EP0824591A1 - Transgenic carnations exhibit prolonged post-harvest life - Google Patents

Abstract

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-occuring carnation plants.

Description

TRANSGENIC CARNATIONS EXHIBIT PROLONGED POST-HARVEST LIFE

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.

Bibliographic details of the publications referred to hereinafter in the specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs) referred to herein in relation to nucleotide and amino acid sequences are defined after the Bibliography.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The flower industry strives to develop new and different varieties of flowering plants, with improved characteristics ranging from disease and pathogen resistance to altered inflorescence and improved post-harvest cut-flower survival. Although classical breeding techniques have been used with some success, improvements in one characteristic are often achieved at the expense of one or more other important characteristics. Recombinant DNA technology provides a means whereby precise improvements are able to be made to one characteristic of a particular cultivar or cultivars, without altering any other commercially- valuable trait. Substantial effort has therefore been directed towards the exploitation of recombinant DNA technology to manipulate the genetic make-up of plants and generate transgenic plants which exhibit desirable characteristics or in which xmdesirable traits are suppressed. One of the characteristics most sought after by consumers of cut-flowers is a prolonged post-harvest vase life. The development of longer-living varieties of the major cut- flower species, including for example carnation, would offer a significant opportunity in a cut-flower market with retail sales in excess of US$25 billion.

Flower senescence is associated with the plant's production of ethylene. Ethylene is directly involved in plant growth and development and its production is strictly regulated. The pathway for ethylene biosynthesis in higher plants, as elucidated by Adams and Yang (1979), involves utilization of the endogenous pool of methionine to create S-adenosyl-methionine (SAM) by the enzyme SAM synthetase. SAM is a ubiquitous component of all living cells and is involved in a variety of metabolic processes. The initial step in ethylene biosynthesis occurs when SAM is converted to 1-aminocyclopropane-l-carboxylic acid (ACC) by the enzyme ACC synthase (ACS). This conversion is essential for ethylene production and often constitutes the rate-limiting step in the pathway. The final step is the subsequent conversion of ACC to ethylene by the enzyme ACC oxidase (ACO), also known as Ethylene Forming Enzyme (EFE). Additional information concerning ethylene biosynthesis may be found in a review by Kende (1993).

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.

As has been shown in tomato (Rottmann et al.; 1991) and Arabidopsis (Liang et al.; 1992), 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.

Furthermore, availability of isolated nucleic acid molecules encoding particular isozymes of the said enzymes permits the manufacture of genetic constructs which can be introduced into carnation plants and afford the possibility of regulating the production of ethylene in such a way as to produce flowers exhibiting a prolonged post-harvest vase life.

Accordingly, 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.

Preferably, the transgenic plant produced by the subject method exhibits one or more of the following properties:

(i) a reduction in production of ACC synthase-specific mRNA or ACC oxidase- specific mRNA;

(ii) a reduction in production of ACC synthase or ACC oxidase enzyme; and/or (iii) delayed senescence of flowers or flower buds cut from said transgenic plant.

In a related embodiment there is provided a method for producing a transgenic carnation plant, said method 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;

(ii) reduction in the production of ACC synthase or ACC oxidase enzyme; (iii) reduction in the production of climacteric ethylene; and/or

(iv) delayed senescence.

Even more particularly, 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.

By "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.

Preferably, the non-full-length fragment is approximately 800-1200 base-pair in length. Preferably, the non-full-length fragment is an internal fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase.

Preferably, 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.

By "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. Furthermore, 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. By "derivative" is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally-occurring enzyme. In this regard, the nucleic acid includes the naturally-occurring nucleotide sequence encoding 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;

(ii) reduction in the production of ACC synthase or ACC oxidase enzyme;

(iii) reduction in the production of climacteric ethylene; and/or

(iv) delayed senescence.

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. More particularly, 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:

(i) reduced levels of ACC synthase-specific mRNA or ACC oxidase below non- transgenic endogenous levels;

(ii) reduced levels of ACC synthase or ACC oxidase enzyme below non-transgenic endogenous levels; and/or

(iii) reduced levels of climacteric ethylene production below non-transgenic endogenous levels;

In a preferred embodiment of the present invention, there is provided a method for producing transgenic carnation plants, said method 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:

(i) reduction in the production of ACC synthase-specific mRNA or ACC oxidase- specific mRNA;

(ii) reduction in the production of ACC synthase or ACC oxidase enzyme; (iii) reduction in the production of climacteric ethylene; and/or

(iv) delayed senescence.

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.

More particularly, the flowers of the said transgenic plants exhibit one or more of the following properties:

(i) reduced levels of ACC synthase-specific mRNA or ACC oxidase-specific mRNA below non-transgenic endogenous levels; (ii) reduced levels of ACC synthase or ACC oxidase enzyme below non-transgenic endogenous levels;

(iii) reduced levels of climacteric ethylene production below non-transgenic endogenous levels; and/or (iv) delayed senescence.

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 Agrobacteήum- edizted transfer.

Accordingly, another aspect of the present invention provides an isolated 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. In one embodiment, such mutants may also be functional, meaning that they exhibit at least some ACC synthase or ACC oxidase activity. In all cases, 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. The expressions "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.

As used herein, the term "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. The term "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. The term "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.

The expression "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. Such 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. Alternatively, 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.

In accordance with the present invention, 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.

Accordingly, 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. Preferably, 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.

In a preferred embodiment, 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. In one embodiment, the plasmid is pWTT2160 mAgrobacterium tumefaciens. In a further embodiment, the plasmid is pCGP407 in Escherichia coli. The 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.

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

In the Figures:

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.

Figure 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; 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; car ACS - the nucleic acid molecule encoding carnation ACC synthase; nos 3' = the terminator region from the Agrobacteήum 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). 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 Agrobacteήum 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 ³²P-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

White Sim; Crowley Sim; Ember Rose and Scania, respectively. The negative control (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 π) 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 ³²P-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. In lane 2, 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 HindJΩ. 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 (lOμg/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 deteα 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:

(A) non-transgenic control Scania flower, 0 days post-harvest;

(B) non-transgenic control Scania flower, 4 days post-harvest;

(C) non-transgenic control Scania flower, 7 days post-harvest;

(D) transgenic ACC synthase sense-suppressed Scania flower, 0 days post-harvest; (E) transgenic ACC synthase sense-suppressed Scania flower, 4 days post-harvest; and (F) transgenic ACC synthase sense-suppressed Scania flower, 11 days post-harvest.

