Abstract:
A new cultivar of  Dianthus  plant named ‘FLORIAMETRINE’ is characterized inter alia by altered inflorescence with respect to tissue and/or organelles including flowers or flower parts. This trait sets ‘FLORIAMETRINE’ apart from all other existing varieties, lines, strains or sports of  Dianthus.  In particular,  Dianthus  ‘FLORIAMETRINE’ has bright purple/violet flowers.

Description:
Latin name of the genus and species claimed:  Dianthus Caryophyllus.    
     Variety denomination: FLORIAMETRINE. 
     FIELD OF THE INVENTION 
     The present invention relates generally to the field of genetic modification of plants. More particularly, the present invention is directed to genetically-modified carnation plants expressing unique color phenotypes in selected parts of the plants. 
     BACKGROUND OF THE INVENTION 
     The flower or ornamental plant industry strives to develop new and different varieties of flowers and/or plants. An effective way to create such novel varieties is through the manipulation of flower color. Classical breeding techniques have been used with some success to produce a wide range of colors for almost all of the commercial varieties of flowers and/or plants available today. This approach has been limited, however, by the constraints of a particular species&#39; gene pool and for this reason it is rare for a single species to have the full spectrum of colored varieties. For example, the development of novel colored varieties of plants or plant parts such as flowers, foliage and stems would offer a significant opportunity in both the cut flower and ornamental markets. In the flower or ornamental plant industry, the development of desired (including novel) colored varieties of carnation is of particular interest. This includes not only different colored flowers but also anthers and styles. 
     Flower color is predominantly due to three types of pigment: flavonoids, carotenoids and betalains. Of the three, the flavonoids are the most common and contribute a range of colors from yellow to red to blue. The flavonoid molecules that make the major contribution to flower color are the anthocyanins, which are glycosylated derivatives of cyanidin and its methylated derivative peonidin, delphinidin and its methylated derivatives petunidin and malvidin and pelargonidin. Anthocyanins are localized in the vacuole of the epidermal cells of petals or the vacuole of the sub epidermal cells of leaves. 
     The flavonoid pigments are secondary metabolites of the phenylpropanoid pathway. The biosynthetic pathway for the flavonoid pigments (flavonoid pathway) is well established, (Holton and Cornish,  Plant Cell  7:1071-1083, 1995; Mol et al.,  Trends Plant Sci.  3:212-217, 1998; Winkel-Shirley,  Plant Physiol.  126:485-493, 2001a; and Winkel-Shirley,  Plant Physiol.  127:1399-1404, 2001b, Tanaka and Mason, In  Plant Genetic Engineering,  Singh and Jaiwal (eds.) SciTech Publishing Llc., USA, 1: 361-385, 2003, Tanaka et al.,  Plant Cell, Tissue and Organ Culture  80: 1-24, 2005, Tanaka and Brugliera,  In Flowering and Its Manipulation, Annual Plant Reviews  Ainsworth (ed.), Blackwell Publishing, UK, 20: 201-239, 2006). Three reactions and enzymes are involved in the conversion of phenylalanine to p-coumaroyl-CoA, one of the first key substrates in the flavonoid pathway. The enzymes are phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA (provided by the action of acetyl CoA carboxylase (ACC) on acetyl CoA and CO 2 ) with one molecule of p-coumaroyl-CoA. This reaction is catalyzed by the enzyme chalcone synthase (CHS). The product of this reaction, 2′,4,4′,6′, tetrahydroxy-chalcone, is normally rapidly isomerized by the enzyme chalcone flavanone isomerase (CHI) to produce naringenin. Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK). 
     The pattern of hydroxylation of the B-ring of DHK plays a key role in determining petal color. The B-ring can be hydroxylated at either the 3′, or both the 3′ and 5′ positions, to produce dihydroquercetin (DHQ) or dihydromyricetin (DHM), respectively. Two key enzymes involved in this part of the pathway are flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′-hydroxylase (F3′5′H), both members of the cytochrome P450 class of enzymes. 
     The production of colored anthocyanins from the dihydroflavonols (DHK, DHQ, DHM), involves dihydroflavonol-4-reductase (DFR) leading to the production of the leucoanthocyanidins. The leucoanthocyanidins are subsequently converted to the anthocyanidins, pelargonidin, cyanidin and delphinidin. These flavonoid molecules are unstable under normal physiological conditions and glycosylation at the 3-position, through the action of glycosyltransferases, stabilizes the anthocyanidin molecule thus allowing accumulation of the anthocyanins. 
     The substrate specificity shown by DFR can regulate the anthocyanins that a plant accumulates. Petunia and cymbidium DFRs do not reduce DHK and thus they do not accumulate pelargonidin-based pigments (Forkmann and Ruhnau,  Z Naturforsch C.  42c, 1146-1148, 1987, Johnson et al.,  Plant Journal,  19, 81-85, 1999). Many important floricultural species including iris, delphinium, cyclamen, gentian, cymbidium are presumed not to accumulate pelargonidin due to the substrate specificity of their endogenous DFRs (Tanaka and Brugliera, 2006, supra). 
