Transgenic plants having altered anthocyann levels

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',5'-hydroxylase thereby permitting the manipulation of intermediates in the flavonoid pathway.

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',5'-hydroxylase thereby permitting the 
manipulation of intermediates in the flavonoid pathway. 
BACKGROUND OF THE INVENTION 
The flower industry strives to develop new and different varieties of 
flowering plants with improved characteristics ranging from disease and 
pathogen resistance to altered inflorence. Although classical breeding 
techniques have been used with some success this approach has been limited 
by the constraints of a particular species' gene pool. It is rare, for 
example, for a single species to have a full spectrum of coloured 
varieties. Accordingly, substantial effort has been directed towards 
attempting to generate transgenic plants exhibiting the desired 
characteristics. The development of blue varieties of the major cutflower 
species rose, carnation and chrysanthemum, for example, would offer a 
significant opportunity in both the cutflower and ornamental markets. 
Flower colour is predominantly due to two types of pigment: flavonoids and 
carotenoids. Flavonoids contribute to a range of colours from yellow to 
red to blue. Carotenoids impart an orange or yellow tinge and are commonly 
the only pigment in yellow or orange flowers. The flavonoid molecules 
which make the major contribution to flower colour are the anthocyanins 
which are glycosylated derivatives of cyanidin, delphinidin, petunidin, 
peonidin, malvidin and pelargonidin, and are localised in the vacuole. The 
different anthocyanins can produce marked differences in colour. Flower 
colour is also influenced by co-pigmentation with colourless flavonoids, 
metal complexation, glycosylation, acylation, methylation and vacuolar pH 
(Forkmann, 1991). 
The biosynthetic pathway for the flavonoid pigments (hereinafter referred 
to as the "flavonoid pathway") is well established and is shown in FIG. 1 
(Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and de 
Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed 
step in the pathway involves the condensation of three molecules of 
malonyl-CoA with one molecule of p-coumaroyl-CoA. This reaction is 
catalysed by the enzyme chalcone synthase (CHS). The product of this 
reaction, 2',4,4',6'-tetrahydroxychalcone, is normally rapidly isomerized 
to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). 
Naringenin is subsequently hydroxylated at the 3 position of the central 
ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK). 
The B-ring of dihydrokaempferol can be hydroxylated at either the 3', or 
both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and 
dihydromyricetin (DHM), respectively. Two key enzymes involved in this 
pathway are flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. The 
flavonoid 3'-hydroxylase acts on DHK to produce DHQ and on naringenin to 
produce eriodictyol. The flavonoid 3',5'-hydroxylase (hereinafter referred 
to as 3',5'-hydroxylase) is a broad spectrum enzyme catalyzing 
hydroxylation of naringenin and DHK in the 3' and 5' positions and of 
eriodictyol and DHQ in the 5' position (Stotz and Forkmann, 1982), in both 
instances producing pentahydroxyflavanone and DHM, respectively. The 
pattern of hydroxylation of the B-ring of anthocyanins plays a key role in 
determining petal colour. 
Because of the aforesaid gene pool constraints, many of the major cutflower 
species lack the 3',5'-hydroxylase and consequently cannot display the 
range of colours that would otherwise be possible. This is particularly 
the case for roses, carnations and chrysanthemums, which constitute a 
major proportion of the world-wide cutflower market. There is a need, 
therefore, to modify plants and in particular roses, carnations and 
chrysanthemums, to generate transgenic plants which are capable of 
producing the 3',5'-hydroxylase thereby providing a means of modulating 
DHK metabolism, as well as the metabolism of other substrates such as DHQ, 
naringenin and eriodictyol. Such modulation influences the hydroxylation 
pattern of the anthocyanins and allows the production of anthocyanins 
derived from delphinidin, thereby modifying petal colour and allowing a 
single species to express a broader spectrum of flower colours. There is a 
particular need to generate transgenic plants which produce high levels of 
anthocyanins derived from delphinidin. In accordance with the present 
invention, gene constructs are generated and used to make transgenic 
plants which express high levels of delphinidin and/or its derivatives 
relative to non-transgenic plants of the same species. It has been 
determined in accordance with the present invention that genetic 
constructs which comprise a promoter from a gene encoding a flavonoid 
pathway enzyme operably linked to a flavonoid 3',5'-hydroxylase are 
capable of directing expression of high levels of delphinin-derived 
anthocyanins. The production of these high levels of delphinidin and 
related molecules is particularly useful in developing a range of plants 
exhibiting altered flower color properties. 
