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(11) Patent Number: KE 447   

(45) Date of grant:  17/06/2011           

(51) lnt.CI.S:A OlH 5//00, C 12N 15//09

(21)Application Number:KElP! 2009/ 001016

(22) Filing Date:19/06/2008

(30) Priority data: 2007-162959  20/06/2007  JP

(86)  PCT data PCT/JP08/061593 19/06/2008 wo 20081156206 24112/2008
 
(73) Owner:INTERNATIONAL FLOWER DEVELOPMENTS PTY.LTD of  1 Park Drive, Bundoora, 3083, Victoria,Australia

(72) Inventor:TANAKA, Y oshikazu of c/o Suntory Limited, Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan; KATSUMOTO, Yukihisa of c/o Suntory Limited, Research Center, 1-1-1,

W akayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan; MIZUTANI, Masako of c/o Suntory Limited, Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan; FUKUI, Yuko of c/o Suntory Limited, Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan; NAKAMURA, Noriko of c/o Suntory Limited, Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan; and TOGAMI, Junichi of c/o Suntory Limited, Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 6188503, Japan.

(74) Agent/address for correspondence:  Waruinge & Waruinge Advocates, P. 0. BOX 82384-00200, Nairobi
 

(54)Tit1e: ROSE CONTAINING FLAVONE AND MALVIDIN, AND METHOD FOR PRODUCTION THEREOF.

(57) Abstract: Disclosed is a rose characterized by containing a flavone and a malvidin by a genetic recombination technique. Typically, the flavone and the malvidin are produced by the expression of a flavone synthesis enzyme gene and the expression of a flavonoid 3',5'-hydroxylase gene originated from pansy (Viola x wittrockiana) and an anthocyanin methyltransferase gene, respectively, all introduced into the rose. The flavone synthesis enzyme gene may be one originated from a plant belonging to the family Scrophulariaceae, such as one originated from common snapdragon (Scrophulariaceae, Antirrhinum majus) and one originated from torenia (Scrophulariaceae, Torenia hybrida). The flavonoid 3',5'-hydroxylase gene may be one originated from pansy (Viola x wittrockiana). The anthocyanin methyltransferase gene may be one originated from torenia (Scrophulariaceae, Torenia hybrida).
DESCRIPTION

ROSE  CONTAINING  FLAVONE  AND  MALVIDIN  AND  METHOD

FOR  PRODUCTION  THEREOF

5

Technical  Field

The present invention relates to an artificially made rose containing a flavone and malvidin. The invention further relates to a method for modifying rose

10    petal color by a co-pigmentation effect which is produced by adding a flavone and malvidin by genetic engineering, and particularly to a method for altering petal color toward blue.


15    Background  Art

Flowers are plant reproductive organs that are required for production of seeds for subsequent generations. Formation of seeds requires adhesion ot pollen onto the pistils, and fertilization. Pollen is

20    usually carried by insects such as bees and butterfl~0s, by hummingbirds, and rarely by bats. The role of iJ_u,~0r petals is to attract these organisms that carry pollen,

and plants have developed modifications to flower color, shape and coloring pattern for this purpose.

25 Since flower color is also the most important trait for ornamental flowers, flowers of various colors have traditionally been bred by cross-breeding. However, it . is rare for one plant variety to have different flower colors, and for example, crossbreeding has not produced

30    any purple to blue varieties for rose (Rosa hybr_ida), carnation (Dianthus caryophyllus), chrysanthemum (Chrysanthemum morifolium) or lily (Lilium spp.), or

bright red varieties for Japanese garden iris (Iris ensata Thumb.) or gentian (Gentiana triflora).

35 Light yellow to red or blue flower colors are generally due to the presence of flavonoids and anthocyanins (colored glucosid~s belonging ~o the

flavonoid class). Flavonoids are common secondary metabolites of plants, and they have a basic C6C3C6 backbone and are synthesized from phenylalanine and malonyl~CoA, as shown below. They are classified as

5    flavones, flavonols, etc. according to the oxidation states of the C-rings.


COS..CoA                        wtiHOH   
¢    3xMalonyi-CoA        ~OH           
        \~    HO    OH 0        -- Glc•    I "'    -    ,.. I   
OH        CHS            C4'GT  AS    --    0 Aurone   
Coumaroy)-CoA    2',4',6',4-Telrahydroxychalcone        OH       
                ~CHI        011    HO        OH       
        HO~•O-va                       
        \        W-;--+        I           
                OH 0    FNS    OH    o Flavones   
        oH   FLS \        Na~g;;~            ~ .o.lr0~       
HO#:_W~~:_           
    HO~~\Jl       
0        F3'H        Woo    F3'5'H    Oil    0           
                Ill   0                           
Dihydroquercetin    Dihydrokaempferol        Dihydromyricelin       
~ DFR        ~ DFR            ~ DFR           
~ ANS        ~ ANS            pNS    OH    3'GT   
        011                Oil                   
        OH                            OH       
                                        5GT   
HO        1111                HO        OH       
                                        -AT   
                                           
        H        011                        3RAT   
OH                            011            MT   
                                           
                                           
Cyanidin    Pelargonidin    Delphinidin

OH

HO

OH    0

Kaempferol

OH



OH    0

Quercetin

OH

OH

HO
OH

OH    0
Myricetin

Flavonols

HOW'"I

OH    0

Apigenin

OH



OH    0

Luteolin

OH

HO
OH

0

Tricetin

Flavones
 


Flavonoids absorb ultraviolet rays and remove radicals, and their original function is therefore believed to be protection of plant bodies from various

5    forms of stress. They have also received attention in recent years as healthy components (see Harborne and Williams 2000 Phytochemistry 55, 481-504).

Several  hundred  molecular  species  of  colored

anthocyanins  are  known,   and  of  the  chromophoric

10    anthocyanidins, the most common are the following 6 types: (1) pelargonidin abundant in orange to red flowers, (2) cyanidin and peonidin abundant in red to crimson flowers, and (3) delphinidin, petunidin and malvidin abundant in violet to blue flowers.
 
The anthocyanin structure has a major effect on color. An increased number of hydroxyl groups on the B

5    ring of the anthocyanin results in a greater degree of blue. Delphinidin-type anthocyanins are bluer than pelargonidin-type anthocyanins and cyanidin-type

anthocyanins. Biosynthesis of flavonoids including anthocyanins is highly conserved across plant species.

10    Flavonoids are biosynthesized in the cytosol, and after addition of sugars and acyl groups, they are transported to the vacuoles and accumulated (see Tanaka et al. 2005 Plant Cell, Tissue and Organ Culture 80,1-24 and Tanaka and Brugliera 2006 Ainsworth, ed. Flowering and its

15 manipulation, pp.201-239, Blackwell Publishing Ltd.). The structural genes of the enzymes involved in the

biosynthesis have all been cloned. Creating recombinant plants therefore allows modification of the structures and amounts of flavonoids that are accumulated in flowers

20    by artificial expression of their genes, thereby altering the flower color (Tanaka et al. 2005 Plant Cell, Tissue and Organ Culture 80,1-24, Tanaka and Brugliera 2006 Ainsworth, Flowering and its manipulation, pp.201-239,
Blackwell Publishing Ltd.). For example; for carnations or roses that cannot produce delphinidin in the petals, the flavonoid 3',5'-hydroxylase (hereinafter abbreviated as "F3'5'H") gene necessary for synthesis of delphinidin

5    has been expressed to produce delphinidin to create an artificial blue flower (see Tanaka 2006 Phytochemistry Reviews 5, 283-291).

Such methods of artificially modifying plant metabolism are sometimes called "metabolic engineering".

10    In order to modify metabolism for accumulation of a substance of interest expression of the gene of the enzyme that produces the substance of interest in a recombinant plant is possible, but in many cases

competition  with  endogenous  enzymes  of  the  same  plant

15    results in little or absolutely no accumulation of the substance of interest, and therefore no industrially

useful  trait  is  obtained.

For  example,   petunias   (Petunia  hybrida)   do  not

accumulate  pelargonidin  due  to  the  specificity  of

20    dihydroflavonol reductase (hereinafter abbreviated a~ "DFR"), and therefore no natural varieties exist with

orange-colored  flowers.

While  orange  petunias  that  accumulate  pelargonidin

by  transfer  of  the  DFR  gene  from  roses  or  the  like  have

25    been reported, the accumulation of pelargonidin requires the use of petunia varieties lacking the genes for flavonoid 3'-hydroxylase (hereinafter abbreviated as

"F3'H"),    F3'5'H  and  flavonol  synthase   (hereinafter

abbreviated  as  "FLS")   that  compete  with  DFR,   because  no

30    change in phenotype is observed when the rose DFR gene is transferred into petunias that do not lack these genes

(see Tanaka and Brugliera 2006 Ainsworth, Flowering and its manipulation, pp.20l-239, Blackwell Publishing Ltd.). Consequently, it cannot be predicted whether a compound

35    of interest will be accumulated to exhibit the desired phenotype simply by transferring a gene of interest.

In  addition,  metabolic  engineening  often  produces
unpredictable results. For example, when expression of the flavone synthase gene was inhibited in torenia

(Torenia hybrida), the flavone content was reduced and accumulation of flavanones was observed. Accumulation of

5    flavanones would be expected to result in an increased anthocyanin content, but in actuality the anthocyanin content decreased (Ueyama et al. Plant Science, 163, 253-263, 2002). It is therefore difficult to predict changes

in metabolites, and persistent modifications have been 10 necessary to obtain desired phenotypes.

Anthocyanins bound with higher numbers of aromatic acyl groups also appear bluer due to an intramolecular copigment effect. Anthocyanins with two or more aromatic acyl groups are known as polyacylated anthocyanins, and

15    they exhibit a stable blue color (see Harborne and Williams 2000 Phytochemistry 55, 481-504).

The  color  of  a  flower  changes  not  only  by  the

structure of the anthocyanin pigments themselves as the essential pigments, but also due to copresent flavonoids

20 (also known as copigments), metal ions, and the pH of the vacuoles. For example, flavones or flavonols are typical copigments that form sandwich-like stacking with anthocyanins and render the anthocyanins bluing and deepening color effects (see Goto (1987) Prog. Chern. Org.

