Patent Application: US-55331596-A

Abstract:
the present invention relates generally to transgenic flowering plants . more particularly , the present invention is directed to transgenic rose , carnation and chrysanthemum plants genetically modified to enable expression of flavonoid 3 &# 39 ;, 5 &# 39 ;- hydroxylase thereby permitting the manipulation of intermediates in the flavonoid pathway .

Description:
eriodictyol and dihydroquercetin were obtained from carl roth kg and naringenin was obtained from sigma . dihydromyricetin was chemically synthesized from myricetin ( extra synthese , france ) by the method of vercruysse et al . ( 1985 ). 3 h !- naringenin ( 5 . 7 ci / mmole ) and 3 h !- dihydroquercetin ( 12 . 4 ci / mmole ) were obtained from amersham . all enzymes were obtained from commercial sources and used according to the manufacturer &# 39 ; s recommendations . dh5α supe44 , δ ( laczya - argf ) u169 , φ80laczδm15 , hsdr17 ( r k -, m k +), reca1 , enda1 , gyra96 , thi - 1 , rela1 , deor . ( hanahan , 1983 and brl , 1986 ). the disarmed agrobacterium tumefaciens strains aglo ( lazo et al ., 1991 ) and lba4404 ( hoekema et al ., 1983 ) were obtained from dr r ludwig , department of biology , university of california , santa cruz , usa and calgene , inc . calif ., usa , respectively . the armed agrobacterium tumefaciens strain icmp 8317 was obtained from dr richard gardner , centre for gene technology , department of cellular and molecular biology , university of auckland , new zealand . plants were grown in specialised growth rooms with a 14 hr day length at a light intensity of 10 , 000 lux minimum and a temperature of 22 to 26 . plasmid pcgp90 was constructed by cloning the cdna insert from pcgp602 ( international patent application pct / au92 / 00334 ; publication number wo 93 / 01290 ) in a sense orientation behind the mac promoter ( comai et al ., 1990 ) of pcgp293 . the binary expression vector pcgp293 was derived from the ti binary vector pcgn1559 ( mcbride and summerfelt , 1990 ). plasmid pcgn1559 was digested with kpni and the overhanging 3 &# 39 ; ends were removed with t4 dna polymerase according to standard protocols ( sambrook et al ., 1989 ). the vector was then further digested with xbai and the resulting 5 &# 39 ; overhang was repaired using the klenow fragment of dna polymerase i . the vector was then re - ligated to give pcgp67 . a 1 . 97 kb psti fragment containing the mac promoter , mas terminator and various cloning sites ( comai et al ., 1990 ) was isolated from pcgp40 and inserted into the pst1 site of pcgp67 to give pcgp293 . plasmid pcgp40 was constructed by removing the gus gene ( jefferson et al ., 1987 ) as a bamhi - saci fragment from pcgn7334 and replacing it with the bamhi - saci fragment from pbluescribe m13 that includes the multicloning site . plasmid pcgn7334 ( obtained from calgene , inc . ca , usa ), was constructed by inserting the fragment containing the chimaeric mac - gus - mas gene into the xhoi site of pcgn7329 ( comai et al ., 1990 ). the bamhi - kpni fragment containing the above - mentioned cdna insert was then isolated from pcgp602 and ligated with a bamhi / kpni fragment of pcgp293 . correct insertion of the insert in pcgp90 was established by restriction analysis of dna isolated from gentamycin resistant transformants . the binary expression vector pcgp812 was derived from the ti binary vector pcgn1558 ( mcbride and summerfelt , 1990 ). a 5 . 2 kb xhoi fragment containing the chimaeric mas - 35s - gus - ocs gene was isolated from pkiwi 101 jannsen and gardner , 1989 ) and sub - cloned into the xhoi site of pbluescript ks to give pcgp82 . the 5 . 2 kb fragment was then re - isolated by hindiii / kpni digestion and sub - cloned into the hindiii / kpni sites of pcgn1558 to give pcgp83 . plasmid pcgp83 was restricted with kpni and the overhanging 3 &# 39 ; ends were removed with t4 dna polymerase according to standard protocols ( sambrook et al ., 1989 ). a smai - bamhi adaptor ( pharmacia ) was then ligated to the flushed kpni sites to give bamhi &# 34 ; sticky &# 34 ; ends . a 3 . 