# Patent

## Title
Method for producing rose with altered petal colors

## Abstract

A method for producing a rose characterized by artificially suppressing the rose endogenous metabolic pathway and expressing the pansy gene coding for flavonoid 3′,5′-hydroxylase.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a National Phase Patent Application of International Application Number PCT/JP2004/011958, filed on Aug. 13, 2004, which claims priority of Japanese Patent Application Number 2003-293121, filed on Aug. 13, 2003, and Japanese Patent Application Number 2004-192034, filed on Jun. 29, 2004.
INCORPORATION BY REFERENCE
The material in the text file entitled “10567931SEQLISTING.txt,” amended Feb. 3, 2011, and being 38,000 bytes in size, is herein incorporated by reference in its entirety.
TECHNICAL FIELD
The present invention relates to a new method for producing a rose with altered petal colors. More specifically, it relates to a method for producing a rose by artificially inhibiting the endogenous metabolic pathway of rose, and expressing the gene coding for pansy flavonoid 3′,5′-hydroxylase and the gene coding for dihydroflavonol reductase which reduces dihydromyricetin.
BACKGROUND ART
Flower petals perform the role of attracting pollinators such as insects and birds, which transport plant pollen, and therefore flower colors, shapes, patterns and odors have evolved in tandem with pollinators (Honda, T. et al., Gendai Kagaku, May, 25-32 (1998)). Probably as a result of this, it is rare for a single species of flower to exhibit several different colors, and for example, rose or carnation varieties exhibiting violet to blue colors do not exist, while iris or gentian varieties exhibiting bright red colors do not exist. Because flower color is the most important aspect of petals for purposes of appreciation as well, flowers of different colors have traditionally been bred by crossbreeding. The rose, known as the “queen of flowers” and having high commercial value, has also been crossbred throughout the world.
For example, the current yellow rose cultivar was created by crossbreeding of Rosa foetida, originating from western Asia, with a non-yellow rose variety. However, because flower color is determined by the genetic capacity of the plant, there has been a limit to the flower colors that can currently be produced in cross-bred strains whose available genetic sources are restricted (Tanaka et al. Plant Cell Physiol. 39, 1119-1126, 1998; Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999). Among these, the cultivation of blue roses has been thought impossible and has been considered the “holy grail” of colors (Oba, H., “Bara no Tanjo”, 1997, Chukoshinsho; Suzuki, M., “Shokubutsu Bio no Mahou: Aoi Bara mo Yume dewanakuhatta”, 1990, Kodansha Bluebacks; Saisho, H., “Aoi Bara”, 2001, Shogakkan).
Although “blue rose” varieties currently exist, these are actually pale violet roses. The first improved variety of “blue rose” by crossbreeding is said to have been the light-violet shaded grey-colored “Grey Pearl” created in 1945. The light-violet pink-colored “Staring Silver” was later created in 1957, and these varieties were crossed to produce several pale violet roses such as “Blue Moon” (1964) and “Madam Violet” (1981). These pale violet roses and other roses were then utilized in further breeding to create light-grey-colored roses such as “Seiryu” (1992) and “Blue Heaven” (2002), which were hailed as new types of “blue roses”.
However, these flower colors are not actually blue but merely greyish-dull pink, and despite many years of breeding efforts, there is still no example of a truly “blue” rose. In horticultural industry, the group of colors from violet to blue is generally considered “blue” according to the RHSCC (The Royal Horticultural Society Colour Chart). It is an aim of the present invention to create rose plants having flower colors falling within the “violet group”, “violet-blue” group and “blue group” according to the Royal Horticultural Society Colour Chart.
Flower colors derive mainly from the three compound groups of anthocyanins, carotenoids and betalains, but it is the anthocyanins, having the widest absorption wavelength range (from orange to blue), that are responsible for blue color. Anthocyanins belong to the flavonoid family and are biosynthesized by the metabolic pathway shown in FIG. 1. Anthocyanins are normally localized in the vacuoles of epithelial cells. The color shade of anthocyanins (i.e. flower color) depends largely on the structure of the anthocyanins, with more numerous hydroxyl groups on the B ring resulting in a bluer color. Hydroxylation of the B ring is catalyzed by flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Absence of F3′H and F3′5′H activity leads to synthesis of pelargonidin (orange to red colors), presence of F3′H activity leads to synthesis of cyanidin (red to rouge colors) and presence of F3′5′H activity leads to synthesis of delphinidin (violet color).
These anthocyanidins are modified with sugars and acyl groups to produce an assortment of anthocyanins. Generally speaking, a larger number of modifying aromatic acyl groups correlates to bluer anthocyanins. Anthocyanins also produce quite different colors depending on the vacuole pH and the copresent flavonols and flavones or metal ions (Saito, N., Tanpakushitsu Kakusan Kouso, 47 202-209, 2002; Broullard and Dangles, In the flavonoids: Advances in Research since 1986 (Ed. by Harborne) Capmann and Hall, London pp. 565-588; Tanaka et al. Plant Cell Physiol. 39 1119-1126, 1998; Mol et al., Trends in Plant Science 3, 212-217, 1998; Mol et al., Curr. Opinion Biotechnol. 10, 198-201 1999).
Rose flower petal anthocyanins are derivatives of pelargonidin, cyanidin and peonidin, whereas no delphinidin derivatives are known (Biolley and May, J. Experimental Botany, 44, 1725-1734 1993; Mikanagi Y., Saito N., Yokoi M. and Tatsuzawa F. (2000) Biochem. Systematics Ecol. 28:887-902). This is considered to be the main reason for the lack of blue roses. Existing roses have been created by crossbreeding of crossable related rose species (R. multiflora, R. chinensis, R. gigantean, R. moschata, R. gallica, R. whichuraiana, R. foetida, etc.).
The fact that no blue rose has been achieved in spite of repeated efforts at crossbreeding is attributed to the lack of delphinidin production ability by rose-related varieties. Production of delphinidin in rose petals would require expression of F3′5′H in the petals as mentioned above, but F3′5′H is believed to be non-expressed in the petals of rose and rose-related varieties. Thus, it is likely impossible to obtain a blue rose by accumulating delphinidin in the petals through crossbreeding. It is known that trace amounts of the blue pigment rosacyanin are found in rose petals and its chemical structure has been determined (Japanese Unexamined Patent Publication No. 2002-201372), but no reports are known regarding augmentation of rosacyanin to create a blue rose, and no findings have been published on the rosacyanin biosynthesis pathway or the relevant enzymes or genes.
Examples of blue or violet colors produced by biological organisms also include indigo plant-produced indigo (for example, Appl. Microbiol. Biotechnol. February 2003, 60(6):720-5) and microbially-produced violacein (J. Mol. Microbiol. Biotechnol. October 2000 2 (4):513-9; Org. Lett., Vol. 3, No. 13, 2001, 1981-1984), and their derivation from tryptophan and their biosynthetic pathways have been studied.
Blue pigments based on gardenia fruit-derived iridoid compounds (S. Fujikawa, Y. Fukui, K. Koga, T. Iwashita, H. Komura, K. Nomoto, (1987) Structure of genipocyanin G1, a spontaneous reaction product between genipin and glycine. Tetrahedron Lett. 28 (40), 4699-700; S. Fujikawa, Y. Fukui, K. Koga, J. Kumada, (1987), Brilliant skyblue pigment formation from gardenia fruits, J. Ferment. Technol. 65 (4), 419-24) and lichen-derived azulenes (Wako Pure Chemical Industries Co., Ltd.) are also known, but no reports are known of expressing these in plant flower petals to produce blue-colored flowers.
It has been expected that a blue rose could be created by transferring the F3′5′H gene expressed by other plants into rose and expressing it in rose petals (Saisho, H., “Aoi Bara”, 2001, Shogakkan). The F3′5′H gene has been obtained from several plants including petunia, gentian and Eustoma russellianum (Holton et al. Nature 366, 276-279, 1993; Tanaka et al. Plan Cell Physiol. 37, 711-716 1996; WO93/18155). There are also reports of transformed varieties of rose (for example, Firoozababy et al. Bio/Technology 12:609-613 (1994); U.S. Pat. No. 5,480,789; U.S. Pat. No. 5,792,927; EP 536,327 A1; US 20010007157 A1).
Actual transfer of the petunia F3′5′H gene into rose has also been reported (WO93/18155, WO94/28140).
However, it has not been possible to obtain a blue rose, and it is believed that obtaining a blue rose will require a modification which alters the metabolism of flower pigments suited for rose.
On the other hand, it has been confirmed that transfer of the F3′5′H gene into red carnation, which produces pelargonidin instead of delphinidin, leads to accumulation of both pelargonidin and delphinidin, but that the flower color is only altered to a slightly purplish red (WO94/28140). This result suggests that it is not possible to obtain a “blue” carnation simply by expression of F3′5′H, and that it is necessary to inhibit the metabolic pathway to endogenous synthesis of pelargonidin by carnation.
In order to avoid competition with the carnation endogenous metabolic pathway (reduction of dihydrokaempferol (DHK) by dihydroflavonol reductase (DFR)), a variety lacking DFR was selected from among white carnations. The F3′5′H gene and petunia DFR (which is known to efficiently reduce dihydromyricetin (DHM) without reducing DHK) gene were transferred into carnation. This resulted in one case of successfully obtaining a recombinant carnation with a delphinidin content of about 100% and a blue-violet flower color previously not found in carnation (Tanpakushitsu Kakusan Kouso, Vol. 47, No. 3, p 225, 2002). Thus, further modification was necessary to realize a blue carnation flower, in addition to accumulating delphinidin by expression of the F3′5′H gene.
DFR has already been cloned from several plants (petunia, tobacco, rose, Torenia, snapdragon, transvaal daisy, orchid, barley, corn, etc.) (Meyer et al., Nature 330, 677-678, 1987; Helariutta et al., Plant Mol. Biol. 22, 183-193 1993; Tanaka et al., Plant Cell Physiol. 36, 1023-1031; Johnson et al., Plant J. 19, 81-85, 1999). Substrate specificity of the DFR gene differs depending on the plant variety, and it is known that the petunia, tobacco and orchid DFR genes cannot reduce DHK, whereas the petunia DFR gene most efficiently reduces DHM among the dihydroflavonols (Forkmann et al., Z. Naturforsch. 42c, 1146-1148, 1987; Johnson et al. Plant J. 19, 81-85, 1999). Nevertheless, no cases have been reported for expression of these DFR genes in rose.
As a means of avoiding competition with the endogenous metabolic pathway or between the enzyme and the exogenous gene-derived enzyme such as F3′5′H, as mentioned above, the gene may be transferred into a variety lacking the gene. Also, it is known that expression of the target gene can be artificially inhibited by deletion methods involving homologous recombination of the target gene, but because of the low frequency of homologous recombination and the limited number of suitable plant varieties, this has not been implemented in practice (for example, Nat. Biotechnol. 2002, 20:1030-4).
Inhibition methods on the transcription level include the antisense method using antisense RNA transcripts for mRNA of the target gene (van der Krol et al., Nature 333, 866-869, 1988), the sense (cosuppression) method using transcripts of RNA equivalent to mRNA of the target gene (Napoli et al., Plant Cell 2, 279-289, 1990) and a method of using duplex RNA transcripts corresponding to mRNA of the target gene (RNAi method; Waterhouse et al., Pro. Natl. Acad. Sci. USA 95, 13959-13964, 1998).
Numerous successful examples of these three methods have been published. For rose, cosuppression of chalcone synthase (CHS) gene which is necessary for synthesis of anthocyanins was reported to successfully alter flower color from red to pink (Gutterson HortScience 30:964-966 1995), but this CHS suppression is incomplete and therefore it has not been possible to totally suppress anthocyanin synthesis to obtain a white flower stock.