25    Natl. Prod. 52). Flavones can thus be considered colorless copigment components. For example, isovitexin, a type of flavone, exhibits a copigment effect for anthocyanins in Japanese garden iris (Iris ensata

Thunb.). Isovitexin  also  stabilizes  anthocyanins,  thus

30    producing a stabilizing effect on Japanese garden iris flower color (see Yabuya et al. Euphytica 2000 115, 1-5)

Flavones usually exhibit stronger copigment effects than flavonols. For example, analysis of genetically modified carnations has indicated a stronger copigment

35    effect for flavones than flavonols (see Fukui et al. Phytochemistry, 63, 15-23, 2003). Accumulation of flavones is therefore important for creating blue flower


color. However, not all plants can produce flavones, and it is known that roses and petunias do not accumulate flavones. In addition to flower color, it is known that flavones play a role in absorption of ultraviolet rays,

5    countering various types of stress, and interaction with microorganisms, and that plants with new traits can be obtained through synthesis of flavones (as a patent document relating to a gene coding for flavone synthase, see Japanese Unexamined Patent Publication No. 2000-

10    279182). However, as yet no examples of flavone-expressing roses have been known.

Flavones  are  synthesized  from  flavanones  by  reaction

catalyzed  by  flavone  synthase.    Specifically,   apigenin  is

synthesized  from  naringenin,   luteolin  is  synthesized  from

15    eriodictyol and tricetin is synthesized from pentahydroxyflavanone. Flavone synthase exists in two forms, flavone synthase I and flavone synthase II. Both

catalyze  the  same  reaction,   but  are  different  types  of

enzymes.    Flavone  synthase  I   is  a  2-oxoglutaric  acid-

20    dependent dioxygenase (see Britsch et al. (1981) Z. Naturforsch 36c pp. 742-750 and Britsch (1990) Arch. Biochem. Biophys. 282 pp. 152-160), while flavone synthase II is a cytochrome P450-type monooxygenase. The

structural  gene  of  flavone  synthase  II  can  be  obtained

25    from torenia, snapdragon, perilla (Perilla frutescens), gerbera (Gerbera hybrida) and gentian (see Tanaka and Brugliera 2006 Ainsworth, Flowering and its manipulation,

pp.201-239,   Blackwell  Publishing  Ltd.).

Flavone  synthesis  is  predictable  when  the  flavone

30    synthase gene is expressed in genetically modified plants that do not produce flavones. However, when the torenia flavone synthase gene is expressed in petunias, it has

been  reported  that  the  deep  violet  color  of  the  flower

becomes  faint   (Tsuda  et  al.   Plant  Biotechnology,   21,   377-

35    386, 2004). It has also been reported that expression of the gentian-derived flavone synthase gene in tobacco results in flavone synthesis but, likewise, results in a
 
fainter flower color (Nakatsuka et al. 2006, Molecular Breeding 17:91-99). Thus, blue flower color is not always obtained even when flavones are synthesized. The reason for the lack of copigment effect could be an

5    unsuitable ratio of the anthocyanin and flavone contents or unsuitable modification of the anthocyanins and flavones with sugars and acyl groups. These results suggest that it is not possible to increase the blueness of flower color simply by expressing the flavone synthase

10    gene  and  accumulating  flavones.

Roses are the most popular of flowering plants, and they have been cultivated since ancient times. Artificially modified varieties have also been produced in the past several hundred years. Roses have therefore

15    been obtained containing flavonoids such as pelargonidin, cyanidin and flavonols. In recent years as well, roses have been created by genetic modification techniques to

produce delphinidin that is not naturally found in roses. However, no flavone-accumulating roses have yet been

20    obtained, either by cross-breeding or by genetic modification. In addition, no roses have yet been obtained that accumulate both a flavone and malvidin.


Disclosure  of  the  Invention

25 The major advantage of using genetic modification for breeding of plants is that, unlike cross-breeding, it allows modifications to plants that cannot be achieved by cross-breeding, and modifications using genes from other organisms. That is, genetic modification allows any gene

30    of an organism of a different species to be transferred into a plant such as a rose, to impart a new ability to the plant. However, unlike model plants such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum L.), the functioning of transferred genes in

35    roses is largely dependent on the source of the gene and the promoter used.

According  to  W02005/017147,   transfer  of  the
flavonoid 3',5'-hydroxylase gene (F3'5'H) into roses resulted in no expression in the genetically modified rose and no detection of delphinidin when the gene was derived from petunia or gentian, but interestingly, when

5    the gene was derived from pansy it was expressed and imparted to roses the new ability to produce delphinidin. In roses, therefore, it cannot be easily inferred which genes derived from which plant varieties will function when transferred.


10 When a gene is transferred into chrysanthemums as well, it is difficult to predict whether the gene will function in the chrysanthemums, and it is known that transferred genes lose their function as recombinant chrysanthemums age. The 358 promoter of cauliflower

15    mosaic virus, which is often used for transfer of foreign genes in recombinant plants, has been reported to function in gentian (see Mishiba et al. Plant Journal

2005,    44:541-556).

While  it  can  be  assumed  that  synthesis  of  flavones

20    in roses can be easily achieved by expressing the flavone synthase gene, it is not easy to predict whether to express the dioxygenase-type or the cytochrome P450-type

flavone    synthase,  or  which  plant  source  should  be  used

for  the  flavone  synthase  gene,   and  therefore  trial  and

25    error is necessary. The copigment effect is a phenomenon produced when anthocyanins and flavones or flavonols are

copresent in a certain concentration in the vacuoles, and it has been demonstrated that this requires the flavone or flavonol copigments to undergo glycosY.lation or other

30    modification more adapted to the condition of glycosylation or other modification of the anthocyanin color sources (see Nature. 2005 Aug 11; 436(7052) :791 and Nature, 358, 515-518 (1992)).

For  expression  of  the  necessary  color  tone  it  is

35    necessary for the anthocyanins and flavones/flavonols to be in the optimal structural combination, and this requires trial and error in regard to what sort of

modifications should exist in the copresent anthocyanins and flavones/flavonols. In qddition, because flavanones such as naringenin are rapidly hydroxylated by flavanone 3-hydroxylase (hereinafter abbreviated as "F3H") in

5    natural roses, flavones are not necessarily synthesized from flavanones even if flavone synthase is functioning

in  the  rose.

It is therefore an object of the present invention to provide roses comprising appropriate pigments for

10    expression  of  desired  color  tones  in  the  roses.

As a result of much research directed toward solving the problems mentioned above, the present inventors have completed this invention upon finding that desired color tone expression can be accomplished by artificially

15    adding  flavones  and  malvidin  to  roses.

Specifically, the present invention provides the following:

1. A rose characterized by comprising a flavone and malvidin added by a genetic modification method.

20 2. A rose according to 1 above, which comprises a flavone and malvidin by expression of pansy (Viola x wittrockiana) flavonoid 3',5'-hydroxylase and anthocyanin methyl transferase.

3.  A  rose  according  to  l   or  2  above,   which  comprises

25    malvidin, a flavone and delphinidin by expression of an anthocyanin methyltransferase gene, a flavone synthase gene and the pansy (Viola x wittrockiana) flavonoid 3',5'-hydroxylase gene.

4.    A  rose  according  to  any  one  of  1  to  3  above,

30    wherein the flavone synthase gene is a flavone synthase gene derived from the family Scrophulariaceae.

5. A rose according to 4 above, wherein the flavone synthase gene derived from the family Scrophulariaceae is a flavone synthase gene derived from snapdragon of the

35    family Scrophulariaceae (Scrophulariaceae, Antirrhinum majus) .

6.    A  rose  according  to  4  above,   wherein  the  flavone

synthase gene derived from the family Scrophulariaceae is a flavone synthase gene derived from torenia of the family Scrophulariaceae (Scrophulariaceae, Torenia

hybrida).

5 7. A rose according to 5 above, wherein the flavone synthase gene derived from snapdragon of the family Scrophulariaceae is a gene coding for:

(1) flavone synthase having the amino acid sequence listed as SEQ ID NO: 2,

10 (2) flavone synthase having the amino acid sequence listed as SEQ ID NO: 2 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino acids,


15 (3) flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 2, or

(4) flavone synthase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide

20    sequence of SEQ ID NO: 1 under highly stringent conditions.

8. A rose according to 6 above, wherein the flavone synthase gene derived from torenia of the family

Scrophulariaceae  is  a  gene  coding  for:

25 (1) flavone synthase having the amino acid sequence listed as SEQ ID NO: 4,

(2) flavone synthase having the amino acid sequence listed as SEQ ID NO: 4 modified by an addition or deletion of one or several amino acids and/or

30    substitution of one or several amino acids by other amino acids,

(3)    flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 4, or
 

35 (4) flavone synthase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 3 under highly stringent
 

conditions.

9. A rose according to any one of 2 to 8 above, wherein the pansy flavonoid 3',5'-hydroxylase gene is a gene coding for:

5 (l) flavonoid 3',5'-hydroxylase having the amino acid sequence listed as SEQ ID NO: 8,

(2) flavonoid 3',5'-hydroxylase having the amino acid sequence listed as SEQ ID NO: 8 modified by an addition or deletion of one or several amino acids and/or

10    substitution of one or several amino acids by other amino acids,

(3)    flavonoid 3',5'-hydroxylase having an amino acid sequence with at least 90% sequence identity to the amino

acid  sequence  listed  as  SEQ  ID  NO:   8,   or

15 (4) flavonoid 3',5'-hydroxylase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 7 under highly stringent conditions.

10 .. A  rose  according  to  any  one  of  2  to  9,  wherein

20    the anthocyanin methyltransferase gene is a gene coding for:

(1)    methyltransferase having the amino acid sequence listed as SEQ ID NO: 10,

(2)    methyltransferase  having  the  amino  acid  sequence

25    listed as SEQ ID NO: 10 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

acids,

(3)    methyltransferase  having  an  amino  acid  sequence  with

30    at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 10, or

(4)    methyltransferase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 9 under highly stringent
 

35    conditions.

11. A rose according to any one of 2 to 10 above, wherein the flower color is changed with respect to the


host before transfer of the anthocyanin methyltransferase gene, flavone synthase gene and pansy 3',5'-hydroxylase gene.