8 kb bglii fragment containing the chimaeric mac - hf1 - mas gene from pcgp807 ( described below ) was ligated with the bamhi &# 34 ; sticky &# 34 ; ends of pcgp83 to yield pcgp812 ( fig2 ). the plasmid pcgp807 was constructed by ligating the 1 . 8 kb bamhi - kpni fragment containing the above - mentioned hf1 cdna insert from pcgp602 with bamhi - kpni ends of pcgp40 . the binary vector pcgp485 was derived from the ti binary vector pcgn1547 ( mcbride and summerfelt , 1990 ). a chimaeric gene was constructed consisting of ( i ) the promoter sequence from a chs gene of snapdragon ; ( ii ) the coding region of the above - mentioned cdna insert from pcgp602 from petunia , and ( iii ) a petunia phospholipid transferase protein ( pltp ) terminator sequence . the chs promoter consists of a 1 . 2 kb gene fragment 5 &# 39 ; of the site of translation initiation ( sommer and saedler , 1986 ). the petunia cdna insert consists of a 1 . 6 kb bcli / fspi fragment from the cdna clone of pcgp602 ( international patent application pct / au92 / 00334 ; publication number wo 93 / 01290 ). the pltp terminator sequence consists of a 0 . 7 kb smai / xhoi fragment from pcgp13δ bam ( holton , 1992 ), which includes a 150 bp untranslated region of the transcribed region of the pltp gene . the chimaeric chs / cdna insert / pltp gene was cloned into the psti site of pcgn1547 to create pcgp485 . plasmid pcgp176 international patent application pct / au92 / 00334 ; publication number wo 93 / 01290 ) was digested with ecori and spei . the digested dna was filled in with klenow fragment according to standard protocols ( sambrook et al ., 1989 ), and self - ligated . the plasmid thereby obtained was designated pcgp627 . an xbai / kpni digest of pcgp627 yielded a 1 . 8 kb fragment which was ligated with a 14 . 5 kb fragment obtained by xbai / kpni digestion of pcgp293 . the plasmid thus created was designated pcgp628 . plasmid pcgp293 ( described above in example 2 ) was digested with xbai and the resulting 5 &# 39 ; overhang was filled in using klenow fragment according to standard protocols ( sambrook et al ., 1989 ). it was then digested with hindiii . during this procedure , the mac promoter ( comai et al ., 1990 ) was deleted . a 0 . 8 kb petunia chs - a promoter from pcgp669 ( described below ) was ligated into this backbone as a blunt - ended ecori / hindiii fragment . this plasmid product was designated pcgp672 . an xbai / asp718 digestion of pcgp807 ( described in example 3 , above ) yielded a 1 . 8 kb fragment containing the hf1 cdna , which was ligated with a 16 . 2 kb xbai / asp718 fragment from pcgp672 . the plasmid thus created was designated pcgp653 . a promoter fragment of the chs - a gene was amplified by pcr , using the oligonucleotides chsa - 782 and chsa + 34 as primers ( see sequences below ) and petunia hybrida v30 genomic dna as template . the pcr product was cloned into ddt - tailed pbluescript ( holton and graham , 1991 ) and the orientation of the gene fragment verified by restriction enzyme mapping . the plasmid thus created was designated pcgp669 . the oligonucleotide primers were designed to the published sequence of the petunia chs - a promoter ( koes , 1988 ). construction of pcgp484 was identical to that for pcgp485 , outlined above in example 4 , except that pcgp484 contains the 3 . 5 kb psti fragment ( containing the chimaeric gene chs - hf1 - pltp ) in the opposite orientation . the plasmid pcgp1458 was contructed using the 10 kb binary vector pbin19 ( bevan , 1984 ) as the backbone . plasmid pbin19 was digested with ecori and the resulting 5 &# 39 ; overhang was filled in using klenow fragment , according to standard protocols ( sambrook et al ., 1989 ). plasmid pcgp485 was digested with psti to remove the chimaeric chs / cdna insert / pltp gene as a 3 . 