EP0824591A4 - Illets transgeniques presentant une vie prolongee apres recolte - Google Patents

Illets transgeniques presentant une vie prolongee apres recolte

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Publication number
EP0824591A4
EP0824591A4 EP96911869A EP96911869A EP0824591A4 EP 0824591 A4 EP0824591 A4 EP 0824591A4 EP 96911869 A EP96911869 A EP 96911869A EP 96911869 A EP96911869 A EP 96911869A EP 0824591 A4 EP0824591 A4 EP 0824591A4
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EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
sequence
seq
transgenic
Prior art date
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EP96911869A
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German (de)
English (en)
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EP0824591A1 (fr
Inventor
Michael Zenon Michael
Michael Wayne Graham
Edwina Cecily Cornish
Neal Ira Gutterson
William Tinsley Tucker
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Florigene Pty Ltd
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Florigene Pty Ltd
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Publication of EP0824591A1 publication Critical patent/EP0824591A1/fr
Publication of EP0824591A4 publication Critical patent/EP0824591A4/fr
Withdrawn legal-status Critical Current

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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
    • 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)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N15/8249—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 ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates generally to transgenic plants which exhibit prolonged post- harvest life properties. More particularly, the present invention is directed to transgenic carnation plants modified to reduce expression of one or more enzymes associated with the ethylene biosynthetic pathway. Flowers of such carnation plants do not produce ethylene, or produce ethylene in reduced amounts, and are, therefore, capable of surviving longer post- harvest than flowers of non-genetically modified, naturally-occurring carnation plants.
  • SAM S-adenosyl-methionine
  • Regulation of the genes encoding these enz mes determines the temporal and spatial patterns of ethylene biosynthesis. This regulation is complex and varies among different species and different tissues as well as in response to different stimuli. Therefore, the ability to control the level of either of these enzymes, but especially the level of ACC synthase since this enzyme controls the production of ethylene, affords control of ethylene levels and, hence, regulation of plant development characteristics controlled by ethylene. These include seed germination; abscission; stress and wound response; fruit ripening and leaf and flower senescence.
  • carnation ACC synthase is encoded by a multigene family (Park et al; 1992), which helps explain the differential regulation of its various isozymes at different developmental stages in various tissues.
  • Availability of isolated nucleic acid molecules encoding, or complementary to sequences encoding, carnation ACC synthase or ACC oxidase permits the manufacture of recombinant materials, such as genetic constructs, useful for controlling the level of these enzymes in plants.
  • the genetic constructs can be introduced into carnation plants, thereby affording the possibility of regulating the plants' production of ethylene.
  • one aspect of the present invention contemplates a method for producing a transgenic plant exhibiting reduced production of climacteric ethylene, compared to its non- transgenic parent or a non-transgenic plant of the same species, said method comprising introducing into a cell or cells of a plant a genetic construct comprising a nucleic acid molecule encoding, or complementary to a sequence encoding ACC synthase or ACC oxidase or a derivative of said nucleic acid molecule, and regenerating a transgenic plant from said cell or cells.
  • the transgenic plant produced by the subject method exhibits one or more of the following properties:
  • a method for producing a transgenic carnation plant comprising introducing into said plant a genetic construct containing an isolated nucleic acid molecule encoding, or complementary to the sequence encoding, ACC synthase or ACC oxidase, or a derivative of said nucleic acid molecule characterized in that said transgenic plant exhibits one or more of the following properties: (i) reduction in the production of ACC synthase-specific mRNA or ACC oxidase- specific mRNA;
  • the present invention contemplates a method for producing a transgenic carnation plant exhibiting prolonged post-harvest life properties, said method comprising introducing into said carnation plant a genetic construct comprising a non-full- length fragment of a nucleic acid molecule encoding ACC synthase or ACC oxidase.
  • climacteric ethylene is meant the developmentally-regulated production of ethylene which induces a series of chemical events leading to ripening or senescence of an organ.
  • the term was originally used to describe the metaboUc state of ripening fruit, but also applies to the senescence of carnation flowers.
  • a peak of production of climacteric ethylene by a control plant can be readily seen in Figure 9.
  • the non-full-length fragment is approximately 800-1200 base-pair in length.
  • the non-full-length fragment is an internal fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase.
  • the non-full-length fragment is inserted in the sense orientation such that reduction of ACC synthase or ACC oxidase expression is by co-suppression.
  • the genetic constructs of the present invention comprise an isolated nucleic acid molecule encoding, or complementary to the sequence encoding, ACC synthase or ACC oxidase, or a derivative of said nucleic acid molecule and where necessary comprise additional genetic sequences such as promoter and terminator sequences which regulates expression of the molecule in the transgenic plants. " When the genetic construct is DNA it may be cDNA or genomic DNA. The ACC synthase or ACC oxidase genetic sequences are preferably from carnation plants. However, the present invention extends to similar genetic sequences from other plants such as related flowering plants and which have a genetic sequence capable of acting via antisense or co-suppression methods.
  • nucleic acid molecule as used herein is meant any contiguous series of nucleotide bases specifying a sequence of amino acids in ACC synthase or ACC oxidase.
  • the nucleic acid may encode the full-length enzyme or a derivative thereof.
  • the nucleic acid molecule may not encode a full-length ACC synthase or ACC oxidase but is of sufficient length to down regulate an endogenous ACC synthase or ACC oxidase gene by co- suppression or antisense.
  • 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 ACC synthase or ACC oxidase 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 chemical equivalent of the ACC synthase or ACC oxidase, 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) reduction in the production of ACC synthase-specific mRNA or ACC oxidase- specific mRNA;
  • a derivative of the subject nucleic acid molecule is also considered to encompass a genetic molecule capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:3 under low stringency conditions at 30°C.
  • Reference to low stringency conditions includes hybridising DNA with 50% formamide at 30 °C.
  • Alternative conditions such as medium and high stringency conditions may also be employed depending on the derivative.