     In carnation, the DFR enzyme is capable of metabolizing two dihydroflavonols to leucoanthocyanidins which are ultimately converted through to anthocyanins pigments that are responsible for flower color. DHK is converted to leucopelargonidin, the precursor to pelargonidin-based pigments, giving rise to apricot to brick-red colored carnations. DHQ is converted to leucocyanidin, the precursor to cyanidin-based pigments, producing pink to red carnations. Carnation DFR is also capable of converting DHM to leucodelphinidin (Forkmann and Ruhnau, 1987 supra), the precursor to delphinidin-based pigments. However, naturally occurring carnation lines do not contain a F3′5′H enzyme and therefore do not synthesize DHM. 
     Nucleotide sequences encoding F3′5′Hs have been cloned (see International Patent Application No. PCT/AU92/00334 incorporated herein by reference and Holton et al.,  Nature,  366:276-279, 1993 and International Patent Application No. PCT/AU03/01111 incorporated herein by reference). These sequences were efficient in modulating 3′, 5′ hydroxylation of flavonoids in petunia (see International Patent Application No. PCT/AU92/00334 and Holton et al., 1993 supra), tobacco (see International Patent Application No. PCT/AU92/00334), carnations (see International Patent Application No. PCT/AU96/00296 incorporated herein by reference) and roses (see International Patent Application No. PCT/AU03/01111). 
     Carnations are one of the most extensively grown cut flowers in the world. 
     There are thousands of current and past cut-flower varieties of cultivated carnation. These are divided into three general groups based on plant form, flower size and flower type. The three flower types are standards, sprays and midis. Most of the carnations sold fall into two main groups, the standards and the sprays. Standard carnations are intended for cultivation under conditions in which a single large flower is required per stem. Side shoots and buds are removed (a process called disbudding) to increase the size of the terminal flower. Sprays and/or miniatures are intended for cultivation to give a large number of smaller flowers per stem. Only the central flower is removed, allowing the laterals to form a ‘fan’ of flowers. 
     Spray carnation varieties are popular in the floral trade, as the multiple flower buds on a single stem are well suited to various types of flower arrangements and provide bulk to bouquets used in the mass market segment of the industry. 
     Standard and spray cultivars dominate the carnation cut-flower industry, with approximately equal numbers sold of each type in the USA. In Japan, spray-type varieties account for 70% of carnation flowers sold by volume, whilst in Europe spray-type carnations account for approximately 50% of carnation flowers traded through out the Dutch auctions. The Dutch auction trade is a good indication of consumption across Europe. 
     Whilst standard and midi-type carnations have been successfully manipulated genetically to introduce new colors (Tanaka and Brugliera, 2006, supra; see International Patent Application No. PCT/AU96/00296), this has not been applied to spray carnations. There is an absence of blue color in color-assortment in carnation, only recently filled through the introduction of genetically-modified standard-type carnation varieties. However, standard-type varieties cannot be used for certain purposes, such as bouquets and flower arrangements where a large number of smaller carnation flowers are needed, such as hand-held arrangements, and small table settings. 
     One particular spray carnation which is particularly commercially popular is the Kortina Chanel line of carnations ( Dianthus caryophyllus  cv. Kortina Chanel). The variety has excellent growing characteristics and a moderate to good resistance to fungal pathogens such as  Fusarium.  There are a number of varieties which have been released as “sports” of Kortina Chanel. These include Kortina, Royal Red Kortina, Cerise Kortina and Dusty Kortina. However, before the advent of the present invention, purple/blue spray carnations were not available. 
     SUMMARY OF THE INVENTION 
     The following traits represent the characteristics of the new  Dianthus  cultivar ‘FLORIAMETRINE’. These traits distinguish this cultivar from other commercial varieties. ‘FLORIAMETRINE’ may exhibit phenotypic differences with variations in environmental, climatic and cultural conditions, without any variance in genotype.
         1.  Dianthus  ‘FLORIAMETRINE’ exhibits pronounced spray habit.   2.  Dianthus  ‘FLORIAMETRINE’ blooms profusely.   3.  Dianthus  ‘FLORIAMETRINE’ exhibits bright purple/violet flowers (RHS N78A).   4.  Dianthus  ‘FLORIAMETRINE’ exhibits green (RHS 137A) foliage.   5. At maturity, the height of the foliage mound of  Dianthus  ‘FLORIAMETRINE’ is 89 cm. The mature width about 15 to 18 cm.   6.  Dianthus  ‘FLORIAMETRINE’ is a perennial.   7.  Dianthus  ‘FLORIAMETRINE’ is suitable for use as a flowering plant in pots, containers, window boxes and the garden, but is primarily suited for the production of cut flowers.   8.  Dianthus  ‘FLORIAMETRINE’ is not hardy and is grown in a greenhouse.       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
       The accompanying color drawing illustrates the overall appearance of the new variety  Dianthus  ‘FLORIAMETRINE’ showing colors as true as reasonably possible to obtain in colored reproductions of this type. Colors in the drawing may differ from the color values cited in the detailed botanical description, which accurately describe the actual colors of the new variety ‘FLORIAMETRINE’. 