12. A rose according to 11 above, wherein the change 5 in flower color is a change toward blue.

13. A rose according to 11 or 12 above, wherein the change in flower color is a change such that the hue

angle (8) according to the L*a*b color system chromaticity diagram approaches 270° which is the blue axis.

10 14. A rose according to 11 above, wherein the change in flower color is a change such that the minimum value of the reflection spectrum of the petal shifts toward the longer wavelength end.

15. A rose portion, descendant, tissue, vegetative 15 body or cell having the same properties as a rose

according  to  any  one  of  1  to  14  above.

16. A method for modifying the flower color of a rose by a co-pigmentation effect produced by adding a flavone and malvidin by a genetic modification technique.

20 17. The method according to 16 above, wherein the co-pigmentation effect is an effect of changing the flower color toward blue.


Best Mode for Carrying Out the Invention 25 Definition of terms

The term "rose", as used throughout the present specification, is a general name for an ornamental plant which is a deciduous shrub of the order Rosales, family Rosaceae, genus Rosa, and it is not limited to any

30    specific variety and includes the entire plant or a portion thereof usually containing the flower.

A  reference  to  a  "portion,  descendant,   tissue,

vegetative body or cell" of a "rose", as used throughout the present specification, means any thing derived from a

35    "rose" so long as it retains the desired genetic trait of a "rose" according to the invention, and it is not limited to any particular entity.

The phrase "highly stringent conditions", as used throughout the present specification means, for example, conditions of heating the antisense strand and the target nucleic acid overnight at 55°C in a solution comprising 6

5 X sse (1 X sse composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 x Denhardt, 100 ~g/ml denatured fragmented salmon sperm DNA and 50% formamide, and rinsing under conditions of 0.1 x sse and/or conditions of 60°C or above, and specifically it refers to

10    any conditions under which the nonspecific signal of the background is essentially absent.

The phrase "hue angle (8) according to the L*a*b color system chromaticity diagram", as used throughout the present specification, refers to the hue angle (8)

15 standardized by the 1976 Commission internationale de l'eclairage (CIE) and adopted in Japan as JIS8729, where 0° is the red direction, 90° is the yellow direction, 180° is the green direction and 270° is the blue direction. Flower color can be represented by a combination of this

20 hue angle and RHS (Royal Horticultural Society) color chart data.

Transfer of flavone synthesis gene, flavonoid 3',5' - hydroxylase gene and anthocyanin methyltransferase gene

The  gene  for  flavone  synthase  II  derived  from

25    perilla was transferred into rose by a known procedure, together with the pansy F3'5'H gene. As a result, no flavones were detected in roses into which the perilla

flavone  synthase  II  gene  had  been  transferred,   indicating

that  the  gene  does  not  function  in  rose.    On  the  other

30    hand, flavone was detected in roses into which torenia or snapdragon flavone synthase II genes had been transferred, indicating that the flavone synthesis genes do function in rose.

Flavone-accumulating  roses  not  found  in  the  prior

35    art were thus created. The flavone content (%) of the total flavonoids may be 1% or greater, preferably 5% or
greater, more preferably 10% or greater and most preferably 30% or greater. Roses accumulating both anthocyanins and flavones have relatively bluer colors compared to roses containing only the same anthocyanins,

5    thus suggesting that flavone accumulation contributes to the new trait of blueness.

In  addition,   it  was  found  that  when  an  anthocyanin

with  a  methylated  B  ring   (anthocyanins  including

malvidin)    and  a  flavone  are  copresent,   a  higher  copigment

10    effect is exhibited than when a delphinidin-containing anthocyanin and a flavone are copresent, and that by transferring a methyltransferase gene for the B ring in addition to the flavone synthase II gene and pansy F3'5'H

gene,    it  is  possible  to  accumulate  methylated

15    delphinidin-type anthocyanins and flavones in rose petals, thus resulting in a bluer color of the rose petals.

By  a  hybridization  test  it  was  also  found  that  the

trait of accumulating both delphinidin-type anthocyanins 20 and flavones is transmitted to progeny.

These findings indicate that in plants that do not accumulate flavones or do not accumulate methylated anthocyanins such as malvidins in the petals, a bluer color shade of the petals can be produced by causing

25    these compounds to be accumulated simultaneously. For this purpose it is preferable for the host to be a plant that normally does not accumulate flavones or malvidin, such as a rose.

The  enzymes  associated  with  the  invention  are

30    typically enzymes having specific amino acid sequences listed in the Sequence Listing. However, it is well

known that desired enzyme activity can be maintained not only with the natural amino acid sequence of an enzyme, but also with the same amino acid sequence having

35    modifications in regions other than the regions associated with the enzyme activity. Consequently, the enzymes of the invention include proteins having the

amino acid sequences specified by the SEQ ID NOs which are modified by an addition or deletion of one or several amino acids and/or by substitution of one or several amino acids with other amino acids, and still maintaining

5    the original enzyme activities, and also proteins having amino acid sequences with at least 90% sequence identity to the specific amino acid sequences specified by the SEQ ID NOs, and maintaining the original enzyme activities.

It  is  known  that  for  any  gene  coding  for  a  certain

10    enzyme, there is a high probability that nucleic acid that hybridizes with the gene under highly stringent conditions will code for an enzyme having the same activity as that enzyme. Thus, enzymes encoded by nucleic acids that hybridize with nucleic acids having

15    the nucleotide sequences specified by the SEQ ID NOs under highly stringent conditions, and having the desired enzyme activities, are also included as enzymes according to the invention.

The  following  genes  may  therefore  be  mentioned  as

20    enzyme  genes  within  the  scope  of  the  invention.

(A)    Snapdragon (Antirrhinum majus) flavone synthase gene A gene coding for:

(1)    flavone  synthase  having  the  amino  acid  sequence

listed  as  SEQ  ID  NO:   2,

25 (2) flavone synthase having the amino acid sequence listed as SEQ ID NO: 2 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino acids,


30 (3) flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 2, or

(4) flavone synthase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide

35    sequence of SEQ ID NO: 1 under highly stringent conditions.

(B)    Torenia   (Torenia  hybrida)   flavone  synthase  gene

A  gene  coding  for:

(1)    flavone synthase having the amino acid sequence listed as SEQ ID NO: 4,

(2)    flavone  synthase  having  the  amino  acid  sequence

5    listed as SEQ ID NO: 4 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

acids,

(3)    flavone  synthase  having  an  amino  acid  sequence  with

10    at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 4, or

(4)    flavone  synthase  encoded  by  nucleic  acid  that

hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 3 under highly stringent

15    conditions.

(C)    Perilla (Perilla frutescens) flavone synthase gene A gene coding for:

(1)    flavone synthase having the amino acid sequence listed as SEQ ID NO: 6,

20 (2) flavone synthase having the amino acid sequence listed as SEQ ID NO: 6 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino acids,


25 (3) flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 6, or

(4) flavone synthase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide

30    sequence of SEQ ID NO: 5 under highly stringent conditions.

(D)    Pansy (Viola x wittrockiana) 3',5'-hydroxylase gene A gene coding for:

(1)    3',5'-hydroxylase  having  the  amino  acid  sequence

35    listed  as  SEQ  ID  NO:   8,

(2)    3',5'-hydroxylase having the amino acid sequence listed as SEQ ID NO: 8 modified by an addition or

deletion of one or several amino acids and/or substitution of one or several amino acids by other amino acids,

(3)    3' ,5'-hydroxylase  having  an  amino  acid  sequence  with

5    at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 8, or

(4)    3',5'-hydroxylase  encoded  by  nucleic  acid  that

hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 7 under highly stringent

10    conditions.

(E)    Torenia (Torenia hybrida) methyltransferase gene A gene coding for:

(1)    methyltransferase  having  the  amino  acid  sequence

listed  as  SEQ  ID  NO:   10,

15 (2) methyltransferase having the amino acid sequen~e listed as SEQ ID NO: 10 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino acids,


20 (3) methyltransferase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 10, or

(4) methyltransferase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide

25    sequence of SEQ ID NO: 9 under highly stringent conditions.


Examples

The  present  invention  will  now  be  explained  in

30    greater detail by the following examples. However, these examples are merely for the purpose of illustration of

the invention and are not intended to restrict the scope of the invention in any way.

Example 1: Simulation of flavone copigment effect with 35 anthocyanins

Anthocyanins were prepared first for simulation of the flavone copigment effect with anthocyanins. Cyanin

was extracted and purified from petals of the rose variety "Rote Rose" (rose cv. "Rote Rose"). Delphin was obtained by alkali hydrolysis of the pigment extracted from petals of the verbena variety "Tapien Violet"

5 (verbena cv. "Tapien Violet" or verbena variety Sunmaref TP-V ("Tapien Violet") ("Tapien" is a Trade Mark registered in Japan)), followed by purification. Malvin and luteolin 7-0-glucoside were purchased from Funakoshi Corp.


10 The flavone (luteolin 7-0-glucoside) was added to each anthocyanin prepared in this manner, at 0, 1, 2 and 4 equivalent molar concentrations in a buffering solution at pH 5.0, and the absorption spectra were measured. The anthocyanins used were cyanin (cyanidin 3,5-diglucoside),

15    delphin (delphinidin 3,5-diglucoside) and malvin (malvidin 3,5-diglucoside). The anthocyanin

concentrations  for  cyanin,  delphin  and  malvin  were  1  mM.

As shown in Tables 1 and 2, addition of the flavone increased the absorbance of the anthocyanin aqueous

20    solutions and the degree of change (absorbance ratio) was greatest with malvin. The absorption maxima (Amax) were
also shifted toward the long wavelength end with addition of the flavone. The degree of change was greatest with malvin, and then with delphin. Upon evaluation of the

25    color shade value based on the L*a*b* color system, addition of the flavone was found to produce a bluer color shade and increased chroma. This effect was most notable with malvin. That is, it was demonstrated that

the  luteolin  7-0-glucoside  copigment  effect  was  exhibited

30    to  the  greatest  extent  with  malvin.

