Patent Application: US-200913140389-A

Abstract:
the invention relates to genetically engineered plants with altered inflorescence . plants such as spray carnations are transformed with a non - indigenous flavonoid 3 ′, 5 ′ hydroxylase and dihydroflavanol - 4 - reductase in conjunction with a genetic suppressor of indigenous dfr . preferably the substrate specificity of the indigenous dfr is different to the non - indigenous dfr in order to enhance the colour of the inflorescence .

Description:
bp , black pansy ; nt , nucleotide ; aa , amino acid ; pet , petunia ; carn , carnation ; thmt , s - adenosylmethionine : anthocyanin 3 ′, 5 ′ methyltransferase from torenia ; ans , anthocyanin synthase ; chs , chalcone synthase ; 3 ′ 5 ′ amt , s - adenosylmethionine : anthocyanin 3 ′, 5 ′ methyltransferase ; fns , flavone synthase ; thfns , flavone synthase from torenia . fig1 is a schematic representation of the biosynthesis pathway for the flavonoid pigments showing production of the anthocyanidin 3 - glucosides that occur in most plants that produce anthocyanins . enzymes involved in the pathway have been indicated as follows : pal = phenylalanine ammonia - lyase ; c4h = cinnamate 4 - hydroxylase ; 4cl = 4 - coumarate : coa ligase ; chs = chalcone synthase ; chi = chalcone flavanone isomerase ; f3h = flavanone 3 - hydroxylase ; dfr = dihydroflavonol - 4 - reductase ; ans = anthocyanidin synthase , 3gt = udp - glucose : flavonoid 3 - o - glucosyltransferase ; other abbreviations include : dhk = dihydrokaempferol , dhq = dihydroquercetin , dhm = dihydromyricetin . fig2 is a diagrammatic representation of the binary plasmid pcgp3360 . chimeric . the construction of pcgp3360 is described in example 1 . selected restriction endonuclease sites are marked . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . refer to table 2 for a description of gene elements . fig3 is a diagrammatic representation of the binary plasmid pcgp3366 . chimeric . the construction of pcgp3366 is described in example 1 . selected restriction endonuclease sites are marked . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . in this figure “ ds carndfr ”= the camv 35s : ds carndfr : 35s 3 ′ expression cassette . refer to table 2 for a description of gene elements . fig4 is a diagrammatic representation of the binary plasmid pcgp3601 . chimeric . the construction of pcgp3601 is described in example 1 . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . in this figure “ ds carndfr ”= the camv 35s : ds carndfr : 35s 3 ′ expression cassette . refer to table 2 for a description of gene elements . fig5 is a diagrammatic representation of the binary plasmid pcgp3605 . chimeric . the construction of pcgp3605 is described in example 1 . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . in this figure “ ds carndfr ”= the camv 35s : ds carndfr : 35s 3 ′ expression cassette and “ thmt ”= camv 35s : thmt : 35s 3 ′ expression cassette . refer to table 2 for a description of gene elements . fig6 is a diagrammatic representation of the binary plasmid pcgp3616 . chimeric . the construction of pcgp3616 is described in example 1 . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . in this figure “ ds carndfr ”= the camv 35s : ds carndfr : 35s 3 ′ expression cassette . refer to table 2 for a description of gene elements . fig7 is a diagrammatic representation of the binary plasmid pcgp3607 . chimeric . the construction of pcgp3607 is described in example 1 . abbreviations include lb = left border from a . tumefaciens ti plasmid , rb = right border region from a . tumefaciens ti plasmid , tetr = antibiotic , tetracycline resistance gene complex . in this figure “ ds carndfr ”= the camv 35s : ds carndfr : 35s 3 ′ expression cassette and “ thfns ”= e35s 5 ′: thfns : petd8 3 ′ expression cassette . refer to table 2 for a description of gene elements . as used in the subject specification , the singular forms “ a ”, “ an ” and “ the ” include plural aspects unless the context clearly dictates otherwise . thus , for example , reference to “ a plant ” includes a single plant , as well as two or more plants ; reference to “ an anther ” includes a single anther as well as two or more anthers ; reference to “ the invention ” includes a single aspect or multiple aspects of an invention ; and so on . the present invention contemplates genetically modified plants such as carnation plants and in particular spray carnations exhibiting altered inflorescence . the altered inflorescence may be in any tissue or organelle including flowers , petals , anthers and styles . particular inflorescence contemplated herein includes a color in the range of red - purple to blue color such as a purple to blue color including mauve . the color determination is conveniently measured against the royal horticultural society ( rhs ) color chart ( rhscc ) and includes colors 77a , 77b , n80b , 81a , 81b , 82a , 82b , 88d and colors in between or proximal to either end of the above range . the term “ inflorescence ” is not to be narrowly construed and relates to any colored cells , tissues organelles or parts thereof , as well as flowers and petals . hence , one aspect of the present invention is directed to a genetically modified plant exhibiting altered inflorescence in selected tissue , the plant comprising expressed genetic material encoding at least one f3 ′ 5 ′ h enzyme and at least one dfr enzyme and expressing genetic material which down regulates a plant &# 39 ; s indigenous dfr gene . the “ plant ” includes a parent plant and its progeny which carry on the genetic modification . in particular , the present invention provides a genetically modified plant exhibiting altered inflorescence , the plant or its progeny comprising expressed genetic material encoding at least one non - indigenous flavonoid 3 ′, 5 ′ hydroxylase ( f3 ′ 5 ′ h ) enzyme and at least one non - indigenous dihydroflavonol 4 - reductase ( dfr ) enzyme and expressing genetic material which down regulates expression of the plant &# 39 ; s indigenous dfr gene . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous s - adenosylmethionine : anthocyanin 3 ′, 5 ′ methyltransferase ( thmt ) and / or a flavone synthase ( thfns ). the genetic material which down regulates the plant &# 39 ; s indigenous dfr gene comprises , in one embodiment , sense and anti - sense nucleotide sequences corresponding to the plant &# 39 ; s indigenous dfr gene or mrna ( ds plantdfr ). the ds plantdfr molecule is a chimeric construct of sense and anti - sense genetic material from the dfr genomic dna or cdna corresponding to the indigenous dfr gene or its mrna in the host plant . the “ indigenous ” dfr is the dfr normally resident in the host plant prior to genetic manipulation . a non - indigenous enzyme or gene includes a gene or other genetic material which has been introduced into a plant or a parent of the plant by genetic engineering or plant breeding practices . the ds plantdfr molecule when expressed down - regulates via ptgs the dfr gene in the host plant . the ds plantdfr molecule may be from carnation ( ds carndfr ), chrysanthemum ( ds chrysdfr ), rose ( ds rosedfr ), gerbera ( ds gerbdfr ), dianthus ( ds diandfr ), petunia ( ds petdfr ) or from an ornamental or horticultural plant ( ds plantdfr ). other ds plantdfr &# 39 ; s may come from lisianthus , tulip , lily , geranium , petunia , iris , torenia , begonia , cyclamen , nierembergia , catharanthus , pelargonium , orchid , grape , apple , euphorbia or fuchsia . in a particular embodiment , the plant is a carnation . accordingly , another aspect of the present invention is directed to a spray carnation exhibiting altered inflorescence in selected tissue , the spray carnation comprising expressed genetic material encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing of at least one ds carndfr molecule . the ds carndfr , when expressed , down regulates expression of the plant &# 39 ; s indigenous dfr gene . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . hence , a further aspect of the present invention is directed to a spray carnation exhibiting altered inflorescence in selected tissue , the spray carnation comprising expressed genetic material encoding at least one non - indigenous f3 ′ 5 ′ h enzyme , at least one non - indigenous dfr enzyme and at least one non - indigenous thmt and / or thfns and expressing of at least one ds carndfr molecule . whilst the present invention encompasses any spray carnation , a carnation of the cerise westpearl line is particularly useful including sports thereof . useful sports of cerise westpearl include westpearl . accordingly , another aspect of the present invention is directed to a genetically modified cerise westpearl spray carnation plant line or sports thereof exhibiting tissues of a purple to blue color , the carnation comprising expressed genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . more particularly , the present invention provides a genetically modified cerise westpearl plant ( cw )/ pcgp3366 ( also referred to as cw / 3366 or cerise westpearl / 3366 ) line exhibiting altered inflorescence , the line comprising an expressed genetic sequence encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . even more particularly , the present invention provides a genetically modified cerise westpearl plant ( cw )/ pcgp3601 ( also referred to as cw / 3601 or cerise westpearl / 3601 ) line exhibiting altered inflorescence , the line comprising an expressed genetic sequence encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . still more particularly , the present invention provides a genetically modified cerise westpearl plant ( cw )/ pcgp3605 ( also referred to as cw / 3605 or cerise westpearl / 3605 ) line exhibiting altered inflorescence , the line comprising an expressed genetic sequence encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . even still more particularly , the present invention provides a genetically modified cerise westpearl plant ( cw )/ pcgp3616 ( also referred to as cw / 3616 or cerise westpearl / 3616 ) line exhibiting altered inflorescence , the line comprising an expressed genetic sequence encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . yet more particularly , the present invention provides a genetically modified cerise westpearl plant ( cw )/ pcgp3607 ( also referred to as cw / 3607 or cerise westpearl / 3607 ) line exhibiting altered inflorescence , the line comprising an expressed genetic sequence encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . in each of the above - mentioned aspects , the plant and its progeny may further comprise genetic material encoding a non - indigenous thmt and / or thfns . examples of cerise westpearl transgenic lines include # 25958 ( florigene moonberry ( trade mark )) and line # 25947 ( florigene moonpearl ( trade mark )). additional genetically modified carnations contemplated herein include the spray carnations westpearl , kortina chanel , vega , barbara and artisan and the standard carnations cinderella , dark rendezvous , miledy . other genetically modified plants contemplated herein include chrysanthemums , roses , gerberas , lisianthus , tulip , lily , geranium , petunia , iris , torenia , begonia , cyclamen , nierembergia , catharanthus , pelargonium , orchid , grape , apple , euphorbia or fuchsia and other ornamental or horticultural plants . another aspect of the present invention is directed to a genetically modified chrysanthemum plant exhibiting tissues of a purple to blue color , the chrysanthemum comprising expressed genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds chrysdfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . still another aspect of the present invention is directed to a genetically modified rose plant exhibiting tissues of a purple to blue color , the rose comprising expressed genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and expressing at least one ds rosedfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . yet another aspect of the present invention is directed to a genetically modified gerbera plant exhibiting tissues of a purple to blue color , the gerbera comprising expressed genetic sequences encoding at least one f3 ′ 5 ′ h enzyme and at least one dfr enzyme and expressing at least one ds gerbdfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . yet another aspect of the present invention is directed to a genetically modified ornamental or horticultural plant exhibiting tissues of a purple to blue color , the ornamental or horticultural plant comprising expressed genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one dfr enzyme and expressing at least one ds plantdfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . the term “ purple to blue color ” includes mauve . the ds plantdfr , ds chrysdfr , ds rosedfr , ds gerbdfr , ds petdfr and ds diandfr comprise sense and anti - sense genomic or cdna fragments of the gene encoding the host plant &# 39 ; s dfr . expression of this molecule results in down - regulation of the indigenous dfr gene in the host plant . similar comments apply in relation to ds plantdfr &# 39 ; s from other host plants . the genetic sequence may be a single construct carrying the nucleotide sequences encoding the f3 ′ 5 ′ h enzymes and the dfr enzyme or multiple genetic constructs may be employed . in addition , the genetic sequences may be integrated into the genome of a plant cell or it may be maintained as an extra - chromosomal artificial chromosome . still furthermore , the generation of a spray carnation expressing at least one f3 ′ 5 ′ h enzyme and at least one dfr enzyme and expressing at least one ds carndfr molecule may be generated by recombinant means alone or by a combination of conventional breeding and recombinant dna manipulation . the genetic sequences are “ expressed ” in the sense of being operably linked to a promoter and other regulatory sequences resulting in transcription and translation to produce f3 ′ 5 ′ h and dfr enzymes . hence , another aspect of the present invention contemplates a method for producing a genetically modified plant such as a spray carnation exhibiting altered inflorescence , the method comprising introducing into regenerable cells of a plant such as a spray carnation plant expressible genetic material encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene and regenerating a plant therefrom or obtaining progeny from the regenerated plant . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . similar methodologies are contemplated herein from chrysanthemums , rose , gerbera and ornamental plants . the plant may then undergo various generations of growth or cultivation . hence , reference to a genetically modified spray carnation includes progeny thereof and sister lines thereof as well as sports thereof . another aspect of the present invention provides a method for producing a genetically modified plant such as a spray carnation line exhibiting altered inflorescence , the method comprising selecting a plant such as a spray carnation comprising expressible genetic material encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one dfr enzyme and incorporation of at least one ds carndfr molecule which down regulates expression of the plant &# 39 ; s indigenous dfr gene and crossing this plant with another plant such as a spray carnation comprising genetic material encoding the other of at least one f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds carndfr molecule and then selecting f1 or subsequent generation plants which express the genetic material . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . nucleotide sequences encoding non - indigenous f3 ′ 5 ′ h and dfr enzymes relative to a host plant may be from any source including viola sp , petunia sp , salvia sp , lisianthus sp , gentiana sp , sollya sp , clitoria sp , kennedia sp , campanula sp , lavandula sp , verbena sp , torenia sp , delphinium sp , solanum sp , cineraria sp , vitis sp , babiana stricta , pinus sp , picea sp , larix sp , phaseolus sp , vaccinium sp , cyclamen sp , iris sp , pelargonium sp , liparieae , geranium sp , pisum sp , lathyrus sp , catharanthus sp , malvia sp , mucuna sp , vicia sp , saintpaulia sp , lagerstroemia sp , tibouchina sp , plumbago sp , hypocalyptus sp , rhododendron sp , linum sp , macroptilium sp , hibiscus sp , hydrangea sp , cymbidium sp , millettia sp , hedysarum sp , lespedeza sp , asparagus sp , antigonon sp , pisum sp , freesia sp , brunella sp or clarkia sp , etc . for example , in one embodiment , the f3 ′ 5 ′ h enzyme comes from viola sp . the dfr may come again from the same or different plant species . for example in one embodiment the dfr enzyme comes from petunia . in another embodiment the dfr comes from iris . the sense and anti - sense fragments forming the hairpin loop of the ds carndfr comes from carnation ( embl accession number z67983 , genbank accession number gi : 1067126 ) or the functional equivalent from chrysanthemum , rose , gerbera or ornamental plant . since the aim of the ds carndfr is to down regulate the indigenous carnation dfr gene via rnai mediated silencing various fragments of the endogenous carnation dfr sequence may be used ( see international patent application no . pct / ib99 / 00606 , wesley et al , plant j , 27 , 581 - 590 , 2001 , ossowski et al , plant j , 53 , 674 - 690 , 2008 ). for example , in one embodiment a 300 bp fragment is used in a sense and anti - sense direction . the intron in the ds carndfr comes from petunia dfr - a intron 1 ( beld et al , plant mol . biol . 13 : 491 - 502 , 1989 ), however , any intron that is able to be processed in carnation can be used . in another embodiment , no intron is used . again , the same comments apply for ds plantdfr molecules generically . the present invention provides for the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of genetic material which down regulates a plant &# 39 ; s indigenous dfr gene in the manufacture of a carnation or sports thereof exhibiting altered inflorescence including tissue having a purple to violet to blue color . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds carndfr molecule in the manufacture of a spray carnation plant such as a cerise westpearl carnation or sports thereof exhibiting altered inflorescence including tissue having a purple to blue color . in another embodiment , the present invention contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds dfr ( directed at silencing of the indigenous dfr gene ) molecule in the manufacture of a genetically modified plant selected from a rose , chrysanthemum , gerbera , tulip , lily , orchid , lisianthus , begonia , torenia , geranium , petunia , nierembergia , pelargonium , iris , impatiens , cyclamen grape , apple , euphorbia or fuchsia or other ornamental or horticultural thereof exhibiting altered inflorescence including tissue having a purple to blue color . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . plant cells may require to be transformed with two or more genetic constructs each carrying one or more of the various genes . the range “ purple to blue color ” includes mauve . cut flowers , tissue culturable cells , regenerable cells , parts of plants , seeds , reproductive material ( including pollen ) are all encompassed by the present invention . as indicated above , nucleotide sequences encoding f3 ′ 5 ′ h and dfr enzymes may all come from the same species of plant or from two or more different species . f3 ′ 5 ′ h nucleotide sequence from viola sp and a dfr from a petunia sp and carnation are particularly useful in the practice of the present invention . the nucleotide sequences encoding the f3 ′ 5 ′ h enzymes and the dfr enzymes and the respective amino acid sequences are defined in table 1 . nucleic acid molecules encoding f3 ′ 5 ′ hs are also provided in international patent application no . pct / au92 / 00334 and holton et al , 1993 supra . these sequences have been used to modulate 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 ) and carnations ( see international patent application no . pct / au96 / 00296 ). nucleotide sequences of f3 ′ 5 ′ h from other species such as viola , salvia and sollya have been cloned ( see international patent application no . pct / au03 / 01111 ). any of these sequences may be used in combination with a promoter and / or terminator . the present invention particularly contemplates f3 ′ 5 ′ h encoded by seq id no : 1 and a dfr encoded by seq id no : 3 and a carnation dfr ( z67983 , gi : 1067126 ) ( seq id no : 9 ) or a nucleotide sequence capable of hybridizing to any of seq id nos : 1 or 3 or 9 or a complementary form thereof under low or high stringency conditions or which has at least about 70 % identity to seq id no : 1 or 3 or 9 after optimal alignment . for the purposes of determining the level of stringency to define nucleic acid molecules capable of hybridizing to seq id no : 1 , seq id no : 3 , seq id no : 9 or their complementary forms , low stringency includes and encompasses from at least about 0 % to at least about 15 % v / v formamide and from at least about 1m to at least about 2 m salt for hybridization , and at least about 1 m to at least about 2 m salt for washing conditions . generally , low stringency is from about 25 - 30 ° c . to about 42 ° c . the temperature may be altered and higher temperatures used to replace the inclusion of formamide and / or to give alternative stringency conditions . alternative stringency conditions may be applied where necessary , such as medium stringency , which includes and encompasses from at least about 16 % v / v to at least about 30 % v / v formamide and from at least about 0 . 5 m to at least about 0 . 9 m salt for hybridization , and at least about 0 . 5 m to at least about 0 . 9 m salt for washing conditions , or high stringency , which includes and encompasses from at least about 31 % v / v to at least about 50 % v / v formamide and from at least about 0 . 01 m to at least about 0 . 15 m salt for hybridization , and at least about 0 . 01 m to at least about 0 . 15 m salt for washing conditions . in general , washing is carried out t m = 69 . 3 + 0 . 41 ( g + c ) % ( marmur and doty , j . mol . biol . 5 : 109 , 1962 ). however , the t m of a duplex dna decreases by 1 ° c . with every increase of 1 % in the number of mismatch base pairs ( bonner and laskey , eur . j . biochem . 46 : 83 , 1974 ). formamide is optional in these hybridization conditions . particular levels of washing stringency include as follows : low stringency is 6 × ssc buffer , 1 . 0 % w / v sds at 25 - 42 ° c . ; a moderate stringency is 2 × ssc buffer , 1 . 0 % w / v sds at a temperature in the range 20 ° c . to 65 ° c . ; high stringency is 0 . 2 to 2 × ssc buffer , 0 . 1 %- 1 . 0 % w / v sds at a temperature of at least 65 ° c . reference to at least 70 % identity includes 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 and 100 % identity . the comparison may also be made at the level of similarity of amino acid sequences of seq id no : s : 2 , 4 or 10 . hence , nucleic acid molecules are contemplated herein which encode an f3 ′ 5 ′ h enzyme or dfr having at least 70 % similarity to the amino acid sequence set forth in seq id nos : 2 or 4 10 . again , at least 70 % similarity includes 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 and 100 % similarity or identity . the nucleic acid molecule encoding the f3 ′ 5 ′ h and dfr enzymes and expression of the ds cam dfr molecule includes one or more promoters and / or terminators . in one embodiment , a promoter is selected which directs expression of a f3 ′ 5 ′ h and / or a dfr nucleotide sequence in tissue having a higher ph . in an embodiment , the promoter sequence is native to the host carnation plant to be transformed or may be derived from an alternative source , where the region is functional in the host plant . other sources include the agrobacterium t - dna genes , such as the promoters for the genes encoding enzymes for biosynthesis of nopaline , octapine , mannopine , or other opines ; promoters from plants , such as promoters from genes encoding ubiquitin ; tissue specific promoters ( see , e . g ., u . s . pat . no . 