         FIG. 1  is a photographic representation of the flower. Colors may appear different from the actual colors due to light reflection but are as accurate as possible by conventional photography. 
         FIG. 2  is a diagrammatic representation of the binary plasmid pCGP2442. Selected restriction endonuclease sites (AscI, PacI, PmeI) are marked. Abbreviations include LB=Left Border from  A. tumefaciens  Ti plasmid; RB=Right Border region from  A. tumefaciens  Ti plasmid; TetR=tetracycline resistance gene complex. 
         FIG. 3  is a photographic representation of a high resolution scan of a Southern blot autoradiograph showing 10 μg of EcoRI-treated genomic DNA from the transgenic carnation line 19907, in comparison to 10 μg of EcoRI-treated genomic DNA from the carnation lines Kortina Chanel, Vega and Purple Spectro, hybridized with the NtALS probe. 
         FIG. 4  is a photographic representation of the ‘Kortina Chanel’ control on the left and the cultivar ‘FLORIAMETRINE’ on the right. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention relates to a new and distinct cultivar of carnation that is grown for use as a flowering plant for pots and containers. The new cultivar is known botanically as  Dianthus caryophyllus  and is referred to hereinafter by the cultivar name ‘FLORIAMETRINE’. 
     ‘FLORIAMETRINE’ is a complex transgenic plant comprising genetic sequences encoding at least two F3′5′H molecules and at least one DFR. The vector pCGP2442 used to transform meristematic cells contains a chimeric AmCHS 5′: Salivia F3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene and the 35S 5′: SuRB selectable marker gene cassette of the plasmid pWTT2132. 
     The new variety originated in vitro by  Agrobacterium tumefaciens -mediated transformation of meristematic cells of the Kortina Chanel (unpatented) carnation with the pCGP2442 vector at Florigene Pty Ltd., in Bundoora, Victoria, Australia. Cuttings of  Dianthus caryophyllus  cv. Kortina Chanel were obtained from Van Wyk and Son Flower Supply, Victoria or Propagation Australia, Queensland, Australia. Transgenic plants containing the chimeric AmCHS 5′: SaliviaF3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, and a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene were successfully generated from the cells. In addition to these genes, the plants also contains genes for acetolactate synthase resistance (SuRB) transformation selection markers. The transformation and regeneration process is described in International Patent Application No. PCT/US92/02612; International Patent Application No. PCT/AU96/00296; and Lu et al., Bio/Technology 9: 864-868, 1991, the contents of each of which are incorporated by reference. 
     The primary focus of the carnation generation program was to produce new cultivars of carnations which exhibited a selected and desired purple/violet color in the spray background. The term ‘FLORIAMETRINE’ was selected because of its pronounced production of delphinidin or delphinidin-based molecules pigments. 
     The new variety was selected from a group of 74 transgenic lines of which only three produced flowers with a significant shift in color into the violet, purple/violet range. ‘FLORIAMETRINE’ is essentially similar to the parent in the morphological aspects of the flower, but can be further distinguished from the parent throughout the accumulation of pigment in the filaments and anthers of the flower. This is a new phenotype of the transgenic line. Some styles and anthers of ‘FLORIAMETRINE’ also have a shift in color to light purple, whereas the styles and anthers from flowers of the parent line were a cream-white color. 
     The new variety was originally selected in vitro as a regenerated shoot from a ‘Kortina Chanel’ carnation meristematic cell that had been transfected with  Agrobacterium tumefaciens  AGL0 (Lazo et al., Bio/technology 9:963-967, 1991) carrying the plasmid pCGP2442. 
     Asexual reproduction of the new cultivar was first accomplished in 2007 in a cultivated area of Bundoora, Victoria, Australia. The method of asexual propagation used was vegetative cuttings. Since that time the characteristics of the new cultivar have been determined stable and are reproduced true to type in successive generation of asexual reproduction. 
     BOTANICAL DESCRIPTION OF THE PLANT 
     The following is a detailed description of the new cultivar ‘FLORIAMETRINE’. Data was collected from plants grown indoors in Bundoora, Victoria, Australia. The color determinations are in accordance with the 2001 edition of The Royal Horticultural Society (R.H.S.) Colour Chart except where general color terms of ordinary dictionary significance are used. Growing conditions are typical to other species, sports and lines of  Dianthus.  
     Botanical classification:  Dianthus caryophyllus.      Species:  Caryophyllus.      Common name: Kortina Chanel.   Commercial classification:  Dianthus caryophyllus  19907.   Type: Perennial.   Use: Used as a flowering plant for pots and containers.   Parentage: ‘FLORIAMETRINE’ is a transgenic plant that resulted from the transformation of  D. caryophyllus  with the transformation vector, pCGP2442.   