SUMMARY OF THE INVENTION 
Accordingly, one aspect of the present invention contemplates a transgenic 
plant selected from rose, carnation and chrysanthemum or progeny or 
flowering parts thereof wherein said plant carries a genetic construct 
comprising a promoter from a gene encoding an enzyme of the flavonoid 
pathway operably linked to a gene encoding a flavonoid 3',5'-hydroxylase 
wherein said transgenic plant produces higher levels of anthocyanins 
derived from delphinidin relative to non-transgenic plants of the same 
species. 
Preferably, the flavonoid 3',5'-hydroxylase is of petunia, verbena, 
delphinum, grape, iris, freesia, hydrangea, cyclamen, potato, pansy, egg 
plant, lisianthus or campanula origin. 
Most preferably, the flavonoid 3',5'-hydroxylase is of petunia origin. 
The gene construct of the present invention comprises a nucleic acid 
molecule encoding a sequence encoding 3',5'-hydroxylase and where 
necessary comprises additional genetic sequences such as promoter and 
terminator sequences which allow expression of the molecule in the 
transgenic plant. When the gene construct is DNA it may be cDNA or genomic 
DNA. Preferably, the DNA is in the form of a binary vector comprising a 
chimaeric gene construct which is capable of being integrated into a plant 
genome to produce the transgenic plant of the present invention. The 
chimaeric gene construct carries a plant promoter from a gene encoding an 
enzyme of the flavonoid pathway. A preferred promoter is from the gene 
encoding chalcone synthase (CHS) and is referred to herein as the "CHS 
promoter". The CHS promoter is particularly preferred since it directs the 
high level expression of genetic sequences operably linked down stream 
thereof. The most preferred binary vectors are pCGP484, pCGP485, pCGP653 
and pCGP1458. pCGP484 was deposited with the Australian Government 
Analytical Laboratories (AGAL), 1 Suakin Street, Pymble, NSW 2073, 
Australia, on Aug. 14, 1998 and has been accorded Accession No. NM 
98/07526. pCGP485 was deposited at the same depository on the same date 
and accorded Accession No. NM 98/07527. pCGP653 and pCGP1458 were also 
deposited with the Australian Government Analytical Laboratories on Aug. 
14, 1998 and accorded Accession Nos. NM 98/07524 and NM 98/07525, 
respectively. 
By "nucleic acid molecule" as used herein is meant any contiguous series of 
nucleotide bases specifying a sequence of amino acids in 
3',5'-hydroxylase. The nucleic acid may encode the full length enzyme or a 
functional derivative thereof. By "derivative" is meant any single or 
multiple amino acid substitutions, deletions, and/or additions relative to 
the naturally-occurring enzyme. In this regard, the nucleic acid includes 
the naturally-occurring nucleotide sequence encoding 3',5'-hydroxylase or 
may contain single or multiple nucleotide substitutions, deletions and/or 
additions to said naturally-occurring sequence. The terms "analogues" and 
"derivatives" also extend to any functional chemical equivalent of the 
3',5'-hydroxylase, the only requirement of the said nucleic acid molecule 
being that when used to produce a transgenic plant in accordance with the 
present invention said transgenic plant exhibits one or more of the 
following properties: 
(i) production of 3',5'-hydroxylase-specific mRNA; 
(ii) production of 3',5'-hydroxylase protein; 
(iii) production of delphinidin and/or its derivatives; and/or 
(iv) altered flower color. 
More particularly, said transgenic plant exhibits one or more of the 
following properties: 
(i) increased levels of 3',5'-hydroxylase-specific mRNA above 
non-transgenic endogenous levels; 
(ii) increased production of 3',5'-hydroxylase protein; 
(iii) elevated levels of production of delphinidin and/or its derivatives 
above non-transgenic endogenous levels; and/or 
(iv) altered flower color. 
The nucleic acid molecules used herein may exist alone or in combination 
with a vector molecule and preferably an expression-vector. Such vector 
molecules replicate and/or express in eukaryotic and/or prokaryotic cells. 
Preferably, the vector molecules or parts thereof are capable of 
integration into the plant genome. The nucleic acid molecule may 
additionally contain a sequence useful in facilitating said integration 
and/or a promoter sequence capable of directing expression of the nucleic 
acid molecule in a plant cell. The nucleic acid molecule and promoter may 
be introduced into the cell by any number of means such as by 
electroporation, micro-projectile bombardment or Agrobacterium-mediated 
transfer. 