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;

(D) transgenic ACC synthase sense-suppressed Red Corso flower, 0 days post-harvest;

(E) transgenic ACC synthase sense-suppressed Red Corso flower, 7 days post-harvest; and

(F) transgenic ACC synthase sense-suppressed 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:

(A) non-transgenic control Ember Rose flower, 0 days post-harvest;

(B) non-transgenic control Ember Rose flower, 4 days post-harvest;

(C) non-transgenic control Ember Rose flower, 7 days post-harvest; (D) transgenic ACC synthase sense-suppressed Ember Rose flower, 0 days post-harvest;

(E) transgenic ACC synthase sense-suppressed Ember Rose flower, 4 days post-harvest; and

(F) transgenic ACC synthase sense-suppressed Ember Rose flower, 7 days post-harvest. Original colour plates are available for inspection from the Applicant.

Figure 13(A)-13(D) is a black and white reproduction of colour photographic plates representing a:

(A) non-transgenic control Crowley Sim flower, 0 days post-harvest;

(B) non-transgenic control Crowley Sim flower, 4 days post-harvest;

(C) transgenic ACC synthase sense-suppressed Crowley Sim flower, 0 days post-harvest; and (D) transgenic ACC synthase sense-suppressed Crowley Sim flower, 4 days post-harvest.

Original colour plates are available for inspection from the Applicant. Figure 14(A)-14(C) is a black and white reproduction of colour photographic plates representing:

(A) one non-transgenic control White Sim flower (on the left of the photograph), and three ACC synthase sense-suppressed transgenic flowers at 0 days post-harvest; (B) one non-transgenic control White Sim flower (on the left of the photograph), and three

ACC synthase sense-suppressed transgenic flowers at 11 days post-harvest; and (C) one non-transgenic control White Sim flower (on the left of the photograph), and three ACC synthase sense-suppressed transgenic flowers at 20 days post-harvest.

All flowers were kept in distilled water and under controlled light and temperature conditions following harvest. The non-transgenic control flower has inrolled and is senescing by 11 days post-harvest and is completely senesced by 20 days post-harvest, while the control flowers remain fresh at 20 days post-harvest. Original colour plates are available for inspection from the Applicant.

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

Biological Reagents

All restriction enzymes and other reagents were obtained from commercial sources and used generally according to the manufacturer's recommendations.

The cloning vector pBluescript II (KS+) was obtained from Stratagene.

EXAMPLE 2 Bacterial Strains

The bacterial strains used were:

Escherichia coli :

XLl-Blue supE44, hsdR17 (r_k-, m_k+), recAl, ≤ndAl, gy_rA96 (Nal¹), ϊhi-1, relAl, lac-, [F'prαAB, lacjj, lacZΔM15, TnlO(tef)] (Bullock et al.,19%7).

DH5α su_PE44 Δ(kcZYA-ΔrgF)U169 ø80dlacZΔM15 hsdB_lZ(r_k-, m_k+), recAl. endAl_r gyτA96 (Nal¹), thi-1. relAl. deoR (Hanahan, 1983 and BRL, 1986). JM 83 F ιaA(lac-proAB) ipsL (Str*)[ø80dΔ(lacZ)M15] (Vieira and Messing, 1982) JM 109 F'traD36 lac !^■» Δ( cZ M15., prΩΔ⁺E⁺/el4(McrA) A( c-proAIi) ϊhi gyrA96 (Nal^r) endAl hsdR17 (r_k-, m_k+) relAl ≤upE recAl

(Yanisch-Perron et^/., 1985)

Agrobacteήum tumefaciens : AGLO Lzzo etal. (1991) EHA101 Hood et al. (1984)

EXAMPLE 3 Growth Conditions

Unless otherwise stated, plants were grown in specialised growth rooms with a 14 h day length at a light intensity of 10,000 lux minimum and a temperature of 22 to 26°C. EXAMPLE 4 Isolation of a carnation ACC synthase (ACS) clone from cv. White Sim a. Polymerase Chain Reaction Primers

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 :

5' ATGGGT(C/T)TNGCNGAAAATCAGC 3' SEQ ID NO:l 5' A(G/A)CANACNCG(A/G)AACCANCCNGG 3' SEQ ID NO:2

b. Isolation of an ACS clone from carnation flowers

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.

EXAMPLE 6 Isolation of a carnation ACC synthase (ACS) clone from cv. Scania

An alternative approach was used to isolate another ACS cDNA clone, this time from the cultivar Scania.

a. Polymerase Chain Reaction Primers A petunia ACC synthase cDNA fragment from cv. Old Glory Blue was prepared using PCR. Primers were synthesized based on known coding sequence from the tomato ACS cDNA, pcW4A, of van der Straeten et al. (1990). The primer sequences employed were:

5' CGGGATCCGCTACTAATGAAGAGCATGGC 3' SEQ ID NO:5 5' GCGGTACCAGGTGACGAAAGTGGTGACA 3' SEQ ID NO:6

b. Isolation of an A CS clone from petunia flowers

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.

c. Construction of a carnation cv. Scania cDNA library

A cDNA library was constructed using mRNA from senescing carnation petals of the cv.

Scania and the Lambda ZAP cDNA cloning vector (Stratagene). The cDNA was generated by oligo(dT) priming of PolyA⁺-enriched RNA using Maloney's Murine Leukaemia Virus Reverse Transcriptase (MMLV) (BRL). 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).

d. Heterologous screening of carnation cDNA library

A 1,380 bp petunia ACC synthase- encoding PCR fragment was ³²P-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.

From the heterologous screening, 10 cDNA clones were isolated. Analysis of five of these clones showed that they all represented the same gene. The longest of the clones contained an insert of approximately 1,820 bp.

EXAMPLE 7 Sequence analysis of carnation cv. Scania ACS cDNA clone

The longest clone, approximately 1,820 bp, was sequenced on both strands. It was found to be 99.6% similar to the nucleotide sequence of the cDNA encoding ACC synthase from carnation cv. White Sim, isolated by Park et al. (1992) (see Example 5, above). 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.

Homology searches against Genbank, SWISS-PROT and EMBL databases were performed using the FASTA and LFASTA programmes (Pearson and Lipman, 1988). Alignment and comparison of the carnation cv.s White Sim and Scania ACC synthase sequences with five other sequences as follows: petunia; tomato; orchid; arabidopsis; zucchini, can be seen in Figure 1. Alignments were performed using the Clustal V programme (Higgins and Sharp, 1989; Higgins et al, 1991). Percentage similarities ranged from 99.6%, between the carnation cultivars, to 65.1% between carnation and zucchini.

EXAMPLE 8 Construction of pWTT2160

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. One such resulting vector was given the designation pWTT2160, and is shown in Figure 2.