  • the transgenic carnation plant carries flowers or flower buds which, when cut from the carnation plant, exhibit prolonged post-harvest life properties as well as one or more of the following properties:
  • a method for producing transgenic carnation plants comprising introducing into said plants a genetic construct containing an isolated nucleic acid molecule encoding, or complementary to the sequence encoding, a non-full-length portion of ACC synthase or ACC oxidase, characterized in that the flowers of the said transgenic plants exhibit one or more of the following properties:
  • the present invention further extends to such transgenic plants having one or more of the above-mentioned properties and to cut flowers or cut parts from said plants including flower buds from said plants.
  • the flowers of the said transgenic plants exhibit one or more of the following properties:
  • Reference herein to the level of ACC synthase enzyme relates to a reduction of 30% or more, or more preferably of 30-50%, or even more preferably 50-75% or still more preferably 75% or greater below the normal endogenous or existing levels of enzyme. Such reduction may be referred to as "modulation" of ACC synthase or ACC oxidase enzyme activity. It is possible that modulation is at the level of transcription, post-transcriptional stability or translation of the ACC synthase or ACC oxidase genetic sequences.
  • 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 Agrobacte ⁇ um- edizted transfer.
  • nucleic add molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding a carnation ACC synthase or ACC oxidase or a mutant, derivative, part, fragment, homologue or analogue of said ACC synthase or ACC oxidase.
  • mutants may also be functional, meaning that they exhibit at least some ACC synthase or ACC oxidase activity.
  • the nucleic acid molecules are capable of suppressing ACO or ACS gene expression, mediated by the nucleic acid molecule being in one or the other orientation relative to its or another promoter; i.e. by sense suppression or antisense suppression.
  • ACC synthase and ACC oxidase include reference to polypeptides and proteins having ACC synthase or ACC oxidase activity as well as any mutants, derivatives, parts, fragments, homologues or analogues of such polypeptides or proteins and which have ACC synthase or ACC oxidase activity.
  • a molecule having ACC synthase or ACC oxidase activity may also be a fusion polypeptide or protein between a polypeptide or protein having ACC synthase or ACC oxidase activity and an extraneous peptide, polypeptide or protein.
  • isolated nucleic acid molecule is meant to include a genetic sequence in a non-naturally-occurring condition. Generally, this means isolated away from its natural state or formed by procedures not necessarily encountered in its natural environment. More specifically, it includes nucleic acid molecules formed or maintained in vitro, including genomic DNA fragments, recombinant or synthetic molecules and nucleic acids in combination with heterologous nucleic acids such as heterologous nucleic acids fused or operably-linked to the genetic sequences of the present invention.
  • isolated nucleic acid molecule also extends to the genomic DNA or cDNA, or part thereof constituting ACC synthase or ACC oxidase or a mutant, derivative, part, fragment, homologue or analogue of ACC synthase or ACC oxidase, whether in sense or in reverse orientation relative to its or another promoter. It further extends to naturally-occurring sequences following at least a partial purification relative to other nucleic acid sequences.
  • isolated nucleic acid molecule as used herein is understood to have the same meaning as a "nucleic acid isolate”. In a particular embodiment, mutants and other like variants of ACC synthase or ACC oxidase retain at least some ACC synthase or ACC oxidase activity and are therefore considered functional.
  • genetic sequences is used herein in its most general sense and encompasses any contiguous series of nucleotide bases specifying directly, or via a complementary series of bases, a sequence of amino acids comprising an ACC synthase or ACC oxidase molecule including a polypeptide or protein having ACC synthase or ACC oxidase activity.
  • a sequence of amino acids may constitute a full-length ACC synthase such as is set forth in, for example, SEQ ID NO:3 or a truncated form thereof or a mutant, derivative, part, fragment, homologue or analogue thereof.
  • the amino acid sequence may comprise part of, for example, these sequences or all or part of the sequences set forth in SEQ ID NO:3, as can be seen in SEQ ID NO:4.
  • the amino acid sequence may alternatively constitute ACC oxidase as set forth in SEQ ID NO:7.
  • the present invention encompasses nucleic acid molecules encoding the above-mentioned amino acid sequences as well as nucleic acid molecules encoding amino acid sequences having at least about 60%, more preferably about 70%, even more preferably about 80%, and still more preferably about 90%, or above, similarity to the amino acid sequences set forth in either SEQ ID NO:3 or SEQ ID NO:7.
  • a nucleic acid molecule encoding, or complementary to the sequence encoding, ACC synthase or ACC oxidase may be introduced into and expressed in a transgenic carnation, thereby providing a means whereby the production of climacteric ethylene by flowers of the said plant may be reduced to below naturally-occurring levels. This allows the onset of flower senescence to be prevented or delayed and flowers to exhibit a prolonged vase life following harvest. Background information on antisense and sense suppression technologies can be found in US Patent Number 5,107,065 and in US Patent Numbers 5,034,323; 5,231,020 and 5,283,184, respectively.
  • the present invention provides a method for producing a transgenic flowering plant wherein the flowers exhibit reduced levels of ethylene production below non-transgenic levels, said method comprising introducing into a cell of a carnation plant, a genetic construct comprising a nucleic acid molecule encoding, or complementary to the sequence encoding, ACC synthase or ACC oxidase under conditions permitting the integration of said nucleic acid molecule into the plant's genome, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the transcription of the nucleic acid molecule into the ACC synthase-specific mRNA or ACC oxidase-specific mRNA and, if necessary, the further translation of the ACC synthase mRNA or ACC oxidase-specific mRNA into the enzyme ACC synthase or ACC oxidase.
  • the introduced genetic construct comprises a non-full-length segment of a nucleic acid molecule encoding ACC synthase or ACC oxidase.
  • This aspect of the present invention extends to flowers cut or otherwise severed from said transgenic plants, including parts of flowers and parts of transgenic plants carrying flowers or flower buds.
  • the present invention further extends to functionally-equivalent methods for achieving the production of a transgenic carnation plant and flowers therefrom exhibiting the said characteristics.
