AU703841B2 - Transgenic carnations exhibit prolonged post-harvest life - Google Patents

Description

WO 96/35792 PCT/AU96/00286 -1- TRANSGENIC CARNATIONS EXHIBIT PROLONGED POST-HARVEST

LIFE

The present invention relates generally to transgenic plants which exhibit prolonged postharvest 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 postharvest 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 commerciallyvaluable 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 undesirable traits are WO 96/35792 PCT/AU96/00286 -2suppressed. 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 cutflower 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-aminocydopropane-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 enzymes 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 WO 96/35792 PCT/AU96/00286 -3in 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 nontransgenic 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: a reduction in production of ACC synthase-specific mRNA or ACC oxidasespecific 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 WO 96/35792 PCT/AU96/00286 -4said transgenic plant exhibits one or more of the following properties: reduction in the production of ACC synthase-specific mRNA or ACC oxidasespecific 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-fulllength 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 metabolic 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 WO 96/35792 PCT/AU96/00286 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 cosuppression 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: reduction in the production of ACC synthase-specific mRNA or ACC oxidasespecific 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.

WO 96/35792 PCT/AU96/00286 -6- 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: reduced levels of ACC synthase-specific mRNA or ACC oxidase below nontransgenic 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: reduction in the production of ACC synthase-specific mRNA or ACC oxidasespecific 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: reduced levels of ACC synthase-specific mRNA or ACC oxidase-specific mRNA below non-transgenic endogenous levels; WO 96/35792 PCT/AU96/00286 -7- (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 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 Agrobacterium-mediated transfer.

Accordingly, another aspect of the present invention provides an isolated nucleic acid 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 WO 96/35792 PCT/AU96/00286 -8other 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 ofnucleotide 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 WO 96/35792 PCT/AU96/00286 -9sequence of amino acids may constitute afull-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 WO 96/35792 PCT/AU96/00286 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 in Agrobacterium tumefaciens.

WO 96/35792 PCT/AU96/00286 11 In a further embodiment, the plasmid is pCGP407 in Escherichia coli. The microorganisms Escherichia coli strain XL1-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 Z18952); 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; SurB 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 Agrobacterium 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 White Sim are compared with sequences fromArabidopsis thaliana, tomato (Holdsworth et al., 1987; EMBL WO 96/35792 PCT/AU96/00286 -12accession number X 04792); orchid (Nadeau et al., 1993; Genbank accession number L 07912); apple (Dong 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 Agrobacterium tumefaciens mannopine synthase gene; 35S the promoter region form the cauliflower mosaic virus gene; NPT II neomycin phosphotransferase II; tml 3' the tml terminator region, DNA sequences 11207-10069, from pTiA6 (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 "P-labelled-760 base pair fragment derived from the ALS coding region.

Filters were washed in 0.2 x SSC/1% w/v SDS at 65oC. 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 1-4 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.

WO 96/35792 PCT/AU96/00286 13- 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 I) 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 mI. The Southern blot was probed with a P-labelled EcoRI DNA fragment from the coding sequence of the NPT II gene. Filters were washed in 0.1 x SSC, 0.1% w/v SDS at 65-C. The bands indicate single or multiple copies of the DNA derived from pCGP407 have been integrated into the genome of the plants. 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 nontransformed White Sim. Eight independent transgenic lines are shown. Filters were probed with a FP-labelled HindII DNA fragment from the acetolactate synthase gene coding region, and washed for 30 min in 2 x SSC, 1% w/v SDS at 650C, followed by 2 x 30 min in 0.2 x SSC, 1% w/v SDS at Figure 8 is an autoradiographic representation of a Northern blot of ACC oxidase mRNA and ACC oxidase antisense RNA isolated from petals. Total RNA (10/g/lane) was analysed from day 0 petals of control, non-transgenic White Sim (lane transgenic Scania (lane 3) and transgenic White Sim (lane 5) flowers; and day 5 petals of control, non-transgenic White Sim (lane transgenic Scania (lane 4) and transgenic White Sim (lane 6) flowers. Also analysed was total RNA isolated from transgenic Scania (lane 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 detect antisense ACC WO 96/35792 PCT/AU96/00286 -14oxidase 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 0 C for 1 hour and, in the case of antisense ACO, finally in 0.1 x SSC/0.1% w/v SDS at 65 0 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: non-transgenic control Scania flower, 0 days post-harvest; non-transgenic control Scania flower, 4 days post-harvest; non-transgenic control Scania flower, 7 days post-harvest; transgenic ACC synthase sense-suppressed Scania flower, 0 days post-harvest; transgenic ACC synthase sense-suppressed Scania flower, 4 days post-harvest; and 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: non-transgenic control Red Corso flower, 0 days post-harvest; non-transgenic control Red Corso flower, 7 days post-harvest; ~111 WO 96/35792 PCT/AU96/00286 non-transgenic control Red Corso flower, 9 days post-harvest; transgenic ACC synthase sense-suppressed Red Corso flower, 0 days post-harvest; transgenic ACC synthase sense-suppressed Red Corso flower, 7 days post-harvest; and 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: non-transgenic control Ember Rose flower, 0 days post-harvest; non-transgenic control Ember Rose flower, 4 days post-harvest; non-transgenic control Ember Rose flower, 7 days post-harvest; transgenic ACC synthase sense-suppressed Ember Rose flower, 0 days post-harvest; transgenic ACC synthase sense-suppressed Ember Rose flower, 4 days post-harvest; and 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: non-transgenic control Crowley Sim flower, 0 days post-harvest; non-transgenic control Crowley Sim flower, 4 days post-harvest; transgenic ACC synthase sense-suppressed Crowley Sim flower, 0 days post-harvest; and transgenic ACC synthase sense-suppressed Crowley Sim flower, 4 days post-harvest.