In accordance with the present invention, a nucleic acid molecule encoding 
3',5'-hydroxylase may be introduced into and expressed in a transgenic 
plant selected from the list consisting of rose, carnation and 
chrysanthemum thereby providing a means to convert DHK and/or other 
suitable substrates into anthocyanin derivatives of anthocyanidins such as 
petunidin, malvidin and especially delphinidin. The production of these 
anthocyanins may contribute to the production of a variety of shades of 
blue colour or blue-like colour or may otherwise modify flower colour by 
diverting anthocyanin production away from pelargonidin, cyanidin and 
peonidin and their derivatives and towards delphinidin and its 
derivatives. Expression of the nucleic acid sequence in the plant may be 
constitutive, inducible or developmental. The expression "altered flower 
color" means any alteration in flower colour relative to the 
naturally-occurring flower colour taking into account normal variations 
between flowerings. Preferably, the altered flower color includes 
production of various shades of blue, purple or pink colouration different 
to those in the non-transgenic plant. 
The present invention also contemplates a method for producing a transgenic 
flowering plant exhibiting elevated levels of production of delphinidin 
and/or its derivatives above non-transgenic endogenous levels, said method 
comprising introducing into a cell of a plant selected from the list 
consisting of rose, carnation and chrysanthemum, a nucleic acid molecule 
encoding a sequence encoding 3',5'-hydroxylase under conditions permitting 
the eventual expression of said nucleic acid molecule, regenerating a 
transgenic plant from the cell and growing said transgenic plant for a 
time and under conditions sufficient to permit the expression of the 
nucleic acid molecule into the 3',5'-hydroxylase enzyme. The present 
invention is also directed to a method for producing a transgenic plant 
selected from rose, carnation and chrysanthemum, said method comprising 
introducing into said plant a gene construct containing a nucleic acid 
sequence encoding a flavonoid 3',5'-hydroxylase characterised in that said 
transgenic plant produces higher levels of anthocyanin derived from 
delphinidin relative to non-transgenic plants of the same respective 
species. 
In a preferred embodiment, the transgenic flowering plant exhibits altered 
flower color properties coincident with elevated levels of delphinidin 
production, and the altered flower color includes the production of blue 
flowers or other bluish shades depending on the physiological conditions 
of the recipient plant. In certain plant species it may be preferable to 
select a "high pH line", such being defined as a variety having a higher 
than average petal vacuolar pH. The origin of the recombinant 
3',5'-hydroxylase or its mutants and derivatives may include, petunia, 
verbena, delphinium, grape, iris, freesia, hydrangea, cyclamen, potato, 
pansy, lisianthus, campanula or eggplant. 
Consequently, the present invention extends to a transgenic rose, carnation 
or chrysanthemum plant containing all or part of a nucleic acid molecule 
representing 3',5'-hydroxylase and/or any homologues or related forms 
thereof and in particular those transgenic plants which exhibit elevated 
3',5'-hydroxylase-specific mRNA and/or elevated production of delphinidin 
derivatives and/or altered inflorence properties. The transgenic plants, 
therefore, contain a stably-introduced nucleic acid molecule comprising a 
nucleotide sequence encoding the 3',5'-hydroxylase enzyme. The invention 
also extends to progeny from such transgenic plants and also to 
reproduction material therefor (e.g. seeds). Such seeds, especially if 
coloured, will be useful inter alia as proprietary tags for plants. 
The present invention is further described by reference to the following 
non-limiting Figures and Examples.

DETAILED DESCRIPTION OF THE INVENTION 
EXAMPLE 1 
Materials 
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). .sup.3 H!-naringenin (5.7 Ci/mmole) and .sup.3 
H!-dihydroquercetin (12.4 Ci/mmole) were obtained from Amersham. All 
enzymes were obtained from commercial sources and used according to the 
manufacturer's recommendations. 
The Eschenchia coli strain used was: 
DH5.alpha. supE44, .DELTA.(lacZYA-ArgF)U169, .phi.80lacZ.DELTA.M15, hsdR17 
(r.sub.k -, m.sub.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. 
The cloning vector pBluescript was obtained from Stratagene. 
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. 
EXAMPLE 2 
Construction of pCGP 90 
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' 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' 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. 
EXAMPLE 3 
Construction of pCGP 812 
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' 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 "sticky" ends. A 3.8 kb BglII fragment 
containing the chimaeric Mac-Hf1-mas gene from pCGP807 (described below) 
was ligated with the BamHI "sticky" ends of pCGP83 to yield pCGP812 (FIG. 
2). 
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. 
EXAMPLE 4 
Construction of pCGP 485 
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' 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 pCGP13.DELTA. 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. 
EXAMPLE 5 
Construction of pCGP 628 
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. 