     
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                 Plant Description 
               
             
          
           
               
                 Bloom period 
                 All year 
               
               
                 Plant habit 
                 Spray type carnation 
               
               
                 Plant height 
                 Average plant height at flowering— 
               
               
                   
                 891 mm 
               
               
                 Plant width 
                 About 150 to 180 mm at flowering 
               
               
                 Plant hardiness 
                 Not tested for hardiness 
               
               
                 Root system 
                 Fine fibrous root system 
               
               
                 Propagation 
                 Vegetative propagation 
               
               
                 Cultural requirements 
                 Grown hydroponically in a 
               
               
                   
                 greenhouse. Plants fertilized via 
               
               
                   
                 drip irrigation system 
               
               
                 Pests and diseases 
                 Susceptible to known  Dianthus  pest 
               
               
                   
                 and diseases 
               
               
                 Time and Temperature needed to 
                 About 3 to 4 weeks to produce 
               
               
                 produce a rooted cutting 
                 rooted cuttings, bench heat: 18-22° 
               
               
                   
                 C., Air temp approximately 15 to 
               
               
                   
                 22° C. 
               
               
                 Crop time 
                 Average days to flowering: 107. 
               
               
                 Stem shape 
                 Cylindrical, Average stem length 
               
               
                   
                 782 mm, Average stem diameter at 
               
               
                   
                 5th node: 6 mm 
               
               
                 Stem surface 
                 Glabrous and glaucous 
               
               
                 Stem color 
                 137B 
               
               
                 Branching 
                 Little branching from the axils of 
               
               
                   
                 lower leaves 
               
               
                 Internode length 
                 Average length of 5th internode: 
               
               
                   
                 73 mm 
               
               
                 Node color 
                 192D 
               
               
                 Node dimensions 
                 About 6 mm diameter and about 3 
               
               
                   
                 mm in length 
               
             
          
           
               
                 Foliage 
               
             
          
           
               
                 Type 
                 Evergreen 
               
               
                 Shape 
                 Linear 
               
               
                 Division 
                 Simple 
               
               
                 Apex 
                 Acute 
               
               
                 Base 
                 Decurrent 
               
               
                 Venation 
                 Not prominent 
               
               
                 Margins 
                 Entire 
               
               
                 Attachment 
                 Sheathing 
               
               
                 Arrangement 
                 Opposite and spiraling up stem 
               
               
                 Surfaces 
                 Glaucous 
               
               
                 Leaf dimensions 
                 3rd leaf from flower, Average 
               
               
                   
                 length: 40.5 mm, Average width: 7 
               
               
                   
                 mm 
               
               
                 Leaf color 
                 137A 
               
               
                 Fragrance 
                 Absent 
               
             
          
           
               
                 Flowers 
               
             
          
           
               
                 Inflorescence 
                 Cymose 
               
               
                 Flower type 
                 Saliform, double and symmetrical 
               
               
                 Flower dimensions 
                 Average corolla height: 22.5 mm, 
               
               
                 (including calyx) 
                 Average calyx height: 32.5 mm 
               
               
                 Fragrance 
                 Absent 
               
               
                 Bud color 
                 191B 
               
               
                 Anthocyanin 
                 Present 
               
               
                 Bud dimensions 
                 Average bud length: 26.4 mm, 
               
               
                   
                 Average bud width: 9 mm 
               
               
                 Bud shape 
                 Cylindrical 
               
               
                 Petals 
                 Persistent, apopetalous, overlapping 
               
               
                 Petal number 
                 Average number of petals: 27 
               
               
                 Petal margin 
                 Denate 
               
               
                 Petal shape 
                 Obtetoid 
               
               
                 Petal surface 
                 Glabrous 
               
               
                 Petal dimensions 
                 Average petal length: 47 mm, 
               
               
                   
                 Average petal width: 22 mm 
               
               
                 Ground color of blade 
                 N78A 
               
               
                 Color of band around centre 
                 N78A 
               
               
                 Color of middle of strap 
                 145C 
               
               
                 Color of base of strap 
                 145D 
               
               
                 Calyx dimensions 
                 Average calyx length: 32.5 mm, 
               
               
                   
                 Average calyx diameter at apex: 
               