    Patent document 1: Japanese Unexamined Patent Publication No. 2002-201372
    Patent document 2: WO93/18155
    Patent document 3: U.S. Pat. No. 5,480,789
    Patent document 4: U.S. Pat. No. 5,792,927
    Patent document 5: EP 536 327 A1
    Patent document 6: US 20010007157 A1
    Patent document 7: WO94/28140
    Non-patent document 1: Honda T. et al. Gendai Kagaku, May, 25-32 (1998)
    Non-patent document 2: Tanaka et al. Plant Cell Physiol. 39, 1119-1126, 1998
    Non-patent document 3: Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999
    Non-patent, document 4: Oba, H., “Bara no Tanjo”, 1997, Chukoshinsho
    Non-patent document Suzuki, M., “Shokubutsu Bio no Mahou: Aoi Bara mo Yume dewanakunatta”, 1990, Kodansha Bluebacks
    Non-patent document 6: Saisho, H., “Aoi Bara”, 2001, Shogakkan
    Non-patent document 7: Saito, N., Tanpakushitsu Kakusan Kouso, 47 202-209, 2002
    Non-patent document 8: Broullard et al. In the flavonoids: Advances in Research since 1986 (Ed by Harborne) Capmann and Hall, London pp 565-588
    Non-patent document 9: Tanaka et al. Plant Cell Physiol. 39 1119-1126, 1998
    Non-patent document 10: Mol et al, Trends in Plant Science 3, 212-217 1998
    Non-patent document 11: Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999
    Non-patent document 12: Biolley and May, J. Experimental Botany, 44, 1725-1734 1993
    Non-patent document 13: Mikanagi Y, et al. (2000) Biochem Systematics Ecol. 28:887-902
    Non-patent document 14: Appl. Microbiol. Biotechnol. 2003 February; 60(6):720-5
    Non-patent document 15: J. Mol. Microbiol. Biotechnol. 2000 October; 2 (4): 513-9
    Non-patent document 16: Org. Lett., Vol. 3, No. 13, 2001, 1981-1984
    Non-patent document 17: S. Fujikawa, et al. (1987) Tetrahedron Lett. 28 (40), 4699-700
    Non-patent document 18: S. Fujikawa, et al. (1987) J. Ferment. Technol. 65 (4), 419-24
    Non-patent document 19: Holton et al. Nature 366, 276-279, 1993
    Non-patent document 20: Tanaka et al. Plant Cell Physiol. 37, 711-716 1996
    Non-patent document 21: Firoozababy et al. Bio/Technology 12:609-613 (1994)
    Non-patent document 22: Tanpakushitsu Kakusan Kouso, Vol. 47, No. 3, p 225, 2002
    Non-patent document 23: Meyer et al. Nature 330, 677-678, 1987
    Non-patent document 24: Helariutta et al. Plant Mol. Biol. 22 183-193 1993
    Non-patent document 25: Tanaka et al. Plant Cell Physiol. 36, 1023-1031
    Non-patent document 26: Johnson et al. Plant J. 19, 81-85, 1999
    Non-patent document 27: Forkmann et al. Z. Naturforsch. 42c, 1146-1148, 1987
    Non-patent document 28: Nat Biotechnol 2002, 20:1030-4
    Non-patent document 29: van der Krol et al. Nature 333, 866-869, 1988
    Non-patent document 30: Napoli et al. Plant Cell 2, 279-289, 1990
    Non-patent document 31: Waterhouse et al. Pro. Natl. Acad. Sci. USA 95, 13959-13964 1998
    Non-patent document 32: Gutterson HortScience 30:964-966 1995


Non-patent document 33: Suzuki, S., “Bara, Hanazufu”, Shogakkann, p. 256-260, 1990
DISCLOSURE OF THE INVENTION
As mentioned above, rose flower colors have been successfully altered by transferring the F3′5′H gene into rose and expressing it in the petals. In carnation, the F3′5′H gene and petunia DFR gene have been expressed in DFR-deficient varieties to create blue-violet carnations. However, a “blue rose” has not yet been created. It is therefore an object of the present invention to provide a rose which blossoms with a blue flower.
The invention thus provides (1) a method for producing a rose characterized by artificially suppressing the rose endogenous metabolic pathway and expressing the pansy gene coding for flavonoid 3′,5′-hydroxylase.
The invention further provides (2) a method for producing a rose characterized by artificially suppressing the rose endogenous metabolic pathway, and expressing the pansy gene coding for flavonoid 3′,5′-hydroxylase and the gene coding for dihydroflavonol reductase.
The invention still further provides (3) a method for producing a rose characterized by artificially suppressing expression of rose endogenous dihydroflavonol reductase, and expressing the pansy gene coding for flavonoid 3′,5′-hydroxylase and the gene coding for dihydroflavonol reductase derived from a plant other than rose.
The invention still further provides (4) a method for producing a rose characterized by artificially suppressing expression of rose endogenous flavonoid 3′-hydroxylase and expressing the pansy gene coding for flavonoid 3′,5′-hydroxylase.
The aforementioned pansy gene coding for flavonoid 3′,5′-hydroxylase is, for example, the gene listed as SEQ ID NO: 1 or SEQ ID NO: 3. The gene coding for dihydroflavonol reductase is preferably derived from iris, Nierembergia, petunia, orchid, gentian or Eustoma russellianum. 
The invention still further provides (5) a rose obtained by the production method according to any one of (1) to (4) above, or a progeny or tissue thereof having the same properties as the rose.
The invention still further provides (6) a rose obtained by the production method according to any one of (1) to (4) above, or a progeny or tissue thereof, wherein the petal color of the rose is violet, blue-violet or blue.
The invention further provides (7) a rose according to (6) above, or a progeny or tissue thereof, wherein the petal color of the rose belongs to the “Violet group”, “Violet-Blue” group or “Blue group” according to the Royal Horticultural Society Colour Chart (RHSCC).
The invention further provides (8) a rose according to (7) above, or a progeny or tissue thereof, wherein the petal color of the rose belongs to “Violet group” 85a or 85b according to the Royal Horticultural Society Colour Chart (RHSCC).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the flavonoid biosynthesis pathway.
CHS: Chalcone synthase, CHI: Chalcone isomerase FNS: Flavone synthase, F3H: Flavanone 3-hydroxylase
F3′H: Flavonoid 3′-hydroxylase
F3′5′H: Flavonoid 3′5′-hydroxylase, FLS: Flavonol synthase
DFR: Dihydroflavonol 4-reductase
ANS: Anthocyanidin synthase, AS: Aurone synthase
C2′GT: Chalcone 2′-glucosyl transferase
FIG. 2 shows the structure of plasmid pBERD1.
FIG. 3 shows the structure of plasmid pBPDBP2.
FIG. 4 shows the structure of plasmid pBPDBP8.
FIG. 5 shows the structure of plasmid pSPB461.
FIG. 6 shows the structure of plasmid pSPB472.
FIG. 7 shows the structure of plasmid pSPB130.
FIG. 8 shows the structure of plasmid pSPB919.
FIG. 9 shows the structure of plasmid pSPB920.
FIG. 10 shows the structure of plasmid pSPB1106.