EXAMPLE 6 
Construction of pCGP 653 
Plasmid pCGP293 (described above in Example 2) was digested with XbaI and 
the resulting 5' 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). 
CHSA-782 
5' GTTTTCCAAATCTTGACGTG 3' 
CHSA+34 
5' ACGTGACAAGTGTAAGTATC 3' 
EXAMPLE 7 
Construction of pCGP 484 
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. 
EXAMPLE 8 
Construction of pCGP 1458 
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' 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' 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. 
EXAMPLE 9 
Transformation of E. coli and A. tumefaciens 
Transformation of the Escherichza coli strain DH5.alpha.-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 .mu.g of plasmid DNA to 100 .mu.L of competent Agrobacterium 
tumefaciens cells prepared by inoculating a 50 mL MG/L (Garfinkel and 
Nester, 1980) culture and growing for 16 h with shaking at 28. The cells 
were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM 
CaCl.sub.2 /15% (v/v) glycerol. The DNA-Agrobacterium mixture was frozen 
by incubation in liquid N.sub.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 .mu.g/mL gentamycin. Cells of 
A. tumefaciens carrying pCGP1458 were selected on MG/L agar plates 
containing 100 .mu.g/mL kanamycin. The presence of the plasmid was 
confirmed by Southern analysis of DNA isolated from the 
gentamycin-resistant transformants. 
EXAMPLE 10 
Transformation of Diantbus caryophyllus 
a. Plant Material 
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. 
b. Co-cultivation of Agrobacterium and Dianthus Tissue 
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.times.10.sup.8 cells/mL next day before inoculation. 
Dianthus tissue was co-cultivated with Agrobacterium on Murashige and 
Skoog's (1962) medium (MS) supplemented with 3% sucrose (w/v), 5 mg/L 
.alpha.-naphthalene acetic acid (NAA), 20 .mu.M acetosyringone and 0.8% 
Difco Bacto Agar (pH 5.7). 
c. Recovery of Transgenic Dianthus Plants 
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 
.mu.E 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.degree. C. under a 14 hour 
photoperiod (200 .mu.E mercury halide light). 
EXAMPLE 11 
Transformation of Rosa hybrida 
1. Rosa hybrida cv Royalty 
Plant tissues of the rose cultivar Royalty were transformed according to 
the method disclosed in PCT 91/04412, having publication number 
WO92/00371. 
2. Rosa hybrida cv Kardinal 
a. Plant Material 
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. 
b. Co-cultivation of Agrobacterium and Rosa shoot Tissue 
Agrobacterium tumefaciens strains ICMP 8317 (Janssen and Gardner 1989) and 
AGL0, containing the binary vector pCGP812, was maintained at 4.degree. 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.times.10.sup.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 .mu.l 
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.degree. 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.times.10.sup.8 
cells/mL was prepared the next day by dilution in liquid MG/L. 
c. Recovery of Transgenic Rosa Plants 
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 .mu.E 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 .mu.E mercury halide light). After 1-2 
weeks potted plants were moved to glasshouse (Day/Night temperature: 
25-28/14) and grown to flowering. 
EXAMPLE 12 
Transformation of Chrysanthemum morifolium 
a. Plant Material 
Chrysanthemum morifolium (cv. Blue Ridge, Pennine Chorus) cuttings were 
obtained from F & 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. 
b. Co-cultivation of Agrobacterium and Chrysanthemum Tissue 
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 .mu.l 
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.times.10.sup.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. 
c. Recovery of Transgenic Chrysanthemum Plants 
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 .mu.E cool white fluorescent light) at 
23.+-.2.degree. 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.degree. C. under a 14 hour photoperiod (300 .mu.E 
mercury halide light). After 2 weeks potted plants were moved to 
glasshouse (Day/Night temperature : 25-28.degree. C./14.degree. C.) and 
grown to flowering. 
EXAMPLE 13 
Southern Analysis 
a. Isolation of Genomic DNA from Dianthus 
DNA was isolated from tissue essentially as described by Dellaporta et al., 
(1983). The DNA preparations were further purified by CsCl buoyant density 
centrifugation (Sambrook et al., 1989). 
b. Isolation of Genomic DNA from Chrysanthemum 
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.sub.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. 
c. Isolation of Genomic DNA from Rosa 
DNA was extracted by grinding tissue in the presence of liquid N.sub.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.degree. C. Chloroform (200 .mu.l ) was 
added and the mixture incubated at 65.degree. 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 
.mu.l TE buffer (pH 8.0). 
d. Southern Blots 
The genomic DNA (10 .mu.g) 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.times.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. 