Table  1

Absorption maxima of anthocyanin aqueous solutions with flavone addition

(Amax:    units:  nm)

5

~tion 0 1 equiv 2 equiv 4 equiv Anthocyanin
Cyanin
522.5    540.5    546.0    545.0
(Cyanidin  3,5-diglucoside)
Delphin
526.0    564.0    569.0    569.5
(Delphinidin  3,5-diglucoside)
Malvin
528.5    568.5    570.5    572.5
(Malvidin  3,5-diglucoside)




Table  2

10    Absorbance  ratios  at  Amax  with  respect  to  no  flavone

addition

    Flavone    addition        0    1  cquiv    2  equiv    4   equiv   
Anthocyanin                       
-                               
Cyanin    3,5-diglucoside)---------    1.000    2.044    2.425    2.363   
(Cyanidin                       
Delphin            l.    000    2.917    4.248    4.798   
(Delphinidin  3,5-diglucoside)                       
                       
Malvin            1.    000    5.194    7.775    9.219   
(Malvidin    3,5-diglucoside)                       
                           


15

Example 2 (reference example): Transfer of pansy F3'5'H#40 gene and perilla flavone synthase gene into rose variety "Lavande"

The  perilla  flavone  synthase  gene-containing  plasmid

20    pYFS3 described in Japanese Unexamined Patent Publication No. 2000-279182 was digested with Xbai and then blunt ended and digested with BamHI to obtain an approximately 1.8 kb perilla flavone synthase gene fragment.

Separately,  pSPB906  described  in  W02005/017147  was

25    digested with Xhoi and then blunt ended and further digested with BamHI. The perilla flavone synthase gene fragment was inserted between the flush ends and the


BamHI cleavage site to obtain plasmid 906-pYFS3. Plasmid 906-pYES3 comprises the perilla flavone synthase gene between the El 2 35S promoter and 08 terminator (both described in W02005/017147).

5 A plasmid obtained by inserting a fragment of the pansy F3'5'H#40 gene, cut out from pCGP1961 described in W02005/017147 by partial digestion with BamHI and Xhoi, at the BamHI and Sali sites of pSPB176 reported by Ueyama et al. (Ueyama et al. Plant Science, 163, 253-263, 2002),

10    was designated as pSPB575. At the Asci site of this plasmid there was inserted an approximately 3.4 kb perilla flavone synthase gene expression cassette

obtained  by  digesting  the  aforementioned  plasmid  906-

pYFS3  with  Asci.    Of  the  obtained  plasmids,   the  vector

15    having the F3'5'H#40 gene expression cassette and the perilla flavone synthase expression cassette linked in the same direction was designated as pSPB1310. This

plasmid constitutively expresses the pansy F3'5'H#40 gene and the perilla flavone synthase gene in plants.

20 Plasmid pSPB1310 constructed in this manner was transferred into the mauve rose variety "Lavande", and 55 transformants were obtained. Delphinidin accumulation was confirmed in 49 of 50 pigment-analyzed transformants, with a maximum delphinidin content of 70% (average: 26%).

25    However, absolutely no flavones were detected, and it was therefore concluded that the perilla flavone synthase gene does not function in rose cells.

The analysis values for representative transformants are shown in Table 3 below.
                       
                                        Table    3                           
                                                                       
Plant        Del        Anthocyanidin    Flavonol            Flavone   
                        (mg/g)            (mg/g)            (mg/g)   
No.    (%)                                                   
                        Del    Cya    Pel    M        Q        K    Tri    Lut    Api    Total   
Control    0.0        0.000        0.078    0.000    0.000    0.451    0.078    0.000    0.000    0.000    0.000   
1    70.3    0.105        0.045    0.000    0.253    0.152    0.017    0.000    0.000    0.000    0.000   
2    67.1    0.098        0.048    0.000    0. 379    0. 291        0.026    0.000    0.000    0.000    0.000   
3        50.7    0.060        0.058    0.000    0.326    0.289    0.013    0.000    0.000    0.000    0.000   
4    60.6    0.050        0.033    0.000    0.216    0.188    0.007    0.000    0.000    0.000    0.000   
5    66.1    0.073        0.037    0.000    0.608    0.380    0.045    0.000    0.000    0.000    0.000   
6    67.7    0.055        0.026    0.000    0.536    0.319    0.039    0.000    0.000    0.000    0.000   
7        56.9    0.062        0.047    0.000    0.253    0.201    0.009    0.000    0.000    0.000    0.000   
8    52.5    0.109        0.099    0.000    0.307    0.438    0.034    0.000    0.000    0. 000    0.000   
9        50.4    0.073        0. 072    0.000    0.281    0. 362    0.013    0.000    0.000    0.000    0.000   
10    61.9    0.085        0.052    0.000    0.228    0.192    0.008    0.000    0.000    0.000    0.000   
Control:    Lavande  control

Del: Delphinidin, Cya: Cyanidin, Pel: Pelargonidin, M: Myricetin, Q: Quercetin, K: Kaempferol, Tri: Tricetin, Lut: Luteolin, Api: Apigenin
5    Del(%):   Proportion  of  delphinidin  in  total  anthocyanidins



Example 3: Transfer of pansy F3'5'H#40 gene and torenia flavone synthase gene into rose variety "Lavande"

10 A plasmid obtained by inserting the torenia flavone synthase gene reported by Akashi et al. (Plant Cell Physiol 40, 1182-1186, 1999) at the EcoRI and Xhoi sites of plasmid pBluescript II SK(-) was designated as pSPB426. After digestion of this plasmid with Kpni, it

15    was blunt ended and further digested with BamHI to obtain an approximately 1.7 kb torenia flavone synthase gene fragment. Separately, pSPB906 described in W02005/017147 was digested with Xhoi and then blunt ended and further digested with BamHI. The torenia flavone synthase gene

20    fragment was inserted between the blunt ends and the BamHI cleavage site to obtain plasmid 906-426.

A plasmid obtained by inserting a fragment of the pansy F3'5'H#40 gene, cut out from pCGP1961 described in

W02005/017147  by  partial  digestion  with  BamHI  and  Xhoi,

obtained by digesting the aforementioned plasmid 906-426 with Asci. Of the obtained plasmids, the vector having the F3'5'H#40 gene expression cassette and the torenia flavone synthase expression cassette linked in the same

5    direction was designated as pSPB1309. This plasmid constitutively expresses the pansy F3'5'H#40 gene and the torenia flavone synthase gene in plants.

Plasmid  pSPB1309  constructed  in  this  manner  was

transferred  into  the  mauve  rose  variety  nLavanden,   and  50

10    transformants were obtained. Delphinidin accumulation was confirmed in 36 of 38 pigment-analyzed transformants, with a maximum delphinidin content of 45% (average: 12%)

Also, novel accumulation of flavones (luteolin and apigenin) was confirmed in 35 transformants, due to the

15    action of the torenia flavone synthase gene. At maximum, the total amount of flavones was a high content of 1.68 mg per 1 g of fresh petal weight.

The analysis values for representative transformants are shown in Table 4 below.

20

Table  4

Plant    Del    Anthocyanidin        Flavonol        Flavone   
            (mg/g)                (mg/g)        (mg/g)   
No.    (%)                                   
                                                       
        Del        Cya    Pel    M        Q    K    Tri    r,ut    Api    Total   
                                                   
Control    0.0    0.000    0.078    0.000    0.000    0.451        0.078    0.000    0.000    0.000   0.000   
1    10.1   0.012    0    .104    0.000   0.000    0.489        0.010   0.000    0.086   0.000   0.086   
2    9.6   0.008    0    . 079    0.000    0.000        0.446        0. 048    0.000    0.089    0.000   0.089   

3    10.4   0.009   0.079   0.000   0.071   0.651   0.264   0.000   0.020   0.000   0.020

4    4 4. 9   0.031   0.038   0.000   0.000   0.359   0.027   0.000   1.684   0.000   1. 684

5    33.2   0. 014   0.027   0.000   0.000   0.203   0.009   0.000   1.171   0.009   1.180
6    37.3   0.013   0.021    0.000   0.000   0.121    0.012   0.000    0.997    0.007   l. 003
7    39.0    0. 013    0.021    0.000    0.000    0.000    0.029    0.000    1    .153   0.008    1.161
8    35.8    0.024   0.043    0.000    0.000    0.205    0.000    0.000    1    . 642    0.010    1.652
9    36.1    0.013    0.024    0.000    0.000    1.223   0.006    0.000    0. 785    0.000    0.785
10    32.2    0.010    0.020   0.000    0.000    0.171    0.027    0.000   0. 917    0.007    0.924

Control:    Lavande  control

Del: Delphinidin, Cya: Cyanidin, Pel: Pelargonidin, M: Myricetin, Q: Quercetin, K: Kaempferol, Tri: Tricetin, Lut: Luteolin, Api: Apigenin
25    Del(%):   Proportion  of  delphinidin  in  total  anthocyanidins



Example  4:  Transfer  of  pansy  F3'5'H#40  gene  and  torenia

flavone    synthase  gene  into  rose  variety  nwKS124n
 
Plasmid pSPB1309 described in Example 3 was transferred into the salmon-pink rose variety "WKS124", and 40 transformants were obtained. Delphinidin accumulation was confirmed in 26 of 27 pigment-analyzed

5    transformants, with a maximum delphinidin content of 96% (average: 81%). Also, novel accumulation of flavones

(tricetin, luteolin and apigenin) was confirmed in 26 transformants, due to the action of the torenia flavone synthase gene. At maximum, the total amount of flavones

10    was a high content of 4.41 mg per 1 g of fresh petal weight.

The analysis values for representative transformants are shown in Table 5 below.