BEST MODE FOR CARRYING OUT THE INVENTION
Several reasons may be postulated for a lack of blue color in rose even with production of delphinidin. The stability, solubility and color of anthocyanins varies depending on modification with acyl groups and sugars. Specifically, it is known that an increased number of aromatic acyl groups results in greater blueness. Also, formation of complexes between flavonol and flavone copigments and anthocyanins produce a blue color and shift the maximum absorption wavelength toward the longer wavelength end while also increasing the absorbance. Anthocyanin color is also dependent on pH. Since a lower pH tends toward redness and a more neutral pH produces blueness, the flower color depends on the pH of the vacuoles in which the anthocyanins are localized. In addition, formation of metal chelates in the copresence of metal ions such as Al3+ and Mg2+ can significantly affect flower color as well. Trial and error and assiduous research led to the proposal for a modification whereby the proportion of delphinidin in flower petals is increased.
First, it was attempted to create a blue rose by the same method used to create a blue-violet carnation. Specifically, it was attempted to analyze white rose variety 112 and identify a DFR-deficient line, but unlike carnation, no completely DFR-deficient line could be obtained. This is presumably due to the fact that carnation is diploid while ordinarily cultivated rose is tetraploid, such that it is difficult to find a line deficient in a single gene.
Next, the pansy F3′5′H gene and petunia DFR gene were transferred into the white flower variety Tineke and accumulation of delphinidin was detected, but the amount was minimal and a blue rose was not obtained.
According to the present invention, the DFR gene, an enzyme participating in the rose endogenous flavonoid synthesis pathway, is artificially suppressed by a gene engineering technique, and the pansy F3′5′H gene is expressed while a dihydromyricetin-reducing DFR gene is also expressed, in order to increase the delphinidin content to roughly 80-100% of the total anthocyanidins in the flower petals, thereby allowing realization of a blue rose.
The dihydromyricetin-reducing DFR genes used in this case were derived from iris (Iridaceae), Nierembergia (Solanaceae) and petunia (Solanaceae), but as other dihydromyricetin-reducing DFR gene sources there may be mentioned non-pelargonidin-accumulating plants such as tobacco (Solanaceae), cyclamen (Primulaceae), delphinium (Ranunculaceae), orchid (Orchidaceae), gentian (Gentianaceae), Eustoma russellianum (Gentianaceae) and the like (Forkmann 1991, Plant Breeding 106, 1-26; Johnson et al., Plant J. 1999, 19, 81-85). The DFR genes used for the present invention are genes that preferentially reduce dihydromyricetin.
According to the invention, the flavonoid 3′-hydroxylase (F3′H) gene, an enzyme participating in the rose endogenous flavonoid synthesis pathway, is artificially suppressed by a gene engineering technique, and the pansy F3′5′H gene is expressed, in order to increase the delphinidin content to roughly 80-100% of the total anthocyanidins in the flower petals, thereby allowing realization of a blue rose.
The roses obtained according to the invention have hitherto non-existent flower colors, and the invention can provide roses with flower colors belonging not only to the red-purple group, purple group and purple-violet group but also to the violet group, violet-blue group and blue group, according to the Royal Horticultural Society Colour Chart.
EXAMPLES
The present invention will now be explained in greater detail by the following examples. Unless otherwise specified, the molecular biological protocols used were based on Molecular Cloning (Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Example 1
Flower Color Measuring Method
The flower petal color shade was evaluated by measurement using a CM2022 spectrophotometric colorimeter (Minolta Japan) with a 10° visual field and a D65 light source, and analysis using SpectraMagic color control software (Minolta Japan). The Royal Horticultural Society Colour Chart (RHSCC) number is the nearest color as compared against Color Classification System Version 2.1.1 (The Japan Research Institute Co., Ltd.; Japanese Unexamined Patent Publication No. 2002-016935), based on the color value (CIE L*a*b* color system) obtained by visual discrimination and measurement with the device mentioned above. This system may be used for objective selection of the nearest RHSCC number.
Upon measuring the color shades of flower petals of cultivars conventionally referred to as “blue roses” and determining the nearest colors according to the RHSCC by this method, it was determined that Blue Moon and Madam Violet were 186d (Greyed-Purple group), Lavande was 186c (Greyed-Purple group), Seiryu was 189d (Greyed-Green group) and Blue Heaven was 198d (Greyed-Green group). These cultivars are called blue roses but are classified in “Grey” groups according to RHSCC number and therefore do not exhibit the blue color which is the object of the present invention.
Example 2
Flavonoid Analysis
1) Extraction of Flower Petal Color
A 0.5 g portion of freeze-dried rose petals was subjected to extraction in 4 ml of 50% acetonitrile (CH3CN) containing 0.1% TFA for 20 minutes under ultrasonic vibration and then filtered with a 0.45 μm filter. High-performance liquid chromatography (HPLC) of the anthocyanins in the extract was conducted under the following conditions. Isocratic elution was carried out using an RSpak DE-413L (4.6 mmφ×25 cm, Shoko Co., Ltd.) column with a flow rate of 0.6 ml/min, and a mobile phase at a linear concentration gradient of 10%→50% CH3CN/H2O containing 0.5% trifluoroacetic acid (TFA) for 15 minutes followed by 50% CH3CN/H2O containing 0.5% TFA for 10 minutes. Detection was performed using an SPD-M10A photodiode array detector (Shimadzu Laboratories), with detection in the wavelength range of 600-250 nm and calculation of the abundance ratio of each anthocyanin based on the 520 nm absorbance area.
2) Anthocyanidin Analysis
A 0.2 ml portion of the filtrate was dried completely under reduced pressure in a glass test tube and dissolved in 0.2 ml of 6N hydrochloric acid (HCl), and subjected to hydrolysis at 100° C. for 20 minutes. The hydrolyzed anthocyanidins were extracted with 0.2 ml of 1-pentanol, and the organic layer was analyzed by HPLC under the following conditions. The column used was an ODS-A312 (6 mmφ×15 cm, YMC Co., Ltd.), and elution was performed at a flow rate of 1 ml/min using a CH3COOH:CH3OH:H2O=15:20:65 solution as the mobile phase.
Detection was performed by spectral measurement at 600-400 nm using an SPD-M10A photodiode array detector (Shimadzu Laboratories), identification based on absorption maximum (λmax) and retention time (RT), and quantitation based on 520 nm absorbance area. The retention time and λmax of delphinidin and cyanidin under these HPLC conditions were 4.0 min, 5.2 min and 534 nm, 525 nm, respectively. Delphinidin hydrochloride and cyanidin hydrochloride purchased from Funakoshi Co., Ltd. were used as samples for identification and quantitation.
3) Flavonol Analysis
A 0.2 ml portion of the flower petal-extracted filtrate was dried to hardness under reduced pressure in a 1.5 ml Eppendorf tube and dissolved in 0.2 ml of 0.1 M potassium phosphate buffer (KPB) at pH 4.5, and then 6 units of β-glucosidase (Shinnihon Kagaku Co., Ltd.) and 1 unit of naringenase (Sigma Chemical Co., MO, USA) were added and the mixture was kept at 30° C. for 16 hours. After the reaction, 0.2 ml of 90% CH3CN was added to the enzyme reaction solution to terminate the reaction. The solution was filtered with a 0.45 μm filter and subjected to HPLC under the following conditions.
Isocratic elution was carried out using a Develosil C30-UG-5 (4.6 mmφ×15 cm, Nomura Chemical Co., Ltd.) column with a flow rate of 0.6 ml/min, and a mobile phase at a linear concentration gradient of 18%-+63% CH3CN/H2O containing 0.1% TFA for 10 minutes followed by 63% CH3CN/H2O containing 0.1% TFA for 10 minutes. Detection was performed using an SPD-M10A photodiode array detector, with detection in the wavelength range of 400-250 nm. The R.T. and λmax of kaempferol and quercetin under these conditions were 11.6 min, 365 nm and 10.3 min, 370 nm, respectively. Kaempferol and quercetin purchased from Funakoshi Co., Ltd. were used as samples for quantitation based on the A330 nm area.
Example 3
pH Measurement Method
Approximately 2 g of rose petals frozen at −80° C. for 1 hour or longer was pressed with a homogenizer to obtain the petal juice. The pH was measured by connecting a 6069-10C microelectrode (Horiba Laboratories) to a pH meter (F-22, Horiba Laboratories).
Example 4
Transformation of Rose
Several methods have been reported for transformation of roses (for example, Firoozababy et al. Bio/Technology 12:609-613 (1994); U.S. Pat. No. 5,480,789; U.S. Pat. No. 5,792,927; EP 536,327 A1; US 20010007157 A1), and transformation may be carried out by any of these techniques. Specifically, rose calli taken from aseptic seedling leaves were immersed for 5 minutes in a bacterial suspension of Agrobacterium tumefaciens Ag10 (Lazo et al., Bio/Technology 9:963-967, 1991), the excess bacterial suspension was wiped off with sterile filter paper, and the calli were transferred to subculturing medium and cocultivated for 2 days in a dark room.
After subsequently rinsing with MS liquid medium containing 400 mg/L carbenicillin, the calli were transferred to selection/elimination medium prepared by adding 50 mg/L kanamycin and 200 mg/L carbenicillin to subculturing medium. Upon repeating transfer and cultivation of the portions which grew normally in selection medium without growth inhibition, the kanamycin-resistant calli were selected out. The kanamycin-resistant transformed calli were cultivated in redifferentiation medium containing 50 mg/L kanamycin and 200 mg/L carbenicillin to obtain kanamycin-resistant shoots. The obtained shoots were rooted in 1/2MS medium and then habituated. The habituated plants were potted and then cultivated in a closed greenhouse until blooming.
Example 5
Obtaining Rose Flavonoid Gene
A cDNA library derived from Kardinal rose variety flower petals was screened using the petunia DFR gene (described in WO96/36716) as the probe, to obtain rose DFR cDNA was which designated as pCGP645. The details have already been reported (Tanaka et al., Plant Cell Physiol. 36, 1023-1031 1995).
Likewise, the same library was screened with the petunia chalcone synthase-A (CHS-A) gene (Koes et al., Gene (1989) 81, 245-257) and the anthocyanidin synthase (ANS) gene (Martin et al., Plant J., (1991) 1, 37-49) according to a publicly known procedure (Tanaka et al., Plant Cell Physiol. 36, 1023-1031 1995), to obtain rose chalcone synthase (CHS) and anthocyanidin synthase (ANS) homologs which were designated as pCGP634 and pCGP1375, respectively. The nucleotide sequence for rose CHS is listed as SEQ ID NO: 5, and the nucleotide sequence for rose ANS is listed as SEQ ID NO: 6.
Example 6
Screening for White Rose
For creation of a blue cultivar by gene recombination, cultivars lacking only the DFR gene may be selected, in order to avoid competition between the endogenous anthocyanin synthesis pathway and the introduced genes (particularly the F3′5′H gene), and the petunia DFR gene and F3′5H gene transferred into those cultivars (WO96/36716).
A screening was conducted among the numerous existing white rose varieties, for those lacking only the DFR gene and normally expressing other anthocyanin biosynthesis enzyme genes. The cause of flower color whitening is believed to be occasional mutation or deletion of structural genes involved in anthocyanin biosynthesis, and occasional loss of transcription regulating factors which control transcription of structural genes involved in anthocyanin biosynthesis. Roses lacking DFR gene mRNA were examined according to the method described in WO96/36716.
First, 112 primarily white rose lines were analyzed for flavonoid composition of the flower petals by the method described in Example 1, and lines with high accumulation of flavonols were selected. The pH of each petal juice was then measured and 80 cultivars with relatively high pH values were chosen as primary candidates.
RNA was then extracted from petals of these cultivars. The RNA extraction was accomplished by a publicly known method (Tanaka et al., Plant Cell Physiol. 36, 1023-1031, 1995). The obtained RNA was used to examine the presence or absence of mRNA corresponding to the rose DFR gene (Tanaka et al., Plant Cell Physiol. 36, 1023-1031, 1995) and the rose anthocyanidin synthase (ANS) gene. RT-PCR was performed and eight cultivars (WKS-11, 13, 22, 36, 43, White Killarney, Tsuru No. 2, Tineke) having low endogenous expression of DFR mRNA and normal ANS mRNA levels were selected.
RT-PCR was carried out with a Script First-strand Synthesis System for RT-PCR (Invitrogen) using RNA obtained from petals of each cultivar. The DFR mRNA was detected using DFR-2F (5′-CAAGCAATGGCATCGGAATC-3′) (SEQ ID NO: 13) and DFR-2B (5′-TTTCCAGTGAGTGGCGAAAGTC-3′) (SEQ ID NO: 14) primers, and the ANS mRNA was detected using ANS-2F (5′-TGGACTCGAAGAACTCGTCC-3′) (SEQ ID NO: 15) and ANS-2B (5′-CCTCACCTTCTCCCTTGTT-3′) (SEQ ID NO: 16) primers.
These eight cultivars showed lower levels of DFR mRNA and normal levels of ANS mRNA in Northern blotting (Table 1), and their cultivating properties were excellent. Two of the transformable cultivars (Tineke, WKS36) were decided on for actual transfer of the delphinidin-producing construct.