  • the present invention is exemplified by generation of transgenic carnation plants of the varieties Red Corso; Ember Rose; Crowley Sim; White Sim; Scania, containing introduced ACC synthase and/or ACC oxidase genetic sequences.
  • the use of these cultivars in no way limits the applicability of the invention described herein, and the results obtained from these transgenic cultivars are generally applicable to other carnation cultivars.
  • the transgenic carnation plant produces flowers which exhibit delayed senescence properties coincident with reduced levels of climacteric ethylene production. Consequently, the present invention extends to a transgenic carnation plant containing all or part of a nucleic acid molecule representing ACC synthase or ACC oxidase and/or any homologues or related forms thereof and in particular those transgenic plants which produce flowers exhibiting reduced ACC synthase- or ACC oxidase-specific mRNA and/or reduced ACC synthase or ACC oxidase levels and/or reduced ethylene production and/or delayed senescence properties.
  • the transgenic plants therefore, contain a stably-introduced nucleic acid molecule comprising a nucleotide sequence encoding the ACC synthase or ACC oxidase enzyme.
  • the invention extends to flowers cut from such transgenic plants and to seeds derived from same.
  • Another aspect of the present invention is directed to a prokaryotic or eukaryotic organism carrying a genetic sequence encoding an ACC synthase or ACC oxidase extrachromasomally in plasmid form.
  • the plasmid is pWTT2160 mAgrobacterium tumefaciens.
  • the plasmid is pCGP407 in Escherichia coli.
  • microorganisms Escherichia coli strain XL 1 -Blue and Agrobacterium tumefaciens strain EHA101 containing the plasmids pCGP407 and pWTT2160, respectively, were deposited with the Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, New South Wales, 2037, Australia on May 1, 1995 under Accession Numbers N95/26121 and N95/26122, respectively.
  • Figure 1 is an alignment of nucleotide sequences for ACC synthase-encoding cDNAs from a variety of species. Carnation sequences from cultivars White Sim and Scania are compared with sequences from petunia (EMBL accession number Z 18952); tomato (van der Straeten et al., 1990); orchid (Genbank accession number L07882); Arabidopsis thaliana (Liang et al, 1992) and zucchini (Sato et al, 1991). Alignments were performed for the coding regions of the sequences using the Clustal V programme of Higgins et al. , 1991. Translation initiation and termination codons are underlined. Asterisks indicate conserved nucleotides.
  • FIG. 2 is a diagrammatic representation of the binary expression vector pWTT2160, construction of which is described in Example 4.
  • Tc resistance the tetracycline resistance gene
  • LB - left border the RB - right border
  • 5 «rB - the coding region and terminator sequences for the acetolactate synthase gene
  • 35S - the promoter region from the cauliflower mosaic virus 35S gene
  • nos 3' the terminator region from the Agrobacte ⁇ um tumefaciens nopaline synthase gene. Selected restriction enzyme sites are indicated.
  • Figure 3 is an alignment of nucleotide sequences for ACC oxidase-encoding cDNAs from a variety of plant species. Carnation sequences from cultivars Scania and hite Sim are compared with sequences from Arabidopsis tbaliana, tomato (Holdsworth et al., 1987; EMBL accession number X 04792); orchid (Nadeau et al., 1993; Genbank accession number L 07912); apple pong et al., 1992); petunia (Wang and Woodson, 1992); sunflower (Liu and Reid, unpublished; Genbank accession number L 29405) and geranium (Wang etal., 1994).
  • Arabidopsis tbaliana tomato (Holdsworth et al., 1987; EMBL accession number X 04792); orchid (Nadeau et al., 1993; Genbank accession number L 07912); apple pong et al., 1992); petunia (
  • Alignments were performed for the coding regions of the sequences using the Clustal V programme of Higgins et al., 1991. Translation initiation and termination codons are underlined. Asterisks indicate conserved nucleotides. Asterisks indicate conserved nucleotides.
  • Figure 4 is a diagrammatic representation of the binary expression vector pCGP407, construction of which is described in Example 8.
  • Gm the gentamycin resistance gene; RB - right border, LB - left border, car ACO - the nucleic acid molecule encoding carnation ACC oxidase: MAC - the mannopine synthase promoter enhanced with cauliflower mosaic virus 35S gene sequences; mas 3' - the terminator region from the Agrobacte ⁇ um tumefaciens mannopine synthase gene; 35S - the promoter region form the cauliflower mosaic virus 35S gene; NPT 13 « neomycin phosphotransf erase II; tml 3' - the tml terminator region, DNA sequences 11207-10069, from pT ; A6 (Barker et al., 1983). Selected restriction enzyme sites are indicated.
  • Figure 5 is an autoradiographic representation of a Southern hybridization of DNA isolated from leaf tissue from a number of different carnation cultivars, which had been transformed with a genetic construct (pWTT2160) containing the acetolactate synthase gene (ALS), as selectable marker, and an internal fragment of the nucleic acid molecule encoding ACC synthase.
  • Carnation genomic DNA was digested with EcoRI and the Southern blot was probed with a 32 P-labelled-760 base pair fragment derived from the ALS coding region. Filters were washed in 0.2 x SSC/1% w/v SDS at 65°C. Numbers 1-4 represent cultivars
  • N is non-transformed White Sim. Multiple bands in lanes 14 indicate where copies of DNA derived from pWTT2160 have been integrated into the genome of plants. No bands were detected in the non-transformed negative control.
  • Figure 6 is an autoradiographic representation of a Southern hybridization of DNA isolated from leaf tissue from the carnation cultivars White Sim and Scania, which had been transformed with a genetic construct (pCGP407) containing the neomycin phosphotransferase (NPT ⁇ ) gene as selectable marker, and a nucleic acid molecule defining ACC oxidase, in reverse orientation relative to the promoter.
  • pCGP407 containing the neomycin phosphotransferase (NPT ⁇ ) gene as selectable marker, and a nucleic acid molecule defining ACC oxidase, in reverse orientation relative to the promoter.
  • Carnation genomic DNA was digested with the restriction enzyme Hind HI.