Original colour plates are available for inspection from the Applicant.

WO 96/35792 PCT/AU96/00286 -16- Figure 14(A)-14(C) is a black and white reproduction of colour photographic plates representing: 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; 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 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.

WO 96/35792 WO 9635792PCT/AU96/00286 17 EXAMPLE 1 Biological Reagents All restriction enzymes and other reagents were obtained from commercial sources and used generally according to the manufacturer's recommendlations.

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

EXAMPLE 2 Bacterial Strains The bacterial strains used were: Escherichia coli XL1-Blue mupE44, hsdR17 =ndA1, ggrA96 (Nalr), thi-1, r1Al, 1ac-, [F'ptmAB, lacI2, IauZAM15, TnlO(tetr)] (Bullock et al., 1987).

supE44 A~aKZYA-ArgF)Ul69 o80dLaZAM15 hsdR17(rk-, mk+), rxcA, endA 1, =~A96 (Nall), thi.1, =eI&1, dcoR (1-anahan, 1983 and BRL, 1986).

JM 83 FaraAolac-proAB) rpaL (Str )[o80dA(LicZ)M15] (Vieira and Messing, 1982) JM 109 Fta3 w AIZ M xAB+e4(cA)AkpDR thij gyrA2 (Nal') end~l hsdR17 mk =JlAI supE44 recAI (Yanisch-Perron et al., 1985) Agrobacterium tumefrejens: AGLO Lazo et al. (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.

WO 96/35792 PCT/AU96/00286 18- 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)TNGCNGAAAATCAGC3' SEQ ID NO:1 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 (ones 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 reversetranscriptase-PCR of PolyA' RNA from ethylene-treated carnation flowers.

EXAMPLE Sequence analysis of carnation cv. White Sim ACS cDNA done The approximately 1,100 bp carnation ACS cDNA fragment was cloned into the vector pBluescript II and the terminal nucleotides were sequenced using SEQ ID NO:1 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 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.

WO 96/35792 PCT/AU96/00286 -19- (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: CGGGATCCGCTACTAATGAAGAGCATGGC 3' SEQ ID 5' GCGGTACCAGGTGACGAAAGTGGTGACA 3' SEQ ID NO:6 b. Isolation ofan A CS clone from petuniaflowers 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 CsC1 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 nudeotide sequence of the PCR product confirmed that it encoded a polypeptide similar to the deduced translation product of the corresponding region from tomato pcW4A 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 WO 96/35792 PCT/AU96/00286 DNA Polymerase I (Klenow fragment), blunted, and linkers were added to create EcoRIcompatible 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 "P-labelled and used to screen the 60,000 plaques of the senescing carnation cv. Scania petal cDNA library (Example 6c., above), under conditions of low stringency: the filters were hybridized in formamide at 30 0 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 0

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, WO 96/35792 PCT/AU96/00286 -21- 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 pWTI2160 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) 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 promoter together with the SurB gene (tobacco acetolactate synthase) allowing selection of chlorsulfuron-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 et al., 1989) To transfer the binary vector pWTT2160 (see Figure 2) from E. coli to Agrobacterium 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 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 jg/mL gentamycin and 100/g/mL rifampicin at 28°C.

WO 96/35792 PCT/AU96/00286 -22- EXAMPLE Transformation of Dianthus caryophyllus with partial ACC synthase sequence a. Plant Material Dianthus caryophyllus (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 cocultivation.

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 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 a-naphthalene acetic acid (NAA); 30g/L sucrose; 0.25 w/v Gelrite Gellan Gum (Schweizerhall), pH 5.8. All phytohormones were added after autoclaving. Cultures were incubated in a growth chamber with a 16-hour photoperiod /E/mVs) 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 subcultured monthly on fresh shoot multiplication medium. After 3-4 sub-cultures, shoot cultures which multiplied at a high rate were established; each shoot with 3-4 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.

WO 96/35792 PCT/AU96/00286 -23b. Co-cultivation ofAgrobacterium atndDianthus Tissue Agrobacterium tumefaciens strain AGLO (Lazo et al., 1991), containing the binary vector pWTT2160, was maintained at 4oC 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.