TABLE 1








Flavonols





(mg/g


Cultivar
petal)

RT-PCR














name
Q
K
Total
pH
DFR
CHS
ANS

















WKS-36
0.082
8.095
8.177
4.81
−
+
+


White
1.343
6.113
7.456
4.7
+
+
+


Killarney


Tsuru No. 2
0.715
5.188
5.903
4.7
+
+
+


WKS-11
2.028
0.475
2.503
4.51
+
+
+


Tineke
0.097
4.337
4.434
4.45
−
+
+


WKS-13
0.320
3.993
4.313
4.45
−
+
+


WKS-22
0.145
10.469
10.614
4.41
−
+
+


WKS-43
0.045
2.104
2.149
4.07
−
+
+





+: mRNA detected at same level as colored rose (Rote Rose cultivar)


−: mRNA detected at lower level than colored rose (Rote Rose cultivar)


Q: Quercetin,


K: kaempferol






Example 7
Transfer of Rose DFR Gene into Tineke
Plasmid pE2113 (Mitsuhara et al., Plant Cell Physiol. 37, 49-59, 1996) comprises the enhancer sequence repeat-containing cauliflower mosaic virus 35S (E1235S) promoter and the nopaline synthase terminator. This plasmid was digested with SacI and the ends were blunted using a Blunting Kit (Takara). The DNA fragment was ligated with an 8 bp SalI linker (Takara) and the obtained plasmid was designated as pUE5.
Plasmid pUE5 was digested with HindIII and EcoRI to obtain an approximately 3 kb DNA fragment, which was introduced into pBin19 (Bevan M., Binary Agrobacterium Vector for plant transformation. Nucl. Acid Res. 12. 8711-21, 1984) previously digested with HindIII and EcoRI, to obtain plasmid pBE5. Next, pCGP645 was digested with BamHI and XhoI to obtain a DNA fragment containing full-length rose DFR cDNA. This was ligated with pBE5 digested with BamHI and XhoI to construct pBERD1 (FIG. 2). The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pBERD1 (FIG. 2) was transferred into the white rose cultivar “Tineke”, and 18 transformants were obtained. Flower color was altered in six of the obtained transformants. Pigment analysis of two plants in which a clear color change from white to pink was observed confirmed accumulation of cyanidin and pelargonidin in both (Table 2). These results suggested that the Tineke cultivar is a cultivar lacking the DFR gene.









TABLE 2






Cya
Pel


Plant No.
(mg/g)
(mg/g)







1
0.014
0.005


2
0.014
0.006





Cya: Cyanidin, Pel: Pelargonidin






Example 8
Transfer of Pansy F3′5′H Gene (#18) and Petunia DFR Gene into Tineke
RNA was extracted from young budding pansy (Black Pansy variety) petals by the method of Turpen and Griffith (BioTechniques 4:11-15, 1986), and Oligotex-dT (Qiagen) was used for purification of polyA+RNA. This polyA+RNA and a λZAPII/GigapackII Cloning Kit (Stratagene) were used to construct a cDNA library from the young budding pansy petals. After transferring approximately 100,000 pfu of phage plaques grown on an NZY plate onto a Colony/PlaqueScreen (DuPont), treatment was conducted by the manufacturer's recommended protocol. The plaques were 32P-labeled and screened using petunia Hf1cDNA (pCGP602, Holton et al., Nature, 366, p 276-279, 1993) as the probe.
The membrane was subjected to pre-hybridization for 1 hour at 42° C. in hybridization buffer (10% (v/v) formamide, 1 M NaCl, 10% (w/v) dextran sulfate, 1% SDS), and then the 32P-labeled probe was added to 1×106 cpm/ml and hybridization was performed for 16 hours at 42° C. The membrane was then rinsed for 1 hour in 2×SSC, 1% SDS at 42° C., fresh rinsing solution was exchanged, and rinsing was again performed for 1 hour. The rinsed membrane was exposed on a Kodak XAR film together with an intensifying screen, and the hybridization signal was detected.
The results of cDNA analysis demonstrated that the two obtained cDNA had high identity with petunia Hf1. The two cDNA types were designated as pansy F3′5′H cDNA, BP#18 (pCGP1959) and BP#40 (pCGP1961). The nucleotide sequence for #18 is listed as SEQ ID NO: 1, and its corresponding amino acid sequence is listed as SEQ ID NO: 2, the nucleotide sequence for #40 is listed as SEQ ID No. 3, and its corresponding amino acid sequence is listed as SEQ ID NO: 4. BP#18 and BP#40 have 82% identity on the DNA level. Also, BP#18 and BP#40 both exhibit 60% identity with petunia Hf1 and 62% identity with petunia Hf2 (Holton et al., Nature, 366, p 276-279, 1993), on the DNA level.
Separately, plasmid pUE5 was digested with EcoRI and the ends were blunted using a Blunting Kit (Takara), and the obtained DNA fragment was ligated with an 8 bp HindIII linker (Takara), producing a plasmid which was designated as pUE5H. There was recovered an approximately 1.8 kb DNA fragment obtained by subjecting plasmid pCGP1959 containing pansy F3′5′H #18 cDNA to complete digestion with BamHI and partial digestion with XhoI. The plasmid obtained by ligation of this with pUE5H digested with BamHI and XhoI was designated as pUEBP18.
Separately, a DNA fragment containing petunia DFR cDNA was recovered by digestion of pCGP1403 (WO96/36716) with BamHI and XhoI, and this DNA fragment was ligated with pBE5 that had been digested with BamHI and XhoI, to prepare pBEPD2. Next, pUEBP18 was partially digested with HindIII and an approximately 2.8 kb DNA fragment was recovered containing the E1235S promoter, pansy F3′5′H #18 cDNA and the nos terminator. This fragment was ligated with a DNA fragment obtained by partial digestion of pBEPD2 with HindIII to obtain a binary vector plasmid pBPDBP2 (FIG. 3). This plasmid was introduced into Agrobacterium tumefaciens Ag10.
Plasmid pBPDBP2 (FIG. 3) was transferred into the white rose cultivar “Tineke”, and 40 transformants were obtained. Flower color was altered in 23 of the obtained transformants, and pigment analysis confirmed accumulation of delphinidin in 16 of the 19 analyzed transformants (Table 3). The delphinidin content was 100% at maximum (average: 87%), but the maximum amount of pigment was very low at 0.035 mg per gram of petals and the flower color was only altered from RHS Color Chart 158d (Yellow-White group) to 56a (Red group) or 65b (Red-Purple group), while no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.