               
                   
                 14.5 mm 
               
               
                 Calyx color 
                 138B 
               
               
                 Anthocyanin 
                 Absent 
               
               
                 Sepals 
                 Average number of sepals: 6 
               
               
                 Fused or Unfused 
                 Unfused 
               
               
                 Sepal color 
                 138B 
               
               
                 Anthocyanin 
                 Absent 
               
               
                 Peduncle dimensions 
                 Average peduncle length: 33.6 mm, 
               
               
                   
                 Average peduncle width: 2 mm 
               
               
                 Peduncle color 
                 138A 
               
               
                 Peduncle surface 
                 Glaucous 
               
               
                 Epicalyx 
                 Present 
               
               
                 Bracts 
                 1 pair in number (2 individual 
               
               
                   
                 bracts) 
               
               
                 Bracts dimensions 
                 About 3 mm by about 20 mm 
               
               
                 Bract color 
                 138A 
               
               
                 Anthacyanin 
                 Absent 
               
               
                 Bracteoles 
                 1 or 2 pair 
               
               
                 Dimensions 
                 About 3 mm by about 25 mm 
               
               
                 Anthocyanin 
                 Absent 
               
               
                 Stipules 
                 Absent 
               
               
                 Stipules dimensions 
                 N/A 
               
               
                 Stipule color 
                 N/A 
               
               
                 Anthacyanin 
                 N/A 
               
               
                 Lastiness of flowers 
                 14 days 
               
             
          
           
               
                 Reproductive Organs 
               
             
          
           
               
                 Stamens 
                 Average number of stamens: 10 
               
               
                 Stamen dimensions 
                 Average length of stamen: 21.5 mm 
               
               
                 Stamen color 
                 Upper: N80C, Lower: N155B 
               
               
                 Anther number 
                 Average of normal anthers: 2, 
               
               
                   
                 Average of abnormal anthers: 6 
               
               
                 Anther attachment 
                 Dorsifixed 
               
               
                 Anther color 
                 N80C 
               
               
                 Anther dimensions 
                 Average anther length: 1.84 mm, 
               
               
                   
                 Average anther width: 0.68 mm 
               
               
                 Pollen 
                 Little pollen 
               
               
                 Pistil 
                 One that divides into 2 above the 
               
               
                   
                 ovary 
               
               
                 Pistil dimensions 
                 Average pistil length: 34 mm 
               
               
                 Styles 
                 Average No: 2, Average length: 26 
               
               
                   
                 mm 
               
               
                 Style color 
                 N155B 
               
               
                 Stigma number 
                 Single 
               
               
                 Stigma shape 
                 A single stigma 
               
               
                 Stigma color 
                 N155B 
               
               
                 Height above petals 
                 Stigma does not protrude above 
               
               
                   
                 petals 
               
               
                 Ovary postion 
                 Superior 
               
               
                 Ovary dimensions 
                 Average ovary height: 8 mm, 
               
               
                   
                 Average ovary width: 5.5 mm 
               
               
                 Ovary shape 
                 Obovoid 
               
               
                 Ovary color 
                 Upper: 145A, Lower: 155A 
               
               
                 Seed 
                 Absent 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Floriametrine 
                 Kortina Chanel control 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Description 
               
             
          
           
               
                 Bloom period 
                 All year 
                 All year 
               
               
                 Plant habit 
                 Spray type carnation 
                 Spray type carnation 
               
               
                 Plant height 
                 Average plant height at  
                 Average plant height at 
               
               
                   
                 flowering —895 mm 
                 flowering —853 mm 
               
               
                 Plant width 
                 150 to 180 mm at 
                 150 to 180 mm at 
               
               
                   
                 flowering 
                 flowering 
               
               
                 Plant hardiness 
                 Not tested for hardiness 
                 Not tested for hardiness 
               
               
                 Root system 
                 Fine fibrous root system 
                 Fine fibrous root system 
               
               
                 Propagation 
                 Vegetative propagation 
                 Vegetative propagation 
               
               
                 Cultural  
                 Grown hydroponically 
                 Grown hydroponically in 
               
               
                 requirements 
                 in a greenhouse. Plants  
                 a greenhouse. Plants 
               
               
                   
                 fertilized via drip 
                 fertilized via drip 
               
               
                   
                 irrigation system 
                 irrigation system 
               
               
                 Pests and diseases 
                 Susceptible to known 
                 Susceptible to known 
               
               
                   
                   Dianthus  pest and 
                   Dianthus  pest and 
               
               
                   
                 diseases 
                 diseases 
               
               
                 Time and  
                 3 to 4 weeks to produce  
                 3 to 4 weeks to produce 
               
               
                 Temperature 
                 rooted cuttings, bench 
                 rooted cuttings, bench 
               
               
                 needed to 
                 heat: 18-22° C., Air 
                 heat: 18-22° C., Air temp 
               
               
                 produce a  
                 temp approx. 15 to 
                 approx. 15 to 22° C. 
               