Acetosyringone was added to the Agrobacterium suspension to a final concentration of 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-dichlorophenoxyacetic acid 100 /zM 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 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 100/MM 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 mg/L BAP, 0.5 mg/L 2,4-D, 1 g/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v WO 96/35792 PCT/AU96/00286 -24- Gelrite. After 2 weeks, explants were transferred to fresh MS medium containing 0.16 mg/L thidiazuron (TDZ), 0.5 mg/L indolbutyric acid (IBA), 2 /g/L chlorsulfuron, 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 yg/L chlorsulfuron, 500 mg/L ticarcillin, 025% w/v Gelrite. Shoots which survived 3 /g/L chlorsulfuron were transferred to MS medium supplemented with 3% w/v sucrose, 500 mg/L ticarcillin, 5 jug/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 /g/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 Szg/L chlorsulfuron, 500 mg/L ticarcillin and 0.25% w/v Gelrite for 2-4 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 /E/m 2 /s cool white fluorescent light) at 23 2°C. Normalized shoots, approximately 1.5-2 cm tall, were rooted on 3 g/kg IBA rooting powder and acclimatised under mist. A soil mix containing 75% perlite/25% peat was used for acclimation, which was carried out at 23°C under a 14 hour photoperiod (200 /E/m 2 /s mercury halide light) and typically lasted 34 weeks. Plants were fertilized with a carnation mix containing lg/L CaNO,and 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 ig/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 WO 96/35792 PCT/AU96/00286 mg/L TDZ; 30g/L sucrose; 0.25% w/v Gelrite; 500 mg/L carbenicillin and 3 1/g/L chlorsulfuron, pH 5.8. If small shoot clusters had formed after 2-3 weeks, they were separated into 2-4 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 31/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) done from cv. Scania a. Preparation of P-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 oligo-dT, 50mM Tris-HC pH 8.0, 75mM KC1, 30mM MgCl2, 10mM DTT, 0.5 mg/mL actinomycin D, 200AM dATP, 200/iM dGTP, 200/AM dTTP, 2.5,/M dCTP, 100J/Ci [a- 3 2

P]-

dCTP (Bresatec, 3000Ci/mmol), 40 units ribonuclease 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 0 C. The mixture was then neutralised by addition of HC1 to a concentration of 0.2M. Unincorporated [a- 32 p]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

b. "P-Labelling ofDNA fragments DNA fragments (50 to 100 ng) were radioactively labelled with 50 ,Ci of 3 2 P-dCTP using an oligolabelling kit (Bresatec). Unincorporated [a- 2 P]dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

WO 96/35792 PCT/AU96/00286 -26c. 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 cloning 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 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 0 C for 3 x 30 min. Filters were then exposed to Kodak XAR film with an intensifying screen at -700C for 16 hours. Clones which hybridized with the in rolling petal cDNA, but not with the day 0 cDNA, were selected for further investigation.

EXAMPLE 12 Sequence analysis of carnation cv. Scania ACO cDNA clone 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 etal., 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 The 1,156 bp carnation cv.

Scania ACO sequence is presented as SEQ ID NO:7.

WO 96/35792 PCT/AU96/00286 -27- 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 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 selection 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 XL1-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 Mg of plasmid DNA to 100 /L of competent Agrobacterium tumefaciens cells prepared by inoculating a 50 mL MG/L (Garfinkel and Nester, 1980) culture and growing for 16 h with shaking at 28 0 C. The cells were then pelleted and resuspended in 0.5 mL of v/v 100 mM CaCl/15% v/v glycerol. The DNA-Agrobacterium mixture was frozen by incubation in liquid N,for 2 min and then allowed to thaw by incubation at 37 0 C for WO 96/35792 PCT/AU96/00286 -28min. 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 0

C.

Cells of A. tumefaciens carrying pCGP407 were selected on MG/L agar plates containing 100 g/mL gentamycin. The presence of the plasmid was confirmed by Southern analysis of DNA isolated from the gentamycin-resistant transformants.

EXAMPLE Transformation of Dianthus caryophyllus with ACC oxidase a. Plant Material Dianthus caryophyllus (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 ofAgrobacterium 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 jM acetosyringone and 0.25% w/v Gelrite (pH 5.7).

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 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 WO 96/35792 PCT/AU96/00286 -29selection medium and care was taken at this stage to remove axillary shoots from stem explants. After 6-8 weeks on selection 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., 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 elongation. 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) soaked in hydroponic mix (Kandreck and Black, 1984) was used for acclimation, which typically lasted 4-5 weeks. Plants were acclimatised at 23"C under a 14-hour photoperiod (200 ,E/m2/s mercury halide light).

EXAMPLE 16 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).

b. Southern Blots The genomic DNA (10 1g) was digested with EcoRI (for sense ACS) or HindIII (for antisense ACO) and electrophoresed 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 in denaturing solution (1.5 M NaCl/0.5 M NaOH) for 1 to 1.5 hours, neutralized in 0.5 M Tris-HC1 (pH 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.

WO 96/35792 PCT/AU96/00286 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.

EXAMPLE 17 Northern Analysis Total RNA was isolated from tissue that had been frozen in liquid N, and ground to a fine powder using a mortar and pestle. An extraction buffer of 4 M guanidinium isothiocyanate, mM Tris-HC1 (pH 20 mM EDTA, 0.1% v/v Sarkosyl, was added to the tissue and the mixture was homogenized for 1 minute using a polytron at maximum speed. The suspension was filtered through Miracloth (Calbiochem) and centrifuged in a JA20 rotor for minutes at 10,000 rpm. The supernatant was collected and made to 0.2 g/ mL CsCI w/v.

Samples were then layered over a 10 mL cushion of 5.7 M CsC1, 50 mM EDTA (pH 7.0) in 38.5 mL Quick-seal centrifuge tubes (Beckman) and centrifuged at 42,000 rpm for 12-16 hours at 230C in a Ti-70 rotor. Pellets were resuspended in TE/SDS (10 mM Tris-HC1 (pH 1 mM EDTA, 0.1% w/v SDS) and extracted with phenol:chloroform*isoamyl alcohol (25:24:1) saturated in 10 mM EDTA (pH The RNA was then maintained as an ethanol precipitate, and appropriate aliquots pelleted prior to use.