EXAMPLE 9

Transformation of E. coli and A. tumefaciens with pWTT2160 Escherichia coli strains JM 83 (Vieira and Messing, 1982) and JM 109 (Yanisch-Perron et al., 1985), used for routine manipulations, were transformed according to standard procedures (Sambrook etal, 1989) .

To transfer the binary vector pWTT2160 (see Figure 2) from E. coli to Agrobacteήum 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 μg/mL gentamycin and lOO g/mL rifampicin at 28°C. EXAMPLE 10 Transformation of Dianthus caryopbyllus with partial ACC synthase sequence a. Plant Material

Dianthus caryopbyllus (cvs. Crowley Sim, Scania, Dark Pierrot, Ember Rose, Laguna, 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.

For cv. White Sim, 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. Shoot tip meristems were isolated, nodes of approximately 1 cm in length were cut from the stem, and both were cultured, at a density of 10-12/standard Petri dish, on a shoot multiplication medium consisting of Murashige and Skoog's (1962) medium (MS) supplemented with B5 vitamins (Gamborg et al, 1968); 590 mg/L 2-[N-morpholino] ethane sulphonate (MES); 1 mg/L benzylaminopurine (BAP); 0.02 mg/L α-naphthalene acetic acid (NAA); 30g/L sucrose; 0.25 % w/v Gelrite Gellan Gum (SchweizerhaU), pH 5.8. All phytohormones were added after autoclaving. Cultures were incubated in a growth chamber with a 16-hour photoperiod (—30 μE/m²/s) at 24 ± 1° C. The light source was always above the cultures, as heat from light below caused condensation and resulted in poor regeneration and multiplication. Each meristem produced a few vitrified shoots within two weeks. These were excised and sub- cultured monthly on fresh shoot multiplication medium. After 34 sub-cultures, shoot cultures which multiplied at a high rate were established; i.e.: each shoot with 34 leaves produced a cluster of shoots with a total of 20-25 leaves within a month. These were used routinely as a source of leaf explants for transformation. b. Co-cultivation of Agrobacterium and Dianthus Tissue

Agrobacteήum 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 10⁸ cells/mL with liquid MS medium, before inoculation. Acetosyringone was added to the Agrobacterium suspension to a final concentration of 20μM. 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 μM acetosyringone and 0.25% w/v Gelrite (pH 5.7).

For co-cultivation with the Dianthus cultivar White Sim, Agrobacterium tumefaciens strain EHA101 (Hood et al., 1984) containing the binary vector pWTT2160 was taken from frozen samples in glycerol, cultured for 2 days at 28°C in the dark on solid L-broth (Ausubel et al., 1992) containing the appropriate antibiotics for selection, and suspended overnight in liquid MinA (Ausubel et al. , 1992) for inoculation. 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 20μM.

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.

c. Recovery of Transgenic Dianthus Plants

For selection of transformed stem tissue, the top 6-8 mm of 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 μg/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite. After 2 weeks, explants were transferred to fresh MS medium containing 0.16 mg/L thidiazuron (TDZ), 0.5 mg/L indolbutyric acid (EBA), 2 μg/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. After 3 weeks, healthy adventitious shoots were transferred to hormone-free MS medium containing 3% w/v sucrose, 3 μg/L chlorsulfuron, 500 mg/L ticarcillin, 025% w/v Gelrite. Shoots which survived 3 μg/L chlorsulfuron were transferred to MS medium supplemented with 3% w/v sucrose, 500 mg/L ticarcillin, 5 μg/L chlorsulfuron and 0.25% w/v Gelrite for shoot elongation.

After 2-3 weeks, leaves were pulled from the shoots which had survived selection and were placed on a regeneration medium consisting of MS medium supplemented with 0.22 mg/L TDZ, 0.5 mg/L IBA, 3 μζ L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite, to obtain shoot regeneration in the presence of selection. Regenerated shoots were transferred to hormone-free MS medium containing 5 g/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite for 24 weeks, then to hormone-free MS medium containing 200 mg/L ticarcillin and 0.4% w/v Gelrite, in glass jars, for normalization. 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 μE/m²/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 μE/m²/s mercury halide light) and typically lasted 34 weeks. Plants were fertilized with a carnation mix containing lg/L CaN0₃and 0.75 g/L of a mixture of microelements plus N:P:K in the ratio 4.7:3.5: 29.2.

For selection of transformed leaf tissue, 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 μg/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 μg/L chlorsulfuron, pH 5.8. If small shoot clusters had formed after 2-3 weeks, they were separated into 24 sections. After another three weeks, regenerated shoots were harvested; leaves of the regenerated shoots were pulled apart and plated on fresh regeneration medium to undergo secondary regeneration. Transformed, vitrified shoots regenerated from the leaves within three weeks. To normalize, they were transferred to hormone-free MS medium containing 1% TC agar and 3 g/L chlorsulfuron and cultured for three weeks in plates and for an additional three weeks in Magenta™ GA-7 cubes. Within 2-3 weeks normal shoots formed and were rooted in hormone-free MS medium containing 0.2% w/v Gelrite. Rooted plants were transferred to soil, hardened off gradually, and then transferred to greenhouse conditions.

EXAMPLE 11 Isolation of carnation ACC oxidase (ACO) clone from cv. Scania a. Preparation of ³²P-labelled probes

Twenty micrograms of total 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 [α-³²P]- dCTP (Bresatec, 3000Ci/mmol), 40 units ribonudease inhibitor (Promega), and 600 units MMLV reverse transcriptase (BRL) and incubated for 1 hour at 37°C. 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 [α-³²P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

b. "P-Labelling of DNA fragments

DNA fragments (50 to 100 ng) were radioactivdy labelled with 50 μCi of [α-³²P dCTP using an oligolabelling kit (Bresatec). Unincorporated [α-³²P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column. c. Differential Screening of carnation cv. Scania cDNA library

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.

EXAMPLE 12 Sequence analysis of carnation cv. Scania ACO cDNA done

Several senescence-associated cDNA clones were identified. The DNA sequence of one of the clones, a 1,156 bp sequence designated pCGP363, had 68% homology to the DNA sequence of a tomato cDNA clone, pTOM13, associated with ethylene production and fruit ripening. Later, pTOM13 was identified as encoding ACC oxidase (Hamilton et al, 1991; Holdsworth etcd., 1987; Spanu et al, 1991). 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.