               
                 rooted cutting 
                 22° C. 
                   
               
               
                 Crop time 
                 Average days to 
                 Average days to 
               
               
                   
                 flowering: 107. 
                 flowering: 108 
               
               
                 Stem shape 
                 Cylindrical, Ave stem 
                 Cylindrical, Ave stem 
               
               
                   
                 length 782 mm, Ave 
                 length 713 mm, Ave. 
               
               
                   
                 stem diameter at 5 th   
                 stem diameter at 5 th  node: 
               
               
                   
                 node: 6 mm 
                 6.7 mm 
               
               
                 Stem surface 
                 Glabrous and glaucous 
                 Glabrous and glaucous 
               
               
                 Stem color 
                 137B 
                 137B 
               
               
                 Branching 
                 Little branching from 
                 Little branching from the 
               
               
                   
                 the axils of lower leaves 
                 axils of lower leaves 
               
               
                 Internode length 
                 Average length of 5 th   
                 Average length of 5 th   
               
               
                   
                 internode: 73 mm 
                 internode: 73 mm 
               
               
                 Node color 
                 192D 
                 192D 
               
               
                 Node dimensions 
                 6 mm diameter and 
                 6 mm diameter and 3 mm 
               
               
                   
                 3 mm in length 
                 in length. 
               
             
          
           
               
                 Foliage 
               
             
          
           
               
                 Type 
                 Evergreen 
                 Evergreen 
               
               
                 Shape 
                 Linear 
                 Linear 
               
               
                 Division 
                 simple 
                 simple 
               
               
                 Apex 
                 Acute 
                 Acute 
               
               
                 Base 
                 Decurrent 
                 Decurrent 
               
               
                 Venation 
                 Not prominent 
                 Not prominent 
               
               
                 Margins 
                 Entire 
                 Entire 
               
               
                 Attachment 
                 Sheathing 
                 Sheathing 
               
               
                 Arrangement 
                 Opposite and spiraling up 
                 Opposite and spiraling 
               
               
                   
                 stem 
                 up stem 
               
               
                 Surfaces 
                 Glaucous 
                 Glaucous 
               
               
                 Leaf dimensions 
                 3 rd  leaf from flower, Ave 
                 3 rd  leaf from flower, Ave 
               
               
                   
                 length: 40.5 mm, Ave 
                 length: 39 mm, Ave 
               
               
                   
                 width: 7 mm 
                 width: 8 mm 
               
               
                 Leaf color 
                 137A 
                 137A 
               
               
                 Fragrance 
                 Absent 
                 Absent 
               
             
          
           
               
                 Flowers 
               
             
          
           
               
                 Inflorescence 
                 Cymose 
                 Cymose 
               
               
                 Flower type 
                 Saliform, double and 
                 Saliform, double and 
               
               
                   
                 symmetrical 
                 symmetrical 
               
               
                 Flower dimensions  
                 Ave. corolla height: 
                 Ave corolla height: 
               
               
                 including 
                 22.5 mm, Ave calyx 
                 23.5 mm, Ave. calyx 
               
               
                 calyx) 
                 height: 32.5 
                 height: 31.5 mm 
               
               
                 Fragrance 
                 Absent 
                 Absent 
               
               
                 Bud color 
                 191B 
                 191B 
               
               
                 Anthocyanin 
                 Present 
                 Present 
               
               
                 Bud dimensions 
                 Ave bud length: 
                 Ave bud length: 24.9 mm, 
               
               
                   
                 26.4 mm, Ave bud 
                 Ave bud width: 9.9 mm 
               
               
                   
                 width: 9 mm 
                   
               
               
                 Bud shape 
                 Cylindrical 
                 Cylindrical 
               
               
                 Petals 
                 Persistent, apopetalous, 
                 Persistent, apopetalous, 
               
               
                   
                 overlapping 
                 overlapping 
               
               
                 Petal number 
                 Ave number of petals: 
                 Ave number of petals: 32 
               
               
                   
                 27 
                   
               
               
                 Petal margin 
                 Denate 
                 Denate 
               
               
                 Petal shape 
                 Obtetoid 
                 Obtetoid 
               
               
                 Petal surface 
                 Glabrous 
                 Glabrous 
               
               
                 Petal dimensions 
                 Ave petal length: 
                 Ave petal length: 47 mm, 
               
               
                   
                 47 mm, Ave petal 
                 Ave petal width: 22 mm 
               
               
                   
                 width: 22 mm 
                   
               
               
                 Ground color  
                 N78A 
                 65A 
               
               
                 of blade 
                   
                   
               