5 kb fragment . the 3 &# 39 ; overhang resulting from psti digestion was removed with t4 dna polymerase and this fragment was then ligated into the filled in ecori site of the plasmid pbin19 . transformation of the escherichza coli strain dh5α - cells with one or other of the vectors pcgp812 , pcgp90 , pcgp485 , pcgp628 , pcgp653 , pcgp484 or pcgp1458 was performed according to standard procedures ( sambrook et al ., 1989 ) or inoue et al ., ( 1990 ). the plasmid pcgp812 , pcgp90 , pcgp485 , pcgp628 , pcgp653 , pcgp484 or pcgp1458 was introduced into the appropriate agrobacterium tumefaciens strain by adding 5 μg of plasmid dna to 100 μl of competent agrobacterium tumefaciens cells prepared by inoculating a 50 ml mg / l ( garfinkel and nester , 1980 ) culture and growing for 16 h with shaking at 28 . 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 min and then allowed to thaw by incubation at 37 for 5 min . the dna / bacterial mixture was then placed on ice for a further 10 min . the cells were then mixed with 1 ml of mg / l media and incubated with shaking for 16 h at 28 . cells of a . tumefaciens carrying either pcgp812 , pcgp90 , pcgp485 , pcgp628 , pcgp653 or pcgp484 were selected on mg / l agar plates containing 100 μg / ml gentamycin . cells of a . tumefaciens carrying pcgp1458 were selected on mg / l agar plates containing 100 μg / ml kanamycin . the presence of the plasmid was confirmed by southern analysis of dna isolated from the gentamycin - resistant transformants . dianthus caryophyllus , ( cv . crowley sim , red sim , laguna ) cuttings were obtained from van wyk and son flower supply , victoria , australia . the outer leaves were removed and the cuttings were sterilized briefly in 70 % ( v / v ) ethanol followed by 1 . 25 % ( w / v ) sodium hypochlorite ( with tween 20 ) for 6 minutes and rinsed three times with sterile water . all the visible leaves and axillary buds were removed under the dissecting microscope before co - cultivation . agrobacterium tumefaciens strain aglo ( lazo et al ., 1991 ), containing any one of the binary vectors pcgp90 , pcgp812 , pcgp485 or pcgp653 , was maintained at 4 on mg / l ( garfinkel and nester , 1980 ) agar plates with 100 mg / l gentamycin . a single colony was grown overnight in liquid mg / l broth and diluted to 5 × 10 8 cells / ml next day before inoculation . dianthus tissue was co - cultivated with agrobacterium on murashige and skoog &# 39 ; s ( 1962 ) medium ( ms ) supplemented with 3 % sucrose ( w / v ), 5 mg / l α - naphthalene acetic acid ( naa ), 20 μm acetosyringone and 0 . 8 % difco bacto agar ( ph 5 . 7 ). co - cultivated tissue was transferred to ms medium supplemented with 1 mg / l benzylaminopurine ( bap ), 0 . 1 mg / l naa , 150 mg / l kanamycin , 500 mg / l ticarcillin and 0 . 8 % difco bacto agar ( selection medium ). after three weeks , explants were transferred to fresh selection medium and care was taken at this stage to remove axillary shoots from stem explants . after 6 - 8 weeks on selection medium healthy adventitious shoots were transferred to hormone free ms medium containing 3 % sucrose , 150 mg / l kanamycin , 500 mg / l ticarcillin , 0 . 8 % difco bacto agar . at this stage gus histochemical assay ( jefferson , 1987 ) and / or npt ii dot - blot assay ( mcdonnell et al ., 1987 ) was used to identify transgenic shoots . transgenic shoots were transferred to ms medium supplemented with 3 % sucrose , 500 mg / l ticarcillin and 0 . 4 % ( w / v ) gelrite gellan gum ( schweizerhall ) for root induction . all cultures were maintained under a 16 hour photoperiod ( 120 μe cool white fluorescent light ) at 23 ± 2 . when plants were rooted and reached 4 - 6 cm tall they were acclimatised under mist . a mix containing a high ratio of perlite ( 75 % or greater ) soaked in hydroponic mix ( kandreck and black , 1984 ) was used for acclimation , which typically lasts 4 - 5 weeks . plants were acclimatised at 23 ° c . under a 14 hour photoperiod ( 200 μe mercury halide light ). plant tissues of the rose cultivar royalty were transformed according to the method disclosed in pct 91 / 04412 , having publication number wo92 / 00371 . kardinal shoots were obtained from van wyk and son flower supply , victoria , australia . leaves were removed and the remaining shoots ( 5 - 6 cm ) were sterilized in 1 . 