  • the Southern blot was probed with a 32 P-labelled EcoRI DNA fragment from the coding sequence of the NPT II gene. Filters were washed in 0.1 x SSC, 0.1% w/v SDS at 65°C.
  • the bands indicate single or multiple copies of the DNA derived from pCGP407 have been integrated into the genome of the plants.
  • the Scania plant #705 shows 6 copies of the NPT II gene and White Sim plant #2373B, in lane 5, has a single copy of NPT II. No bands were detected in the non-transformed negative control. The size of the fragments detected is indicated in kilobases on the left-hand side of the figure.
  • Figure 7 is an autoradiographic representation of a Northern blot of RNA isolated from lateral shoot tissue from carnations transformed with pWTT2160. The control is non- transformed White Sim. Eight independent transgenic lines are shown. Filters were probed with a ⁇ P-labelled HindJ ⁇ . DNA fragment from the acetolactate synthase gene coding region, and washed for 30 min in 2 x SSC, 1% w/v SDS at 65°C, followed by 2 x 30 min in 0.2 x SSC, 1% w/v SDS at 65°C.
  • Figure 8 is an autoradiographic representation of a Northern blot of ACC oxidase mRNA and ACC oxidase antisense RNA isolated from petals.
  • Total RNA (lO ⁇ g/lane) was analysed from day 0 petals of control, non-transgenic White Sim (lane 1), transgenic Scania (lane 3) and transgenic White Sim (lane 5) flowers; and day 5 petals of control, non-transgenic White Sim (lane 2), transgenic Scania (lane 4) and transgenic White Sim (lane 6) flowers. Also analysed was total RNA isolated from transgenic Scania (lane 7), transgenic White Sim (lane 8) day 5 flowers which had been exposed to ethylene (150ppm) for the preceding 18 h.
  • Filters were hybridised with either a strand-specific antisense RNA probe, to detect ACC oxidase mRNA, or a strand-specific sense ACC oxidase RNA probe to dete ⁇ antisense ACC oxidase RNA, and washed in 2 x SSC/1% w/v SDS at 65°C for 1 hour followed by 0.2 x SSC/1% w/v SDS at 65°C for 1 hour and, in the case of antisense ACO, finally in 0.1 x SSC/0.1% w/v SDS at 65°C for 1 hour. Ribonuclease treatment was incorporated.
  • Figure 9 shows a graph of ethylene production in carnation flowers. Flowers of carnation cvs. Scania and White Sim were placed in a gas-tight chamber for three hours each day after harvest. The ethylene content of a gas sample taken from the chamber was measured using gas chromatography, as described in Example 19. Ethylene measurements are expressed as nanolitres of ethylene produced per gram of flower tissue (not including stem) per hour. Values for the control, non-transgenic flowers are the average of ethylene measurements from nine individual flowers. The transgenic Scania and White Sim values are averaged from 3 flowers each.
  • Figure 10(A)-10(F) is a black and white reproduction of colour photographic plates representing a:
  • the transgenic flower remains fresh at 11 days post-harvest, while the non-transgenic control has inrolled by day 4 and is completely senesced by 7 days post-harvest.
  • Original colour plates are available for inspection from the Applicant.
  • Figure 11(A)-11(F) is a black and white reproduction of colour photographic plates representing a: (A) non-transgenic control Red Corso flower, 0 days post-harvest; (B) non-transgenic control Red Corso flower, 7 days post-harvest; (C) non-transgenic control Red Corso flower, 9 days post-harvest;
  • the transgenic flower remains fresh at 9 days post-harvest, while the non-transgenic control has inrolled and completely senesced by 7 days post-harvest.
  • Original colour plates are available for inspection from the Applicant.
  • Figure 12(A)-12(F) is a black and white reproduction of colour photographic plates representing a:
  • Figure 13(A)-13(D) is a black and white reproduction of colour photographic plates representing a:
  • Figure 14(A)-14(C) is a black and white reproduction of colour photographic plates representing:
  • Figure 15 is a black and white reproduction of a colour photographic plate representing one non-transgenic control Scania flower (on the left of the photograph), and one antisense ACC oxidase transgenic Scania flower, taken at 6 days post-harvest. Vase life measurements were carried out in distilled water and under controlled light and temperature conditions. An original colour plate is available for inspection from the Applicant.
  • Figure 16 is a black and white reproduction of a colour photographic plate representing one non-transgenic control White Sim flower (on the right of the photograph), and one antisense ACC oxidase transgenic White Sim flower, taken at 8 days post-harvest. The flowers were kept in distilled water and under controlled light and temperature conditions following harvest. An original colour plate is available for inspection from the Applicant.
  • EXAMPLE 1
  • the cloning vector pBluescript II (KS+) was obtained from Stratagene.
  • the bacterial strains used were:
  • a carnation ACC synthase (ACS) cDNA clone from cv. White Sim was prepared using a reverse-transcriptase Polymerase Chain Reaction (PCR) method. PCR primers were synthesized based on highly-conserved regions occurring within the approximately 1,500 base pair (bp) coding sequence. An approximately 1,100 bp fragment was obtained after amplification.
  • the primer sequences employed were :
  • RNA was isolated from carnation cv. White Sim petals harvested at the fully open stage and then exposed to 1 part per million ethylene overnight to induce climacteric ethylene synthesis.
  • a standard phenol lysis method was used for the RNA isolation (Jones et al, 1985).
  • PolyA + RNA was prepared from the total RNA preparation using standard oligo(dT) cellulose chromatography (Aviv and Leder, 1972). The reverse-transcriptase reaction and subsequent PCR amplification were performed according to Ausubel et al. , 1992.
  • a fragment of the predicted size of approximately 1,100 bp was obtained after reverse- transcriptase-PCR of PolyA + RNA from ethylene-treated carnation flowers.