RNA samples were electrophoresed through 2.2 M formaldehyde/1.2% w/v agarose gels using running buffer containing 40 mM morpholino-propanesulphonic acid (pH 5 mM sodium acetate, 0.1 mM EDTA (pH The RNA was transferred to Hybond-N filters (Amersham) as described by the manufacturer and probed with 2 P-labelled cDNA fragment 8 cpm/pg, 2 x 10 cpm/mL). Prehybridization (1 h at 42 0 C) and hybridization (16 h at 420C) was carried out in 50% v/v formamide, 1 M NaCI, 1% w/v SDS, 10% w/v dextran sulphate, 100 /g/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 0 C for 1 hour. In the case of antisense ACO, however, filters were also washed in 0.1 x SSC/0.1% w/v SDS at 65°C for 1 hour. All filters were exposed to Kodak XAR film WO 96/35792 PCT/AU96/00286 -31with an intensifying screen at -70 0 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 radioactively labelled with 50 pCi of [a-"P]-dCTP using an oligolabelling kit (Bresatec). Unincorporated [a-P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

EXAMPLE 19 Transformation of Dianthus cultivars The genetic contructs contained in the plasmids pWTT2160 and pCGP407 were introduced into various varieties of carnation using Agrobacterium-mediated 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 nontransgenic controls. For example, measurements of ethylene production, using a Varian model 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 capacity to produce ethylene. The graph in Figure 9 shows ethylene evolution by transgenic WO 96/35792 PCT/AU96/00286 -32and 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 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 either a sense transcript and the co-suppression technology disclosed in US Patent Numbers 5,034,323; 5,231,020 and 5,238,184, or an antisense transcript and the antisense technology disclosed 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 cycle in controlled conditions, (1000 lux, 22°C, 65% relative 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), respectively. In each case, the transgenic carnation flower appears fresher for longer, when WO 96/35792 PCT/AU96/00286 -33compared 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 completely 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.

WO 96/35792 PCT/AU96/00286 -34-

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WO 96/35792 PCT/AU96/00286 -38- SEQUENCE LISTING GENERAL INFORMATION: 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:

ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: PCT INTERNATIONAL FILING DATE: 09-MAY-1996 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: PN2862 (AU) FILING DATE: 09-MAY-1995 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: HUGHES DR, E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 WO 96/35792 PCT/AU96/00286 -39- INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGGGT(C/T)TNG CNGAAAATCA GC 22 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: A(G/A)CANACNCG (A/G)AACCANCCN GG 22 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1942 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 134..1684 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGTCTTAATC TTTGTCACTT TACAACAACA TAAATTATAT TCTCAATCAT TTTCTCTATA TATATATATA TACCCTCCAT TTTTCCTACT CCCCCTCCAC AAAAAATATA ATAATAGTGA 120 WO 96/35792 PCT/AU96/00286 GTGTGTAATC AAA ATG GGT TCT TAT AAG GGT GTT TAC GAC CGT GAA ATT Met Gly Ser Tyr Lys Giy Val Tyr Asp Arg Giu Ile 1 5 CTT TCA AAA ATC GCT ACG AAC GAT GGC CAT GGT GAG AAT TTG GAG TAC Leu Ser Lys Ile Ala Thr Asn Asp Gly His Giy Glu Asn Leu Giu Tyr 20 169 217 TTT GAT GGG TGG AAA GCT TAT GAT Phe Asp Gly Trp Lys Ala Tyr Asp 35 AGA GAT CCT Arg Asp Pro CAT TCT ACC AAG His Ser Thr Lys TCT AAT GGC GTT Ser Asn Gly Val CAA ATG GGT CTC Gin Met Gly Leu GAA AAT CAG CTT Glu Asn Gin Leu

TGC

Cys 265 313 361 TTC GAT TTA GTT Phe Asp Leu Val GAG TGG CTA CTC Glu Trp Leu Leu AAC CCA CAA GCC Asn Pro Gin Ala TCA ATT Ser Ile TGT ACC AAC Cys Thr Asn GAT TAT CAT Asp Tyr His

GAA

Glu GGT GTA AAT AAG Gly Val Asn Lys ATG GAT ATT GCC Met Asp Ile Ala ATT TTT CAG Ile Phe Gin AAA TTT ATG Lys Phe Met 409 GGT TTG CCC GAG TTT AGA AGT GCT GTG Gly Leu Pro Giu Phe Arg Ser Ala Val 457 505 GGG AAG Gly Lys 110 GCA AGA GAT GAG Ala Arg Asp Glu

AAA

Lys 115 GTC ATA TTC AAT Val Ile Phe Asn

CCA

Pro 120 GAT AGA ATT GTA Asp Arg Ile Val

ATG

Met 125 AGT GGT GGA GCC Ser Gly Gly Ala

AGT

Ser 130 GCA AGT GAA ACT CTT TTG TTT TGC TTG GCC Ala Ser Giu Thr Leu Leu Phe Cys Leu Ala AAC CCC GGT GAC Asn Pro Gly Asp

GCC

Ala 145 TTT TTA ATT CCG Phe Leu Ile Pro

TCT

Ser 150 CCT TAT TAT CCC Pro Tyr Tyr Pro GCA TTT Ala Phe 155 AAC CGC GAT Asn Arg Asp TGC TCG AGC Cys Ser Ser 175 GCA TAT GAA Ala Tyr Glu 190 CGG TGG AGA ACT Arg Trp Arg Thr GTA AAT TTA ATC Val Asn Leu Ile CCA TTT ACT Pro Phe Thr 170 TTA CAA TCG Leu Gin Ser TCG AAT AAT TTC Ser Asn Asn Phe