               
                 Color of band  
                 N78A 
                 65A 
               
               
                 around centre 
                   
                   
               
               
                 Color of middle  
                 145C 
                 145C 
               
               
                 of strap 
                   
                   
               
               
                 Color of base  
                 145D 
                 145D 
               
               
                 of strap 
                   
                   
               
               
                 Calyx dimensions 
                 Ave calyx length: 
                 Ave calyx length: 31.5 mm, 
               
               
                   
                 32.5 mm, Ave calyx 
                 Ave calyx diameter at 
               
               
                   
                 diameter at apex: 
                 apex: 14.9 mm 
               
               
                   
                 14.5 mm 
                   
               
               
                 Calyx color 
                 138B 
                 138B 
               
               
                 Anthocyanin 
                 Absent 
                 Absent 
               
               
                 Sepals 
                 Ave number of sepals: 
                 Ave number of sepals: 
               
               
                   
                 6 
                 5.4 
               
               
                 Fused or Unfused 
                 Unfused 
                 Unfused 
               
               
                 Sepal color 
                 138B 
                 138B 
               
               
                 Anthocyanin 
                 absent 
                 absent 
               
               
                 Peduncle  
                 Ave peduncle length: 
                 Ave peduncle length: 
               
               
                 dimensions 
                 33.6 mm, Ave peduncle 
                 45.2 mm, Ave peduncle 
               
               
                   
                 width: 2 mm 
                 width: 2.4 
               
               
                 Peduncle color 
                 138A 
                 138A 
               
               
                 Peduncle surface 
                 Glaucous 
                 Glaucous 
               
               
                 Epicalyx 
                 Present 
                 Present 
               
               
                 Bracts 
                 1 pair in number (2 
                 1 pair in number (2 
               
               
                   
                 individual bracts) 
                 individual bracts) 
               
               
                 Bracts dimensions 
                 3 mm × 20 mm 
                 3 mm × 20 mm 
               
               
                 Bract color 
                 138A 
                 138A 
               
               
                 Anthacyanin 
                 absent 
                 absent 
               
               
                 Bracteoles 
                 1 or 2 pair 
                 1 or 2 pair 
               
               
                 Dimensions 
                 3 mm × 25 mm 
                 3 mm × 25 mm 
               
               
                 Anthocyanin 
                 Absent 
                 Absent 
               
               
                   
               
             
          
         
       
     
     The  Dianthus  ‘FLORIAMETRINE’ is now described by the following non-limiting Examples. 
     EXAMPLE 1 
     GENERATION OF  DIANTHUS  ‘FLORIAMETRINE’ 
     In order to increase the levels of delphinidin-based anthocyanins and therefore increase the chance of violet/purple/blue color in the Kortina Chanel spray carnation flowers, a novel construct was prepared that included the use of two F3′5′H chimeric genes and a petunia DFR gene. 
     The DFR genomic fragments used in this application were isolated from petunia. The petunia DFR enzyme is only capable of using DHQ and DHM as a substrate, but not DHK (Holton and Cornish, 1995 supra). This ensures that most or all of the anthocyanidin produced is delphinidin. 
     The F3′5′H coding sequences in the chimeric genes used in the new construct were from pansy (carnANS 5′: BP F3′5′H #18: carnANS 3′ in pCGP2205) and salvia (AmCHS 5′: Salvia F3′5′H #47: petD8 3′ in pCGP2122) as these represent the two expression cassettes that were the most efficient in producing the highest levels of delphinidin in the Kortina Chanel spray carnation. 
     Preparation of the Transformation Vector, pCGP2442 
     The transformation vector pCGP2442 ( FIG. 2 ) contains a chimeric AmCHS: Salvia F3′5′H#47: petD8 3′ gene in tandem with a petunia genomic DFR-A gene, a chimeric carnANS 5′: BPF3′5′H#18: carnANS 3′ gene and the 35S 5′: SuRB selectable marker gene cassette of the plasmid pWTT2132 (see International Patent Application No. PCT/AU03/01111 incorporated herein by reference). 
       Agrobacterium tumefaciens  Strains and Transformations 
     The disarmed  Agrobacterium tumefaciens  strain used was AGL0 (Lazo et al., 1991 supra). 
     Plasmid DNA was introduced into the  Agrobacterium tumefaciens  strain AGL0 by adding 5 μg of plasmid DNA to 100 μL of competent AGL0 cells prepared by inoculating a 50 mL LB culture (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 1989) and incubation for 16 hrs with shaking at 28° C. The cells were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM CaCl 2 /15% (v/v) glycerol. The DNA- Agrobacterium  mixture was frozen by incubation in liquid N 2  for 2 minutes and then allowed to thaw by incubation at 37° C. for 5 minutes. The DNA/bacterial mix was then placed on ice for a further 10 minutes. The cells were then mixed with 1 mL of LB (Sambrook et al., 1989 supra) media and incubated with shaking for 16 hrs at 28° C. Cells of  A. tumefaciens  carrying the plasmid were selected on LB agar plates containing appropriate antibiotics such as 50 μg/mL tetracycline or 100 μg/mL gentamycin. The confirmation of the plasmid in  A. tumefaciens  was done by restriction endonuclease mapping of DNA isolated from the antibiotic-resistant transformants. 
     Plant transformations were as described in International Patent Application No. PCT/US92/02612 or International Patent Application No. PCT/AU96/00296 or Lu et al.,  Bio/Technology  9: 864-868, 1991 each incorporated herein by reference. 
     Cuttings of  Dianthus caryophyllus  cv. Kortina Chanel were obtained from Van Wyk and Son Flower Supply, Victoria or Propagation Australia, Queensland, Australia. 
     EXAMPLE 2 
     DETECTION OF THE SURB CHIMERIC GENE FROM THE TRANSFORMATION VECTOR PGP2442 IN  DIANTHUS  ‘FLORAMETRINE’ PLANTS 
     In order to determine stable transformation of  Dianthus caryophyllus  with the T-DNA from the transformation vector pCGP2442, transgenic plants were analyzed by Southern blot. The results are shown in  FIG. 3 . 
     Preparation of Genomic DNA and Southern Analysis 
     Genomic DNA was isolated from leaf tissues as described by Dellaporta et al.,  Molecular Biology Reporter  1(14):19-21, 1983. The genomic DNA (10 μg) was digested for 48 hours using 120 units of the restriction endonuclease EcoRI at 37° C. DNA fragments were separated by electrophoresis through a 0.8% w/v agarose gel. The DNA was transferred to Hybond NX membrane (Amersham) as described (Sambrook et al., 1989 supra). 
     The following samples were analyzed:
         1. HindIII-treated λDNA standard markers (size range: 23.13, 9.42, 6.56, 4.36, 2.32, 2.03 kb),   2. 10 μg of EcoRI-treated genomic DNA from transgenic carnation line 19907 (FLORIAMETRINE),   3. 10 μg of EcoRI-treated genomic DNA from non-transgenic carnation parental line, Kortina Chanel,   4. 10 μg of EcoRI-treated genomic DNA from non-transgenic carnation line, Vega; and   5. 10 μg of EcoRI-treated genomic DNA from non-transgenic carnation line, Purple Spectro.       