25 % ( w / v ) sodium hypochlorite ( with tween 20 ) for 5 minutes followed by three rinses with sterile water . isolated shoot tips were soaked in sterile water for 1 hour and precultured for 2 days on ms medium containing 3 % sucrose , 0 . 1 mg / l bap , 0 . 1 mg / l kinetin , 0 . 2 mg / l gibberellic acid , 0 . 5 % ( w / v ) polyvinyl pyrrolidone and 0 . 25 % gelrite gellan gum , before co - cultivation . agrobacterium tumefaciens strains icmp 8317 ( janssen and gardner 1989 ) and agl0 , containing the binary vector pcgp812 , was maintained at 4 ° c . on mg / l agar plates with 100 mg / l gentamycin . a single colony from each agrobacterium strain was grown overnight in liquid mg / l broth . a final concentration of 5 × 10 8 cells / ml was prepared the next day by dilution in liquid mg / l . before inoculation , the two agrobacterium cultures were mixed in a ratio of 10 : 1 ( agl0 / pcgp812 : 8317 / pcgp812 ). a longitudinal cut was made through the shoot tip and an aliquot of2 μl of the mixed agrobacterium cultures was placed as a drop on the shoot tip . the shoot tips were co cultivated for 5 days on the same medium used for preculture . agrobacterium tumefaciens strain agl0 , containing the binary vector pcgp1458 , was maintained at 4 ° c . on mg / l agar plates with 100 mg / l kanamycin . a single colony from each agrobacterium strain was grown overnight in liquid mg / l broth . a final concentration of 5 × 10 8 cells / ml was prepared the next day by dilution in liquid mg / l . after co - cultivation , the shoot tips were transferred to selection medium . shoot tips were transferred to fresh selection medium every 3 - 4 weeks . galls observed on the shoot tips were excised when they reached 6 - 8 mm in diameter . isolated galls were transferred to ms medium containing 3 % sucrose , 25 mg / l kanamycin , 250 mg / l cefotaxime and 0 . 25 % gelrite gellan gum for shoot formation . shoots regenerated from gall tissue were isolated and transferred to selection medium . gus histochemical assay and callus assay were used to identify transgenic shoots . transgenic shoots were transferred to ms medium containing 3 % sucrose , 200 mg / l cefotaxime and 0 . 25 % gelrite gellan gum for root induction . all cultures were maintained under 16 hour photoperiod ( 60 μe cool white fluorescent light ) at 23 ± 2 . when the root system was well developed and the shoot reached 5 - 7 cm in length the transgenic rose plants were transferred to autoclaved debco 514110 / 2 potting mix in 8 cm tubes . after 2 - 3 weeks plants were replanted into 15 cm pots using the same potting mix and maintained at 23 under a 14 hour photoperiod ( 300 μe mercury halide light ). after 1 - 2 weeks potted plants were moved to glasshouse ( day / night temperature : 25 - 28 / 14 ) and grown to flowering . chrysanthemum morifolium ( cv . blue ridge , pennine chorus ) cuttings were obtained from f & amp ; i baguley flower and plant growers , victoria , australia . leaves were removed from the cuttings , which were then sterilized briefly in 70 % ( v / v ) ethanol followed by 1 . 25 % ( w / v ) sodium hypochlorite ( with tween 20 ) for 3 minutes and rinsed three times with sterile water . internodal stem sections were used for co - cultivation . agrobacterium tumefaciens strain lba4404 ( hoekema et al ., 1983 ), containing any one of the binary vectors pcgp90 , pcgp484 , pcgp485 or pcgp628 , was grown on mg / l agar plates containing 50 mg / l rifampicin and 10 mg / l gentamycin . a single colony from each agrobacterium was grown overnight in the same liquid medium . these liquid cultures were made 10 % with glycerol and 1 ml aliquots transferred to the freezer (- 80 ). a 100 - 200 μl aliquot of each frozen agrobacterium was grown overnight in liquid mg / l containing 50 mg / l rifampicin and 10 mg / l gentamycin . a final concentration of 5 × 10 8 cells / ml was prepared the next day by dilution in liquid ms containing 3 % ( w / v ) sucrose . stem sections were co - cultivated , with agrobacterium containing any one of lba4404 / pcgp90 , lba4404 / pcgp484 , lba4404 / pcgp485 or lba4404 / pcgp628 , on co - cultivation medium for 4 days . after co - cultivation , the stem sections were