AAA

Lys 180 ATC ACT AAG GAA Ile Thr Lys Glu

GCC

Ala 185 GAC GCC CTT Asp Ala Leu

AAA

Lys 195 AAG AAC ATC AAA Lys Asn Ile Lys

GTT

Val 200 AAG GGT ATT ATC Lys Gly Ile Ile ACA AAC CCG TCA AAT Thr Asn Pro Ser Asn CCC TTA GGA ACG GTC CTA GAC AAG GAC ACC Pro Leu Gly Thr Val Leu Asp Lys Asp Thr 215 220 WO 96/35792 PCT/AU96/00286 -41- CTA AAA ATG TTA Leu Lys Met Leu ACA TTC GTA AA T Thr Phe Val Asn AAA AAT ATA CAC Lys Asn Ile His CTT GTG Leu Val 235 TGT GAC GAG Cys Asp Glu AGT GTT GCT Ser Val Ala 255

ATA

Ile 240 TAT GCA ACC ACA GTA TTT AAT TCG CCG Tyr Ala Thr Thr Val Phe Asn Ser Pro 245 AGC TTT ATA Ser Phe Ile 250 CAA GAC CTT Gin Asp Leu GAG GTT ATA AAG Glu Val Ile Lye

GAC

Asp 260 ATG CCT CAT GTA Met Pro His Val

AAT

Asn 265 GTT CAT Vai His 270 ATT TTA TAT AGT Ile Leu Tyr Ser TCC AAG GAC ATG Ser Lye Asp Met ATG CCG GGC TTT Met Pro Giy Phe

AGG

Arg 285 GTT GGG ATC ATT Val Gly Ile Ile TCT TAT AAT GAC Ser Tyr Asn Asp

CGT

Arg 295 GTC GTC TCA ACT Vai Val Ser Thr

GCT

Ala 300 985 1033 1081 CGT CGA ATG TCG Arg Arg Met Ser

AGT

Ser 305 TTT GGA CTT GTT Phe Gly Leu Vai

TCT

Ser 310 TCT CAA ACT CAG Ser Gin Thr Gin TTT ATG Phe Met 315 ATC GCG GCA Ile Ala Ala GAG AGT AGA Glu Ser Arg 335

TTG

Leu 320 CTC TCA GAT GAT Leu Ser Asp Asp

GAT

Asp 325 TTT GTT AGA CGA Phe Val Arg Arg TTC TTG GTT Phe Leu Val 330 ACA AGC GAG Thr Ser Glu 1129 1177 GAC AGA CTC TTT Asp Arg Leu Phe

CGA

Arg 340 AGG CAC CAG CAT Arg His Gin His

TTC

Phe 345 CTG GCT Leu Ala 350 AAG ATA GGA ATA Lys Ile Gly Ile

GGA

Gly 355 TGC CTC CAA GGA Cys Leu Gin Gly

AAC

Asn 360 GCG GCA TTG TTT Ala Ala Leu Phe TGG ATG GAT TTG Trp Met Asp Leu

AGG

Arg 370 CAT CTA TTA GAC His Leu Leu Asp

GAA

Glu 375 GCA ACG GTT GAA Ala Thr Val Glu 1225 1273 1321 GAG TTA AAG TTA Glu Leu Lys Leu AGA GTG ATC ATC Arg Val Ile Ile GAA GTG AAA ATC Glu Val Lys Ile AAT GTG Asn Val 395 TCA COG GGT Ser Pro Gly TGC TTT GCC Cys Phe Ala 415

TCG

Ser 400 TCC TTC CTG TGC Ser Phe Leu Cys

TCT

Ser 405 GAG CCA GGG TGG Glu Pro Gly Trp TTT AGG GTT Phe Arg Val 410 CTC AAT CGA Leu Asn Arg 1369 1417 AAC ATG GAC AAT Asn Met Asp Asn

GCG

Ala 420 ACC TTA GAC GTT Thr Leu Asp Val

GCA

Ala 425 ATT AGG Ile Arg 430 TCT TTT GTA Ser Phe Val ACC CGT GGA AGG GTG GAC AAT TCA ACA ATG ACA 1465 Thr Arg 435 Gly Arg Val Asp Asn Ser Thr Met Thr WO 96/35792 PCT/AU96/00286 -42-

ACA

Thr 445 ACA TCA GCA AGA Thr Ser Ala Arg GCA GCA GCA ACA ACA ACA ACA ACA ACA ACA Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr 450 c;q 1513 1561 ACA ACA ACA ACA Thr Thr Thr Thr

ACA

Thr 465 ACA ACA ACA ACG Thr Thr Thr Thr

ACA

Thr 470 ATT AAG AAG AAA Ile Lye Lye Lye CGA GGG Arg Gly 475 CAA ATG GAG Gin Met Glu TTA ATG TCA Leu Met Ser 495 CGA CTT AGC TTC Arg Leu Ser Phe

AAC

Asn 485 AAT CGA AGA TTC Asn Arg Arg Phe GAA GAC GGT Glu Asp Gly 490 CCT ATG CCT Pro Met Pro 1609 1657 CCT CAT AGC ATC Pro His Ser Ile TTA TCT CCT CAC Leu Ser Pro His