5 , 459 , 252 to conkling et al ; wo 91 / 13992 to advanced technologies ); promoters from plant viruses ( including host specific viruses ), or partially or wholly synthetic promoters . numerous promoters that are potentially functional in carnation plants ( see , for example , greve , j . mol . appl . genet . 1 : 499 - 511 , 1983 ; salomon et al , embo , j . 3 : 141 - 146 , 1984 ; garfinkel et al , cell 27 : 143 - 153 , 1983 ; barker et al , plant mol . biol . 2 : 235 - 350 , 1983 ); including various promoters isolated from plants ( such as the ubi promoter from the maize obi - 1 gene , see , e . g ., u . s . pat . no . 4 , 962 , 028 ) and viruses ( such as the cauliflower mosaic virus promoter , camv 35s ). in other embodiments the promoter is amchs 5 ′, rosechs 5 , carnans 5 ′ and / or petdfr 5 ′ ( from pet gen dfr ) with corresponding terminators petd8 3 ′, nos 3 , carn ans 3 ′ and petdfr 3 ′ ( from pet gen dfr ), respectively . the promoter sequences may include cis - acting sequences which regulate transcription , where the regulation involves , for example , chemical or physical repression or induction ( e . g ., regulation based on metabolites , light , or other physicochemical factors ; see , e . g ., wo 93 / 06710 disclosing a nematode responsive promoter ) or regulation based on cell differentiation ( such as associated with leaves , roots , seed , or the like in plants ; see , e . g . u . s . pat . no . 5 , 459 , 252 disclosing a root - specific promoter ). other cis - acting sequences which may be employed include transcriptional and / or translational enhancers . these enhancer regions are well known to persons skilled in the art , and can include the atg initiation codon and adjacent sequences . the nucleic acid molecule ( s ) encoding at least one f3 ′ 5 ′ h enzyme and at least one dfr enzyme and incorporation of at least one ds carndfr molecule , in combination with suitable promoters and / or a terminators is / are used to modulate the activity of a flavonoid molecule in a spray carnation . reference herein to modulating the level of a delphinidin - based molecule relates to an elevation or reduction in levels of up to 30 % or more particularly of 30 - 50 %, or even more particularly 50 - 75 % or still more particularly 75 % or greater above or below the normal endogenous or existing levels of activity . the term “ inflorescence ” as used herein refers to the flowering part of a plant or any flowering system of more than one flower which is usually separated from the vegetative parts by an extended internode , and normally comprises individual flowers , bracts and peduncles , and pedicels . as indicated above , reference to a “ transgenic plant ” may also be read as a “ genetically modified plant ” and includes a progeny or hybrid line ultimately derived from a first generation transgenic plant . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds carndfr molecule which down regulates expression of an indigenous dfr gene in the manufacture of a spray carnation such as a cerise westpearl carnation or sports thereof exhibiting altered inflorescence including tissue having a purple to blue color . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds chrysdfr molecule which down regulates expression of an indigenous dfr gene in the manufacture of a chrysanthemum plant or sports thereof exhibiting altered inflorescence including tissue having a purple to blue color . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds rosedfr molecule which down regulates expression of an indigenous dfr gene in the manufacture of a rose plant or sports thereof exhibiting altered inflorescence including tissue having a purple to blue color . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds gerbdfr molecule which down regulates expression of an indigenous dfr gene in the manufacture of a gerbera plant or sports thereof exhibiting altered inflorescence including tissue having a purple to blue color . the present invention also contemplates the use of genetic sequences encoding at least one non - indigenous f3 ′ 5 ′ h enzyme and at least one non - indigenous dfr enzyme and incorporation of at least one ds plantdfr molecule which down regulates expression of an indigenous dfr gene in the manufacture of a plant exhibiting altered inflorescence including tissue having a purple to blue color . in an embodiment , the plant and its progeny , further comprise genetic material encoding a non - indigenous thmt and / or thfns . the genetic material may comprise a single or multiple constructs . the “ purple to blue color ” includes mauve . a cultivation business model is also provided , the model comprising generating a genetically modified spray carnation plant as described herein , providing platelets , seeds , regenerable cells , tissue culturable cells or other material to a grower , generating commercial sale numbers of plants , and providing cut flowers to retailers or wholesalers . the present invention is further described by the following non - limiting examples . in these examples , materials and methods as outlined below were employed : methods followed were as described in sambrook et al , molecular cloning : a laboratory manual , cold spring harbor laboratories , cold spring harbor , n . y ., usa , 1989 or sambrook and russell , molecular cloning : a laboratory manual 3 rd edition , cold spring harbor laboratories , cold spring harbor , n . y ., usa , 2001 or plant molecular biology manual ( 2 nd edition ), gelvin and schilperoot ( eds ), kluwer academic publisher , the netherlands , 1994 or plant molecular biology labfax , croy ( ed ), bios scientific publishers , oxford , uk , 1993 . the cloning vectors pbluescript and pcr script were obtained from stratagene , usa . pcr72 . 1 was obtained from invitrogen , usa . supe44 , δ ( laczya - argf ) u169 , ( ø801aczδm15 ), hsdr17 ( r k − , m k + ), reca1 , enda1 , gyra96 , thi - 1 , rela1 , deor . ( hanahan , j . mol . biol . 166 : 557 , 1983 ) supe44 , hsdr17 ( r k − , m k + ), reca1 , enda1 , gyra96 , thi - 1 , rela1 , lac − ,[ f ′ proab , laci q , laczδm15 , tn10 ( tet r )] ( bullock et al , biotechniques 5 : 376 , 1987 ). ompt hsds ( rb - mb -) dcm + tet r gal enda hte [ argu iley leuw cam r ] m15 e . coli is derived from e . coli k12 and has the phenotype nal s , str s , rif s , thi − , ara + , gal + , mtl − , f − , reca + , uvr + , lon + . transformation of the e . coli strains was performed according to the method of inoue et al , gene 96 : 23 - 28 , 1990 . the disarmed agrobacterium tumefaciens strain used was agl0 ( lazo et al , bio / technology 9 : 963 - 967 , 1991 ). 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 , 1989 supra ) and incubation 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 1 ml 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 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 . dna ligations were carried out using the amersham ligation kit or promega ligation kit according to procedures recommended by the manufacturer . fragments were generally isolated on a 1 % ( w / v ) agarose gel and purified using the qiaex ii gel extraction kit ( qiagen ) or bresaclean kit ( bresatec , australia ) following procedures recommended by the manufacturer . overhanging 5 ′ ends were repaired using dna polymerase i klenow fragment according to standard protocols ( sambrook et al , 1989 supra ). overhanging 3 ′ ends were repaired using bacteriophage t4 dna polymerase according to standard protocols ( sambrook et al , 1989 supra ). shrimp alkaline phosphatase ( sap ) [ usb ] was typically used to remove phosphoryl groups from cloning vectors to prevent re - circularization according to the manufacturer &# 39 ; s recommendations . unless otherwise specified , pcr conditions using plasmid dna as template included using 2 ng of plasmid dna , 100 ng of each primer , 2 μl , 10 mm dntp mix , 5 μl 10 × taq dna polymerase buffer , 0 . 