DNA sequencing of this and other clones was performed essentially by the method of Sanger et al. (1977) using the Sequenase enzyme (USB, version 2.1). The 1,156 bp carnation cv. Scania ACO sequence is presented as SEQ ID NO:7. Homology searches against Genbank, SWISS-PROT and EMBL databases were again performed using the FASTA and LFASTA programmes (Pearson and Lipman, 1988). Alignment and comparison of the carnation cv. Scania ACC oxidase sequence with eight other sequences as follows: carnation cv. White Sim; Arabidopsis thaliana; tomato; orchid; apple; petunia; sunflower and geranium, can be seen in Figure 3. Alignments were performed using the Clustal V programme (Higgins et al, 1991). Percentage similarities ranged from 95%, between carnation cultivars, to 72 % between carnation and for geranium.

EXAMPLE 13 Construction of pCGP 407

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).

EXAMPLE 14

Transformation of E. coli and A. tumefaciens with pCGP407

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 μg of plasmid DNA to 100 μL 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 N₂for 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 μg/mL gentamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the gentamycin-resistant transformants.

EXAMPLE 15 Transformation of Dianthus caryopbyllus with ACC oxidase

a. Plant Material

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.

b. Co-cultivation of Agrobacterium and Dianthus Tissue

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 μM acetosyringone and 0.25% w/v Gelrite (pH 5.7).

c. Recovery of Transgenic Dianthus Plants

For sdection of transformed stem tissue, 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). 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 sdection medium 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. At this stage, 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/m²/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 μE/m²/s mercury halide light).

EXAMPLE 16 5 Southern Analysis a. Isolation of Genomic DNA from Dianthus

DNA was isolated from tissue essentially as described by Dellaporta et al. , (1983) . The DNA preparations were further purified by CsCl buoyant density centrifugation (Sambrook et al, 1989). 0 b. Southern Blots

The genomic DNA (10 μg) was digested with EcoRI (for sense ACS) or HinάSlL (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. Southern analysis of putative transgenic Dianthus plants obtained after selection on either chlorsulfuron or kanamycin confirmed the integration of the appropriate chimaeric gene into the genome, as shown in Figures 5 and 6.

5 EXAMPLE 17

Northern Analysis

Total RNA was isolated from tissue that had been frozen in liquid N₂ and ground to a fine powder using a mortar and pestle. An extraction buffer of 4 M guanidinium isothiocyanate, 50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.1% v/v Sarkosyl, was added to the tissue and

10 the mixture was homogenized for 1 minute using a polytron at maximum speed. The suspension was filtered through Miradoth (Calbiochem) and centrifuged in a J A20 rotor for 10 minutes at 10,000 rpm. The supernatant was collected and made to 0.2 g/ mL CsCl w/v. Samples were then layered, over a 10 mL cushion of 5.7 M CsCl, 50 mM EDTA (pH 7.0) in 38.5 mL Quick-seal centrifuge tubes (Beckman) and centrifuged at 42,000 rpm for 12-16

15 hours at 23°C in a Ti-70 rotor. Pellets were resuspended in TE/SDS (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% w/v SDS) and extracted with phenol:chloroform:isoamyl alcohol (25:24:1) saturated in 10 mM EDTA (pH 7.5). The RNA was then maintained as an ethanol precipitate, and appropriate aliquots pelleted prior to use.

20 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 ³²P-labelled cDNA fragment (10^s cpm/ g, 2 x 10^s cpm/mL). Prehybridization (1 h at 42°C) and hybridization (16 h at

25 42°C) was carried out in 50% v/v formamide, 1 M NaCl, 1% w/v SDS, 10% w/v dextran sulphate, 100 μg/mL salmon sperm DNA.

Filters were washed in 2 x SSC/1% w/v SDS at 65°C for 1 hour and then 0.2 x SSC/1% w/v

SDS at 65°C for 1 hour. In the case of antisense ACO, however, filters were also washed in

30 0.1 x SSC/0.1% w/v SDS at 65°C for 1 hour. All filters were exposed to Kodak XAR film with an intensifying screen at -70°C for 48 hours.

Northern analysis of sense ACS plants indicated that the ALS transgene was expressed in the leaves of six of the eight lines assayed (see Figure 7).

Northern analysis of antisense ACO plants indicated that petals from transgenic Scania and White Sim flowers produce only very low levels of ACO and ACS mRNA at days 4 to 6, the time when inrolling would occur in normal, control flowers (see Figure 8).

EXAMPLE 18

^»P-Labelling of DNA Probes

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

EXAMPLE 19 Transformation of Dianthus cultivars

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. For example, 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.

EXAMPLE 20 Prolonged post-harvest survival

The introduction of one or more additional copies of either the ACC synthase or ACC oxidase DNA sequences into a plant's genome is capable of having a marked effect on the post-harvest life of the cut-flower. It has been possible to suppress the expression of the endogenous gene, using άther a sense transcript and the co-suppression technology disclosed in US Patent Numbers 5,034,323; 5,231,020 and 5238,184, or an antisense transcript and the antisense technology disdosed in US Patent Number 5,107,065, thereby generating transformed carnation flowers which produce significantly reduced levels of climacteric ethylene. These flowers exhibit post-harvest survival times often in excess of twice the normal vase-life of their non-transformed equivalents, and in the absence of the usual treatment with chemicals such as the environmentally-toxic silver thiosulphate.

Exemplification of the "long-life" phenotype, using the sense ACS approach, is shown in Figures 10(A)-10(F), 11(A)-11(F), 12(A)-12(F), and 13(A)-13(D).

All flowers were kept in water and under 12h day/night cyde in controlled conditions, (1000 lux, 22°C, 65% rdative humidity) following harvest. 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. Comparable results have been obtained for the cultivars Red Corso; Ember Rose and Crowley Sim, as seen in Figures 11(A)-11(F), 12(A)-12(F), and 13(A)-13(D), respectivdy. In each case, the transgenic carnation flower appears fresher for longer, when compared with the non-transgenic control.

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. By contrast, 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.

Furthermore, 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. A transgenic, "long-life" flower of Scania, compared with a non-transgenic control flower at 6 days post-harvest, can be seen in Figure 15. Figure 16 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.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ALLRAD NO.1 PTY LTD and FLORIGENE INVESTMENTS PTY LTD

(ii) TITLE OF INVENTION: TRANSGENIC CARNATIONS EXHIBIT PROLONGED POST-HARVEST LIFE

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVIES COLLISON CAVE

(B) STREET: 1 LITTLE COLLINS STREET

(C) CITY: MELBOURNE

(D) STATE: VICTORIA

(E) COUNTRY: AUSTRALIA

(F) ZIP: 3000

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT INTERNATIONAL

(B) FILING DATE: 09-MAY-1996

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: PN2862 (AU)

(B) FILING DATE: 09-MAY-1995

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HUGHES DR, E JOHN L

(C) REFERENCE DOCKET NUMBER: EJH EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 92542777