15                            Table  5                                                   
                                                                                                   
    Plant    Del    Anthocyanidin    Flavonol                    Flavone   
                (mg/g)                (mg/g)                            (mg/g)           
    No.    (%)                                                               
                Del    Cya    Pel    M        Q        K    Tri    Lut    Ap_i_    Total   
    Control    0.    0    0.000    0.006    0. 073    0.000        0. 076            3.312    0.000        0.000        0.000    0.000   
    1    84.8    0.326    0.045    0.014    0. 42'/        0.026            0. 797    0.941        0.122        0.394    1. 456   
    2    86    .6    0.567    0.084    0.003    0.806        0.096    0.218    2.148        1. 863            0.395    4.406   
    3    82.4    0.191    0.029    0. 011    0.000        0.139    0.626    1. 095        0.055        0.838    1. 988   
    4    83.4    0.448    0.083    0.007    0.000        0.037    0.434    1.157        0.131        0.406    1. 774   
    5    80    .1    0.340    0. 072    0.012    0.185        0. 064    0.735    0. 872        0.111        0.401    1. 384   
    6    83    .5    0.362    0. 065    0.007    0.000        0.090    0. 676    1. 642        0.229        0.777    2.647   
    7    88    .5    0.895    0.111    0.006    0.000        0.095    0.288    1.501        0.113        0.046    1.660   
    8    87    .3    0.862    0.123    0.003    0.275        0.092    0.200    1.286        0.127        0.082    1. 495   
    9    89    .6    0.252    0.029    0.001    0.126        0.049    0. 097    2.558        0.332        0.295    3.184   
    10    81    .3    0.101    0.022    0.001    0.065        0.031    0.146    1.822        0.215        0.405    2.442   
Control:    WKS124  control

Del: Delphinidin, Cya: Cyanidin, Pel: Pelargonidin, M: Myricetin, Q: Quercetin, K: Kaempferol, Tri: Tricetin, Lut: Luteolin, Api: Apigenin Del(%): Proportion of delphinidin in total anthocyanidins

20

Example 5: Transfer of pansy F3'5'H#40 gene, torenia flavone synthase gene and torenia anthocyanin methyltransferase gene into rose variety "WKS124"

25 Plasmid pSPB1309 described in Example 3 was treated with Paci for cleavage at the Paci site present near the linkage point between the torenia flavone synthase expression cassette and the pansy F3'5'H#40 gene expression cassette (m:ore specifically, located near the

3'-end of the 08 terminator of the flavone synthase expression cassette) and at the Paci site in the vector multicloning site, to cut out the pansy F3'5'H*40 gene expression cassette.

5 Separately, the binary vector pSPB1530 having the torenia methyltransferase gene expression cassette, described in W02003-062428, was cut with Paci and the aforementioned pansy F3'5'H*40 expression cassette was inserted therein in the same direction as the

10    methyltransferase gene expression cassette. This plasmid was designated as TMT-BP40.

Separately, plasmid pSPB1309 was cleaved with Asci to cut out the torenia flavone synthase expression

cassette.    This  was  inserted  into  the  Asci  site  of  TMT-

15    BP40 in the same direction as the previous expression cassettes, and the obtained plasmid was designated as pSFL535. This plasmid constitutively expresses the pansy F3'5'H*40 gene, the torenia methyltransferase gene and the torenia flavone synthase gene in plants.

20 Plasmid pSFL535 obtained in this manner was transferred into the salmon-pink rose variety "WKS124", and 173 transformants were obtained. Accumulation of malvidin (an anthocyanidin that has been methyiated at the 3' and 5' positions of delphinidin) was confirmed in

25    88 of 98 anthocyanidin-analyzed transformants, and the presence of product indicated that the pansy F3'5'H#40

gene and torenia anthocyanin methyltransferase gene were functioning in the rose petals. The malvidin content was a maximum of 84% (average: 50%).

30 Also; novel accumulation of flavones (tricetin, luteolin and apigenin) was confirmed in 77 transformants, due to the action of the torenia flavone synthase gene. At maximum, the total amount of flavones was a high content of 4.58 mg per 1 g of fresh petal weight.

35    Methylated  tricetin  was  detected  in  51  transformants.

The analysis values for representative transformants are shown in Table 6 below.

                                        Table    6                                                       
                                                                                           
    Plant    Del*    Mal            Anthocyanidins        Flavonols                Flavones           
            (%)                (mg/g)                    (mg/g)                    (mg/g)           
    No.    (%)                                                                           
                                                                                                   
                    Del    Cya        Pet    Pel    Peo    Mal    M    Q    K    Tri    Lut    Api        Total   
    Control    0.0        0.0    0.000    0.006    0.000  0.073    0.000    0.000    0.000  0.076  3.312    0.000        0.000  0.000   0.00   
1    97.8        65.0    0.121    0.005    0.079  0.000    0.009    0.397    0.331        0.000  0.000    2.273  0. 623  0.207  3.103   
2    96.9        81.3    0.048    0.005    0.048  0.000    0.014    0.500    0.231        0.000  0.000    3.699  0.762  0.116  4.577   
3    96. 4        83.8    0.014    0.003    0.024  0.000    0.008    0.258    0.209        0.009  0.510    l . 334  0.343  0.538  2.215   
4    87.4        77.9    0.008    0. 026    0.017  0.000    0.007    0.208    0.020        0.000  0.000    3.651    0.451        0.087        4.188   
5    93.2        79.9    0. 011    0.010  0.019  0.000  0.005    0.182  0. 062        0.000  0.000    3. 011    0.278        0.000        3.289   
6    93.2        61.2    0.160    0.014  0.113  0.002  0.042    0.521  0.279        0.000  0.405    l .    329  0.448   0.616  2.393   
7    90.9    63.5    0. 071    0.010  0.048  0.002  0.028    0.275  0.102        0.000  0.145    0. 765    0.299  0.403  l . 468   
8    95.1    64.7   0.165    0.012  0.121  0.002  0.033    0.610  0.280  0.000  0.116    l . 700  0.503  0.465        2.667   
9    8 6. 7        67.5   0.031    0.006  0.033  0.008  0.030    0.225  0. 071  0.000  0. 579    l . 217  0.186        0.980  2.383   
10    93.1    72.7   0.070    0.008   0,067  0.002  0.036    0.486    0.12 6        0.000    0.176    l . 858  0.459  0.545  2.861   
11    85.2        60.9   0.112    0.064  0.065  0.003  0.041    0.443  0.188   0.000  0.221    l . 4 78   0.397  0.530  2.405   
12    93.2    67.1   0.099    0.009  0.075  0.001  0.036    0.447  0.053  0.000  0.023    1.472        0. 2 97        0.058            l . 826   
13    89.2        64.3   0. 072    0.015  0.070  0.002  0.045    0.367  0.108   0.000  0.064  l . 4 73  0.310  0.108  l . 891   
14    90.2        63.3   0.082    0.016  0.080  0.003  0.040    0.383  0.070  0.344  0.094  l . 348    0.308  0.148  l . 803   
    15    87.8        64.4   0.035    0. 011  0.036  0.001  0.025    0.196  0.150  0.000  0.099  l . 863  0.358  0.075  2. 296   
16    92.0    70.7   0.061    0.009  0.055  0.002  0.033    0.383  0.113  0.000  0.067  2.389  0.421  0.237    3.046   

17    91.1    65.2   0.140  0.019  0.117  0.003  0.066  0. 64 8  0.313  0.000  0.191  2.727  0.565  0.133  3.425

18    90.0    63.8   0.056  0.010  0.044  0.001  0.028  0.245  0.161  0.000  0.139  0. 963  0.212  0.056  l . 231

19    89.3    68.3   0.065  0.013  0.067  0.004  0.051  0.430  0.076  0.000  0.042  2.438   0.353  0.134  2.924

20    89.1    63.8   0.060  0.015  0.049  0.002  0.030  0.277  0.224  0.041  0.208  l . 484  0.324  0.215  2.022

Control:    WKS124  control

Del:    Delphinidin,  Cya:  Cyanidin,  Pet:   Petunidin,   Pel:   Pelargonidin,   Peo:  Peonidin,  Mal:  Malvidin,

M:    Myricetin,  Q:  Quercetin,  K:  Kaempferol,   Tri:  Tricetin,  Lut:  Luteolin,  Api:  Apigenin

Del(%):    Proportion  of  delphinidinic  pigments   (delphinidin,  petunidin,  malvidin)   in  total  anthocyanins,

Mal(%):    Proportion  of  malvidin  in  total  anthocyanidins

Example 6: Transfer of pansy F3'5'H#40 gene, torenia flavone synthase gene and torenia anthocyanin methyltransferase gene into rose variety "Lavande"

Plasmid  pSFL535  described  in  Example  5  was

5    transferred into the mauve rose variety "Lavande", and 130 transformants were obtained. Accumulation of

malvidin (an anthocyanidin that has been methylated at the 3' and 5' positions of delphinidin) was confirmed in 37 of 118 anthocyanidin-analyzed transformants, and the

10    presence of product indicated that the pansy F3'5'H#40 gene and torenia anthocyanin methyltransferase gene were functioning in the rose petals. The malvidin content was a maximum of 55.6% (average: 20.5%).

Also,    novel  accumulation  of  flavones   (tricetin,

15    luteolin and apigenin) was confirmed in 78 transformants, due to the action of the torenia flavone synthase gene. At maximum, the total amount of flavones was a high content of 5.11 mg per 1 g of fresh petal weight. In

addition, methylated tricetin or luteolin was detected in 20 20 of the flavone-producing transformants.