CAA

Gin TCA CCC CTT GTT AAA GCA AGA ACA TAAGTCTAAA ATCATGAGTT Ser Pro Leu Val Lye Ala Arg Thr 510 515 1704 ATTAATAATA AATTTATCGA ACCAGTGTGA CGCCATTGAA ACGGTGCGAC GGGAGTTGAA ACGGTGTGAA AGACCACATT CAGATGAAGC ATTATATCTT CTCAACAAAA CATTGAACTT AATATAATTC AATATAACTT CTCTGTAATT TCATGTATAC AAACACTATA AATATGTAGT CATGTGTAAG ATCATTGATA TAGAAAAATA TAAATGATTT TCTGATTTTA AAAAAAAA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1087 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1087 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 1764 1824 1884 1942

ATG

Met 1 GGT CTC GCT GAA AAT CAG CTT TGC TTC GAT Gly Leu Ala Giu Asn Gin Leu Cys Phe Asp TTA GTT ACG GAG TGG Leu Val Thr Glu Trp CTA CTC AAA AAC CCA CAA GCC TCA ATT TGT ACC AAC GAA Leu Leu Lye Asn Pro Gln Ala Ser Ile Cys Thr Asn Glu GGT GTA AAT Gly Val Asn WO 96/35792 PCT/AU96/00286 -43 AAG TTC ATG GAT ATT GCC ATT TTT CAt, GAT TAT CAT GCT Lys kne Met Asp Ile Ala Ile Gin Asp Tyr His Giy TTG CCC GAG Leu Pro Ciu TTT AGA ACT GCT GTG OCA AAA TTT ATG GCG AAG GCA ACA CAT GAG AAA Phe Arg Ser Ala Vai Ala Lys Phe Met Gly Lys Ala Arg Asp Giu Lys s0 cc

GTC

Val ATA TTC AAT CCA Ile Phe Asn Pro

GAT

Asp 70 AGA ATT GTA ATG Arg Ile Val Met

ACT

Ser 75 CGT GGA GCC AGT Gly Gly Ala Ser

CCA

Al a ACT CAA ACT CTT TTG TTT TCC TTC CC Ser Giu Thr Leu Leu Phe Cys Leu Ala CCC GCT CAC GCC Pro Cly Asp Ala TTT TTA Phe Leu ATT CCC TCT Ile Pro Ser ACT CCA CTA Thr Gly Val 115

CCT

Pro 100 TAT TAT CCC CCA Tyr Tyr Pro Ala

TTT

Phe 105 AAC CCC CAT TTA Aen Arg Asp Leu CCC TCG AGA Arg Trp Arg 110 AAT AAT TTC Asn Asn Phe 336 384 AAT TTA ATC CCA Aen Leu Ile Pro

TTT

Phe 120 ACT TGC TCG AC Thr Cys Ser Ser AAA ATC Lys Ile 130 ACT AAG CAA CC Thr Lys Giu Ala

TTA

Leu 135 CAA TCG CCA TAT Gin Ser Ala Tyr

GAA

Ciu 140 GAC GCC CTT AAA Asp Ala Leu Lys 432

AAC

Lye 145 AAC ATC AAA GTT AAG CCT ATT ATC GTC Aen Ile Lye Val Lye Gly Ile Ile Val 150 AAC CCC TCA AAT Aen Pro Ser Asn TTA CCA ACC CTC Leu Cly Thr Val

CTA

Leu 165 GAC AAC CAC ACC Asp Lye Asp Thr

CTA

Leu 170 AAA ATC TTA TTA Lys Met Leu Leu ACA TTC Thr Phe 175 CTA AAT CC AAA AAT ATA CAC CTT Val Aen Ala Lye An Ile His Leu 180 TGT CAC GAG ATA Cys Asp Clu Ile TAT GCA ACC Tyr Ala Thr 190 CTT ATA AAG Val Ile Lye ACA CTA TTT Thr Val Phe 195 AAT TCC CCC AGC Asn Ser Pro Ser

TTT

Phe 200 ATA ACT GTT CCT Ile Ser Val Ala

GAG

Giu 205 CAC ATC Asp Met 210 CCT CAT GTA AAT Pro His Val Asn CAC CTT CTT CAT Asp Leu Val His

ATT

Ile 220 TTA TAT ACT TTC Leu Tyr Ser Leu

TCC

Ser 225 AAC CAC ATO GC Lye Asp Met Gly

ATG

Met 230 CCG CCC TTT ACC Pro Gly Phe Arg

CTT

Val 235 CCC ATC ATT TAC Cly Ile Ile Tyr

TCT

Ser TAT AAT CAC CGT GTC CTC TCA ACT CCT CCT CCA ATC TCC AGT TTT GCA Tyr Aen Asp Arg Val Val Ser Thr Ala Arg Arg Met Ser Ser Phe Gly 245 250 255 WO 96/35792 PCT/AU96/00286 -44- CTT GTT TCT Leu Val Ser GAT GAT TTT Asp Asp Phe 275

TCT

Ser 260 CAA ACT CAG TTT Gin Thr Gin Phe

ATG

Met 265 ATC GCG GCA TTG Ile Ala Ala Leu CTC TCA GAT Leu Ser Asp 270 AGA CTC TTT Arg Leu Phe GTT AGA CGA TTC Val Arg Arg Phe