(B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

ATGGGT(C/T)TNG CNGAAAATCA GC 22

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

A(G/A)CANACNCG (A/G)AACCANCCN GG 22

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1942 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 134..1684

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGTCTTAATC TTTGTCACTT TACAACAACA TAAATTATAT TCTCAATCAT TTTCTCTATA 60 TATATATATA TACCCTCCAT TTTTCCTACT CCCCCTCCAC AAAAAATATA ATAATAGTGA 120 GTGTGTAATC AAA ATG GGT TCT TAT AAG GGT GTT TAC GAC CGT GAA ATT 169 Met Gly Ser Tyr Lys Gly Val Tyr Asp Arg Glu lie 1 5 10

CTT TCA AAA ATC GCT ACG AAC GAT GGC CAT GGT GAG AAT TTG GAG TAC 217 Leu Ser Lys lie Ala Thr Asn Asp Gly His Gly Glu Asn Leu Glu Tyr 15 20 25

TTT GAT GGG TGG AAA GCT TAT GAT AGA GAT CCT TAT CAT TCT ACC AAG 265 Phe Asp Gly Trp Lys Ala Tyr Asp Arg Asp Pro Tyr His Ser Thr Lys 30 35 40

AAT TCT AAT GGC GTT ATT CAA ATG GGT CTC GCT GAA AAT CAG CTT TGC 313 Asn Ser Asn Gly Val lie Gin Met Gly Leu Ala Glu Asn Gin Leu Cys 45 50 55 60

TTC GAT TTA GTT ACG GAG TGG CTA CTC AAA AAC CCA CAA GCC TCA ATT 361 Phe Asp Leu Val Thr Glu Trp Leu Leu Lys Asn Pro Gin Ala Ser lie 65 70 75

TGT ACC AAC GAA GGT GTA AAT AAG TTC ATG GAT ATT GCC ATT TTT CAG 409 Cys Thr Asn Glu Gly Val Asn Lys Phe Met Asp lie Ala lie Phe Gin 80 85 90

GAT TAT CAT GGT TTG CCC GAG TTT AGA AGT GCT GTG GCA AAA TTT ATG 457 Asp Tyr His Gly Leu Pro Glu Phe Arg Ser Ala Val Ala Lys Phe Met 95 100 105

GGG AAG GCA AGA GAT GAG AAA GTC ATA TTC AAT CCA GAT AGA ATT GTA 505 Gly Lys Ala Arg Asp Glu Lys Val lie Phe Asn Pro Asp Arg lie Val 110 115 120

ATG AGT GGT GGA GCC AGT GCA AGT GAA ACT CTT TTG TTT TGC TTG GCC 553 Met Ser Gly Gly Ala Ser Ala Ser Glu Thr Leu Leu Phe Cys Leu Ala 125 130 135 140

AAC CCC GGT GAC GCC TTT TTA ATT CCG TCT CCT TAT TAT CCC GCA TTT 601 Asn Pro Gly Asp Ala Phe Leu lie Pro Ser Pro Tyr Tyr Pro Ala Phe 145 150 155

AAC CGC GAT TTA CGG TGG AGA ACT GGA GTA AAT TTA ATC CCA TTT ACT 649 Asn Arg Asp Leu Arg Trp Arg Thr Gly Val Asn Leu lie Pro Phe Thr 160 165 170

TGC TCG AGC TCG AAT AAT TTC AAA ATC ACT AAG GAA GCC TTA CAA TCG 697 Cys Ser Ser Ser Asn Asn Phe Lys lie Thr Lys Glu Ala Leu Gin Ser 175 180 185

GCA TAT GAA GAC GCC CTT AAA AAG AAC ATC AAA GTT AAG GGT ATT ATC 745 Ala Tyr Glu Asp Ala Leu Lys Lys Asn lie Lys Val Lys Gly lie lie 190 195 200

GTC ACA AAC CCG TCA AAT CCC TTA GGA ACG GTC CTA GAC AAG GAC ACC 793 Val Thr Asn Pro Ser Asn Pro Leu Gly Thr Val Leu Asp Lys Asp Thr 205 210 215 220 CTA AAA ATG TTA TTA ACA TTC GTA AAT GCG AAA AAT ATA CAC CTT GTG 841 Leu Lys Met Leu Leu Thr Phe Val Asn Ala Lys Asn lie His Leu Val 225 230 235

TGT GAC GAG ATA TAT GCA ACC ACA GTA TTT AAT TCG CCG AGC TTT ATA 889 Cys Asp Glu lie Tyr Ala Thr Thr Val Phe Asn Ser Pro Ser Phe lie 240 245 250

AGT GTT GCT GAG GTT ATA AAG GAC ATG CCT CAT GTA AAT CAA GAC CTT 937 Ser Val Ala Glu Val lie Lys Asp Met Pro His Val Asn Gin Asp Leu 255 260 265

GTT CAT ATT TTA TAT AGT TTG TCC AAG GAC ATG GGC ATG CCG GGC TTT 985 Val His lie Leu Tyr Ser Leu Ser Lys Asp Met Gly Met Pro Gly Phe 270 275 280

AGG GTT GGG ATC ATT TAC TCT TAT AAT GAC CGT GTC GTC TCA ACT GCT 1033 Arg Val Gly lie lie Tyr Ser Tyr Asn Asp Arg Val Val Ser Thr Ala 285 290 295 300

CGT CGA ATG TCG AGT TTT GGA CTT GTT TCT TCT CAA ACT CAG TTT ATG 1081 Arg Arg Met Ser Ser Phe Gly Leu Val Ser Ser Gin Thr Gin Phe Met 305 310 315

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

GAG AGT AGA GAC AGA CTC TTT CGA AGG CAC CAG CAT TTC ACA AGC GAG 1177 Glu Ser Arg Asp Arg Leu Phe Arg Arg His Gin His Phe Thr Ser Glu 335 340 345

CTG GCT AAG ATA GGA ATA GGA TGC CTC CAA GGA AAC GCG GCA TTG TTT 1225 Leu Ala Lys lie Gly lie Gly Cys Leu Gin Gly Asn Ala Ala Leu Phe 350 355 360

GTT TGG ATG GAT TTG AGG CAT CTA TTA GAC GAA GCA ACG GTT GAA AGA 1273 Val Trp Met Asp Leu Arg His Leu Leu Asp Glu Ala Thr Val Glu Arg 365 370 375 380

GAG TTA AAG TTA TGG AGA GTG ATC ATC AAT GAA GTG AAA ATC AAT GTG 1321 Glu Leu Lys Leu Trp Arg Val lie lie Asn Glu Val Lys lie Asn Val 385 390 395