  • EXAMPLE S Sequence analysis of carnation cv. White Sim ACS cDNA clone The approximately 1,100 bp carnation ACS cDNA fragment was cloned into the vector pBluescript II (KS+) and the terminal nucleotides were sequenced using SEQ ID NO:l and SEQ ID NO:2 oligonucleotides as sequencing primers. DNA sequencing was performed essentially by the method of Sanger et al. (1977) using the Sequenase enzyme (USB, version 2.1), and showed this approximately 1,100 bp fragment to be part of the climacteric ACS gene of carnation, based on nucleotide sequence similarity to the sequence from Park et al. (1992). The full-length carnation ACS nucleotide sequence is presented as SEQ ID NO:3 and the approximately 1,100 bp internal fragment is presented as SEQ ID NO:4.
  • RNA was isolated from petunia cv. Old Glory Blue senescing flower petals which were producing greater than 5 nL ethylene/gram fresh weight/hour. A standard CsCl cushion method (Sambrook et al., 1989) was used for the RNA isolation. The reverse-transcriptase reaction and subsequent PCR amplification were performed according to Ausubel et al., 1992. A 1,380 bp fragment was obtained after 35 amplification cycles. Determination of the nucleotide sequence of the PCR product confirmed that it encoded a polypeptide similar to the deduced translation product of the corresponding region from tomato pcW4 A cDNA.
  • a cDNA library was constructed using mRNA from senescing carnation petals of the cv.
  • the cDNA was generated by oligo(dT) priming of PolyA + -enriched RNA using Maloney's Murine Leukaemia Virus Reverse Transcriptase (MMLV) (BRL).
  • MMLV Maloney's Murine Leukaemia Virus Reverse Transcriptase
  • the second strand of cDNA was produced with DNA Polymerase I ( lenow fragment), blunted, and linkers were added to create EcoRI- compatible ends.
  • This DNA was then size-selected on a S200 column (Pharmacia) and ligated into Lambda ZAP bacteriophage arms to create a library with 60,000 recombinant phage.
  • This library was amplified to provide a working stock (Sambrook et al. 1989).
  • a 1,380 bp petunia ACC synthase- encoding PCR fragment was 32 P-labelled and used to screen the 60,000 plaques of the senescing carnation cv. Scania petal cDNA library (Example 6c, above), under conditions of low stringency: the filters were hybridized in 50% formamide at 30°C, and washed for 30 min in 5 x SSC, 1% w/v SDS at room temperature, followed by 2 x 30 min in 5 x SSC, 1% w/v SDS at 42°C.
  • the Scania sequence is 133 bp shorter and contains several nucleotide differences, leading to three amino acid changes: serine to glycine at position 131; arginine to glycine at position 381; isoleucine to serine at position 500. It also contains an additional threonine at position 130.
  • the 1,100 bp carnation cv. White Sim ACS cDNA fragment (see Example 5) was inserted between a cauliflower mosaic virus 35S promoter/chlorophyll ab binding protein (Cab) 5' region and the nopaline synthase 3' region (Harpster et al., 1988).
  • the resulting fragment comprising a chimaeric, partial carnation ACS genetic sequence was inserted into T-DNA vectors containing a suitable selectable marker gene, such as one which comprises the 35S promoter together with the S «rB gene (tobacco acetolactate synthase) allowing selection of cMorsulfuron-resistant transformants.
  • a suitable selectable marker gene such as one which comprises the 35S promoter together with the S «rB gene (tobacco acetolactate synthase) allowing selection of cMorsulfuron-resistant transformants.
  • pWTT2160 One such resulting vector was given the designation pWTT2160,
  • EHA101 To transfer the binary vector pWTT2160 (see Figure 2) from E. coli to Agrobacte ⁇ um tumefaciens strain EHA101, the technique of triparental mating (Ditta et al., 1980) was used.
  • E. coli strain NE 47 containing the mobilizing plasmid pRK 2013 (Gutterson et al, 1986), was the helper strain.
  • the EHA101 strain was rifampicin-resistant (Hood et al., 1984), enabling transconjugants to be selected on LB-agar plates (Ausubel et al., 1992) containing 10 ⁇ g/mL gentamycin and lOO g/mL rifampicin at 28°C.
  • Dianthus caryopbyllus (cvs. Crowley Sim, Scania, Dark Pierrot, Ember Rose, Website, Mango, Monte Lisa, Red Corso, Tangerine, Valencia and Ashley) 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 min and rinsed three times with sterile water. All the visible leaves and axillary buds were removed under the dissecting microscope before co- cultivation.
  • stems grown in the greenhouse were harvested, surface-sterilized for 2 min in 75% v/v ethanol followed by 20% v/v commercial bleach + 0.1% v/v Tween-20 for 20 - 30 min, and rinsed three times in sterile water.
  • Agrobacte ⁇ um tumefaciens strain AGLO (Lazo et al, 1991), containing the binary vector pWTT2160, was maintained at 4°C on LB agar plates with 50 mg/L tetracycline. A single colony was grown overnight in liquid LB broth containing 50 mg/L tetracycline. The following day it was diluted to 5 x 10 8 cells/mL with liquid MS medium, before inoculation. Acetosyringone was added to the Agrobacterium suspension to a final concentration of 20 ⁇ M.
  • Dianthus stem tissue was co-cultivated with Agrobacterium for 5 days on MS medium supplemented with 3% w/v sucrose, 0.5 mg/L BAP, 0.5 mg/L 2,4-dichlorophenoxy- acetic acid (2,4-D), 100 ⁇ M acetosyringone and 0.25% w/v Gelrite (pH 5.7).
  • Agrobacterium tumefaciens strain EHA101 Hood et al., 1984
  • pWTT2160 binary vector pWTT2160
  • Bacterial concentration for inoculation of plant tissue was 0.5 - 1.0 x 10' cells/mL.
  • Acetosyringone was added to the Agrobacterium suspension to a final concentration of 20 ⁇ M.
  • Leaves of the cultivar White Sim were isolated by pulling from shoot cultures. For selection with chlorsulfuron it was advantageous to remove only the axillary meristems larger than 1 mm. Leaves were mixed with bacteria for a few minutes, then taken off the mixture and placed on a filter paper on a co-cultivation medium for 5 days.
