic DNA (10 ⁇ g) was digested for 48 hours using 120 units of the restriction endonuclease EcoRI at 37° C. DNA fragments were separated by electrophoresis through a 0.8% w/v agarose gel. The DNA was transferred to Hybond NX membrane (Amersham) as described (Sambrook et al., 1989 supra).
  • the gel was prepared for blotting by a 15 minute depurination step in 0.25 M HCl, two 20 minute washes in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and two 20 minute washes in neutralization solution (0.5 M Tri-HCl, pH 7.5, 0.48 M HCl, 1.5 M NaCl).
  • DNA was capillary transferred to Hybond-NX nylon membrane (Amersham Biosciences, UK) in 20 ⁇ SSC (3 M NaCl, 0.3 M Tris-Na citrate, pH 7.0).
  • a probe corresponding to a 770 bp fragment of the ALS (acetolactate synthase) gene from Nicotiana tabacum (NtALS) was used for Southern blot analysis.
  • the probe fragment was originally generated by PCR and subsequently sub-cloned into an amplification vector (pBluescript II, Stratagene, USA), given a reference number (pCGP1651) and the fragment sequenced. After confirmation of the correct sequence, the DNA fragment was isolated from the source plasmid using the restriction endonuclease HindIII. The fragment was separated by 1% w/v agarose gel electrophoresis and purified using the MinElute Gel Extraction kit and protocol (Qiagen, Australia).
  • DNA fragments (25-50 ng) were labeled with 50 ⁇ Ci of [ ⁇ -32P]-dCTP (PerkinElmer Life and Analytical Sciences, USA) using a Decaprime kit (Ambion, USA). Unincorporated [ ⁇ - 32 P]-dCTP was removed by chromatography on Sephadex G-50 (Fine) columns. The labeled probe fragment was counted using a BioScan radioisotope counter (QC:4000 XER, BioScan, USA).
  • Membranes were pre-hybridized in 10 mL hybridization buffer 50% v/v deionized formamide, 1 M NaCl, 1% w/v SDS and 10% w/v dextran sulfate) at 42° C. for 1 hr. Once denatured, 10,000,000 dpm of 32 P -labeled probe was added to the hybridization solution and hybridization was continued at 42° C. for a further 16 hours. Membranes were washed twice in low stringency buffer (2 ⁇ SSC, 1% w/v SDS) at 65° C. for 30 minutes. Membranes were exposed to Kodax BioMax MS X-Ray film (Kodak, USA) with an intensifying screen at ⁇ 70° C. for 16 hours. The exposed films were automatically developed using a Curix 60 X-ray developer (AGFR-Gevaert Group, Belgium).

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WO2010050605A1 (fr) * 2008-10-27 2010-05-06 インターナショナル フラワー ディベロプメンツ プロプライアタリー リミティド Adn chromosomique issu de cinéraire impliqué dans la synthèse de flavonoïde et son utilisation
JP5477619B2 (ja) * 2009-07-15 2014-04-23 国立大学法人 鹿児島大学 デルフィニウム（Ｄｅｌｐｈｉｎｉｕｍｓｐｐ．）から単離された配糖体化酵素とその利用
US8348929B2 (en) 2009-08-05 2013-01-08 Rocin Laboratories, Inc. Endoscopically-guided tissue aspiration system for safely removing fat tissue from a patient
US8465471B2 (en) 2009-08-05 2013-06-18 Rocin Laboratories, Inc. Endoscopically-guided electro-cauterizing power-assisted fat aspiration system for aspirating visceral fat tissue within the abdomen of a patient
RU2640248C2 (ru) 2012-04-16 2017-12-27 Сантори Холдингз Лимитед Новый ген флавоноид 3,5-гидроксилазы колокольчика и его применение
CN112218525B (zh) 2017-09-12 2023-11-07 丹姿格丹花卉农场 紫盆花植物及其产生方法

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WO1996036716A1 (fr) 1995-05-16 1996-11-21 International Flower Developments Pty. Ltd. Plantes transgeniques presentant des couleurs florales modifiees et procedes permettant de les obtenir
WO2004020637A1 (fr) 2002-08-30 2004-03-11 International Flower Developments Pty. Ltd. Sequences genetiques de flavonoide 3',5'hydroxylase et leurs utilisations
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DE69431748D1 (de) * 1993-03-02 2003-01-02 Du Pont Erhöhung des methionin-gehaltes in pflanzensamen durch expression von 10kd zein aus mais
AUPN903196A0 (en) * 1996-03-29 1996-04-26 Australian National University, The Single-step excision means
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WO1993001290A1 (fr) 1991-07-11 1993-01-21 International Flower Developments Pty. Ltd. Sequences genetiques codant les enzymes du mecanisme d'action des flavonoïdes et leurs utilisations
WO1996036716A1 (fr) 1995-05-16 1996-11-21 International Flower Developments Pty. Ltd. Plantes transgeniques presentant des couleurs florales modifiees et procedes permettant de les obtenir
US6774285B1 (en) * 1996-03-01 2004-08-10 Florigene Limited Nucleic acid sequences encoding flavonoid 3′-hydroxylase and methods of altering flower color therewith
WO2004020637A1 (fr) 2002-08-30 2004-03-11 International Flower Developments Pty. Ltd. Sequences genetiques de flavonoide 3',5'hydroxylase et leurs utilisations

Non-Patent Citations (14)

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Title
Dellaporta et al, "A Plant DNA Minipreparation: Version II," Molecular Biology Reporter 1(14):19-21, 1983.
Forkmann and Ruhnau, "Distinct Substrate Specificity of Dihydroflavonol 4-Reductase from Flowers of Petunia hybrida," Z Naturforsch C. 42c, 1146-1148, 1987.
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JP5500556B2 (ja) 2014-05-21
EP2187729A4 (fr) 2010-09-01
AU2008323629A1 (en) 2009-05-22
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US20100293668A1 (en) 2010-11-18

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