The analysis values for representative transformants are shown in Table 7 below.
                                            Table    7                                       
                                                                               
    Plant    Del    Mal            Anthocyanidins    (mg/g)        Flavonols    (mg/g)        Flavones(mg/g)   
                                                                                   
    No.    (%)    (%)        Del    Cya        Pet    Pel    Pea    Mal    M    Q    K    Tri    LutApi    Total   
                                                                       
                                                                                       
                                                                                       
    Control    0%    0%    0.000    0.109  0.000    0.000  0.000    0.000    0.000        1. 020    0.195  0.000  0.000  0.000  0.000   
    (Lavande)                                           
                                                                                       
    1    20.7%    3.3%    0.012    0.065  0.002    0.001  0.002    0.003    0.097        0.272    0.015  0.289  0.035  0.000  0.324   
    2    45.9%    31.8%    0.003    0.006  0.001    0.000  0.007    0.007    0.024        0.076    0.000  1. 062  0.212  0.009  1. 283   
    3    74.8%    46.5%    0.063  0.023  0.010  0.000  0.041    0.119    0.458        0.285    0.049  0.204  0.022  0.000  0.226   
    4    71. 4%    51.6%    0.018  0.010  0.005  0.000  0.022    0.058    0.292        0.153    0.011  0.139  0.015  0.000  0.153   
    5    70.5%    32.1%    0    .025  0.012  0.007  0.000  0.012    0.027    0.510        0.192    0.033  0.000  0.026  0.000  0.026   
    6    28.9%    4.4%    0    .031    0. 096    0.005  0.000  0.005    0.006    0.268        0.619    0.037  0.262  0.038  0.009  0.310   
7    84.4%    53.4%    0    .036  0.008  0.014  0.000  0.017    0.086    0. 811        0.168    0.000  1. 054  0.086  0.000    1.139   
8    79.8%    53.2%    0    .032  0. 011  0.012  0.000  0.022    0.087    0.316  0.107    0.004  0. 863  0.037  0.000  0.900   
    9    83.3%    55.6%    0.038  0.012  0.012  0.006  0.012    0.100  0.593  0.013    0.000  4.885  0.223  0.000  5 .. 108   
    10    63.0%    33.6%    0    .003  0.002  0.000  0.001  0.001    0.003  0.032  0.000    0.000  3.992  0.219  0.003  4.214   
    11    88.1%    45.8%    0.027  0.004  0.006  0.000  0.005    0.036  0.285  0.060        0.009  2.779  0.077  0.000  2.855   
                               
                               
    12    86.2%    43.4%    0.041  0.009  0. 011  0.000  0.008    0.053  0.412  0.103    0.020  4.243  0.119  0.000  4.363   
                                                                                       
13    73.6%  38.0%  0.019  0.009  0.004  0.000  0.009  0.025  0.227  0.049  0.000  3.794  0.000  0.000  3.794

14    89.3%  49.7%  0.035  0.005  0.007  0.000  0.006  0.052  0.217  0.030  0.000  4.936  0.139  0.000  5.075

15    65.1% 31.1% 0.010 0.007 0.005 0.000 0.007 0.013 0.155 0.050 0.000 3.184 0.128 0.000 3.312 Control: Lavande control

Del:    Delphinidin,   Cya:    Cyanidin,   Pet:    Petunidin,   Pel:    Pelargonidin:   Pea:    Peonidin,  Mal:    Malvidin,

M:    Myricetin,  Q: Quercetin:   K: Kaempferol,   Tri: Tricetin,   Lut: Luteolin,  Api: Apigenin

Del(%):    Proportion  of  delphinidinic  pigments   (delphinidin,  petunidin,  malvidin)   in  total  anthocyanins


Example 7: Transfer of pansy F3'5'H#40 gene, torenia flavone synthase gene and torenia anthocyanin methyltransferase gene into rose variety "WKS82"

Plasmid  pSFL535  described  in  Example  5  was

5    transferred into the mauve rose variety "WKS82", and 250 transformants were obtained. Accumulation of malvidin

(an anthocyanidin that has been methylated at the 3' and 5' positions of delphinidin) was confirmed in 110 of 232 anthocyanidin-analyzed transformants, and the presence of

10    product indicated that the pansy F3'5'H#40 gene and torenia anthocyanin methyltransferase gene were functioning in the rose petals. The malvidin content was a maximum of 65.2% (average: 19.7%).

Also,   novel  accumulation  of  flavones   (tricetin,

15    luteolin and apigenin) was confirmed in 125 transformants, due to the action of the torenia flavone synthase gene. At maximum, the total amount of flavones was a high content of 4.71 mg per 1 g of fresh petal

weight. In addition, methylated tricetin or luteolin was 20 detected in 80 of the flavone-producing transformants.

The analysis values for representative transformants are shown in Table 8 below.

                                        Table    8                       
                                                       
    Plant    Del    Mal        Anthocyanidins   (mg/g)        Flavonols    (mg/g)        Flavones(mg/g)   
                                                               
    No.    (%)    (%)        Del    cya    Pet    Pel    Peo    Mal    M    Q    K    Tri    LutApiTotal   
                                                               
                                                                   
    Control    0%    0%        0.000    0.124  0.000  0.000  0.000  0.000  0.000  1.598    0.081  0.000  0.000  0.000  0.000   
    (WKS82)                           
                                                                   
    1    57.5%    46.1%        0.003    0.006  0.003  0.000  0.018  0.026  0 .. 494  0.750    0.064  0.764  0.000  0.000  0.764   
    2    70.5%    51.0%        0.007    0.005  0.004  0.000    0 0     011    0.028  0.564  0.384    0.055  2.977  0.199  0.000  3.176   
    3    82.1%    65.2%        0.006  0.004  0.005  0.000  0.008  0.042  0.800    0.536    0.115  0.534  0.000  0.000  0.534   
4    75.3%  57.5%        0.004  0.003  0.003  0.000  0.008  0.024  0.387    0.288    0.074  1. 808  0.160  0.000  1.968   
5    55.2%    37.6%        0.005  0.009  0.004  0.000  0.015  0.020  1.054    0.806    0.038  0.114  0.000  0.000  0.114   
    6    48.8%    37.8%  0.004  0.006  0.002  0.000  0.021  0.020  0.700    1. 319    0.148  0.034  0.000  0.000  0.034   
    7    78.4%    62.8%        0.007    0.003  0.004  0.000    0 0     011    0.042  0.577    0.266    0.015  0.302  0.022  0.000  0.324   
    8    54.6%    39.1%        0.006  0.009  0.003  0.000  0.018    0.023    0. 571    0.774    0.045    0.172  0.028  0.000  0.200   
9    73.5%    57.3%        0.009  0.004  0.004  0.000  0.016    0.044    0.866    0 0     511    0.031    0.104  0.000  0.000  0.104   
10    75.9%    57.5%        0.005  0.002  0.002  0.000  0.007  0.022  0.882  0.498    0.151  0.038  0.000  0.000  0.038   
11    69.3%    52.9%        0.007  0.006  0.005  0.000  0.016  0.038  0.825  0. 411    0.029  0.095  0.000  0.000  0.095   
12    71.4%    50.2%        0.013  0.007  0.006  0.000  0.020  0.046  0.721  0.459    0.022  0.075  0.004  0.000  0.080   
13    59.8%    42.2%        0.016  0.014  0.009  0.000  0.044  0.062  1. 540  1. 415    0.202  0.193  0.000  0.000  0.095   
14    67.9%  50.9%  0.006  0.006  0.006  0.000  0.017  0.036  0.829  0.704  0.125  0.000  0.000  0.000  0.200

15    34.4%  13.0%  0.006  0.014  0.003  0.000  0 0     013  0.006  0.230  1.109  0.000  4.155  0.551  0.006  4. 711

control:    WKS82  control

Del:    Delphinidin,  Cya:    Cyanidin,   Pet:    Petunidin,   Pel:    Pelargonidin:   Peo:    Peonidin,  Mal:    Malvidin,

M:    Myricetin,   Q: Quercetin:   K: Kaempferol,   Tri: Tricetin,   Lut: Luteolin,  Api: Apigenin

Del(%):    Proportion  of  delphinidinic  pigments   (delphinidin,  petunidin,  malvidin)   in  total  anthocyanins

Mal(%):    Proportion  of  malvidin  in  total  anthocyanidins
 
Example 8: Transfer of pansy F3'5'H#40 gene, torenia flavone synthase gene and torenia anthocyanin methyltransferase gene into rose variety "WKS110"

Plasmid  pSFL535  described  in  Example  5  was

5    transferred into the mauve rose variety "WKS140", and 74 transformants were obtained. Accumulation of malvidin

(an anthocyanidin that has been methylated at the 3' and 5' positions of delphinidin) was confirmed in 20 of 74 anthocyanidin-analyzed transformants, and the presence of

10    product indicated that the pansy F3'5'H#40 gene and torenia anthocyanin methyltransferase gene were

functioning in the rose petals. The malvidin content was a maximum of 51.3% (average: 33.5%).

Also,    novel  accumulation  of  flavones   (tricetin,

15    luteolin and apigenin) was confirmed in 29 transformants, due to the action of the torenia flavone synthase gene.

At maximum, the total amount of flavones was a high content of 3.04 mg per 1 g of fresh petal weight. In addition, methylated tricetin or luteolin was detected in

20    20  of  the  flavone-producing  transformants.

The analysis values for representative transformants are shown in Table 9 below.

                                    Table    9                                               
                                                                               
    Plant    Del    Mal        Anthocyanidins   (mg/g)        Flavonols    (mg/g)    Flavones(mg/g)   
                                                                                       
    No.    (%)    (%)    Del    Cya    Pet    Pel    Peo    Mal    M    Q        K    Tri    Lut        Api    Total   
                                                                           
                                                                                       
    Control    0%    0%    0.000    0.075  0.000    0.000    0.000    0.000    0.000    2.412        0.271  0.000        0.000    0.000  0.000   
    (WKS140)                                                           
                                                                                       
1    62.0%    31.7%    0.025    0.020        0.015    0.000    0.030    0.042    0.655  1. 085        0.202  2.314        0.305    0.032  2.650   
2    67.3%    38.3%    0.013    0:009        0.009    0.000    0.015  0.029  0.491  0. 627  0.104  1.790        0.227  0.031  2.048   
3    79.6%    34.1%    0.025    0.008        0. 011    0.000    0.008  0.027  0.572    0.555  0.129  2.388        0.237    0.015  2.639   
    4    69.8%    38.9%    0.021    0.012        0. 011    0.000    0.019  0.040    0.589  0. 766    0.165  1. 941        0.282    0.014    2.237   
    5    80.4%    51.3%    0.013    0.005        0.009  0.000    0.010    0.038    0. 513  0.307        0.074    1. 392  0.166    0.018    1. 577   
    6    70.1%    35.8%    0.014  0.008        0.006  0.000    0.010    0.021  0.607    0.538  0.108  1. 297        0.177    0.019    1. 493   

7    67.2%  34.7%  0.020  0. 013  0.009  0.000  0.017  0.031  1.005  0. 717  0.127  1. 805  0.264  0.028  2.097

8    70.0%  36.2%  0.019  0.010  0.009  0.000  0.015  0.029  0.831  0.802  0.143  1.909  0.241  0.027  2.176