TTG

Leu 280 GTT GAG AGT AGA Val Glu Ser Arg

GAC

Asp 285 CGA AGG Arg Arg 290 CAC CAG CAT TTC His Gin His Phe

ACA

Thr 295 AGC GAG CTG GCT Ser Glu Leu Ala ATA GGA ATA GGA Ile Gly Ile Gly

TGC

Cys 305 CTC CAA GGA AAC Leu Gin Gly Asn

GCG

Ala 310 GCA TTG TTT GTT TGG ATG GAT TTG AGG Ala Leu Phe Val Trp Met Asp Leu Arg 315

CAT

His 320 CTA TTA GAC GAA Leu Leu Asp Glu

GCA

Ala 325 ACG GTT GAA AGA Thr Val Glu Arg

GAG

Glu 330 TTA AAG TTA TGG Leu Lys Leu Trp AGA GTG Arg Val 335 1008 ATC ATC AAT Ile Ile Asn TGC TCT GAG Cys Ser Glu 355

GAA

Glu 340 GTG AAA ATC AAT Val Lys Ile Asn TCA CCG GGT TCG Ser Pro Gly Ser TCC TTC CTG Ser Phe Leu 350 1056 CCA GGG TGG TTT Pro Gly Trp Phe AGG GTT TGC T Arg Val Cys 360 1087 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID CGGGATCCGC TACTAATGAA GAGCATGGC WO 96/35792 PCT/AU96/00286 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCGGTACCAG GTGACGAAAG TGGTGACA INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1156 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 53..1015 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AAAACAAATA CAAATACAAA TACAAATACA TTGAATTTGT TAATTAACGA AC ATG Met 1 GCA AAC ATT GTC AAC TTC CCT ATC ATT GAC ATG GAG AAG CTC AAT AAT Ala Asn Ile Val Asn Phe Pro Ile Ile Asp Met Glu Lys Leu Asn Asn TAT AAT GGT Tyr Asn Gly GTT GAG AGG AGT Val Glu Arg Ser GTT TTG GAC CAA Val Leu Asp Gln AAG GAT GCT Lys Asp Ala TGT CAC Cys His GAA CTG Glu Leu AAC TGG GGA TTC TTC CAG GTG GTG AAC CAT AGT TTG TCA CAT Asn Trp Gly Phe Phe Gln Val Val Asn His Ser Leu Ser His ATG GAC AAA GTG GAG AGG Met Asp Lys Val Glu Arg ATG ACA AAA Met Thr Lys 60 GAG CAT TAC Glu His Tyr AAG AAA Lys Lys 247 WO 96/35792 PCT/AU96/00286 -46- TTC AGG GAG CAA AAG TTC AAA GAC AtG Phe Arg Glu Gin Lys Phe Lys Asp Met CAG ACC AAA GGT TTA GTG Gin Thr Lys Gly Leu Val 295 343 TCT GCT GAG TCT CAA GTC AAT GAC ATT GAT TGG GAG AGC Ser Ala Glu Ser Gin Val Asn Asp Ile Asp Trp Glu Ser ACC TTC TAC Thr. Phe Tyr GAT CTC GAC Asp Leu Asp CTT CGT CAT Leu Arg His 100 CGT CCC ACC TCC Arg Pro Thr Ser

AAC

Asn 105 ATC TCC GAG GTC Ile Ser Glu Val

CCT

Pro 110 GAC CAA Asp Gin 115 TAC AGG AAG TTG Tyr Arg Lys Leu AAG GAG TTT GCA Lys Glu Phe Ala

GCC

Ala 125 CAG ATT GAG AGG Gin Ile Glu Arg 439 TCC GAG CAA CTG TTG GAC TTG TTA TGT Ser Glu Gin Leu Leu Asp Leu Leu Cys 135 AAC CTT GGC CTT Asn Leu Gly Leu AAA GGC TAC CTT Lys Gly Tyr Leu

AAG

Lys 150 AAT GCC TTC TAT Asn Ala Phe Tyr

GGT

Gly 155 GCC AAT GGC CCC Ala Asn Gly Pro ACT TTT Thr Phe 160 GGT ACC AAG Gly Thr Lys AAA GGA CTT Lys Gly Leu 180 AGC AAC TAC CCG Ser Asn Tyr Pro

CCT

Pro 170 TGC CCC AAA CCC Cys Pro Lys Pro GAC CTT ATC Asp Leu Ile 175 CTC TTG TTC Leu Leu Phe AGG GCC CAC ACC Arg Ala His Thr GCT GGT GGC ATC Ala Gly Gly Ile CAG GAC GAC AAG GTC AGC Gin Asp Asp Lys Val Ser

GGC

Gly 200 CTC CAG CTC CTC Leu Gin Leu Leu

AAG

Lys 205 GAT GGT CAT TGG Asp Gly His Trp

GTT

Val 210 GAT GTT CCT CCC Asp Val Pro Pro

ATG

Met 215 AAA CAC TCC ATT Lys His Ser Ile

GTT

Val 220 GTT AAC TTG GGG Val Asn Leu Gly CAA CTT GAG GTT Gin Leu Glu Val ACA AAT GGC AAG Thr Asn Gly Lys AAG AGT GTG ATG Lys Ser Val Met CAC CGC His Arg 240 GTG ATA GCG Val Ile Ala AAC CCG GGA Asn Pro Gly 260 ACA GAT GGT AAC Thr Asp Gly Asn ATG TCG ATA GCA Met Ser Ile Ala TCA TTC TAC Ser Phe Tyr 255 TTG GTG GAA Leu Val Glu 823 871 AGT GAT GCC GTG Ser Asp Ala Val