TABLE 3





Plant
Del
Del
Cya
M
Q
K


No.
content (%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
87
0.002
0.000
0.000
0.058
0.354


2
100
0.004
0.000
0.338
0.059
1.921


3
82
0.002
0.001
0.203
0.039
1.382


4
100
0.003
0.000
0.245
0.050
1.840


5
76
0.005
0.001
0.000
0.280
3.288


6
0
0.000
0.000
0.000
0.098
0.409


7
0
0.000
0.001
0.000
0.101
0.358


8
0
0.000
0.001
0.000
0.030
2.277


9
83
0.013
0.003
0.000
0.117
0.841


10
85
0.011
0.002
0.000
0.104
3.300


11
84
0.020
0.004
0.000
0.168
3.137


12
91
0.025
0.002
0.294
0.119
1.252


13
90
0.028
0.003
0.000
0.075
1.912


14
91
0.014
0.001
0.000
0.152
2.667


15
90
0.035
0.004
0.000
0.086
1.616


16
83
0.023
0.005
0.000
0.117
2.267


17
91
0.014
0.001
0.000
0.113
0.825


18
76
0.003
0.001
0.000
0.085
2.351


19
82
0.005
0.001
0.000
0.054
1.616





Del: delphinidin,


M: Myricetin






Example 9
Transfer of Pansy F3′5′H Gene (#40) and Petunia DFR Gene into Tineke
Plasmid pE2113 (Mitsuhara et al., Plant Cell Physiol. 37, 49-59, 1996) was digested with HindIII and XbaI to obtain an approximately 800 bp DNA fragment, which was ligated with pBin19 (Bevan M., Binary Agrobacterium Vector for plant transformation. Nucl. Acid Res. 12. 8711-21, 1984) previously digested with HindIII and XbaI. The obtained plasmid was designated as pCGP1391. Another plasmid, pCGP669 (WO94/21840), contains the petunia chalcone synthase A (CHS-A) gene promoter. This plasmid was digested with EcoRI, blunted and then digested with HindIII.
The approximately 700 bp DNA fragment was ligated with pCGP1391 that had been digested with HindIII and SnaBI, and the obtained plasmid was designated as pCGP1707. Also, there was recovered an approximately 1.8 kb DNA fragment obtained by subjecting plasmid pCGP1961 containing pansy F3′5′H #40 cDNA to complete digestion with BamHI and partial digestion with XhoI. The plasmid obtained by ligation of this with pUE5H digested with BamHI and XhoI was designated as pUEBP40. Plasmid pUEBP40 was digested with EcoRV and XbaI and an approximately 5.5 kb DNA fragment was recovered.
This fragment was ligated with an approximately 700 bp fragment obtained by digesting plasmid pCGP1707 with HindIII, blunting the ends and further digesting with XbaI, to obtain plasmid pUFBP40. Next, pUFBP40 was partially digested with HindIII and an approximately 3.4 kb DNA fragment was recovered containing the cauliflower 35S promoter enhancer, CHS-A promoter, pansy F3′5′H #40 cDNA and the nos terminator. This fragment was ligated with a DNA fragment obtained by partial digestion of pBEPD2 with HindIII to obtain a binary vector plasmid pBPDBP8 (FIG. 4). This plasmid was introduced into Agrobacterium tumefaciens Ag10.
Plasmid pBPDBP8 (FIG. 4) was transferred into the white rose cultivar “Tineke”, and 53 transformants were obtained. Flower color was altered in 17 of the obtained transformants, and pigment analysis confirmed accumulation of delphinidin in 8 of the 9 analyzed transformants (Table 4). The delphinidin content was 93% at maximum (average: 79%), but the maximum amount of pigment was very low at 0.014 mg per gram of petals and the flower color was only altered from RHS Color Chart 158d (Yellow-White group) to 56a (Red group) or 65b (Red-Purple group), while no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained. This suggested that the Tineke variety is not a variety lacking only the DFR gene.













TABLE 4






Del
Del
Cya
M
Q
K


Plant No.
content (%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
0
0.000
0.001
0.000
0.018
2.023


2
9
0.001
0.006
na
na
na


3
93
0.011
0.001
0.000
0.036
2.724


4
86
0.007
0.001
0.000
0.076
2.957


5
71
0.013
0.006
0.000
0.073
2.503


6
87
0.014
0.002
0.000
0.058
3.390


7
78
0.005
0.002
0.000
0.049
1.241


8
47
0.004
0.004
0.000
0.070
1.800


9
78
0.004
0.001
0.000
0.029
2.326





na: no analysis/measurement






Example 10
Transfer of Pansy F3′5′H Gene (#18) and Petunia DFR Gene into WKS36
Plasmid pBPDBP2 (FIG. 3) was transferred into the white rose “WKS36”, and 138 transformants were obtained. Flower color was altered in 10 of the obtained transformants, and accumulation of delphinidin was confirmed in all of the plants (Table 5). The delphinidin content was 91% at maximum (average: 60%), but the maximum amount of pigment was very low at 0.033 mg per gram of petals and the flower color was only altered to very light pink, while no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained. This suggested that the WKS36 variety is not a variety lacking only the DFR gene.













TABLE 5






Del
Del
Cya
M
Q
K


Plant No.
content (%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
60
0.008
0.005
0.381
0.169
2.291


2
40
0.006
0.009
0.633
0.486
2.911


3
54
0.005
0.005
0.654
0.336
3.460


4
43
0.016
0.021
0.000
0.656
2.469


5
53
0.009
0.008
0.404
0.325
2.397


6
53
0.004
0.003
0.498
0.251
2.768


7
45
0.013
0.016
0.000
0.381
1.537


8
83
0.004
0.001
0.000
0.156
1.632


9
80
0.033
0.008
0.000
0.557
3.766


10
91
0.013
0.000
0.000
0.184
2.610









Example 11
Transfer of Pansy F3′5′H Gene (#18) and Petunia DFR Gene into WKS36
A plasmid obtained by replacing the AscI site of plasmid pUCAP (van Engelen et al., Transgenic Research 4, 288-290, 1995) with PacI linker was designated as pUCPP. Separately, an expression cassette prepared by linking the rose chalcone synthase promoter, pansy F3′5′H #18 cDNA and nos terminator was obtained in the following manner.
Chromosomal DNA was extracted from young leaves of the Kardinal rose cultivar (Tanaka et al., Plant Cell Physiol. 36, 1023-1031, 1995). An approximately 100 μg portion of DNA was partially digested with Sau3AI, and approximately 20-kb DNA fragments were recovered by sucrose density gradient.
These were ligated with lambda phage EMBL3 (for example, Stratagene) that had been digested with BamHI, and a chromosomal DNA library was prepared by the manufacturer's recommended protocol. The library was screened by a publicly known method (Tanaka et al., Plant Cell Physiol. 36, 1023-1031, 1995) using rose chalcone synthase cDNA (DNA database: GenBank Accession No. AB038246) as the probe. Among the obtained chalcone synthase chromosome clones, there existed lambda CHS20 which included an approximately 6.4 kb DNA sequence upstream from the start codon of chalcone synthase. The approximately 2.9 kb DNA fragment obtained by digestion of lambda CHS20 with HindIII and EcoRV includes the chalcone synthase promoter region.
This fragment was ligated with a fragment obtained by digestion of pUC19 (Yanisch-Perron C et al., Gene 33:103-119, 1985) with HindIII and SmaI. This was designated as pCGP1116. The sequence of the chalcone synthase promoter region included therein is listed as SEQ ID NO: 21. An approximately 2.9 kb DNA fragment obtained by digestion of pCGP1116 with HindIII and KpnI was ligated with a DNA fragment obtained by digestion of pJB1 (Bodeau, Molecular and genetic regulation of Bronze-2 and other maize anthocyanin genes. Dissertation, Stanford University, USA, 1994) with HindIII and KpnI to obtain pCGP197.
Separately, an approximately 300 bp DNA fragment containing the nopaline synthase terminator, obtained by digestion of pUE5 with SacI and KpnI, was blunted and linked with pBluescriptSK—which had been digested with EcoRV and BamHI and blunted. A plasmid of those obtained in which the 5′ end of the terminator was close to the SalI site of pBluescriptSK—was designated as pCGP1986.
A DNA fragment obtained by digesting pCGP1986 with XhoI, blunting the ends and further digesting with SalI was linked with a DNA fragment obtained by digesting pCGP197 with HindIII, blunting the ends and further digesting with SalI, to obtain pCGP2201.
Next, a DNA fragment obtained by digesting pCGP2201 with SalI and blunting the ends was linked with an approximately 1.7 kb DNA fragment (containing the pansy flavonoid 3′,5′-hydroxylase gene) obtained by digesting pCGP1959 with BamHI and KpnI and blunting the ends. A plasmid of those obtained in which the rose chalcone synthase promoter had been inserted in a direction allowing transcription of the pansy flavonoid 3′,5′-hydroxylase gene in the forward direction was designated as pCGP2203. Plasmid pCGP2203 was recovered by digestion with HindIII and SacI. The DNA fragment was cloned at the HindIII and Sad sites of pUCPP, and the resulting plasmid was designated as pSPB459. Next, plasmid pE2113 was digested with SnaBI and a BamHI linker (Takara) was inserted to obtain a plasmid designated as pUE6.
An approximately 700 bp DNA fragment obtained by digestion of pUE6 with HindIII and BamHI was linked with an approximately 2.2 kb DNA fragment obtained by digestion of pCGP1405 (WO96/36716) with BamHI and BglII and with the binary vector pBinplus (van Engelen et al., Transgenic Research 4, 288-290, 1995) digested with HindIII and BamHI, to obtain pSPB460. An approximately 5 kb DNA fragment obtained by digestion of pSPB459 with PacI was introduced into the PacI site of pSPB460 to obtain pSPB461 (FIG. 5) having the petunia DFR and pansy F3′5′H #18 genes linked in the forward direction on the binary vector. This plasmid is modified for constitutive expression of the petunia DFR gene in plants and specific transcription of the pansy F3′5′H #18 gene in flower petals. The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pSPB461 (FIG. 5) was transferred into the white rose “WKS36”, and 229 transformants were obtained. Flower color was altered in 16 of the obtained transformants, and accumulation of delphinidin was confirmed in all 12 of the pigment-analyzed plants (Table 6). The delphinidin content was 79% at maximum (average: 58%), but the amount of pigment was very low at 0.031 mg per gram of petals and the flower color was only altered to very light pink, while no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained. This suggested that the WKS36 variety is not a variety lacking only the DFR gene.