9    70.9% 37.9% 0.015 0.008 0.008 0.000 0.012 0.027 0.497 0.690 0.106 1. 841 0.265 0.032 2.138 10 69.9% 36.0% 0.018 0.010 0.009 0.000 0.015 0.030 0.544 0.663 0.143 2.102 0.236 0.017 2.355
11    57.4%    31.0%    0. 011    0. 011  0.008  0.000  0.019  0.022    0.386  0.892    0.129  2.088    0.271    0.012    2.372
12    62.9%    32.4%    0.016    0.014    0.010    0.000    0.018    0.028    0.351    0.846    0.114    2.274    0.281    0.009    2.565
13    62.1%    34.1%    0.021    0.018    0.014    0.000    0.030    0.042    0.887    0.789    0.177    1.855  0.389    0.018    2. 262
14    73.7%    37.5%    0.016    0.006    0.004    0.000    0.008    0.021    0.597    0.489    0.081    1. 664    0.158  0.000    1. 821
15    58.6%    28.3%    0.013    0.012    0.007    0.000    0.016    0.019    0.513    1.121    0.166    2.650    0.373    0.015    3.038
Control:    WKS140  control

Del:    Delphinidin,   Cya:    Cyanidin,   Pet:    Petunidin,   Pel:    Pelargonidin:   Peo:    Peonidin,  Mal:    Malvidin,

M:    Myricetin,  Q: Quercetin:  K: Kaempferol,   Tri: Tricetin,   Lut: Luteolin,  Api: Apigenin

Del(%):    Proportion  of  delphinidinic  pigments   (delphinidin,  petunidin,  malvidin)   in  total  anthocyanins

Mal(%):    Proportion  of  malvidin  in  total  anthocyanidins

Example 9: Propagation of flavone and malvidin synthesis ability to progeny - Hybridization between cultivated roses and gene recombinant roses containing transferred pansy F3'5'H#40 gene, torenia flavone synthase gene and

5    torenia  anthocyanin  methyltransferase  gene

In order to investigate the mode of inheritance to progeny for flavone synthesis ability in roses, cross-breeding was carried out using a malvidin- and flavone-producing rose created in Example 5 (plant No. 6 in Table

10    6) as the pollen parent. As the seed parent there was used the medium-sized cultivated rose "Medeo" (floribunda rose variety "Medeo").

Accumulation  of  malvidin  was  confirmed  in  7  of  the

10  pigment-analyzed  transformant  F1  hybrid  progeny  that

15    were obtained, and the presence of product indicated that the pansy F3'5'H#40 gene and torenia anthocyanin methyltransferase gene were functioning in the rose petals. The malvidin content was a maximum of 68.2% (average: 46.6%).


20 On the other hand, novel accumulation of flavones (tricetin, luteolin and apigenin) was confirmed in 8 transformant progeny, due to the action of the torenia flavone synthase gene. At maximum, the total amount of flavones was an extremely high content of 7.35 mg per 1 g

25    of fresh petal weight. In addition, methylated tricetin or luteolin was detected in 6 of the flavone-producing transformant progeny.

The analysis values for representative transformant progeny are shown in Table 10 below.

                                    Table    10                                                       
                                                                                                       
Plant        Del    Mal        Anthocyanidins    (rng/g)        Flavonols    (rng/g)        Flavones (rng/g)       
                                                                                                   
No.        (%)    (%)                                                                                           
                    Del    Cya    Pet    Pel        Peo    Mal    M    Q    K    Tri        Lut    Api    Total   
                                                                               
                                                                                                       
Pollen                                                                                                       
parent        93.2%        61.2%    0.160    0.014    0.113    0.002    0.042        0.521    0.279    0.000    0.405    1.329    0.448    0. 616        2.393   
(Example    5                                                                           
                                                                                                       
Plant  No.6)                                                                                                   
Seed  parent    0%    0%        0.000    0.004    0.000    0.004    0.000        0.000    0.000    0.028    2.323    0.000    0.000    0.000        0.000   
(var.  Medeo)                                                                           
                                                                                                   
1        0.0%    0.0%        0.000    0.015    0.000    0.122    0.000        0.000    0.000    0.000    4.318    0.000    0.000    0.000        0.000   
                                                                               
2        82.6%    49.1%        0.165    0.034    0.085    0.005    0.090        0.367    0. 311    0.039    0.118    1. 596    0.064    0.006        1.666   
3        0.0%    0.0%        0.000    0.001    0.000    0.004    0.000        0.000    0.000    0.000    2.391    0.000    0.000    0.000        0.000   
4        80.1%    50.5%        0.073    0.028    0.064    0.00    0.054        0.233    0.210    0.048                0. 429    0.000    0.000    0.032        0.032   
                                                                                           
5        94.4%    52.8%        0.003    0.001    0.003    0.000    0.000        0.009    0.408    0.069    0.668    2.024    0.152    0.222        2.398   
6        81.8%    34.4%        0.056    0.015    0.034    0.002    0.017        0.065    0.076    0.033    0.202    3.860    0.123    0.039        4.023   
                                                                                           
7        48.2%    0.0%        0.011    0.002    0.011    0.001    0.021        0.000    0. 089    0.038        0.808    1.603    0.117    0.217        1. 937   
8        90.6%    35.5%        0.107    0.016    0. 071    0.002    0.013        0.114    0.080    0.010    0.100    6.497    0.351    0.504        7.351   
9        70.4%    35.8%        0.008    0.003    0.001    0.002    0.003        0.009    0.118    0.038        0.523    2.902    0.137    0.048        3.088   
                                                                                   
                                                                                   
10        91.2%    68.2%        0. 011    0.002    0.012    0.001    0.007        0.068    1.131    0.324        1. 077    1. 031        0.091    0.033        1.154   
                                                                                       
                                                                                       
Del:    Delphlnldln,   Cya:    Cyanldln,   Pet:    Petunldln,   Pel:    Pelargonldln:   Peo:    Peonldln,  Mal:    Malvldln,

M:    Myricetin,   Q: Quercetin:   K: Kaernpferol,   Tri: Tricetin,   Lut: Luteolin,  Api: Apigenin

Del(%):    Proportion  of  delphinidinic  pigments   (delphinidin,   petunidin,  rnalvidin)   in  total  anthocyanins

Mal(%):    Proportion  of  rnalvidin  in  total  anthocyanidins

Example 10: Propagation of flavone synthesis ability to progeny

Cross-breeding of rose variety "WKS124" containing transferred pansy F3'5'H#40 gene and torenia anthocyanin

5    methyltransferase gene, with rose variety "Lavande" containing transferred pansy F3'5'H#40 gene and torenia flavone synthase gene.

In  order  to  investigate  the  mode  of  inheritance  to

progeny  for  flavone  synthesis  ability  in  roses,   cross-

10    breeding was carried out using a flavone-producing line created in Example 3 (plant No. 4 in Table 4) as the pollen parent. As the seed parent there was used transformant WKS124/1532-12-l (described in

W02003/062428),    with  high  accumulation  of  malvidin  in  the

15    petals due to transfer of pSPB1532 into the rose variety WKS124 and the resulting actions of the pansy F3'5'H#40 gene and torenia anthocyanin methy1transferase gene.

Upon pigment analysis of 149 of the obtained transformant progeny, accumulation of flavones (tricetin,

20    luteolin, apigenin) was confirmed in 88 individuals. At maximum, the total amount of flavones was a high content of 4.09 mg per 1 g of fresh petal weight. Also, methylated tricetin was detected in 42 progeny, while methylated luteolin (chrysoeriol (3'-Met-Lut)) was

25    detected in 11. Accumulation of malvidin was confirmed in 129 of the 149 pigment-analyzed progeny. The malvidin content was a maximum of 79% (average: 36%).

The analysis values for representative transformant progeny are shown in Table 11 below.
                            Table    11                                   
                                                                       
    Plant    Del*    Mal            Anthocyanidins            Flavonols            Flavones           
                            (rng/g)                    (rng/g)            (rng/g)           
    No.    (%)    (%)                                                               
                                                                                   
                    Del    Cya    Pet    Pel        Peo    Mal    M    Q    K    Tri    Lut    Api    Total       
    Pollen    44.9    0.0        0.031    0.038    0.000    0.000    0.000    0.000   0.000   0.359   0.027   0.000   1. 684   0.000   1. 684       
    parent                                           
                                                                                   
    Seed    93.2    73.0        0.127    0.    011    0.112    0.003    0.066    0.863   0.365   0.093   0.348   0.000   0.000   0.000   0.000       
    parent                                               
                                                                                   
    1    92.1    69.1        0.032    0.005    0.030    0.000        0. 016    0.186   0.197   0.105   0.090   1.950   0.078   0.059   2.088       
    2    75.3    56.7        0.076    0.048    0.055    0.005    0.121    0.400   0.345   0.081   0.097   2.879   0.156   0.086   3.121       
    3    80.6    60.6        0.041   0.015   0.039    0.004    0.059    0.244   0.000   0.000   0.113   2. 986   0.193   0.000   3.179       
    4    82.4    65.8        0.005    0.002   0.006    0.000    0.009    0.043   0.000   0.131   0.084   2.036   0.066   0.000   2.103       
    5    68.8    56.7   0.010    0.006   0.012    0.013   0.036    0.101   0.000   0.093   0.179   1.740   0.224   0.000   1. 965       
    6    79.3    60.4        0.018    0.009   0.017    0.000   0.029    0.111   0.089   0.053   0.084   1. 956   0.093   0.029   2.078       
    7    86.1%    52.1   0.158    0.044   0.125    0.003   0.069    0.432   0.000   0.118   0.300   3.059   0.363   0.397   3.819       
    8    81.3    59.8   0.026    0.010   0.027    0. 011   0.025    0.149   0.000   0.247   0.226   1. 489   0.232   0.179   1. 900       
    9    79.2    59.2%  0.015   0.008   0.014   0.000   0.022    0.086   0.422   0.398   0.224    2.510    0. 726    0.094    3.330       
    10    82.5   66.8%  0.019   0.008   0.022   0.000   0.038   0.175   0.445   0.559   0.322   2.122   0.446   0.111   2. 678       
    11    73.3    59.3   0.036   0.025   0.037   0.000   0.112    0.306   0.121   0.130   0.000   0.596   0.565   0.066   1.227       
    12    94.0    76.2   0.018    0.002   0.018   0.000   0.010    0.154   0.840   0.426   0.445   3.655   0.306   0.124   4.086       
    13    82.8    62.8        0.009    0.004   0.010   0.000   0.012    0.059   0.394   0.400   0.278   1. 068   0.250   0.096   1. 414       
                                                                                   
14    82.7    58.8   0.119   0.048   0.122   0.011   0.115   0.592   0.327   0.089   0.074   1. 940   0.131   0.085   2.156

15    78.4 61.6 0.009 0.006 0.009 0.000 0.018 0.066 0. 313 0.582 0.370 1. 634 0.423 0.143 2.200 Del: Delphlnldln, Cya: Cyanldln, Pet: Petunldln, Pel: Pelargonldln, Peo: Peonldln, Mal: Malvldln,

M:    Myricetin,   Q: Quercetin,   K: Kaernpfero1,   Tri: Tricetin,  Lut: Luteolin,  Api: Apigenin

Del(%):    Proportion  of  de1phinidinic  pigments   (de1phinidin,   petunidin,  rnalvidin)   in  total  anthocyanins

Mal(%):    Proportion  of  rna1vidin  in  total  anthocyanidins


Example 11: Evaluation of flavone-containing rose flower color

The transformants created in Examples 4 and 5 (host: rose variety "WKS124") were divided into 5 groups: (1)

5    those accumulating delphinidin as the major pigment and containing no flavones, (2) those accumulating delphinidin as the major pigment and containing flavones,

(3) those highly accumulating malvidin as the major pigment and containing no flavones, (4) those highly

10    accumulating malvidin as the major pigment and containing flavones, and (5) host (accumulating pelargonidin as the major pigment), and the color shade of the petals were evaluated using a spectrocolorimeter (n = 10).