transferred to selection medium . after 3 - 4 weeks , regenerating explants were transferred to fresh medium . adventitious shoots which survived the kanamycin selection were isolated and transferred to ms medium containing kanamycin and cefotaxime for shoot elongation and root induction . all cultures were maintained under a 16 hour photoperiod ( 80 μe cool white fluorescent light ) at 23 ± 2 ° c . leaf samples were collected from plants which rooted on kanamycin and southern blot analysis was used to identify transgenic plants . when transgenic chrysanthemum plants reached 4 - 5 cm in length they were transferred to autoclaved debco 51410 / 2 potting mix in 8 cm tubes . after 2 weeks plants were replanted into 15 cm pots using the same potting mix and maintained at 23 ° c . under a 14 hour photoperiod ( 300 μe mercury halide light ). after 2 weeks potted plants were moved to glasshouse ( day / night temperature : 25 - 28 ° c ./ 14 ° c .) and grown to flowering . dna was isolated from tissue essentially as described by dellaporta et al ., ( 1983 ). the dna preparations were further purified by cscl buoyant density centrifugation ( sambrook et al ., 1989 ). dna was isolated from leaf tissue using an extraction buffer containing 4 . 5m guanidinium thiocyanate , 50 mm edta ph 8 . 0 , 25 mm sodium citrate ph 7 . 0 , 0 . 1m 2 - mercaptoethanol , 2 % ( v / v ) lauryl sarcosine . the plant tissue was ground to a fine powder in liquid n 2 following which extraction buffer was added ( 5 ml / g of tissue ) and the solution mixed on a rotating wheel for 16 h . the mixture was then phenol : chloroform : isoamylalcohol ( 50 : 49 : 1 ) extracted twice and the genomic dna precipitated by adding three volumes of ethanol and centrifuging for 15 min at 10 , 000 rpm . dna was extracted by grinding tissue in the presence of liquid n 2 in a mortar and pestle and adding 1 ml of extraction buffer ( 0 . 14m sorbitol , 0 . 22m tris - hcl ph8 . 0 !, 0 . 022m edta , 0 . 8m nacl , 0 . 8 % ( w / v ) ctab , 1 % n - laurylsarcosine ) heated at 65 ° c . chloroform ( 200 μl ) was added and the mixture incubated at 65 ° c . for 15 min . following centrifugation , the supernatant was phenol - chloroform extracted and then added to an equal volume of isopropanol , inverting to mix . this mixture was centrifuged and the pellet washed with 95 % ethanol , re - centrifuged and washed with 70 % ethanol . the pellet was vacuum - dried and resuspended in 30 μl te buffer ( ph 8 . 0 ). the genomic dna ( 10 μg ) was digested for 16 hours with 60 units of ecori and electrophoresed through a 0 . 7 % ( w / v ) agarose gel in a running buffer of tae ( 40 mm tris - acetate , 50 mm edta ). the dna was then denatured in denaturing solution ( 1 . 5m nacl / 0 . 5m naoh ) for 1 to 1 . 5 hours , neutralized in 0 . 5m tris - hcl ( ph 7 . 5 )/ 1 . 5m nacl for 2 to 3 hours and the dna was then transferred to a hybond n ( amersham ) filter in 20 × ssc . southern analysis of putative transgenic dianthus , rosa and chrysanthemum plants obtained after selection on kanamycin confirmed the integration of the appropriate chimaeric gene into the genome . total rna was isolated from tissue that had been frozen in liquid n 2 and ground to a fine powder using a mortar and pestle . an extraction buffer of 4m guanidinium isothiocyanate , 50 mm tris - hcl ( ph 8 . 0 ), 20 mm edta , 0 . 1 % ( v / v ) sarkosyl , was added to the tissue and the mixture was homogenized for 1 minute using a polytron at maximum speed . the suspension was filtered through miracloth ( calbiochem ) and centrifuged in a ja20 rotor for 10 minutes at 10 , 000 rpm . the supernatant was collected and made to 0 . 2 g / ml cscl ( w / v ). samples were then layered over a 10 ml cushion of 5 . 7m cscl , 50 mm edta ( ph 7 . 0 ) in 38 . 5 ml quick - seal centrifuge tubes ( beckman ) and centrifunged at 42 , 000 rpm for 12 - 16 hours at 23 in a ti - 70 rotor . pellets were resuspended in te / sds ( 10 mm tris - hcl ( ph 7 . 5 ), 1 mm edta , 0 . 1 % ( w / v ) sds ) and extracted with phenol : chloroform : isoamyl alcohol ( 25 : 24 : 1 ) saturated in 10 mm edta ( ph 7 . 