TCA CCG GGT TCG TCC TTC CTG TGC TCT GAG CCA GGG TGG TTT AGG GTT 1369 Ser Pro Gly Ser Ser Phe Leu Cys Ser Glu Pro Gly Trp Phe Arg Val 400 405 410

TGC TTT GCC AAC ATG GAC AAT GCG ACC TTA GAC GTT GCA CTC AAT CGA 1417 Cys Phe Ala Asn Met Asp Asn Ala Thr Leu Asp Val Ala Leu Asn Arg 415 420 425

ATT AGG TCT TTT GTA ACC CGT GGA AGG GTG GAC AAT TCA ACA ATG ACA 1465 lie Arg Ser Phe Val Thr Arg Gly Arg Val Asp Asn Ser Thr Met Thr 430 435 440 ACA ACA TCA GCA AGA GCA GCA GCA ACA ACA ACA ACA ACA ACA ACA ACA 1513 Thr Thr Ser Ala Arg Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr Thr 445 450 455 460

ACA ACA ACA ACA ACA ACA ACA ACA ACG ACA ATT AAG AAG AAA CGA GGG 1561 Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr lie Lys Lys Lys Arg Gly 465 470 475

CAA ATG GAG CTT CGA CTT AGC TTC AAC AAT CGA AGA TTC GAA GAC GGT 1609 Gin Met Glu Leu Arg Leu Ser Phe Asn Asn Arg Arg Phe Glu Asp Gly 480 485 490

TTA ATG TCA CCT CAT AGC ATC CTA TTA TCT CCT CAC TCT CCT ATG CCT 1657 Leu Met Ser Pro His Ser lie Leu Leu Ser Pro His Ser Pro Met Pro 495 500 505

CAA TCA CCC CTT GTT AAA GCA AGA ACA TAAGTCTAAA ATCATGAGTT 1704

Gin Ser Pro Leu Val Lys Ala Arg Thr 510 515

ATTAATAATA AATTTATCGA ACCAGTGTGA CGCCATTGAA ACGGTGCGAC GGGAGTTGAA 1764

ACGGTGTGAA AGACCACATT CAGATGAAGC ATTATATCTT CTCAACAAAA CATTGAACTT 1824

AATATAATTC AATATAACTT CTCTGTAATT TCATGTATAC AAACACTATA AATATGTAGT 1884

CATGTGTAAG ATCATTGATA TAGAAAAATA TAAATGATTT TCTGATTTTA AAAAAAAA 1942

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1087 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1087

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4;

ATG GGT CTC GCT GAA AAT CAG CTT TGC TTC GAT TTA GTT ACG GAG TGG 48 Met Gly Leu Ala Glu Asn Gin Leu Cys Phe Asp Leu Val Thr Glu Trp 1 5 10 15

CTA CTC AAA AAC CCA CAA GCC TCA ATT TGT ACC AAC GAA GGT GTA AAT 96 Leu Leu Lys Asn Pro Gin Ala Ser lie Cys Thr Asn Glu Gly Val Asn 20 25 30 AAG TTC ATG GAT ATT GCC ATT TTT CAG GAT TAT CAT GGT TTG CCC GAG 144 Lys Phe Met Asp lie Ala lie Phe Gin Asp Tyr His Gly Leu Pro Glu 35 40 45

TTT AGA AGT GCT GTG GCA AAA TTT ATG GGG AAG GCA AGA GAT GAG AAA 192 Phe Arg Ser Ala Val Ala Lys Phe Met Gly Lys Ala Arg Asp Glu Lys 50 55 60

GTC ATA TTC AAT CCA GAT AGA ATT GTA ATG AGT GGT GGA GCC AGT GCA 240 Val lie Phe Asn Pro Asp Arg lie Val Met Ser Gly Gly Ala Ser Ala 65 70 75 80

AGT GAA ACT CTT TTG TTT TGC TTG GCC AAC CCC GGT GAC GCC TTT TTA 288 Ser Glu Thr Leu Leu Phe Cys Leu Ala Asn Pro Gly Asp Ala Phe Leu 85 90 95

ATT CCG TCT CCT TAT TAT CCC GCA TTT AAC CGC GAT TTA CGG TGG AGA 336 lie Pro Ser Pro Tyr Tyr Pro Ala Phe Asn Arg Asp Leu Arg Trp Arg 100 105 110

ACT GGA GTA AAT TTA ATC CCA TTT ACT TGC TCG AGC TCG AAT AAT TTC 384 Thr Gly Val Asn Leu lie Pro Phe Thr Cys Ser Ser Ser Asn Asn Phe 115 120 125

AAA ATC ACT AAG GAA GCC TTA CAA TCG GCA TAT GAA GAC GCC CTT AAA 432 Lys lie Thr Lys Glu Ala Leu Gin Ser Ala Tyr Glu Asp Ala Leu Lys 130 135 140

AAG AAC ATC AAA GTT AAG GGT ATT ATC GTC ACA AAC CCG TCA AAT CCC 480 Lys Asn lie Lys Val Lys Gly lie lie Val Thr Asn Pro Ser Asn Pro 145 150 155 160

TTA GGA ACG GTC CTA GAC AAG GAC ACC CTA AAA ATG TTA TTA ACA TTC 528 Leu Gly Thr Val Leu Asp Lys Asp Thr Leu Lys Met Leu Leu Thr Phe 165 170 175

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

ACA GTA TTT AAT TCG CCG AGC TTT ATA AGT GTT GCT GAG GTT ATA AAG 624 Thr Val Phe Asn Ser Pro Ser Phe He Ser Val Ala Glu Val He Lys 195 200 205

GAC ATG CCT CAT GTA AAT CAA GAC CTT GTT CAT ATT TTA TAT AGT TTG 672 Asp Met Pro His Val Asn Gin Asp Leu Val His He Leu Tyr Ser Leu 210 215 220

TCC AAG GAC ATG GGC ATG CCG GGC TTT AGG GTT GGG ATC ATT TAC TCT 720 Ser Lys Asp Met Gly Met Pro Gly Phe Arg Val Gly He He Tyr Ser 225 230 235 240

TAT AAT GAC CGT GTC GTC TCA ACT GCT CGT CGA ATG TCG AGT TTT GGA 768 Tyr Asn Asp Arg Val Val Ser Thr Ala Arg Arg Met Ser Ser Phe Gly 245 250 255 CTT GTT TCT TCT CAA ACT CAG TTT ATG ATC GCG GCA TTG CTC TCA GAT 816 Leu Val Ser Ser Gin Thr Gin Phe Met He Ala Ala Leu Leu Ser Asp 260 265 270

GAT GAT TTT GTT AGA CGA TTC TTG GTT GAG AGT AGA GAC AGA CTC TTT 864 Asp Asp Phe Val Arg Arg Phe Leu Val Glu Ser Arg Asp Arg Leu Phe 275 280 285