5 μl taq dna polymerase in a total volume of 50 μl . cycling conditions comprised an initial denaturation step of 5 minutes at 94 ° c ., followed by 35 cycles of 94 ° c . for 20 sec , 50 ° c . for 30 sec and 72 ° c . for 1 minute with a final treatment at 72 ° c . for 10 minutes before storage at 4 ° c . pcrs were performed in a perkin elmer geneamp pcr system 9600 . dna fragments ( 50 to 100 ng ) were radioactively labeled with 50 μci of [ α - 32 p ]- dctp using a gigaprime kit ( geneworks ). unincorporated [ α - 32 p ]- dctp was removed by chromatography on sephadex g - 50 ( fine ) columns or microbiospin p - 30 tris chromatography columns ( biorad ). single colonies were analyzed for inserts by inoculating lb broth ( sambrook et al , 1989 supra ) with appropriate antibiotic selection ( e . g . 100 μg / ml ampicillin or 10 to 50 μg / ml tetracycline etc .) and incubating the liquid culture at 37 ° c . ( for e . coli ) or 29 ° c . ( for a . tumefaciens ) for ˜ 16 hours with shaking . plasmid dna was purified using the alkali - lysis procedure ( sambrook et al , 1989 supra ) or using the wizardplus sv minipreps dna purification system ( promega ) or qiagen plasmid mini kit ( qiagen ). once the presence of an insert had been determined , larger amounts of plasmid dna were prepared from 50 ml overnight cultures using the alkali - lysis procedure ( sambrook et al , 1989 supra ) or qiafilter plasmid midi kit ( qiagen ) and following conditions recommended by the manufacturer . dna sequencing was performed using the prism ( trademark ) ready reaction dye primer cycle sequencing kits from applied biosystems . the protocols supplied by the manufacturer were followed . the cycle sequencing reactions were performed using a perkin elmer pcr machine ( geneamp pcr system 9600 ). sequencing runs were generally performed by the australian genome research facility at the university of queensland , st lucia , brisbane , australia and at the walter and eliza hall institute of medical research , melbourne , australia . sequences were analyzed using a macvector ( trade mark ) application ( version 9 . 5 . 2 and earlier ) [ macvector inc , cary , n . c ., usa ]. homology searches against genbank , swiss - prot and embl databases were performed using the fasta and tfasta programs ( pearson and lipman , proc . natl . acad . sci . usa 85 ( 8 ): 2444 - 2448 , 1988 ) or blast programs ( altschul et al , j . mol . biol . 215 ( 3 ): 403 - 410 , 1990 ). percentage sequence similarities were obtained using lalign program ( huang and miller , adv . appl . math . 12 : 373 - 381 , 1991 ) or clustalw program ( thompson et al , nucleic acids research 22 : 4673 - 4680 , 1994 ) within the macvector ( trade mark ) application ( macvector inc , usa ) using default settings . multiple sequence alignments were produced using clustalw ( thompson et al , 1994 supra ) using default settings . plant transformations were as described in international patent application no . pct / us92 / 02612 incorporated herein by reference or international patent application no . pct / au96 / 00296 or lu et al , bio / technology 9 : 864 - 868 , 1991 . other methods may also be employed . cuttings of dianthus caryophyllus cv . cerise westpearl were obtained from propagation australia , queensland , australia . the royal horticultural society &# 39 ; s color charts , third and / or fifth edition ( london , uk ), 1995 and / or 2007 were used to provide a description of observed color . they provide an alternative means by which to describe the color phenotypes observed . the designated numbers , however , should be taken only as a guide to the perceived colors and should not be regarded as limiting the possible colors which may be obtained . carnation petals consist of 3 zones , the claw , corona and limb ( glimn - lacy and kaufman , botany illustrated , introduction to plants , major groups , flowering plant families , 2 nd ed , springer , usa , 2006 ). in general only the petal limb is colored with the claw being a green color and the corona a white shade ( see fig4 ). reference to carnation petal / flower / inflorescence color generally relates to the color of the carnation petal limb . thin layer chromatography ( tlc ) and high performance liquid chromatography ( hplc ) analysis was performed generally as described in brugliera et al , plant j . 5 : 81 - 92 , 1994 . in general tlc and hplc analysis was performed on extracts isolated from the petal limbs . prior to hplc analysis , the anthocyanin and flavonol molecules present in petal limb extracts were acid hydrolyzed to remove glycosyl moieties from the anthocyanidin or flavonol core . anthocyanidin and flavonol standards were used to help identify the compounds present in the floral extracts . petal extracts were prepared essentially as described in fukui et al , 2003 supra . petal were added to 6 n hcl ( 0 . 2 ml ) and boiled at 100 ° c . for 20 min . the hydrolyzed anthocyanidins were extracted with 0 . 2 ml of 1 - pentanol . hplc analysis of the anthocyanidins was performed using an ods - a312 ( 15 cm × 6 mm , ymc co ., ltd , kyoto , japan ) column , a flow rate of solvent of 1 ml min − 1 , and detection at an absorbance of 600 - 400 nm on a spd - m20a photodiode array detector ( shimadzu co ., ltd ). the solvent system used was as follows : acetic acid : methanol : water = 15 : 20 : 65 . under these hplc conditions , the retention time and λ max of delphinidin were 4 . 0 min and 534 nm , respectively , and these values were compared with those of authentic delphinidin chloride ( funakoshi co ., ltd , tokyo , japan ). the anthocyanidin peaks were identified by reference to known standards , viz delphinidin , petunidin , malvidin , cyanidin and peonidin for tlc or hplc analysis , petal limbs were collected from stage 4 flowers at the stage of maximum pigment accumulation . for northern blot analysis , petals were collected from stage 3 flowers at the stage of maximal expression of flavonoid pathway genes . a summary of promoter , terminator and coding fragments used in the preparation of constructs and the respective abbreviations is listed in table 2 . cerise westpearl is a cerise colored carnation ( rhscc 57d ) it typically accumulates pelargonidin - based pigments (˜ 99 % of total anthocyanin content of 1 . 0 mg / g petal fresh weight ) and therefore lacks f3 ′ h activity and so is presumed mutant in the f3 ′ h gene . hplc analysis results on 2 flowers revealed 1 . 08 mg / g anthocyanin ( 99 % pelargonidin ), 2 . 9 to 4 . 6 mg / g flavonols and 0 . 3 to 0 . 