5 ). following ethanol precipitation the rna pellets were resuspended in te / sds . rna samples were electrophoresed through 2 . 2m formaldehyde / 1 . 2 % ( w / v ) agarose gels using running buffer containing 40 mm morpholinopropanesulphonic acid ( ph 7 . 0 ), 5 mm sodium acetate , 0 . 1 mm edta ( ph 8 . 0 ). the rna was transferred to hybond - n filters ( amersham ) as described by the manufacturer and probed with 32 p - labelled cdna fragment ( 108 cpm / μg , 2 × 10 6 cpm / ml ). prehybridization ( 1 h at 42 ° c ) and hybridization ( 16 h at 42 ° c ) was carried out in 50 % ( v / v ) formamide , 1m nacl , 1 % ( w / v ) sds , 10 % ( w / v ) dextran sulphate . degraded salmon sperm dna ( 100 μg / ml ) was added with the 32 p - labelled probe for the hybridization step . filters were washed in 2 × ssc / 1 % ( w / v ) sds at 65 ° c . for 1 to 2 hours and then 0 . 2 × ssc / 1 % ( w / v ) sds at 65 ° c . for 0 . 5 to 1 hour . filters were exposed to kodak xar film with an intensifying screen at - 70 for 48 hours . northern analysis of dianthus cv . red sim transformed with plasmid pcgp90 indicated that eight of thirteen plants were positive . total rna was extracted from petals ( buds and of flowers 5 days post - harvest ) according to the method of manning , 1991 . dna fragments ( 50 to 100 ng ) were radioactively labelled with 50 μci of α - 32 p !- dctp using an oligolabelling kit ( bresatec ). unincorporated α - 32 p !- dctp was removed by chromatography on a sephadex g - 50 ( fine ) column . prior to hplc analysis the anthocyanin molecules present in petal extracts were acid hydrolysed to remove glycosyl moieties from the anthocyanidin core . the hydroxylation pattern on the b ring of the anthocyanin pigments was determined by hplc analysis of the anthocyanidin core molecule . the hplc system used in this analysis was a hewlett - packard 1050 equipped with a multiwavelength detector ( mwd ). reversed phase chromatographic separations were performed on a spherisorb s5 ods2 cartridge column , 250 mm × 4 mm id . flower pigments were extracted from petal segments ( ca . 50 mg ) with 5 ml of methanol containing 1 % ( v / v ) of aqueous 6m hydrochloric acid . extracts were diluted with water ( 1 : 9 ) and filtered ( millex hv , 0 . 45μ ) prior to injection into the hplc system . crude methanolic extracts ( 100 μl ) obtained in a . above were evaporated to dryness in pierce reacti - vials using a stream of dry nitrogen at room temperature . the residues were dissolved in 200 μl 2m hcl , vials were capped and then heated at 100 ° c . or 30 minutes . hydrolysis mixtures were diluted with water ( 1 : 9 ) and filtered ( millex hv , 0 . 45μ ) prior to hplc analysis . separation of flower pigments was effected via gradient elution using the following system : solvent a : ( triethylamine : conc . h 3 po 4 : h 2 o ) ( 3 : 2 . 5 : 1000 ) detection : mwd with simultaneous data acquisition at 280 , 350 and 546 nm . the anthocyanidin peaks were identified by reference to known standards . an alternative method for the analysis of anthocyanin molecules present in petal extracts is to be found in brugliera et al ., 1994 . hplc analysis is conducted to determine the presence of delphinidin , pelargonidin and cyanidin pigments in samples of carnation , chrysanthemum and rose tissues having been transformed with one or other of the plasmids pcgp90 , pcgp485 , pcgp484 , pcgp628 , pcgp653 or pcgp1458 . representative data of pcgp90 , pcgp485 and pcgp653 in transgenic carnation flowers are shown in table 1 . table 1______________________________________hplc analysis of pcgp90 , pcgp485 and pcgp653 transgenic flowers % del - % pelar - % sample phinidin gonidin cyanidin______________________________________non - transgenic carnation : cultivar : red sim 0 85 . 3 0 . 8transgenic carnation : red sim + pcgp90 ( i ) acc #* 1933 1 . 9 82 . 7 nd **( ii ) acc # 2011 3 . 7 76 . 9 ndred sim + pcgp485 ( i ) acc # 3654b 13 . 0 75 . 1 2 . 3red sim + pcgp653 ( i ) acc # 3660 / 2 18 . 1 71 . 4 3 . 2 ( ii ) acc # 3655 35 . 6 49 . 1 7 . 