CGA AGG CAC CAG CAT TTC ACA AGC GAG CTG GCT AAG ATA GGA ATA GGA 912 Arg Arg His Gin His Phe Thr Ser Glu Leu Ala Lys He Gly He Gly 290 295 300

TGC CTC CAA GGA AAC GCG GCA TTG TTT GTT TGG ATG GAT TTG AGG CAT 960 Cys Leu Gin Gly Asn Ala Ala Leu Phe Val Trp Met Asp Leu Arg His 305 310 315 320

CTA TTA GAC GAA GCA ACG GTT GAA AGA GAG TTA AAG TTA TGG AGA GTG 1008 Leu Leu Asp Glu Ala Thr Val Glu Arg Glu Leu Lys Leu Trp Arg Val 325 330 335

ATC ATC AAT GAA GTG AAA ATC AAT GTG TCA CCG GGT TCG TCC TTC CTG 1056 He He Asn Glu Val Lys He Asn Val Ser Pro Gly Ser Ser Phe Leu 340 345 350

TGC TCT GAG CCA GGG TGG TTT AGG GTT TGC T 1087

Cys Ser Glu Pro Gly Trp Phe Arg Val Cys 355 360

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CGGGATCCGC TACTAATGAA GAGCATGGC 29 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GCGGTACCAG GTGACGAAAG TGGTGACA 28

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1156 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 53..1015

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AAAACAAATA CAAATACAAA TACAAATACA TTGAATTTGT TAATTAACGA AC ATG 55

Met

1

GCA AAC ATT GTC AAC TTC CCT ATC ATT GAC ATG GAG AAG CTC AAT AAT 103 Ala Asn He Val Asn Phe Pro He He Asp Met Glu Lys Leu Asn Asn 5 10 15

TAT AAT GGT GTT GAG AGG AGT CTT GTT TTG GAC CAA ATT AAG GAT GCT 151 Tyr Asn Gly Val Glu Arg Ser Leu Val Leu Asp Gin He Lys Asp Ala 20 25 30

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

GAA CTG ATG GAC AAA GTG GAG AGG ATG ACA AAA GAG CAT TAC AAG AAA 247 Glu Leu Met Asp Lys Val Glu Arg Met Thr Lys Glu His Tyr Lys Lys 50 55 60 65 TTC AGG GAG CAA AAG TTC AAA GAC ATG GTT CAG ACC AAA GGT TTA GTG 295 Phe Arg Glu Gin Lys Phe Lys Asp Met Val Gin Thr Lys Gly Leu Val 70 75 80

TCT GCT GAG TCT CAA GTC AAT GAC ATT GAT TGG GAG AGC ACC TTC TAC 343 Ser Ala Glu Ser Gin Val Asn Asp He Asp Trp Glu Ser Thr. Phe Tyr 85 90 95

CTT CGT CAT CGT CCC ACC TCC AAC ATC TCC GAG GTC CCT GAT CTC GAC 391 Leu Arg His Arg Pro Thr Ser Asn He Ser Glu Val Pro Asp Leu Asp 100 105 110

GAC CAA TAC AGG AAG TTG ATG AAG GAG TTT GCA GCC CAG ATT GAG AGG 439 Asp Gin Tyr Arg Lys Leu Met Lys Glu Phe Ala Ala Gin He Glu Arg 115 120 125

TTA TCC GAG CAA CTG TTG GAC TTG TTA TGT GAG AAC CTT GGC CTT GAG 487 Leu Ser Glu Gin Leu Leu Asp Leu Leu Cys Glu Asn Leu Gly Leu Glu 130 135 140 145

AAA GGC TAC CTT AAG AAT GCC TTC TAT GGT GCC AAT GGC CCC ACT TTT 535 Lys Gly Tyr Leu Lys Asn Ala Phe Tyr Gly Ala Asn Gly Pro Thr Phe 150 155 160

GGT ACC AAG GTC AGC AAC TAC CCG CCT TGC CCC AAA CCC GAC CTT ATC 583 Gly Thr Lys Val Ser Asn Tyr Pro Pro Cys Pro Lys Pro Asp Leu He 165 170 175

AAA GGA CTT AGG GCC CAC ACC GAC GCT GGT GGC ATC ATT CTC TTG TTC 631 Lys Gly Leu Arg Ala His Thr Asp Ala Gly Gly He He Leu Leu Phe 180 185 190

CAG GAC GAC AAG GTC AGC GGC CTC CAG CTC CTC AAG GAT GGT CAT TGG 679 Gin Asp Asp Lys Val Ser Gly Leu Gin Leu Leu Lys Asp Gly His Trp 195 200 205

GTT GAT GTT CCT CCC ATG AAA CAC TCC ATT GTT GTT AAC TTG GGG GAC 727 Val Asp Val Pro Pro Met Lys His Ser He Val Val Asn Leu Gly Asp 210 215 220 225

CAA CTT GAG GTT ATT ACA AAT GGC AAG TAC AAG AGT GTG ATG CAC CGC 775 Gin Leu Glu Val He Thr Asn Gly Lys Tyr Lys Ser Val Met His Arg 230 235 240

GTG ATA GCG CAG ACA GAT GGT AAC AGG ATG TCG ATA GCA TCA TTC TAC 823 Val He Ala Gin Thr Asp Gly Asn Arg Met Ser He Ala Ser Phe Tyr 245 250 255

AAC CCG GGA AGT GAT GCC GTG ATT TAC CCG GCG CCA ACA TTG GTG GAA 871 Asn Pro Gly Ser Asp Ala Val He Tyr Pro Ala Pro Thr Leu Val Glu 260 265 270

AAA GAA GAG GAG AAA TGC AGA GCA TAC CCA AAA TTT GTG TTC GAG GAT 919 Lys Glu Glu Glu Lys Cys Arg Ala Tyr Pro Lys Phe Val Phe Glu Asp 275 280 285 TAC ATG AAT CTC TAC TTA AAG CTC AAG TTC CAA GAG AAG GAG CCC AGG 967 Tyr Met Asn Leu Tyr Leu Lys Leu Lys Phe Gin Glu Lys Glu Pro Arg 290 295 300 305

TTT GAA GCA ATG AAG GCC ATG GAA ACC ACG GGT CCC ATT CCA ACT GCT 1015 Phe Glu Ala Met Lys Ala Met Glu Thr Thr Gly Pro He Pro Thr Ala 310 315 320

TGAAATAATG ATTTGATTTG ATATAATGCA ATGCTTCTCA TCAACCAATT TAAGTATTTC 1075

TAATATACGC CACTCTCATC TCATCTCATA TATTCATATT CATATTATTA GTGTTTGTTG 1135

AATAAGAGCT TCCTTTTAAG T 1156

Claims

CLAIMS:

1. 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.