     Following electrophoresis, the gel was prepared for blotting by a 15 minute depurination step in 0.25 M HCl, two 20 minute washes in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and two 20 minute washes in neutralization solution (0.5 M Tri-HCl, pH 7.5, 0.48 M HCl, 1.5 M NaCl). DNA was capillary transferred to Hybond-NX nylon membrane (Amersham Biosciences, UK) in 20×SSC (3 M NaCl, 0.3 M Tris-Na citrate, pH 7.0). 
     Preparation of Probes 
     A probe corresponding to a 770 bp fragment of the ALS (acetolactate synthase) gene from  Nicotiana tabacum  (NtALS) was used for Southern blot analysis. The probe fragment was originally generated by PCR and subsequently sub-cloned into an amplification vector (pBluescript II, Stratagene, USA), given a reference number (pCGP1651) and the fragment sequenced. After confirmation of the correct sequence, the DNA fragment was isolated from the source plasmid using the restriction endonuclease HindIII. The fragment was separated by 1% w/v agarose gel electrophoresis and purified using the MinElute Gel Extraction kit and protocol (Qiagen, Australia). 
     32P-Labeling of DNA Probes 
     DNA fragments (25-50 ng) were labeled with 50 μCi of [α-32P]-dCTP (PerkinElmer Life and Analytical Sciences, USA) using a Decaprime kit (Ambion, USA). Unincorporated [α- 32 P]-dCTP was removed by chromatography on Sephadex G-50 (Fine) columns. The labeled probe fragment was counted using a BioScan radioisotope counter (QC:4000 XER, BioScan, USA). 
     Hybridization and Detection 
     Membranes were pre-hybridized in 10 mL hybridization buffer 50% v/v deionized formamide, 1 M NaCl, 1% w/v SDS and 10% w/v dextran sulfate) at 42° C. for 1 hr. Once denatured, 10,000,000 dpm of  32 P -labeled probe was added to the hybridization solution and hybridization was continued at 42° C. for a further 16 hours. Membranes were washed twice in low stringency buffer (2×SSC, 1% w/v SDS) at 65° C. for 30 minutes. Membranes were exposed to Kodax BioMax MS X-Ray film (Kodak, USA) with an intensifying screen at −70° C. for 16 hours. The exposed films were automatically developed using a Curix 60 X-ray developer (AGFR-Gevaert Group, Belgium).