  • the co-cultivation medium was the same as the shoot multiplication medium but contained 0.5 mg/L BAP and 0.5 mg/L 2,4-D instead of 1 mg/L BAP; 0.02 mg/L NAA, as well as lOO M acetosyringone. Plates were sealed with parafilm.
  • each co-cultivated stem was cut into 34 mm segments, which were then transferred to MS medium supplemented with 0.5 mg/L BAP, 0.5 mg/L 2,4-D, 1 ⁇ g/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite.
  • MS medium supplemented with 0.5 mg/L BAP, 0.5 mg/L 2,4-D, 1 ⁇ g/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite.
  • explants were transferred to fresh MS medium containing 0.16 mg/L thidiazuron (TDZ), 0.5 mg/L indolbutyric acid (EBA), 2 ⁇ g/L cUorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite and care was taken at this stage to remove axillary shoots from stem explants.
  • TDZ 0.16 mg/L thidiazuron
  • EBA ind
  • Suncaps (Sigma) were placed on top of the glass jars to speed up the normalization of shoots. All cultures were maintained under a 16 h photoperiod (120 ⁇ E/m 2 /s cool white fluorescent light) at 23 ⁇ 2°C. Normalized shoots, approximately 1.5-2 cm tall, were rooted on 3 g/kg IBA rooting powder and acclimatised under mist. A soil mix containing 75% perlite/25% peat was used for acclimation, which was carried out at 23 °C under a 14 hour photoperiod (200 ⁇ E/m 2 /s mercury halide light) and typically lasted 34 weeks. Plants were fertilized with a carnation mix containing lg/L CaN0 3 and 0.75 g/L of a mixture of microelements plus N:P:K in the ratio 4.7:3.5: 29.2.
  • leaves were transferred to a fresh medium consisting of MS medium supplemented with B5 vitamins; 590 mg/L MES: 0.5 mg/L BAP; 0.5 mg/L 2,4-D; 30g/L sucrose; 025 % w/v Gelrite; 500 mg/L carbenicillin and 2 ⁇ g/L chlorsulfuron, pH 5.8, for 2 weeks.
  • Leaf explants were then transferred to a regeneration medium consisting of MS salts supplemented with B5 vitamin; 590 mg/L MES 0.5 mg/L IBA; 0.22 mg/L TDZ; 30g/L sucrose; 0.25% w/v Gelrite; 500 mg/L carbenicillin and 3 ⁇ g/L chlorsulfuron, pH 5.8.
  • RNA was incubated at 100°C for 2 minutes and then cooled on ice for a further 2 minutes.
  • the RNA was added to a reaction mixture containing 20 g/ml oligo-dT, 50mM Tris-HCl pH 8.0, 75mM KCl, 30mM MgCl 2> lOmM DTT, 0.5 mg/mL actinomycinD, 200 M dATP, 200 xM dGTP, 200 *M dTTP, 2.5 M dCTP, 100 Ci [ ⁇ - 32 P]- dCTP (Bresatec, 3000Ci/mmol), 40 units ribonudease inhibitor (Promega), and 600 units MMLV reverse transcriptase (BRL) and incubated for 1 hour at 37°C.
  • 20 g/ml oligo-dT 50mM Tris-HCl pH 8.0, 75mM KCl, 30mM MgCl 2> lOmM DTT, 0.5 mg
  • EDTA and NaOH were added to a final concentration of 50mM and 0.2M, respectively and the mixture was incubated for 20 minutes at 70°C. The mixture was then neutralised by addition of HCl to a concentration of 0.2M. Unincorporated [ ⁇ - 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.
  • a cDNA library was constructed using mRNA from senescing carnation petals of the cv. Scania and the Lambda ZAP cDNA doning vector (Stratagene), as described in Example 6c, above.
  • a differential screening approach was used to isolate cDNA clones representing genes expressed in senescing carnation petals but reduced in flowers at the time of harvest. Thirty thousand colonies were screened at 1,500 colonies per 15cm plate.
  • Duplicate plaque lifts were hybridized with cDNA probes from either (i) day 0 petal or (ii) in rolling petal and washed under high stringency conditions: hybridization on nitrocellulose in 50% v/v formamide, 6 x SSC, 1% w/v SDS at 42°C for 16 h and washing in 0.2 x SSC, 1% w/v SDS at 65°C for 3 x 30 min. Filters were then exposed to Kodak XAR film with an intensifying screen at -70°C for 16 hours. Clones which hybridized with the in rolling petal cDNA, but not with the day 0 cDNA, were sdected for further investigation.
  • the deduced amino acid sequence of 321 amino acids shares 68% identity with the tomato ACO amino acid sequence (Holdsworth et al, 1987), 74.6% identity with apple ACO (Dong et al, 1992) and greater than 99% identity with the ACO sequence from another cultivar of carnation, White Sim (Wang and Woodson, 1991).
  • the Scania sequence differs from that of White Sim only at amino acid residue 147.
  • An alanine in the White Sim sequence is replaced by a glycine in the Scania sequence.
  • Vector pCGP407 was constructed using the standard techniques described in Sambrook et al. (1989). The carnation ACO cDNA fragment, contained within pCGP363 (see Example 12), was inserted in reverse orientation into a binary expression vector, pCGP293 (Brugliera et al., 1994), between the MAC promoter (Comai et al., 1990) and the mas 3' terminator region (from the Agrobacterium mannopine synthase gene). According to Comai et al. (1990), MAC is a strong constitutive promoter.
  • the binary vector pCGP407 contained the neomycin phosphotransferase (NPT II) gene, in addition to the antisense ACO nucleic acid molecule, allowing sdection of transgenic shoots by growth on kanamycin ( Figure 4).
  • NPT II neomycin phosphotransferase
  • Transformation of the Escherichia coli strain XLl-Blue with the vector pCGP407 was performed according to standard procedures (Sambrook et al., 1989) or Inoue et al., (1990).