TABLE 6






Del
Del
Cya
M
Q
K


Plant No.
content (%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
39
0.002
0.004
0.000
0.414
3.744


2
52
0.006
0.005
0.000
0.465
3.363


3
27
0.002
0.005
0.000
0.342
3.703


4
58
0.014
0.010
0.000
0.430
2.780


5
62
0.008
0.005
0.498
0.281
2.189


6
72
0.002
0.001
0.000
0.193
2.391


7
71
0.010
0.004
0.000
0.152
4.021


8
79
0.031
0.008
0.403
0.215
2.660


9
26
0.004
0.011
0.000
0.249
2.331


10
54
0.007
0.006
0.000
0.299
2.085


11
74
0.017
0.006
0.145
0.248
3.505


12
74
0.013
0.005
0.000
0.229
2.005









Example 12
Transfer of Pansy F3′5′H Gene (#18), Petunia DFR Gene and Perilla Anthocyanin β-Glucoside Acyltransferase Gene into WKS36
A gene comprising a start codon added to the perilla hydroxycinnamoyl CoA: anthocyanin β-glucoside acyltransferase (3AT) gene was designated as pSAT208F (Yonekura-Sakakibara et al., Plant Cell Physiol. 41, 495-502, 2000). An approximately 3.9 kb DNA fragment obtained by digestion of pSPB580 (PCT/AU03/00079) with BamHI and XhoI was linked with an approximately 1.8 kb DNA fragment obtained by digestion of pSAT208F with BamHI and XhoI.
The obtained plasmid was digested with AscI, and a DNA fragment was recovered containing the E1235S promoter, the perilla 3AT gene and the petunia phospholipid transfer protein terminator. The DNA fragment was inserted into the AscI site of pSPB461 to obtain plasmid pSPB472 (FIG. 6) having the perilla 3AT, petunia DFR and pansy F3′5′H #18 gene transcription directions in the forward direction. This plasmid is modified for constitutive expression of the perilla 3AT gene and the petunia DFR gene in plants and specific transcription of the pansy F3′5′H #18 gene in flower petals. The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pSPB472 (FIG. 6) was transferred into the white rose “WKS36”, and 75 transformants were obtained. Flower color was altered in four of the obtained transformants, and accumulation of delphinidin was confirmed in all three of the pigment-analyzed plants (Table 7). The delphinidin content was 67% at maximum (average: 49%), but the amount of pigment was very low at 0.011 mg per gram of petals and the flower color was only altered to very light pink, while no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained. This suggested that the WKS36 variety is not a variety lacking only the DFR gene.













TABLE 7





Plant
Del
Del
Cya
M
Q
K


Plant No.
content (%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)







1
32
0.003
0.006
0.219
0.236
1.972


2
67
0.011
0.005
0.520
0.329
3.234


3
46
0.006
0.007
0.000
0.579
3.874









Thus, despite screening of several white roses, it was not possible to obtain a cultivar lacking only the DFR gene. In other words, it was not possible to obtain a blue rose by the method for creation of blue carnation (WO94/28140).
Example 13
Inhibition of Rose DFR Gene by Cosuppression
Plasmid pBERD1 was transferred into the pale violet rose “Lavande”, and 26 transformants were obtained. However, none of the plants exhibited altered flower color, suggesting that it is difficult to inhibit the rose endogenous DFR gene by cosuppression.
Example 14
Screening for Colored Roses
Cultivars for creation of blue roses were then selected from among colored roses. After visually selecting 136 lines from colored rose cultivars with relatively blue shades, 89 of the lines were subjected to pigment analysis. The values obtained for the examined colored roses are shown in Tables 8 to 10.












TABLE 8






Cya
Pel
Peo
Q
K


Name
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)







Lavande
0.078
0.000
0.000
0.451
0.078


Madam Violet
0.055
0.000
0.000
1.780
0.189


Vol de Nuit
0.317
0.003
0.000
2.661
0.316


Blue Moon
0.049
0.000
0.000
1.341
0.119


Seiryu
0.015
0.000
0.000
3.030
1.300


WKS077
1.875
0.008
0.000
1.430
0.247


WKS078
0.211
0.000
0.000
1.286
0.133


WKS079
2.864
0.003
0.000
1.030
0.106


WKS080
0.040
0.000
0.000
0.362
0.047


WKS081
0.032
0.000
0.000
4.480
1.563


WKS082
0.074
0.000
0.000
2.400
0.196


WKS083
0.018
0.405
0.000
0.146
0.962


WKS084
0.055
0.000
0.000
1.269
0.159


WKS087
0.032
0.000
0.000
0.797
0.134


WKS089
0.030
0.000
0.000
1.484
0.317


WKS090
1.571
0.007
0.000
1.346
0.339


WKS091
0.045
0.169
0.000
0.186
0.899


WKS092
0.038
0.002
0.000
1.358
0.135


WKS095
0.015
0.000
0.000
2.945
0.255


WKS096
0.024
0.000
0.000
2.032
0.349


WKS097
0.991
0.002
0.000
1.659
0.185


WKS100
0.051
0.000
0.000
1.410
0.615


WKS101
0.424
0.000
0.000
2.194
0.482


WKS104
0.066
0.000
0.000
2.347
0.424


WKS107
1.202
0.004
0.000
3.134
0.460


WKS114
0.429
0.000
0.000
3.509
0.541


WKS116
0.026
0.000
0.000
3.440
0.868


WKS117
0.027
0.000
0.000
0.227
0.149


WKS121
0.669
0.006
0.000
1.336
0.453


WKS123
0.487
0.003
0.000
3.663
0.826





Peo: Peonidin


















TABLE 9






Cya
Pel
Peo
Q
K


Name
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)







WKS124
0.022
0.045
0.000
0.192
2.012


WKS125
0.187
0.002
0.000
0.349
0.089


WKS126
0.544
0.002
0.000
2.226
0.895


WKS127
1.609
0.008
0.006
2.278
0.528


WKS128
1.844
0.003
0.007
2.576
0.409


WKS129
1.645
0.002
0.006
0.450
0.160


WKS130
1.332
0.008
0.005
1.599
0.525


WKS131
0.582
0.002
0.001
2.460
0.567


WKS132
1.101
0.006
0.000
0.298
0.208


WKS133
2.773
0.003
0.000
1.263
0.230


WKS133
3.487
0.011
0.023
0.414
0.108


WKS134
1.084
0.001
0.002
2.777
0.413


WKS135
0.241
0.007
0.001
0.803
0.113


WKS136
0.637
0.000
0.003
1.451
0.062


WKS137
1.208
0.014
0.002
1.034
1.027


WKS138
1.955
0.006
0.000
3.857
0.855


WKS139
0.285
0.003
0.000
1.363
0.538


WKS140
0.075
0.000
0.000
0.291
0.097


WKS141
0.197
0.000
0.000
0.358
0.045


WKS142
1.906
0.029
0.106
1.890
1.860


WKS143
1.125
0.027
0.020
1.596
1.129


WKS144
2.685
0.484
0.000
0.160
0.184


WKS145
0.948
0.006
0.000
3.086
1.222


WKS146
3.108
0.047
0.000
0.228
0.398


WKS147
0.593
0.003
0.004
3.619
0.924


WKS148
0.059
0.000
0.000
3.113
0.466


WKS149
1.101
0.013
0.000
1.481
1.866


WKS150
0.498
0.562
0.000
0.061
0.156


WKS151
0.947
1.073
0.000
0.038
0.227


WKS152
0.303
1.599
0.000
0.015
0.464





Peo: Peonidin


















TABLE 10






Cya
Pel
Peo
Q
K


Name
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)