EXAMPLE 14 
Northern Analysis 
a. Dianthus and Chrysanthemum RNA 
Total RNA was isolated from tissue that had been frozen in liquid N.sub.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 .sup.32 P-labelled cDNA 
fragment (108 cpm/.mu.g, 2.times.10.sup.6 cpm/mL). Prehybridization (1 h 
at 42.degree. C) and hybridization (16 h at 42.degree. 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 .mu.g/mL) was added with the .sup.32 
P-labelled probe for the hybridization step. 
Filters were washed in 2.times.SSC/1% (w/v) SDS at 65.degree. C. for 1 to 2 
hours and then 0.2 .times.SSC/1% (w/v) SDS at 65.degree. 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. 
b. Rosa RNA 
Total RNA was extracted from petals (buds and of flowers 5 days 
post-harvest) according to the method of Manning, 1991. 
EXAMPLE 15 
.sup.32 P-Labelling of DNA Probes 
DNA fragments (50 to 100 ng) were radioactively labelled with 50 .mu.Ci of 
.alpha.-.sup.32 P!-dCTP using an oligolabelling kit (Bresatec). 
Unincorporated .alpha.-.sup.32 P!-dCTP was removed by chromatography on a 
Sephadex G-50 (Fine) column. 
EXAMPLE 16 
Anthocyanidin Analysis 
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.times.4 mm ID. 
a. Extraction of anthocyanins and flavonoids 
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, 0.45.mu.) prior to 
injection into the HPLC system. 
b. Hydrolysis of anthocyanins 
Crude methanolic extracts (100 .mu.L) 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 .mu.L 2M HCl, vials were 
capped and then heated at 100.degree. C. or 30 minutes. Hydrolysis 
mixtures were diluted with water (1:9) and filtered (Millex HV, 0.45.mu.) 
prior to HPLC analysis. 
c. Chromatography 
Separation of flower pigments was effected via gradient elution using the 
following system: 
Solvent A: (triethylamine: conc. H.sub.3 PO.sub.4 : H.sub.2 O) (3:2.5:1000) 
Solvent B: acetonitrile 
Gradient Conditions: 5% B to 40% B over 20 minutes 
Flow Rate: 1 ml/min 
Temperature: 35.degree. C. 
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 0.8 
TRANSGENIC CARNATION: 
Red Sim + pCGP90 
(i) Acc #* 1933 1.9 82.7 nd** 
(ii) Acc # 2011 3.7 76.9 nd 
Red Sim + pCGP485 
(i) Acc # 3654B 13.0 75.1 2.3 
Red 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 
EXAMPLE 17 
Preparation of Plant Extracts for Assay of 3',5'-Hydroxylase Activity 
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.degree. C. and an aliquot of the 
supernatant assayed for 3',5'-hydroxylase activity. 
3',5'-Hydroxylase Assay 
3',5'-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 .mu.L of plant extract, 5 .mu.L of 50 mM 
NADPH in assay buffer (100 mM potassium phosphate (pH8.0), 1 mM EDTA and 
20 mM 2-mercaptoethanol), and 10.sup.5 dpm .sup.14 C! naringenin in a 
final volume of 200 .mu.L. 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 
.mu.L 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. 
EXAMPLE 18 
Transformation of various cultivars 
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',5'-hydroxylase enzyme activity in addition to 
3',5'-hydroxylated anthocyanins (seen in Example 16), compared with 
non-transgenic controls which do not contain the gene necessary for the 
production of 3',5'-hydroxylase activity. 
EXAMPLE 19 
Carnation cv. Crowley Sim+pCGP 90 
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; "Kan" =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 + + - 
______________________________________ 
EXAMPLE 20 
Carnation cv. Laguna+pCGP 485 
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',5'-hydroxylated anthocyanin derivatives. These 
3',5'-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. 
EXAMPLE 21 
Rose cv. Royalty+pCGP 485/pCGP 628 
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',5'-hydroxylated anthocyanin derivatives. These 
3',5'-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. 
EXAMPLE 22 
Rose cv. Kardinal+pCGP 1458 
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',5'-hydroxylated anthocyanin derivatives. These 
3',5'-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. 
EXAMPLE 23 
Chrysanthemum cv. BlueRidge+pCGP 484/pCGP 485/pCGP 628 
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',5'-hydroxylated anthocyanin 
derivatives. These 3',5'-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. 
EXAMPLE 24 
Altered Inflorescence 
The expression of the introduced flavonoid 3',5'-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'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'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. 
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