  • the plasmid pCGP407 was introduced into Agrobacterium tumefaciens strain AGLO by adding 5 ⁇ g of plasmid DNA to 100 ⁇ L 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°C. The cells were then pelleted and resuspended in 0.5 mL of 85% v/v 100 mM CaCl/15% v/v glycerol. The DNA-Agrobacterium mixture was frozen by incubation in liquid N 2 for 2 min and then allowed to thaw by incubation at 37°C 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°C.
  • Cells of A. tumefaciens carrying pCGP407 were sdected on MG/L agar plates containing 100 ⁇ g/mL gentamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the gentamycin-resistant transformants.
  • Dianthus caryopbyllus (cvs. White Sim and Scania) cuttings were obtained from Van Wyk and Son Flower Supply, Victoria, Australia. The outer leaves were removed and the cuttings were sterilized briefly in 70% v/v ethanol followed by 1.25% w/v sodium hypochlorite (with Tween 20) for 6 minutes and rinsed three times with sterile water. All the visible leaves and axillary buds were removed under the dissecting microscope before co-cultivation.
  • Agrobacterium tumefaciens strain AGLO (Lazo et al., 1991), containing the binary vector pCGP407, was maintained at 4°C on LB agar plates with 50 mg/L tetracycline. A single colony was grown overnight in liquid LB broth containing 50 mg/L tetracycline. The following day it was diluted to 5 x 10" cells/mL with liquid MS medium, before inoculation.
  • Dianthus stem tissue was co-cultivated with Agrobacterium for 5 days on MS medium supplemented with 3% w/v sucrose, 0.5 mg/L BAP, 0.5 mg/L 2,4-D, 100 ⁇ M acetosyringone and 0.25% w/v Gelrite (pH 5.7).
  • the top 6-8 mm of each co-cultivated stem was cut into 3-4 mm segments, which were then transferred to MS medium supplemented with 1 mg/L BAP, 0.1 mg/L NAA, 150 mg/L kanamycin, 500 mg/L ticarcillin and 0.8% Difco Bacto Agar (selection medium).
  • MS medium supplemented with 1 mg/L BAP, 0.1 mg/L NAA, 150 mg/L kanamycin, 500 mg/L ticarcillin and 0.8% Difco Bacto Agar (selection medium).
  • explants were transferred to fresh selection medium and care was taken at this stage to remove axillary shoots from stem explants.
  • healthy adventitious shoots were transferred to hormone-free MS medium containing 3% w/v sucrose, 150 mg/L kanamycin, 500 mg/L ticarcillin, 0.8% Difco Bacto Agar.
  • NPT II dot-blot assay (McDonnell et al, 5 1987) was used to identify transgenic shoots. Transgenic shoots were transferred to MS medium supplemented with 3% w/v sucrose, 500 mg/L ticarcillin and 0.4% w/v Gelrite for shoot dongation. All cultures were maintained under a 16 hour photoperiod (120 E/m 2 /s cool white fluorescent light) at 23 ⁇ 2°C. 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) 0 soaked in hydroponic mix (Kandreck and Black, 1984) was used for acclimation, which typically lasted 4-5 weeks. Plants were acclimatised at 23 °C under a 14-hour photoperiod (200 ⁇ E/m 2 /s mercury halide light).
  • 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). 0 b. Southern Blots
  • the genomic DNA (10 ⁇ g) was digested with EcoRI (for sense ACS) or Hin ⁇ SlL (for antisense ACO) and dectrophoresed through a 0.7% w/v or 0.8% w/v, respectively, agarose gel in a running buffer of TAE (40 mM Tris-acetate, 50 mM EDTA).
  • the DNA was then denatured 5 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 by capillary transfer (Sambrook et al., 1989) in 20 x SSC.
  • Denaturing solution 1.5 M NaCl/0.5 M NaOH
  • Tris-HCl pH 7.5
  • 1.5 M NaCl 1.5 M NaCl
  • RNA samples were electrophoresed through 2.2 M formaldehyde/1.2% w/v agarose gels using running buffer containing 40 mM morpholino-propanesulphonic add (pH 7.0), 5 mM sodium acetate, 0.1 mM EDTA (pH 8.0).
  • the RNA was transferred to Hybond-N filters (Amersham) as described by the manufacturer and probed with 32 P-labelled cDNA fragment (10 s cpm/ g, 2 x 10 s cpm/mL).
  • DNA fragments (50 to 100 ng) were radioactivdy labelled with 50 Ci of [ ⁇ - 32 P]-dCTP using an oligolabelling kit (Bresatec). Unincorporated [ ⁇ - 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.
  • the genetic contracts contained in the plasmids pWTT2160 and pCGP407 were introduced into various varieties of carnation using Agrobacterium- ediatxz gene transfer, as described in Examples 10 and 15, above. Integration of the appropriate DNA into the plant genome was confirmed by Southern analysis of plants obtained after kanamycin or chlorsulfuron selection, as described in Example 16.
  • Plants successfully rendered transgenic, in accordance with the present invention have significantly reduced levels of climacteric ethylene production, compared with non- transgenic controls.
  • measurements of ethylene production, using a Varian modd 3300 gas chromatograph equipped with a Porapak* N column (80C), flame ionization detector and Varian 4400 Integrator indicated that flowers of carnation cvs. Scania and White Sim carrying the introduced antisense ACO genetic construct had a greatly reduced capadty to produce ethylene.
  • the graph in Figure 9 shows ethylene evolution by transgenic and control (non-transgenic) flowers from day of harvest onwards.
  • Control plants produced flowers which synthesized normal amounts of ethylene, showing the expected climacteric rise in ethylene production at the onset of inrolling.
  • Transgenic flowers of carnation cvs. Scania and White Sim produced less than 10% of the level of ethylene produced by control flowers.
  • Figure 10(A)-10(F) shows transgenic carnation flowers of the cultivar Scania at 0, 4, and 11 days post-harvest. Control non-transgenic flowers are shown at 0, 4 and 7 days post-harvest. The transgenic flower still looks fresh at 11 days, while the non-transgenic equivalent already shows petal in-rolling, typical of senescing carnation flowers, at 4 days post-harvest and is totally senesced by 7 days post-harvest.