WKS153
1.178
0.796
0.000
0.020
0.179


WKS154
0.219
0.659
0.000
0.007
0.265


WKS155
0.547
0.006
0.000
1.274
0.073


WKS156
0.851
0.005
0.000
1.139
0.238


WKS157
0.955
0.555
0.000
0.133
1.315


WKS158
0.634
0.005
0.000
0.526
0.219


WKS159
0.106
0.320
0.000
0.034
0.959


WKS160
0.750
0.005
0.000
2.283
0.768


WKS161
0.262
0.419
0.000
0.197
1.115


WKS162
0.039
0.564
0.000
0.041
0.447


WKS163
0.184
0.002
0.000
0.756
0.105


WKS164
0.918
0.012
0.000
1.954
2.832


WKS165
0.097
0.604
0.000
0.026
0.197


WKS166
0.116
0.015
0.000
0.488
0.566


WKS167
0.647
0.002
0.000
2.507
0.499


WKS168
1.109
0.029
0.000
1.797
2.328


WKS169
0.070
0.003
0.000
0.208
1.369


Baby Faurax
2.247
0.022
0.058
4.518
0.580


Indigo
0.891
0.006
0.000
5.781
3.820


Intermezzo
0.040
0.000
0.000
1.075
0.443


James Veitch
1.281
0.004
0.002
2.087
0.923


Lagoon
0.053
0.000
0.000
2.887
0.315


Magenta
0.126
0.000
0.000
1.062
0.191


MRS COLVILLE
1.666
0.012
0.000
3.500
2.940


Mme. Isaac
0.629
0.003
0.000
1.021
0.105


Pereire


Mme. de La
0.869
0.005
0.000
4.994
2.794


Roche-Lambert


Roseraie de
0.364
0.005
1.256
0.156
0.077


L'hay


Rose de Rescht
1.348
0.004
0.000
4.027
0.842


Rose du Roi a
2.556
0.017
0.000
0.968
0.411


Fleurs Pourpres





Peo: Peonidin






Example 15
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-acyltransferase Gene into Lavande
Modification of anthocyanins with aromatic acyl groups can stabilize the anthocyanins and produce a bluer color (for example, WO96/25500). The following experiment was conducted with the goal of producing acylated delphinidin-type anthocyanins.
RNA was obtained from Torenia Summer Wave flower petals, and polyA+RNA was prepared therefrom. A cDNA library was prepared from the polyA+RNA with λZAPII (Stratagene) as the vector, using a directional cDNA library preparation kit (Stratagene) according to the manufacturer's recommended protocol. The major anthocyanin of Torenia is modified with an aromatic acyl group at the 5-position glucose (Suzuki et al., Molecular Breeding 2000 6, 239-246), and therefore anthocyanin acyltransferase is expressed in Torenia petals.
Anthocyanin acyltransferase includes the conserved amino acid sequence Asp-Phe-Gly-Trp-Gly-Lys, and corresponding synthetic DNA can be used as primer to obtain the anthocyanin acyltransferase gene (WO96/25500). Specifically, 10 ng of single-stranded cDNA synthesized for construction of the Torenia cDNA library was used as template, and 100 ng of ATC primer (5′-GA(TC)TT(TC)GGITGGGGIAA-3′, I: inosine) (SEQ ID NO: 17) and 100 ng of oligo dT primer (5′-TTTTTTTTTTTTTTTTTCTCGAG-3′) (SEQ ID NO: 18) were used as primers for PCR with Taq polymerase (Takara, Japan), under the manufacturer's recommended conditions.
The PCR was carried out in 25 cycles of reaction with one cycle consisting of 1 minute at 95° C., 1 minute at 55° C. and 1 minute at 72° C. The approximately 400 bp DNA fragment that was obtained was recovered with Gene Clean II (BIO, 101. Inc.) according to the manufacturer's recommended protocol, and was subcloned in pCR-TOPO. Determination of the nucleotide sequence revealed a sequence homologous to the gentian acyltransferase gene (Fujiwara et al., 1998, Plant J. 16 421-431). The nucleotide sequence was determined by the Dye Primer method (Applied Biosystems), using Sequencer 310 or 377 (both by Applied Biosystems).
The DNA fragment was labeled with DIG using a DIG-labeling detection kit (Japan Roche), and used for screening of a Torenia cDNA library by plaque hybridization according to the manufacturer's recommended protocol. Twelve of the obtained positive signal clones were randomly selected, the plasmids were recovered, and their nucleotide sequences were determined. These exhibited high homology with anthocyanin acyltransferase. The total nucleotide sequence of the cDNA in the clone designated as pTAT7 was determined. The nucleotide sequence is listed as SEQ ID NO: 7, and the corresponding amino acid sequence is listed as SEQ ID NO: 8.
After digesting pBE2113-GUS (Mitsuhara et al., Plant Cell Physiol. 37, 49-59, 1996) with SacI, the ends were blunted and an 8 bp XhoI linker (Takara) was inserted. An approximately 1.7 kb DNA fragment obtained by digesting pTAT7 with BamHI and XhoI was inserted at the BamHI and XhoI sites of this plasmid, to obtain pSPB120. After digesting pSPB120 with SnaBI and BamHI, the ends were blunted and ligation was performed to obtain pSPB120′. Separately, plasmid pCGP1961 containing pansy F3′5′H #40 cDNA was completely digested with BamHI and then partially digested with XhoI to obtain an approximately 1.8 kb DNA fragment which was recovered and ligated with pUE5H previously digested with BamHI and XhoI, to obtain a plasmid which was designated as pUEBP40.
After digesting pUEBP40 with SnaBI and BamHI, the ends were blunted and ligation was performed to obtain pUEBP40′. This plasmid pUEBP40′ was partially digested with HindIII to obtain an approximately 2.7 kb DNA fragment which was recovered and linked with a DNA fragment obtained by partial digestion of pSPB120′ with HindIII. Of the obtained plasmids, a binary vector having the neomycin phosphotransferase gene, pansy F3′5′H #40 gene and Torenia 5AT gene linked in that order in the same direction from the right border sequence on the binary vector, was designated as pSPB130 (FIG. 7). This plasmid is modified for constitutive expression of the pansy F3′5′H #40 gene and the Torenia 5AT gene in plants and specific transcription of the genes in the flower petals. The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pSPB130 (FIG. 7) was transferred into the pale violet rose variety “Lavande”, and 41 transformants were obtained. Accumulation of delphinidin was confirmed in 20 of the 32 pigment-analyzed plants (Tables 11 and 12). The delphinidin content was 71% at maximum (average: 36%). The flower color was altered from RHS Color Chart 186c (Greyed-Purple group) to 79d (Purple group). The proportion of acylated anthocyanins was only about 30% of the total anthocyanins. Upon spectral measurement of the acylated anthocyanins, the maximum absorption wavelength had shifted toward longer wavelength by 4 nm from delphinidin 3,5-diglucoside, but because of the low proportion among the total anthocyanins, no clear effect was achieved for the flower color.














TABLE 11







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















1
0
9
0.005
0.050
na
na
na


2
0
11
0.009
0.069
na
na
na


3
0
10
0.010
0.087
na
na
na


4
0
22
0.028
0.102
na
na
na


5
5
51
0.073
0.069
na
na
na


6
4
57
0.093
0.069
na
na
na


7
5
48
0.039
0.042
na
na
na


8
13
0
0.000
0.065
na
na
na


9
17
9
0.006
0.062
na
na
na


10
26
0
0.000
0.104
na
na
na


11
17
67
0.074
0.036
na
na
na


12
0
0
0.000
0.131
na
na
na


13
0
0
0.000
0.083
na
na
na


14
6
48
0.084
0.092
na
na
na


15
0
20
0.020
0.081
na
na
na


16
42
13
0.020
0.131
0.000
0.637
0.020


17
32
36
0.032
0.058
na
na
na


18
7
0
0.000
0.146
na
na
na


19
0
0
0.000
0.069
na
na
na


20
0
0
0.000
0.142
na
na
na


21
0
0
0.000
0.080
na
na
na





na: no analysis/measurement




















TABLE 12







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















22
0
0
0.000
0.069
na
na
na


23
0
0
0.000
0.057
na
na
na


24
18
4
0.006
0.149
na
na
na


25
17
4
0.008
0.208
na
na
na


26
0
0
0.000
0.188
na
na
na


27
0
0
0.000
0.078
na
na
na


28
17
67
0.090
0.044
na
na
na


29
17
71
0.057
0.024
na
na
na


30
16
40
0.040
0.059
na
na
na


31
21
70
0.082
0.036
0.305
0.062
0.008


32
18
62
0.066
0.040
na
na
na





na: no analysis/measurement






Example 16
Transfer of Pansy F3′5′H Gene (#40) and Torenia anthocyanin 5-acyltransferase gene into WKS100
Plasmid pSPB130 (FIG. 7) was transferred into the pale violet rose variety “WKS100”, and 146 transformants were obtained. Accumulation of delphinidin was confirmed in 56 of the 63 pigment-analyzed plants (Tables 13-15). The delphinidin content was 95% at maximum (average: 44%). The flower color was altered from RHS Color Chart 56d (Red group) to 186d (Greyed-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.