6 mg / g dihydroflavonols accumulating in the petals of cerise westpearl . cerise westpearl is a sport of the pink colored flower westpearl . in order to produce novel purple / blue flowers in the spray carnation background of cerise westpearl , two binary vector constructs were prepared utilizing the pansy f3 ′ 5 ′ h cdna clone and petunia genomic dfr gene with or without a ds carndfr expression cassette . table 3 provides a summary of chimeric f3 ′ 5 ′ h and dfr gene expression cassettes contained in binary vector constructs used in the transformation of cerise westpearl ( see table 2 for an explanation of abbreviations ). the constructs pcgp3601 , 3605 , 3607 , 3616 are all based upon pcgp3366 and have an extra expression cassette that is either a floral specific or constitutive expression of anthocyanin 3 ′ s ′ methyltransferase cdna clone from torenia ( targeting methylating of the delphinidin ) [ pcgp3601 and 3605 ] or floral specific or constitutive expression of a flavone synthase cdna clone from torenia ( targeting producing of the co - pigments , flavones ) [ pcgp3616 and 3607 ]. the transformation vector pcgp3360 ( amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′; pet gen dfr ; 35s 5 ′: surb ) the transformation vector pcgp3360 contains the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ expression cassette and the petunia genomic dfr - a gene along with the 35s 5 ′: surb selectable marker gene . the plasmid pcgp3356 contains a chimeric gene consisting of amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ in a pbluescript backbone . a ˜ 1 . 6 kb fragment harboring the bpf3 ′ 5 ′ h # 40 cdna clone was released from the plasmid pcgp1961 ( see international patent application no . pct / au03 / 01111 ) upon digestion with the restriction endonucleases ecori and kpni . the overhanging ends were repaired and the fragment was purified . the plasmid pcgp725 containing amchs 5 ′: pethf1 : petd8 3 ′ in pbluescript ( described in international patent application no . pct / au03 / 01111 ) was digested with the restriction endonucleases xbai and bamhi to release the backbone vector harboring the amchs 5 ′ and petd8 3 ′ regions . the overhanging ends were repaired and the ˜ 4 . 9 kb fragment was isolated , purified and ligated with the blunt ended bpf3 ′ 5 ′ h # 40 fragment from pcgp1961 ( described above ). correct insertion of the bpf3 ′ 5 ′ h # 40 cdna clone in a sense orientation between the am chs 5 ′ promoter and the pet d8 3 ′ terminator was established by restriction endonuclease analysis of plasmid dna isolated from ampicillin - resistant transformants . the resulting plasmid was designated as pcgp3356 . construction of the intermediate plasmid , pcgp3357 ( amchs 5 ′: bpf3 ′ 5 ′ h # 40 : pet d8 3 ′ in pcgp1988 ) the plasmid pcgp3357 contains a chimeric gene consisting of amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ along with the 35s 5 ′: surb selectable marker gene in the pcgp1988 vector ( see international patent application no . pct / au03 / 01111 ). the plasmid pcgp3356 ( described above ) was digested with the restriction endonuclease psti to release a 3 . 5 kb fragment bearing the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ expression cassette . the resulting 5 ′- overhang was repaired using dna polymerase i ( klenow fragment ) according to standard protocols ( sambrook et al , 1989 supra ). the fragment was purified and ligated with smai ends of the plasmid pcgp1988 ( see international patent application no . pct / au03 / 01111 ). correct insertion of amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ gene in a tandem orientation with respect to the 35s 5 ′: surb selectable marker gene cassette was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3357 . a genomic library was made from petunia hybrida cv . old glory blue dna in the vector λ2001 ( holton , 1992 supra ). approximately 200 , 000 pfu were plated out on nzy plates , lifts were taken onto nen filters and the filters were hybridized with 400 , 000 cpm / ml of 32 p - labeled petunia dfr - a cdna fragment ( described in brugliera et al , 1994 , supra ). hybridizing clones were purified , dna was isolated from each and mapped by restriction endonuclease digestion . a 13 kb sad fragment of one of these clones was isolated and ligated with sad ends of pbluescriptii to create the plasmid pcgp1472 . finer mapping indicated that an ˜ 5 . 3 kb bglii fragment contained the entire petunia dfr - a gene ( beld et al , 1989 supra ). the 5 . 3 kb fragment harboring the pet gen dfr gene was released from the plasmid pcgp1472 upon digestion with the restriction endonuclease bglii . the overhanging ends were repaired and the fragment was purified and ligated with the repaired asci ends of the plasmid pcgp3357 ( described above ). correct insertion of pet gen dfr gene in a tandem orientation with respect to the amchs bpf3 ′ 5 ′ h # 40 : petd8 3 ′ and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3360 ( fig2 ). the transformation vector pcgp3366 ( camv35s : ds carn dfr : 35s 3 ′; pet gen dfr ; amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ; 35s 5 ′: surb ) the transformation vector pcgp3366 contains the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′ expression cassette and the petunia genomic dfr - a ( pet gen dfr ) genes along with a camv35s : ds carn dfr : 35s 3 ′ expression cassette and the 35s 5 ′: surb selectable marker gene . a fragment bearing 180 bp of the petunia dfr - a intron 1 was amplified by pcr using the plasmid pcgp1472 ( described above ) as template and the following primers : the forward primer ( dfrint35s f ) was designed to incorporate the restriction endonuclease recognition sites xhoi and bamhi at the 5 ′- end . the reverse primer ( dfrint35s r ) was designed to incorporate xba i and bglii restriction endonuclease recognition sites at the 3 ′- end of the 180 bp product that was amplified . the resulting 180 by pcr product was then digested with the restriction endonucleases xhoi and xbai and ligated with xhoi / xbai ends of the plasmid prtppoptcafp ( a source of the camv35s promoter and terminator fragments ) ( wnendt et al ., curr genet . 25 : 510 - 523 , 1994 ). correct insertion of the petunia dfr - a intron 1 fragment between the camv35s and 35s 3 ′ fragments of prtppoptcafp was confirmed by restriction endonuclease analysis of plasmid dna isolated from ampicillin - resistant transformants . the resulting plasmid was designated pcgp3359 . isolation of a partial carnation dfr cdna clone has been described in international patent application no . pct / au96 / 00296 . around 120 , 000 pfus of a carnation kortina chanel petal cdna library ( construction of which is described in international patent application no . pct / au97 / 000124 ) were screened using the 32 p - labeled fragments of an ecori / xhoi partial carnation dfr fragment ( see international pct / au96 / 00296 ) as a probe under high stringency hybridization washing conditions . around 20 strongly hybridizing plaques were selected and further purified . of these one ( kcdfr # 17 ) contained a 1 . 3 kb insert and represented a full - length carnation dfr cdna clone with 51 bp of 5 ′ untranslated sequence . the plasmid was designated as pcgp1547 . construction of the intermediate plasmid pcgp3363 ( camv35s : sense partial carnation dfr : petunia dfr intron 1 : 35s 3 ) a fragment bearing ˜ 300 bp of the carnation dfr cdna clone was amplified by pcr using the plasmid pcgp1547 ( described above ) as template and the following primers : the forward primer ( ds carndfr f ) was designed to incorporate the restriction endonuclease recognition sites xbai and xhoi at the 5 ′- end . the reverse primer ( ds carndfr r ) was designed to incorporate bglii and bamhi restriction endonuclease recognition sites at the 3 ′- end of the ˜ 300 bp product that was amplified . the resulting ˜ 300 bp pcr product was then digested with the restriction endonucleases xhoi and bamhi and ligated with xhoi / bamhi ends of the plasmid pcgp3359 ( described above ). correct insertion of the partial carnation dfr fragment in a sense direction between the camv35s and petunia dfr intron 1 fragment of the plasmid pcgp3359 was confirmed by restriction endonuclease analysis of plasmid dna isolated from ampicillin - resistant transformants . the resulting plasmid was designated pcgp3363 . the amplified partial carnation dfr fragment described above was digested with the restriction endonucleases bglii and xbai and ligated with bglii / xbai ends of the plasmid pcgp3363 ( described above ). correct insertion of the partial carnation dfr fragment in an anti - sense direction between the petunia dfr intron 1 and 35s 3 ′ fragments of the plasmid pcgp3363 was confirmed by restriction endonuclease analysis of plasmid dna isolated from ampicillin - resistant transformants . the resulting plasmid was designated pcgp3364 . a ˜ 1 . 