  • Transgenic, "long-life" flowers of the carnation cv. White Sim have also been produced using the sense ACS approach, in accordance with the present invention, as may be seen in Figure 14(A)-14(C).
  • the non-transgenic control White Sim flower (on the left in each photograph) has begun to inroll and senesce by 11 days post-harvest and is completely senesced at 20 days post-harvest.
  • the three ACS sense-suppressed transgenic flowers appear as fresh as new at 11 days post-harvest and are still not in-rolling at 20 days post-harvest.
  • flowers from plants rendered transgenic using antisense ACO have also been produced for the carnation cultivars White Sim and Scania.
  • the level of ACO mRNA has been suppressed and, hence, climacteric ethylene production all but eliminated and carnation flower vase life correspondingly extended.
  • the normal vase life of these flowers is approximately 5 days from day of harvest to the beginning of inrolling.
  • Flowers from transgenic Scania and White Sim had a vase life of 8 to 9 days, after which the petals slowly discoloured and dessicated without displaying the inrolling behaviour typical of carnation flower senescence. All control plants produced flowers of normal senescence phenotype.
  • FIG. 15 shows a photograph of a transgenic, "long-life” White Sim flower next to a flower from a non-transgenic White Sim control plant, both at 8 days post-harvest. The transgenic flower still appears fresh while the control non-transgenic flower has completdy senesced.
  • ATC GCG GCA TTG CTC TCA GAT GAT GAT TTT GTT AGA CGA TTC TTG GTT 1129 lie Ala Ala Leu Leu Ser Asp Asp Asp Phe Val Arg Arg Phe Leu Val 320 325 330
  • AATATAATTC AATATAACTT CTCTGTAATT TCATGTATAC AAACACTATA AATATGTAGT 1884
  • GTA AAT GCG AAA AAT ATA CAC CTT GTG TGT GAC GAG ATA TAT GCA ACC 576 Val Asn Ala Lys Asn He His Leu Val Cys Asp Glu He Tyr Ala Thr 180 185 190
  • TGT CAC AAC TGG GGA TTC TTC CAG GTG GTG AAC CAT AGT TTG TCA CAT 199 Cys His Asn Trp Gly Phe Phe Gin Val Val Asn His Ser Leu Ser His 35 40 45

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Abstract

L'invention concerne généralement des plantes transgéniques présentant des caractéristiques de vie prolongée après leur récolte. Plus particulièrement, l'invention concerne des ÷illets transgéniques modifiés afin de limiter l'expression d'une ou plusieurs enzymes associées à la voie de biosynthèse de l'éthylène. Les fleurs de ces ÷illets ne produisent pas d'éthylène, ou produisent de l'éthylène en quantité limitée et sont, de ce fait, capables de survivre plus longtemps après la récolte que les fleurs d'÷illets naturels, non modifiés génétiquement.
EP96911869A 1995-05-09 1996-05-09 Illets transgeniques presentant une vie prolongee apres recolte Withdrawn EP0824591A4 (fr)

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AUPN2862A AUPN286295A0 (en) 1995-05-09 1995-05-09 Transgenic carnations exhibit prolonged post-harvest life
PCT/AU1996/000286 WO1996035792A1 (fr) 1995-05-09 1996-05-09 ×illets transgeniques presentant une vie prolongee apres recolte

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JP2004500886A (ja) * 2000-06-19 2004-01-15 セネスコ　テクノロジーズ，インコーポレイティド 植物リパーゼをコードするｄｎａ、遺伝子導入植物および植物における老化を制御するための方法
HUE035897T2 (en) 2003-06-23 2018-05-28 Pioneer Hi Bred Int Incorporating the green-capability of a single gene-controlled option into the plant

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WO1991001375A1 (fr) * 1989-07-14 1991-02-07 Imperial Chemical Industries Plc Structures d'adn, cellules et plantes derivees de celles-ci
WO1996007742A1 (fr) * 1994-09-02 1996-03-14 Asgrow Seed Company Plantes transgeniques exprimant les genes de l'oxydase de l'acide 1-aminocyclopropane-1-carboxylique
WO1996021027A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques exprimant le gene de l'acc synthase

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WO1991001375A1 (fr) * 1989-07-14 1991-02-07 Imperial Chemical Industries Plc Structures d'adn, cellules et plantes derivees de celles-ci
WO1996007742A1 (fr) * 1994-09-02 1996-03-14 Asgrow Seed Company Plantes transgeniques exprimant les genes de l'oxydase de l'acide 1-aminocyclopropane-1-carboxylique
WO1996021027A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques exprimant le gene de l'acc synthase

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BOVY,A.G., ET AL.: "genetic modification of the vase-life of carnation", ACTA HORTICULTURAE - SIXTH INTERNATIONAL SYMPOSIUM ON POSTHARVEST PHYSIOLOGY OF ORNAMENTAL PLANTS, vol. 405, 1995, pages 179 - 189, XP002096435 *
HENSKENS,J.A.M., ET AL.: "molecular cloning of two different ACC synthase PCR fragments in carnation flowers and organ-specific expression of the corresponding genes", PLANT MOLECULAR BIOLOGY, vol. 26, 1994, pages 453 - 458, XP002095863 *
MICHAEL M Z ET AL: "CLONING OF ETHYLENE BIOSYNTHETIC GENES INVOLVED IN PETA SENESCENCE OF CARNATION AND PETUNIA, AND THEIR ANTISENSE EXPRESSION IN TRANSGENIC PLANTS", CURRENT PLANT SCIENCE AND BIOTECHNOLOGY IN AGRICULTURE, vol. 16, 1993, pages 298 - 303, XP002014398 *
MICHAEL,M.Z.: "isolation of petal senescence-associated cDNA clones encoding 1-aminocyclopropane-1-carboxylate synthase from Petunia hybrida and Dianthus caryophyllus", EMBL SEQUENCE DATA LIBRARY, 9 December 1992 (1992-12-09), heidelberg, germany, XP002095861 *
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See also references of WO9635792A1 *

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