ATT

Ile 265 TAC CCG GCG CCA Tyr Pro Ala Pro

ACA

Thr 270 AAA GAA GAG GAG AAA TGC AGA Lys Glu Glu Glu Lys Cys Arg 275 280 GCA TAC CCA AAA Ala Tyr Pro Lys

TTT

Phe 285 GTG TTC GAG GAT Val Phe Glu Asp WO 96/35792 PCT/AU96/00286 -47 TAC ATG AAT CTC TAC TTA AAG CTC AAG TTC CAA Tyr Met Asn Leu Tyr Leu Lys Leu Lys Phe Gin 290 295 300 TTT GAA GCA ATG AAG GCC ATG GAA ACC ACG GGT Phe Glu Ala Met Lys Ala Met Glu Thr Thr Gly 310 315 TGAAATAATG ATTTGATTTG ATATAATGCA

ATGCTTCTCA

TAATATACGC CACTCTCATC TCATCTCATA

TATTCATATT

AATAAGAGCT TCCTTTTAAG

T

GAG AAG GAG CCC AGG Glu Lys Glu Pro Arg 305 CCC ATT CCA ACT GCT Pro Ile Pro Thr Ala 320 TCAACCAATT

TAAGTATTTC

CATATTATTA GTGTTTGTTG 967 1015 1075 1135 1156

Claims (5)

1. A method for producing a transgenic ornamental flowering plant exhibiting prolonged post-harvest life compared to its non-transgenic ornamental flowering parent or a non-transgenic ornamental flowering 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 ACC synthase or ACC oxidase or a derivative of said nucleic acid molecule, and regenerating a transgenic ornamental flowering plant from said cell or cells.

2. A method according to claim 1 wherein the transgenic ornamental flowering plant exhibits one or more of the following properties: S(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; (iii) reduced production of climacteric ethylene; and/or (iv) prolonged post-harvest life 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. 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 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 i^ \\I P:\OPER\EJH\54930-96.CLM 8/1/99 -49- length fragment of a nucleic acid molecule 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: 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. Smolecule is approximately 800-1200 bp in length. 8. A method according to claim 1 or 7 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 9. A method according to claim 1 or 7 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:3 under low stringency conditions at 30 0 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. 9. A method according to claim 1 or 7 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. A method according to claim 1 or 7 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 I P:\OPER\EJH54930-96.CLM 8/1/99 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. 11. A method according to claim 1 or 7 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. 12. A method according to claim 1 or 7 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. S 13. A method according to claim 1 or 7 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. 14. 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 ACC synthase or ACC oxidase or a derivative of said nucleic acid molecule and regenerating a transgenic plant from the cell or cells. A method according to claim 14 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 0 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. S 16. A method according to claim 14 wherein the nucleic acid molecule comprises a sequence P\OPEREJ54930-96.CLM 8/1/99 -51- 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. 17. A method according to claim 14 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. 18. A method according to claim 14 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 sequence substantially as set forth in SEQ ID NO:4 or having at least about 40% similarity Sthereto. 1. A method according to claim 14 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. 21. A method according to claim 1 or 6 or 14 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. 22. A transgenic carnation plant comprising a nucleic acid molecule encoding ACC synthase or ACC oxidase or a derivative of said nucleic acid molecule wherein said transgenic plant P:AOPER\EH\54930-96.CLM 8/1/99 -52- exhibits one or more of the following properties: 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) prolonged post-harvest life of flowers or flower buds cut from said transgenic plants. 23. A transgenic plant according to claim 22 wherein the nucleic acid molecule is a non-full length fragment of a nucleic acid molecule encoding ACC synthase or ACC oxidase. o 24. A transgenic plant according to claim 23 wherein the non-full length fragment is approximately 800-1200 base pairs in length. .o 25. A transgenic plant according to claim 24 wherein the non-full length fragment is an internal fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase. 26. 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 a ACC synthase or ACC oxidase wherein flowers or flower buds of said transgenic plant exhibit one or more of the following properties: a reduced level ofACC 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. 27. A transgenic plant according to claim 26 wherein the non-full length fragment of the nucleic acid molecule encoding ACC synthase or ACC oxidase is approximately 800-1200 bp in length. P:\OPER\EJH54930-96.CLM 8/1/99 -53- 28. A transgenic plant according to any one of claims 22 to 26 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. 29. A transgenic plant according to any one of claims 22 to 26 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 0 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. A transgenic plant according to any one of claims 22 to 26 wherein the nucleic acid molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or is a S. 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. 31. A transgenic plant according to any one of claims 22 to 26 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. 32. A transgenic plant according to any one of claims 22 to 26 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. 33. A transgenic plant according to any one of claims 22 to 26 wherein the nucleic acid molecule encodes an amino acid sequence substantially as set forth in SEQ ID NO:7 or having P:\OPER\EJH54930-%.CLM 8/1/99 -54- at least about 40% similarity thereto. 34. A cut flower from a transgenic carnation according to any one of claims 22 to 33. Seeds or other reproductive material from a transgenic carnation according to any one of claims 22 or 33. 36. A method according to any one of claims 1 to 21 or a transgenic plant according to any one of claims 22 to 33 or a cut flower according to claim 34 or seeds or other reproductive material according to claim 35 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 8th day of January, 1999 FLORIGENE LIMITED By DAVIES COLLISON CAVE Patent Attorneys for the Applicants s'^ o o el L.L -1