4 kb fragment bearing the camv35s : ds carn dfr : 35s 3 ′ expression cassette was released from the plasmid pcgp3364 ( described above ) upon digestion with the restriction endonuclease psti . the fragment was purified and ligated with the psti ends of the plasmid pcgp3360 ( described above ) ( fig2 ). correct insertion of camv35s : ds carn dfr : 35s 3 ′ expression cassette in a tandem orientation with respect to the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ; pet gen dfr and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3366 ( fig3 ). the t - dnas of the transformation vectors pcgp3360 and pcgp3366 were introduced into the spray carnation line , cerise westpearl via agrobacterium - mediated transformation . transgenic cells were selected based on their ability to grow and produce roots on media containing the herbicide , chlorsulfuron . transgenic plantlets with roots were removed form media and transferred to soil and grown to flowering in temperature controlled greenhouses in bundoora , victoria , australia . the color of the petal limbs of the transgenic plants were recorded by eye using rhscc and hplc analysis was used to determine the anthocyanidins in the hydrolyzed petal limb extracts . the results are summarized in table 4 . % cc = the percentage of the total number of events produced that had a shift in petal color towards the purple range # hplc = number of individual events of which the anthocyanidins of hydrolyzed petal limb extracts were analyzed by hplc . petals for analysis were selected based on a visible shift in color of the petal from pink into the purple range . % del ( range ) = the range in % of delphinidin detected in the hydrolyzed extracts of the petals for the population of transgenic events av del = the average % of delphinidin detected in the hydrolyzed extracts of the petals for the population of transgenic events del mg / g fw = the range in the amount of delphinidin ( in mg / g of fresh weight ) detected in the hydrolyzed extracts of the petals for the population of transgenic events the results suggest that of the two constructs tested ( pcgp3360 and pcgp3366 ), pcgp3366 resulted in a higher percentage of events that produced flowers with a shift in color to the purple range . furthermore the average delphinidin detected in the hydrolyzed extracts of the petals was higher in pcgp3366 events compared to pcgp3360 events . this was presumably due to the down regulation of the endogenous carnation dfr by the ds carndfr cassette via rnai - mediated silencing leading to decreased competition between the endogenous dfr and the introduced f3 ′ 5 ′ h for the dhk substrate . the introduced petunia dfr ( which is not able to utilise dhk ) subsequently allowed conversion dhm ( product of f3 ′ 5 ′ h reaction on dhk ) to leucodelphinidin and activity by the endogenous anthocyanin pathway enzymes resulted in delphinidin derived pigments accumulating in the petal tissue . to identify spray carnation lines producing petals of a novel color , the colors of petal limbs were compared to mauve / purple carnation lines already in the market place . these included the midi carnation lines florigene moonshadow ( trade mark ) [ 82a , 82b ] and florigene moondust ( 76a ) and the standard carnation lines florigene moonvista ( trade mark ) [ 81a +], florigene moonshade ( trade mark ) [ 81a , 82a ], florigene moonlite ( trade mark ) [ 77d / 82d , 77c , n80b ] and florigene moonaqua ( trade mark ) [ 84a / b ]. twenty two cw / 3366 lines were initially selected as being novel spray carnation lines whilst only one cw / 3360 line was selected as being novel spray carnation line . further trailing with respect to petal color consistency and petal number reduced the list to 11 cw / 3366 lines and no cw / 3360 lines as being novel spray carnation lines with potential for new product lines ( table 5 ). rhscc number = the color code of the petal limbs from the flowers of transgenic carnation lines . “+” alongside an rhscc number highlights that the color is a darker or more intense shade of the selected code delphinidin levels = delphinidin levels detected in hydrolyzed extracts of petal limb tissue as determined by hplc given in percentage of total anthocyanidins and mg / g of fresh weight of petal tissue . further field trial assessments in colombia revealed that lines # 25958 , # 25947 , # 25973 , # 25965 and # 25976 produced novel spray carnation flower colors with consistent and stable colors and good plant growth characteristics . two lines (# 25958 and # 25947 ) were selected for commercialization . line # 25958 was subsequently named florigene moonberry ( trade mark ) and line # 25947 was called florigene moonpearl ( trade mark ). both are being grown in colombia for production of cut flowers to markets around the world . due to the success in obtaining high delphinidin levels in the carnation variety , cerise westpearl using the construct pcgp3366 ( containing at least one f3 ′ 5 ′ h enzyme and at least one dfr enzyme and incorporation of at least one ds carndfr molecule ) the same genes are introduced into other colored carnation cultivars such as but not limited to cinderella , westpearl , vega , artisan , barbara , dark rendezvous , miledy , kortina chanel . the transgenic plants are assessed for flower color as described above and lines with novel flower color ( as compared to controls ) are selected for commercialization . use of the binary vector pcgp3366 as a backbone - addition of other expression cassettes . in order to shift petal color further towards the blue / purple spectrum other genes that modulated anthocyanin or flavonoid composition were added to the pcgp3366 binary vector . these included genes coding for s - adenosylmethionine : anthocyanin 3 ′ 5 ′ methyltransferase ( amt ) activity to modulate the production of methylated anthocyanins such as the production of malvidin and petunidin pigments and genes coding for flavone synthase ( fns ) activity to modulate the production of flavones in carnation . in an attempt to produce anthocyanins based upon malvidin ( the methylated form of delphinidin ) 2 new transformation vectors , pcgp3601 and pcgp3605 , were prepared by addition of amt expression cassettes to the transformation vector , pcgp3366 ( fig3 ). the amt sequence from torenia ( international patent application no . pct / au03 / 00079 ) was used under the control of a floral specific promoter fragment from the ans gene of carnation ( carnans 5 ′) and a constitutive promoter fragment from the cauliflower mosaic virus 35s gene ( camv35s ). the transformation vector , pcgp3601 ( carnans 5 ′: thmt : carnans 3 ; amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ′; pet gen dfr ; camv35s 5 ′: ds carn dfr : 35s 3 ; 35s 5 ′: surb ) the binary construct pcgp3601 contains a carnans 5 ′: thmt : carnans 3 ′ expression cassette in the pcgp3366 binary construct backbone ( described above ) ( fig3 ). a ˜ 1 . 0 kb fragment bearing the torenia amt cdna clone ( thmt ) ( seq id no : 11 ) was released from the plasmid ptmt5 ( described in international patent application no . : pct / jp00 / 00490 ) upon digestion with the restriction endonucleases ecori and asp718 . the overhanging ends were repaired and the purified fragment was ligated with xbai / psti repaired ends of the plasmid pcgp1275 ( described in international patent application no . pct / au2008 / 001700 incorporated herein by reference ). correct insertion of the thmt fragment in between a promoter fragment of the carnation ans gene ( carnans 5 ) and a terminator fragment of the carnation ans gene ( carnans 3 ) was established by restriction endonuclease analysis of plasmid dna isolated from ampicillin resistant transformants . the resulting plasmid was designated as pcgp3431 . a 4 . 