5______________________________________ * acc # = plant accession number ** nd = not detected plant tissue was homogenised in a 10 times volume of ice - cold extraction buffer ( 100 mm potassium phosphate ( ph 7 . 5 ), 1 mm edta , 0 . 25m sucrose , 0 . 25m mannitol , 0 . 1 % ( w / v ) bsa , 0 . 1 mg / ml pmsf , 20 mm 2 - mercaptoethanol and 10 mg / ml polyclar at ). the homogenate was centrifuged at 13 , 000 rpm in a ja20 rotor ( beckman ) for 10 min at 4 ° c . and an aliquot of the supernatant assayed for 3 &# 39 ;, 5 &# 39 ;- hydroxylase activity . 3 &# 39 ;, 5 &# 39 ;- hydroxylase enzyme activity was measured using a modified version of the method described by stotz and forkmann ( 1982 ). the assay reaction mixture typically contained 195 μl of plant extract , 5 μl of 50 mm nadph in assay buffer ( 100 mm potassium phosphate ( ph8 . 0 ), 1 mm edta and 20 mm 2 - mercaptoethanol ), and 10 5 dpm 14 c ! naringenin in a final volume of 200 μl . following incubation at 23 overnight , the reaction mixture was extracted twice with 0 . 5 ml of ethylacetate . the ethyl acetate phase was dried under vacuum and then resuspended in 10 μl of ethyl acetate . the radio - labelled flavonoid molecules were then separated on cellulose thin layer plates ( merck art 5577 , germany ) using a chloroform : acetic acid : water ( 10 : 9 : 1 , v / v ) solvent system . at the completion of the chromatography , the tlc plates were air - dried and the reaction products localised by autoradiography and identified by comparison to non - radioactive naringenin , eriodictyol , dihydroquercetin and dihydromyricetin standards which were run alongside the reaction products and visualized under uv light . the chimaeric genes contained in any one of the constructs pcgp90 , pcgp812 , pcgp628 , pcgp485 , pcgp653 , pcgp484 or pcgp1458 is introduced into plant varieties of rose , carnation and chrysanthemum using agrobacterium - mediated gene transfer , as described in examples 10 , 11 and 12 . integration of the appropriate chimaeric gene into the plant genome is confirmed by southern analysis of plants obtained after kanamycin selection and hplc analysis is used to detect the presence of anthocyanins as described in example 16 , above . plants successfully rendered transgenic and which are able to express the transgene in accordance with the present invention , have significant levels of 3 &# 39 ;, 5 &# 39 ;- hydroxylase enzyme activity in addition to 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanins ( seen in example 16 ), compared with non - transgenic controls which do not contain the gene necessary for the production of 3 &# 39 ;, 5 &# 39 ;- hydroxylase activity . the plasmid pcgp90 was introduced into the carnation cultivar crowley sim using agrobacterium - mediated gene transfer , as described in example 10 . integration of the construct in the plant genome was confirmed by southern analysis of plants obtained after kanamycin selection . nine plants were examined for the presence of the nptii and hf1 genes and for the production of delphinidin . eight of the nine plants analyzed were positive for both nptii and hf1 but hplc analysis was unable to detect any evidence of delphinidin production by these plants ( see table 2 ; &# 34 ; kan &# 34 ; = kanamycin ). table 2______________________________________ # acc # kan hf1 delphinidin______________________________________1 1930a + + - 2 1942b + + - 3 2008b - - - 4 2217a + + - 5 2217b + + - 6 2338a + + - 7 2338b + + - 8 2338c + + - 9 2338d + + - ______________________________________ the plasmid pcgp485 was introduced into the carnation cultivar laguna using agrobacterium - mediated gene transfer , as described in example 10 . integration of the construct in the plant genome was confirmed by southern analysis of plants obtained after kanamycin selection . hplc analysis of the anthocyanin molecules present in petal extracts is carried out according to the procedure set out in example 16 , above , to show the presence of 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanin derivatives . these 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanins are only produced as a result of