the present invention relates to a new and distinct cultivar of carnation that is grown for use , for example , 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 ‘ florijade ’. ‘ florijade ’ is a complex transgenic plant comprising a functional f3 ′, 5 ′ h and a cytochrome b5 in petals . the vector pcgp2355 used to transform meristematic cells contained the carnans 5 ′: pet f3 ′ 5 ′ h : carnans 3 ′ expression cassette along with a amchs 5 ′: pet cytb5 : petd8 3 ′ expression cassette 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 cerise westpearl carnation with the pcgp2355 vector at florigene pty ltd , in bundoora , victoria , australia . cuttings of dianthus caryophyllus cv . cerise westpearl were obtained from propagation australia , queensland , australia . transgenic plants containing a chimeric petunia f3 ′ 5 ′ h gene in tandem with a petunia cytochrome b5 gene were successfully generated from the cells . in addition to these genes , the plants also contained 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 are herein 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 ‘ florijade ’ was selected because of its pronounced production of delphinidin or delphinidin - based pigments . the new variety was selected from a group of 47 transgenic lines . ‘ florijade ’ is essentially similar to the parent in the morphological aspects of the flower , but can be distinguished from the parent through out based on the accumulation of the purple delphinidin - based pigment in the petals of the flower . this is a new phenotype of the transgenic line . the new variety was originally selected in vitro as a regenerated shoot from a ‘ cerise westpearl ’ carnation meristematic cell that had been transfected with agrobacterium tumefaciens agl0 ( lazo et al , bio / technology 9 : 963 - 967 , 1991 ) carrying the plasmid pcgp2355 ( fig2 ). the new variety differs from the ‘ cerise westpearl ’ parent in that its average crop time is 167 days versus 174 . 7 days for the parent ; its internode length averages 73 . 4 mm versus 81 . 6 mm for the parent ; its average number of petals is 35 versus 44 . 7 for the parent ; its ground color of blade and color of band around the center is n78a versus 58b for the parent ; and its average number of stamen is 12 . 3 versus 10 . 3 for the parent . the new variety also differs from other known dianthus cultivars , such as ‘ floriametrine ’ and ‘ floriagate ’. for example , the new variety has a crop time of about 167 days , whereas ‘ floriametrine ’ has an average crop time of about 107 days . the new variety has a node color of about 192d whereas ‘ floriagate ’ has a node color of about 192a . the leaf dimension of the new variety are about 49 . 7 mm by about 6 . 3 mm , whereas the leaf dimension of ‘ floriametrine ’ are about 40 . 5 mm by about 7 mm and the leaf dimensions of ‘ floriagate ’ are about 53 . 9 mm by about 6 . 6 mm . 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 generations of asexual reproduction . the following is a detailed description of the new cultivar ‘ florijade ’. data was collected from plants grown indoors in bundoora , victoria , australia . the royal horticultural society &# 39 ; s colour charts , third and / or fifth edition ( london , uk ), 1995 and / or 2007 were used to provide a description of observed color , 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 : carnation . commercial classification : dianthus caryophyllus 26407 . type : herbaceous perennial . use : used as a flowering plant for pots and containers . parentage : ‘ florijade ’ is a transgenic plant that resulted from the transformation of d . caryophyllus cultivar ‘ cerise westpearl ’ as disclosed in paragraph [ 0025 ] with the transformation vector , pcgp2355 . in order to increase the levels of delphinidin - based anthocyanins and therefore increase the chance of violet / purple / blue color in the cerise westpearl spray carnation flowers , a construct ( pcgp2355 ) was prepared that included the use of a f3 ′ 5 ′ h gene and a cyt b5 gene . the f3 ′ 5 ′ h coding sequence in the chimeric gene ( carnans 5 ′: pet f3 ′ 5 ′ h : carnans 3 ′) used in the construct pcgp2355 was from petunia . the cytochrome b5 coding sequence used in the the chimeric gene ( amchs 5 ′: pet cytb5 : petd8 3 ′) used in the construct pcgp2355 was from petunia . the transformation vector pcgp2355 ( fig2 ) contains the carnans 5 ′: pet f3 ′ 5 ′ h : carnans 3 ′ expression cassette along with a amchs 5 ′: pet cytb5 : petd8 3 ′ expression cassette and the 35s 5 ′: surb selectable marker gene . 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 incubating for 16 hours 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 1ml of lb ( sambrook et al , 1989 supra ) media and incubated with shaking for 16 hours 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 . 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 of which are incorporated herein by reference . cuttings of dianthus caryophyllus cv . cerise westpearl were obtained from queensland , australia . detection of the surb chimeric gene from the transformation vector pcgp2355 in dianthus ‘ florijade ’ plants in order to determine stable transformation of dianthus caryophyllus with the t - dna from the transformation vector pcgp2355 , transgenic plants were analyzed by southern blot . the results are shown in fig3 . 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 ). 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 , 25958 (‘ floriagate ’), 3 . 10 μg of ecori - treated genomic dna from non - transgenic carnation parental line , cerise westpearl , 4 . 10 μg of ecori - treated genomic dna from non - transgenic carnation line , purple spectro , 5 . 10 μg of ecori - treated genomic dna from transgenic carnation line , 19907 (‘ floriametrine ’), and 6 . 10 μg of ecori - treated genomic dna from transgenic carnation line 26407 (‘ florijade ’). following electrophoresis , the gel was prepared for blotting by a 15 minutes depurination step in 0 . 25 m hcl , two 20 minute washes in denaturing solution ( 1 . 5 m nacl , 0 . 5 m naoh ) and two 20 minute washes in neutralization solution ( 0 . 5 m tri - hcl , ph 7 . 5 , 0 . 48 m hcl , 1 . 5 m nacl ). dna was capillary transferred to hybond - nx nylon membrane ( amersham biosciences , uk ) in 20 × ssc ( 3 m nacl , 0 . 3 m tris - na citrate , ph 7 . 0 ). a probe corresponding to a 770 bp fragment of the als ( acetolactate synthase ) gene from nicotiana tabacum ( ntals ) was used for southern blot analysis . the probe fragment was originally generated by pcr and subsequently sub - cloned into an amplification vector ( pbluescript ii , stratagene , usa ), given a reference number ( pcgp1651 ) and the fragment sequenced . after confirmation of the correct sequence , the dna fragment was isolated from the source plasmid using the restriction endonuclease hindiii . the fragment was separated by 1 % w / v agarose gel electrophoresis and purified using the minelute gel extraction kit and protocol ( qiagen , australia ). dna fragments ( 25 - 50 ng ) were labeled with 50 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 ). 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 hour . once denatured , 10 , 000 , 000 dpm of 32p - 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 ).
a new cultivar of dianthus plant named ‘ florijade ’ is characterized , inter alia , by pronounced spray habit , perennial and profuse bloom , and green foliage . dianthus ‘ florijade ’ is suitable for use , for example , as a flowering plant in pots , containers , window boxes , and the garden , but is primarily suited for the production of cut flowers . dianthus ‘ florijade ’ is not hardy and is typically grown in a glasshouse . these traits set ‘ florijade ’ apart from all existing varieties , lines , strains , or sports of dianthus . in particular , dianthus ‘ florijade ’ has bright purple / violet flowers .