4 kb fragment harboring the carnans 5 ′: thmt : carnans 3 ′ expression cassette was isolated from the plasmid pcgp3431 ( described above ) upon digestion with the restriction endonuclease clai . the overhanging ends were repaired and the purified fragment was ligated with the pmei ends of the plasmid pcgp3366 ( described above ) ( fig3 ). correct insertion of the carnans 5 ′: thmt : carnans 3 ′ expression cassette in a tandem orientation with respect to the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 , pet gen dfr ; camv35s 5 ′: ds carn dfr : 35s 3 ′ and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3601 ( fig4 ). the transformation vector , pcgp3605 ( camv35s : ds cam dfr : 35s 3 ; camv35s : thmt : 35s 3 ; pet gen dfr ; amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ; 35s 5 ′: surb ) the binary construct pcgp3605 contains a camv35s : thmt : 35s 3 ′ expression cassette in the pcgp3366 binary construct backbone ( described above ) ( fig3 ). the plasmid ptmt5 ( described in international patent application no . pct / jp00 / 00490 ) was firstly linearized upon digestion with the restriction endonuclease asp718 . the overhanging ends were repaired and a ˜ 1 . 0 kb fragment bearing the torenia amt cdna clone ( thmt ) ( seq id no : 11 ) was then released from the linearized plasmid upon digestion with the restriction endonuclease ecori . the fragment was purified and ligated with xbai ( repaired ends )/ ecori ends of the plasmid prtppoptcafp ( a source of the camv35s promoter and terminator fragments ) ( wnendt et al ., 1994 , supra ). correct insertion of the thmt fragment in a sense orientation between the promoter and terminator fragments of the cauliflower mosaic virus 35s gene ( camv35s and 35s 3 ′ respectively ) was established by restriction endonuclease analysis of plasmid dna isolated from ampicillin resistant transformants . the resulting plasmid was designated as pcgp3097 . a ˜ 1 . 6 kb fragment harboring the camv35s : thmt : 35s 3 ′ expression cassette was isolated from the plasmid pcgp3097 ( described above ) upon digestion with the restriction endonuclease psti . the overhanging ends were repaired and the purified fragment was ligated with the pmei ends of the plasmid pcgp3366 ( described above ) ( fig3 ). correct insertion of the camv35s : thmt : 35s 3 ′ expression cassette in a tandem orientation with respect to the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 , pet gen dfr ; camv35s : ds carn dfr : 35s 3 ′ and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3605 ( fig5 ). in an attempt to produce flavones ( to act as co - pigments ) and high levels of delphinidin in a cerise westpearl background , a further 2 transformation vectors , pcgp3616 and pcgp3607 were prepared by adding fns expression cassettes to the transformation vector , pcgp3366 ( fig3 ). the fns sequence from torenia ( international patent application no . pct / jp00 / 00490 ) ( seq id no : 13 ) was used under the control of a floral specific promoter fragment from the chs gene of rose ( rosechs 5 ) and a constitutive promoter fragment from the cauliflower mosaic virus 35s gene ( camv35s ). the transformation vector , pcgp3616 ( camv35s : ds carn dfr : 35s 3 ; rosechs 5 ′: thfns : nos 3 ; pet gen dfr ; amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ; 35s 5 ′: surb ) the binary construct pcgp3616 contains a rosechs 5 ′: thfns : nos 3 ′ expression cassette in the pcgp3366 binary construct backbone ( described above ) ( fig3 ). a 3 . 2 kb fragment bearing e35s 5 ′: thfns : petd8 3 ′ expression cassette was released from the binary vector plasmid psfl535 ( described in international patent application wo2008 / 156206 ) upon digestion with the restriction endonuclease asci . the fragment was purified and ligated with the asci ends of the 2 . 9 kb plasmid pucap + asci ( the plasmid pucap / asci is a puc19 based cloning vector with extra cloning sites specifically an asci recognition site at either ends of the multicloning site ). correct insertion of the e35s 5 ′: thfns : petd8 3 ′ expression cassette in the puc based cloning vector was established by restriction endonuclease analysis of plasmid dna isolated from ampicillin resistant transformants . the resulting plasmid was designated as pcgp3123 . this plasmid pcgp3123 ( described above ) was linearized upon digestion with the restriction endonuclease bamhi . the overhanging ends were repaired and a fragment bearing the thfns cdna clone was then released after partial digestion of the linearized plasmid with the restriction endonuclease xhoi . the 1 . 7 kb fragment was purified and ligated with smai / xhoi ends of the plasmid pcgp2203 ( rose chs 5 ′: bpf3 ′ 5 ′ h # 18 : nos 3 ′ in pbluescript backbone ) described in international patent application no . pct / au2008 / 001694 . correct insertion of the thfns fragment between the rosechs promoter and nos terminator was established by restriction endonuclease analysis of plasmid dna isolated from ampicillin resistant transformants . the resulting plasmid was designated pcgp3612 . a 4 . 9 kb fragment harboring the rosechs 5 ′: thfns : nos 3 ′ expression cassette was isolated from the plasmid pcgp3612 ( described above ) upon digestion with the restriction endonucleases bglii and noti . the overhanging ends were repaired and the purified fragment was ligated with the pmei ends of the plasmid pcgp3366 ( described above ) ( fig3 ). correct insertion of the rosechs 5 ′: thfns : nos 3 ′ expression cassette in a tandem orientation with respect to the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 , pet gen dfr ; camv35s : ds carn dfr : 35s 3 ′ and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3616 ( fig6 ). the transformation vector , pcgp3607 ( camv35s ′: ds carn dfr : 35s 3 ; e35s 5 ′: thfns : petd8 3 ; pet gen dfr ; amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 ; 35s 5 ′: surb ) the binary construct pcgp3607 contains an e35s 5 ′: thfns : petd8 3 ′ expression cassette in the pcgp3366 binary construct backbone ( described above ) ( fig3 ). a 3 . 2 kb fragment bearing e35s 5 ′: thfns : petd8 3 ′ expression cassette was released from the plasmid pcgp3123 ( described above ) upon digestion with the restriction endonuclease asci . the fragment was purified and ligated with the pmei ends of the plasmid pcgp3366 ( described above ) ( fig3 ). correct insertion of the e35s 5 ′: thfns : petd8 3 ′ expression cassette in a tandem orientation with respect to the amchs 5 ′: bpf3 ′ 5 ′ h # 40 : petd8 3 , pet gen dfr ; camv35s : ds carn dfr : 35s 3 ′ and 35s 5 ′: surb genes was established by restriction endonuclease analysis of plasmid dna isolated from tetracycline - resistant transformants . the resulting plasmid was designated as pcgp3607 ( fig7 ). the t - dnas of the transformation vectors pcgp3601 ( fig4 ), pcgp3605 ( fig5 ), pcgp3607 ( fig7 ) and pcgp3616 ( fig6 ) were introduced into the spray carnation line , cerise westpearl via agrobacterium - mediated transformation . transgenic cells were selected based on their ability to grow and produce roots on media containing the herbicide , chlorsulfuron . transgenic plantlets with roots were removed form media and transferred to soil and grown to flowering in temperature controlled greenhouses in bundoora , victoria , australia . the results are summarized in table 6 . the transgenic plants are assessed for flower color as described above and lines with novel flower color ( as compared to controls ) are selected for commercialization . 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 . brouillard and dangles , in : the flavonoids — advances in research since 1986 , harborne , ( ed ), chapman and hall , london , uk , 1 - 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