the expression of the exogenous dna sequence , ie : the hf1 cdna sequence , introduced via transformation with the binary vector pcgp485 . the plasmids pcgp485 and pcgp628 were introduced into the rose cultivar royalty using agrobacterium - mediated gene transfer , as referred to in example 11 . integration of the construct in the plant genome was confirmed by southern analysis of plants obtained after kanamycin selection . hplc analysis of the anthocyanin molecules present in petal extracts is again carried out according to the procedure set out in example 16 , above , to show the presence of 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanin derivatives . these 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanins are only produced as a result of the expression of the exogenous dna sequence , ie : the hf1 cdna sequence , introduced via transformation with either of the binary vectors pcgp485 or pcgp628 . the plasmid pcgp1458 was introduced into the rose cultivar kardinal using agrobacterium - mediated gene transfer , as described in example 11 . integration of the construct in the plant genome was confirmed by southern analysis of plants obtained after kanamycin selection . hplc analysis of the anthocyanin molecules present in petal extracts is again carried out according to the procedure set out in example 16 , above , to show the presence of 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanin derivatives . these 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanins are only produced as a result of the expression of the exogenous dna sequence , ie : the hf1 cdna sequence , introduced via transformation with the binary vector pcgp1458 . the plasmids pcgp484 , pcgp485 and pcgp628 were introduced into the chrysanthemum cultivar blueridge using agrobacterium - mediated gene transfer , as described in example 12 . integration of the construct in the plant genome was confirmed by southern analysis of plants obtained after kanamycin selection . hplc analysis of the anthocyanin molecules present in petal extracts is again carried out according to the procedure set out in example 16 , above , to show the presence of 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanin derivatives . these 3 &# 39 ;, 5 &# 39 ;- hydroxylated anthocyanins are only produced as a result of the expression of the exogenous dna sequence , ie : the hf1 cdna sequence , introduced via transformation with any one of the binary vectors pcgp484 , pcgp485 or pcgp628 . the expression of the introduced flavonoid 3 &# 39 ;, 5 &# 39 ;- hydroxylase enzyme activity in the transgenic plant is capable of having a marked effect on flower colour . floral tissues in transgenic plants may change from the pale pinks and reds of the non - transgenic control plants to colours ranging from a darker pink / maroon to a blue / purple colour . the colours may also be described in terms of numbers from the royal horticultural society &# 39 ; s colour chart . in general , the changes can be described as moving the colour from the pale - to - mid pink hues of 60c / d - 65c / d , to the darker bluer / purpler hues represented by many , but not all , of the colour squares between 70 and 85 . it should be remembered that other biochemical and physiological conditions will affect the individual outcome and the citing of specific colours should not be interpreted as defining the possible range . in the case of the transgenic carnation flower , accession number 3655 , produced using the plasmid construct pcgp653 described above , an obvious bluing effect on the petals was observed . the normally - orange - red colour of red sim carnation cultivars ( corresponding approximately to 45a / b of the royal horticultural society &# 39 ; s colour chart ) had changed to a blue / purple hue . 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 . bethesda research laboratories . brl puc host : e . coli dh5α ™ competent cells . bethesda res . lab . focus 8 ( 2 ): 9 , 1986 . brugliera , f ., holton , t . a ., stevenson , t . w ., farcy , e ., lu , c - 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