In  both  the  roses  with  delphinidin  as  the  major

15    pigment and the roses with malvidin as the major pigment, a shift in hue angle of the petals toward blue had occurred when flavones were copresent. This tendency was more pronounced in the roses with malvidin as the major pigment, and the reflection spectrum minimum (AMin) was

20    also shifted significantly toward the long wavelength end. These results confirmed that the petal color shade had changed to blue by the copresence of flavones. The results are shown in Table 12 below.
       
    Table    12           
                   
~es   Hue  angle    Reflection   
    spectrum  minimum   
Ge                (AMin)   
Host  No  gene  transfer   (WKS124                   
control)            31.14°    Average:    520iliil   
Pelargonidin  accumulated  as                   
        (=391.14°)    Maximum:    520nm   
main  pigment,  absolutely  no                   
                   
flavones    present                   
Ex.7   (1)   Pansy    F3',5'H                   
Delphinidin  highly  accumulated        Average:   349.03°    Average:    540nm   
as  main  pigment,  absolutely  no    Bluest  value:   344.68°    Maximum:    540nm   
flavones    present                   

(2)    Pansy  F3',5'H  +  torenia

FNS        Average:  343.64°    Average:    540nm   
Delphinidin  highly    accumulated               
        Bluest  value:  337.18°    Maximum:    540nm   
as  main   pigment 1    flavones               
                   
present                   

(3)    Pansy  F3',5'H  +  torenia  MT
Malvidin  highly  accumulated  as    Average:   341.78°    Average:    542run
main  pigment,   absolutely  no    Bluest  value:   336.82°    Maximum:    550run
flavones  present           

(4)    Pansy  F3',5'H  + torenia  MT

+    torenia  FNS Average:   334.45° Average:   551run

Malvidin highly accumulated as Bluest value: 329.84° Maximum: 560nm main pigment, flavones present

Hue    angle   (hue):   The  angle  displacement  for  the  color  tone  in  the

counter-clockwise  direction  from  the  a*   (red  direction)   axis  as  0°  in

5    the L*a*b* color system, for indication of the color position. An angle of 90° is the yellow direction, an angle of 180° is the green direction, an angle of 270° is the blue direction, and an angle of 0° (= 360°) is the red direction. In other words, a numerical value approaching 270° represents a bluer color tone.


10

Industrial  Applicability

According  to  the  invention  it  is  possible  by  genetic

modification  to  add  flavones  and  malvidin  to  roses,   as

15    popular flowering plants used for decoration, in order to alter rose flower color toward blue by a co-pigmentation effect. Roses with blue flower color are expected to be in high commercial demand as ornamental plants.

All  of  the  patent  documents  and  non-patent  technical

20    documents cited in the present specification are hereby incorporated by reference either individually or as a whole.

This  completes  the  explanation  of  the  invention,   but

the invention should be interpreted as encompassing any alterations or modifications such as do not deviate from the gist thereof, and the scope of the invention is not to be considered as based on the description in the

5    examples but rather as defined by the scope of the attached claims.

CLAIMS

A  rose  characterized  by  comprising  a  flavone

and  malvidin  added  by  a  genetic  modification  method.

2.    A  rose  according  to  claim  1,  which  comprises  a

5    flavone and malvidin by expression of pansy (Viola x wittrockiana) flavonoid 3',5'-hydroxylase and anthocyanin

methyl transferase.

3.    A  rose  according  to  claim  1  or  2,   which

comprises  malvidin,   a  flavone  and  delphinidin  by

10    expression of an anthocyanin methyltransferase gene, a flavone synthase gene and the pansy (Viola x wittrockiana) flavonoid 3',5'-hydroxylase gene.

4.    A  rose  according  to  any  one  of  claims  1  to  3,

wherein the flavone synthase gene is a flavone synthase 15 gene derived from the family Scrophulariaceae.

5. A rose according to claim 4, wherein the flavone synthase gene derived from the family Scrophulariaceae is a flavone synthase gene derived from snapdragon of the family Scrophulariaceae

20    (Scrophulariaceae,  Antirrhinum  majus).

6.    A rose according to claim 4, wherein the flavone synthase gene derived from the family Scrophulariaceae is a flavone synthase gene derived from

torenia  of  the  family  Scrophulariaceae   (Scrophulariaceae,

25    Torenia  hybrida) .

7. A rose according to claim 5, wherein the flavone synthase gene derived from snapdragon of the family Scrophulariaceae is a gene coding for

(1)    flavone  synthase  having  the  amino  acid

30    sequence  listed  as  SEQ  ID  NO:   2,

(2) flavone synthase having the amino acid sequence listed as SEQ ID NO: 2 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

35    acids,

(3) flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino

acid  sequence  listed  as  SEQ  ID  NO:   2,   or

(4) flavone synthase encoded by nucleic acid that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 1 under highly stringent

5    conditions.

B.    A rose according to claim 6, wherein the flavone synthase gene derived from torenia of the family Scrophulariaceae is a gene coding for

(1)    flavone  synthase  having  the  amino  acid

10    sequence  listed  as  SEQ  ID  NO:   4,

(2)    flavone  synthase  having  the  amino  acid

sequence listed as SEQ ID NO: 4 modified by an addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

15    acids,

(3) flavone synthase having an amino acid sequence with at least 90% sequence identity to the amino acid sequence listed as SEQ ID NO: 4, or

(4)    flavone  synthase  encoded  by  nucleic  acid

20    that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 3 under highly stringent

conditions.

9. A rose according to any one of claims 2 to 8, wherein the pansy flavonoid 3',5'-hydroxylase gene is a

25    gene  coding  for:

(1) flavonoid 3',5'-hydroxylase having the amino acid sequence listed as SEQ ID NO: 8,

(2) flavonoid 3',5'-hydroxylase having the amino acid sequence listed as SEQ ID NO: 8 modified by an

30    addition or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

acids,

(3) flavonoid 3',5'-hydroxylase having an amino acid sequence with at least 90% sequence identity to the

35    amino  acid  sequence  listed  as  SEQ  ID  NO:   8,   or

(4)    flavonoid  3',5'-hydroxylase  encoded  by

nucleic  acid  that  hybridizes  with  nucleic  acid  having  the


nucleotide sequence of SEQ ID NO: 7 under highly stringent conditions.

10. A rose according to any one of claims 2 to 9, wherein the anthocyanin methyltransferase gene is a gene

5    coding  for:

(1)    methyltransferase  having  the  amino  acid

sequence  listed  as  SEQ  ID  NO:   10,

(2)    methyltransferase  having  the  amino  acid

sequence  listed  as  SEQ  ID  NO:   10  modified  by  an  addition

10    or deletion of one or several amino acids and/or substitution of one or several amino acids by other amino

acids,

(3) methyltransferase having an amino acid sequence with at least 90% sequence identity to the amino

15    acid  sequence  listed  as  SEQ  ID  NO:   10,  or

(4)    methyltransferase  encoded  by  nucleic  acid

that hybridizes with nucleic acid having the nucleotide sequence of SEQ ID NO: 9 under highly stringent conditions.

20 11. A rose according to any one of claims 2 to 10 above, wherein the flower color is changed with respect to the host before transfer of the anthocyanin methyltransferase gene, flavone synthase gene and pansy 3',5'-hydroxylase gene.


25 12. A rose according to claim 11, wherein the change in flower color is a change toward blue.

13. A rose according to claim 11 or 12, wherein the change in flower color is a change such that the hue

angle (6) according to the L*a*b color system chromaticity 30 diagram approaches 270° which is the blue axis.
14. A rose according to claim 11, wherein the change in flower color is a change such that the minimum value of the reflection spectrum of the petal shifts toward the longer wavelength end.

35 15. A rose portion, descendant, tissue, vegetative body or cell having the same properties as a rose according to any one of claims 1 to 14.

16. A method for modifying the flower color of a rose by a co-pigmentation effect produced by adding a flavone and malvidin by a genetic modification technique.
17.    The  method  according  to  claim  16,  wherein  the

5    co-pigmentation effect is an effect of changing the flower color toward blue.


ABSTRACT

The invention provides a rose characterized by comprising a flavone and malvidin added by a genetic

5    modification method. The flavone and rnalvidin are typically produced by expression of a transferred flavone synthase gene, pansy flavonoid 3',5'-hydroxylase gene and anthocyanin methyltransferase gene. The flavone synthase

gene    is,   for  example  a  flavone  synthase  gene  of  the

10    family Scrophulariaceae, and specifically it may be the flavone synthase gene of snapdragon of the family

Scrophulariaceae, or the flavone synthase gene of torenia of the family Scrophulariaceae. The flavonoid 3' ,5' - hydroxylase gene is, for example, the pansy flavonoid

15    3',5'-hydroxylase gene. The anthocyanin methyltransferase gene is, for example, the methyltransferase gene of torenia of the family Scrophulariaceae.

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