2. A method according to claim 1 wherein the transgenic plant exhibits one or more of the following properties:

(i) a reduction in production of ACC synthase-specific mRNA or ACC oxidase- specific mRNA;

(ii) a reduction in production of ACC synthase or ACC oxidase; and/or

(iii) delayed senescence of flowers or flower buds cut from said transgenic plant.

3. A method according to claim 1 or 2 wherein the genetic construct comprises a non-full length fragment of a nucleic acid molecule encoding ACC synthase or ACC oxidase.

4. A method according to claim 3 wherein the non-full length fragment is approximately 800-1200 base pairs in length.

5. A method according to claim 3 wherein the non-full length fragment is an internal fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase.

6. A method according to claim 3 or 4 or 5 wherein reduction in production of ACC synthase-specific mRNA or ACC oxidase-specific mRNA or reduction in production of ACC synthase or ACC oxidase is achieved by co-suppression.

7. A method for producing a transgenic carnation plant having flowers or flower buds which, when cut from said carnation plant, exhibit prolonged post-harvest life properties relative 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 non-full length fragment of a nucleic acid molecule encoding, or complementary to a sequence encoding, ACC synthase or ACC oxidase, and regenerating a plant from said cell or cells wherein flowers of the said transgenic plant exhibit one or more of the following properties:

(i) a reduced level of ACC synthase-specific mRNA or ACC oxidase-specific mRNA below non-transgenic endogenous levels:

(ii) a reduced level of ACC synthase or ACC oxidase below non-transgenic endogenous levels; and/or

(iii) a reduced level of climacteric ethylene production below non-transgenic endogenous levels.

8. A method according to claim 7 wherein the non-full length fragment of the nucleic acid molecule is approximately 800-1200 bp in length and the reduction in ACC synthase-specific mRNA or ACC oxidase-specific mRNA or, reduction in ACC synthase or ACC oxidase or reduction in climacteric ethylene production is by co-suppression.

9. A method according to claim 1 or 8 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO: 3 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:3 under low stringency conditions at 30°C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:3.

10. A method according to claim 1 or 8 wherein the nucleic acid molecule comprises a seq uence of nucleotides substantially as set forth in SEQ ID NO:4 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:4 under low stringency conditions at 30" C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:4.

11. A method according to claim 1 or 8 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:7 under low stringency conditions at 30°C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:7.

12. A method according to claim 1 or 8 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:3 or having at least about 40% similarity thereto.

13. A method according to claim 1 or 8 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least about 40% similarity thereto.

14. A method according to claim 1 or 8 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:7 or having at least about 40% similarity thereto.

15. A method for producing a transgenic flowering carnation plant wherein the flowers exhibit reduced levels of ethylene production relative to levels in its non-transgenic parent plant or a non-transgenic plant of the same species, said method comprising introducing into a cell or cells of a carnation plant, a genetic construct comprising 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 the cell or cells.

16. A method according to claim 15 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:3 under low stringency conditions at 30°C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:3.

17. A method according to claim 15 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:4 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:4 under low stringency conditions at 30° C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:4.

18. A method according to claim 15 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:7 under low stringency conditions at 30° C or is a nucleic acid molecule having a nucleotide sequence having at least about 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:7.

19. A method according to claim 15 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:3 or having at least about 40% similarity thereto.

20. A method according to claim 15 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least about 40% similarity thereto.

21. A method according to claim 15 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:7 or having at least about 40% similarity thereto.

22. A method according to claim 1 or 7 or 15 wherein the genetic construct is plasmid pWTT2160 or plasmid pCGP407 deposited with the Australian Government Analytical Laboratory under Accession Numbers N95/26121 and N95/26122, respectively.

23. A transgenic carnation plant 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 wherein said transgenic plant exhibits one or more of the following properties:

(i) a reduction in the production of ACC synthase-specific mRNA;

(ii) a reduction in the production of ACC synthase enzyme;

(iii) a reduction in the production of climacteric ethylene; and/or

(iv) delayed senescence of flowers or flower buds cut from said transgenic plants.

24. A transgenic plant according to claim 23 wherein the nucleic acid molecule is a non-full length fragment of a nucleic acid molecule encoding ACC synthase or ACC oxidase..

25. A transgenic plant according to claim 24 wherein the non-full length fragment is approximately 800-1200 base pairs in length.

26. A transgenic plant according to claim 25 wherein the non-full length fragment is an internal fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase.

27. A transgenic carnation plant capable of carrying flowers or flower buds with prolonged post-harvest life properties relative to its non-transgenic parent or a non-transgenic part of the same species, said plant comprising a non-full length fragment of a nucleic acid molecule encoding, or complementary to a sequence encoding, a ACC synthase or ACC oxidase wherein flowers or flower buds of said transgenic plant exhibit one or more of the following properties: (i) a reduced level of ACC synthase-specific mRNA or ACC oxidase-specific mRNA below non-transgenic endogenous levels;

(ii) a reduced level of ACC synthase or ACC oxidase enzyme below non-transgenic endogenous levels; and/or

(iii) a reduced level of ethylene production below non-transgenic endogenous levels.

28. A transgenic plant according to claim 27 wherein the non-full length fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase is approximately 800-1200 bp in length and endogenous ACC synthase or ACC oxidase gene expression is reduced by co- suppression.

29. A method according to claim 23 or 27 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO: 3 under low stringency conditions at 30° C or is a nucleic acid molecule having a nucleotide sequence having at least 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:3.

30. A method according to claim 23 or 27 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:4 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO: 4 under low stringency conditions at 30° C or is a nucleic acid molecule having a nucleotide sequence having at least 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:4.

31. A method according to claim 23 or 27 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO: 7 or is a derivative thereof or is a nucleic acid molecule capable of hybridising to the sequence of nucleotides set forth in SEQ ID NO:7 under low stringency conditions at 30°C or is a nucleic acid molecule having a nucleotide sequence having at least 60% similarity to the sequence of nucleotides set forth in SEQ ID NO:7.

32. A transgenic plant according to claim 23 or 27 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:3 or having at least about 40% similarity thereto.

33. A transgenic plant according to claim 23 or 27 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least about 40% similarity thereto.

34. A transgenic plant according to claim 23 or 27 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:7 or having at least about 40% similarity thereto.

35. A cut flower from a transgenic carnation according to any one of claims 23 to 34.

36. Seeds or other reproductive material from a transgenic carnation according to any one of claims 23 to 34.