TABLE 13







Del









Acylation
content
Del
Cya
Pel
M
Q
K


Plant No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























1
20
75
0.036
0.012
0.000
2.944
0.974
0.322


2
16
51
0.027
0.027
0.000
1.685
1.734
0.512


3
13
50
0.024
0.024
0.000
0.000
1.382
1.912


4
23
50
0.037
0.037
0.000
na
na
na


5
9
25
0.013
0.033
0.005
na
na
na


6
10
26
0.034
0.097
0.000
na
na
na


7
13
65
0.053
0.028
0.000
1.936
1.184
0.760


8
13
65
0.044
0.024
0.000
1.622
1.065
0.562


9
14
62
0.033
0.021
0.000
2.096
1.444
0.710


10
14
95
0.137
0.008
0.000
0.000
0.156
1.097


11
10
62
0.036
0.022
0.000
2.025
1.194
0.799


12
5
59
0.054
0.038
0.000
2.194
1.289
0.783


13
9
43
0.033
0.044
0.000
2.542
1.803
0.734


14
9
50
0.030
0.031
0.000
0.020
1.971
0.741


15
1
70
0.066
0.028
0.000
1.652
1.659
0.867


16
0
20
0.008
0.023
0.008
0.308
2.632
1.463


17
1
63
0.068
0.040
0.000
2.037
2.128
1.554


18
21
51
0.037
0.035
0.000
2.659
1.936
1.002


19
0
0
0.000
0.095
0.000
na
na
na


20
0
0
0.000
0.037
0.000
na
na
na


21
0
23
0.026
0.086
0.003
0.182
4.554
3.083


22
4
71
0.110
0.044
0.000
3.265
1.643
1.341


23
12
65
0.051
0.025
0.002
1.356
0.888
0.387


24
6
58
0.038
0.027
0.000
2.374
2.016
0.809


25
5
52
0.044
0.040
0.000
2.651
2.546
1.108





na: no analysis/measurement





















TABLE 14







Del









Acylation
content
Del
Cya
Pel
M
Q
K


Plant No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























26
6
64
0.033
0.019
0.000
2.707
1.546
0.605


27
16
0
0.000
0.041
0.000
na
na
na


28
16
13
0.007
0.050
0.000
0.249
3.359
1.459


29
12
7
0.007
0.095
0.000
na
na
na


30
15
9
0.007
0.069
0.000
na
na
na


31
15
8
0.007
0.081
0.000
na
na
na


32
7
7
0.007
0.094
0.000
na
na
na


33
13
10
0.006
0.055
0.000
na
na
na


34
14
46
0.078
0.090
0.002
na
na
na


35
7
8
0.007
0.078
0.000
na
na
na


36
3
48
0.045
0.039
0.010
3.050
2.304
1.326


37
2
39
0.029
0.046
0.000
na
na
na


38
1
55
0.073
0.059
0.000
1.608
2.138
1.015


39
1
33
0.030
0.063
0.000
na
na
na


40
2
59
0.050
0.035
0.000
3.651
2.727
1.076


41
17
15
0.011
0.061
0.000
na
na
na


42
0
0
0.000
0.048
0.002
na
na
na


43
3
17
0.009
0.046
0.000
na
na
na


44
40
32
0.027
0.058
0.000
na
na
na


45
2
0
0.000
0.031
0.000
na
na
na


46
2
0
0.000
0.038
0.000
na
na
na


47
1
8
0.004
0.048
0.000
na
na
na


48
19
57
0.046
0.034
0.000
2.626
2.165
0.900


49
10
59
0.047
0.032
0.000
1.737
1.901
1.054


50
2
70
0.057
0.024
0.000
1.545
0.880
0.694





na: no analysis/measurement





















TABLE 15







Del









Acylation
content
Del
Cya
Pel
M
Q
K


Plant No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























51
4
10
0.006
0.056
0.000
na
na
na


52
16
12
0.006
0.039
0.002
na
na
na


53
34
84
0.156
0.030
0.000
5.100
1.056
0.511


54
32
89
0.131
0.017
0.000
3.907
0.803
0.431


55
29
89
0.098
0.013
0.000
3.687
0.453
0.226


56
21
83
0.083
0.017
0.000
2.679
0.817
0.431


57
14
8
0.007
0.082
0.000
na
na
na


58
9
44
0.034
0.041
0.002
2.258
2.054
0.672


59
7
51
0.040
0.038
0.000
2.246
2.151
0.765


60
0
7
0.008
0.111
0.000
na
na
na


61
1
48
0.069
0.073
0.000
1.558
1.730
0.565


62
13
0
0.000
0.036
0.000
na
na
na


63
16
14
0.005
0.029
0.000
na
na
na





na: no analysis/measurement






Example 17
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS116
Plasmid pSPB130 (FIG. 7) was transferred into the pale violet rose variety “WKS116”, and 282 transformants were obtained. Accumulation of delphinidin was confirmed in 33 of the 36 pigment-analyzed plants (Tables 16 and 17). The delphinidin content was 80% at maximum (average: 73%). The flower color was altered from RHS Color Chart 196d (Greyed-Green group) to 186d (Greyed-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.














TABLE 16







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















1
1.8
78
0.015
0.004
0.746
0.753
0.507


2
12.7
78
0.097
0.028
1.826
2.352
1.572


3
5.9
78
0.030
0.009
1.000
1.452
0.934


4
0.0
76
0.030
0.010
0.813
0.990
0.480


5
2.6
72
0.038
0.015
1.279
1.835
0.832


6
0.0
72
0.019
0.007
0.839
0.983
0.642


7
3.1
75
0.033
0.011
1.131
1.476
0.877


8
1.9
75
0.028
0.009
0.761
0.977
0.466


9
2.6
76
0.034
0.011
na
na
na


10
2.7
73
0.031
0.011
na
na
na


11
4.4
77
0.033
0.010
1.001
1.003
0.618


12
7.0
74
0.035
0.012
0.849
0.945
0.577


13
9.3
74
0.025
0.009
na
na
na


14
3.2
80
0.044
0.011
1.045
0.959
0.545


15
4.5
75
0.031
0.010
1.115
1.256
0.729


16
10.5
71
0.028
0.012
1.055
1.155
0.670


17
1.7
51
0.016
0.016
0.330
1.537
1.052


18
10.5
77
0.112
0.033
2.008
2.976
2.216


19
0.0
0
0.000
0.010
na
na
na


20
0.0
30
0.007
0.015
na
na
na


21
na
56
0.013
0.010
0.197
1.960
1.463


22
4.4
47
0.006
0.007
na
na
na


23
3.6
77
0.026
0.008
na
na
na





na: no analysis/measurement




















TABLE 17







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















24
7.2
82
0.028
0.006
1.295
1.272
0.805


25
3.5
83
0.035
0.007
na
na
na


26
17.4
26
0.009
0.025
na
na
na


27
39.3
91
0.101
0.010
3.499
0.563
0.178


28
28.2
85
0.047
0.005
na
na
na


29
0.0
0
0.000
0.025
na
na
na


30
10.4
89
0.092
0.012
na
na
na


31
1.9
0
0.000
0.036
na
na
na


32
5.8
76
0.027
0.009
na
na
na


33
16.8
88
0.066
0.009
na
na
na


34
10.5
87
0.103
0.015
na
na
na


35
13.7
38
0.021
0.034
na
na
na


36
18.3
95
0.051
0.003
na
na
na





na: no analysis/measurement






Example 18
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS124
Plasmid pSPB130 (FIG. 7) was transferred into the pale orange rose variety “WKS124”, and 0.50 transformants were obtained. Accumulation of delphinidin was confirmed in 13 of the 15 pigment-analyzed plants (Table 18). The delphinidin content was 95% at maximum (average: 82%). The flower color was altered from RHS Color Chart 52d (Red group) to 71c (Red-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.















TABLE 18







Del








Plant
Acylation
content
Del
Cya
Pel
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























1
0.6
0
0.000
0.013
0.069
na
na
na


2
35.5
75
0.256
0.051
0.034
0.066
0.093
1.190


3
43.0
78
0.385
0.068
0.041
0.039
0.046
1.197


4
44.2
85
0.811
0.120
0.028
0.106
0.094
1.021


5
na
86
0.907
0.123
0.024
0.219
0.066
0.852


6
4.6
0
0.000
0.023
0.075
na
na
na


7
7.9
90
1.498
0.169
0.008
0.905
0.143
0.679


8
8.4
90
1.403
0.146
0.008
0.971
0.145
0.827


9
26.7
88
0.521
0.066
0.003
0.623
0.108
0.853


10
21.9
89
0.504
0.058
0.003
0.636
0.098
0.727


11
26.0
85
0.928
0.145
0.019
0.424
0.152
0.455


12
3.8
95
1.017
0.058
0.000
1.161
0.140
0.262


13
11.6
84
0.939
0.156
0.025
0.748
0.128
0.262


14
38.5
69
0.166
0.071
0.007
0.000
0.059
0.776


15
27.1
55
0.137
0.040
0.074
0.000
0.021
2.330





na: no analysis/measurement






Example 19
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS132
Plasmid pSPB130 (FIG. 7) was transferred into the bright red rose variety “WKS132”, and 24 transformants were obtained. Accumulation of delphinidin was confirmed in 6 of the 7 pigment-analyzed plants (Table 19). The delphinidin content was 43% at maximum (average: 12%). The flower color was altered from RHS Color Chart 57a (Red-Purple group) to 66a (Red-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.












TABLE 19







Del





Plant
Acylation
content
Del
Cya
Pel


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)




















1
1.8
0.4
0.008
1.872
0.009


2
1.0
0.0
0.000
1.409
0.010


3
21.3
11.4
0.237
1.841
0.007


4
6.8
42.5
0.461
0.619
0.006


5
7.6
9.5
0.204
1.936
0.011


6
na
1.3
0.016
1.227
0.007


7
23.7
5.4
0.081
1.407
0.005









Example 20
Transfer of Pansy F3′5′H gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS133
Plasmid pSPB130 (FIG. 7) was transferred into the dark red-violet rose variety “WKS133”, and 16 transformants were obtained. Accumulation of delphinidin was confirmed in all eight of the pigment-analyzed plants (Table 20). The delphinidin content was 34% at maximum (average: 11%). The flower color was altered from RHS Color Chart 53a (Red group) to 61a (Red-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.
















TABLE 20







Del









Plant
Acylation
content
Del
Cya
Pel
Peo
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
























1
10.3
23.7
1.322
4.253
0.009
0.004
0.691
0.792
0.133


2
11.8
33.8
1.192
2.324
0.005
0.003
0.621
0.422
0.093


3
6.1
12.9
0.009
0.060
0.000
0.000
0.102
0.500
0.048


4
3.8
9.1
0.363
3.627
0.005
0.008
na
na
na


5
15.8
2.0
0.078
3.774
0.009
0.000
0.045
0.939
0.472


6
11.5
2.7
0.135
4.771
0.011
0.005
0.046
0.576
0.034


7
13.3
3.0
0.180
5.800
0.009
0.009
0.100
0.937
0.179


8
12.2
3.5
0.161
4.470
0.009
0.009
0.068
0.738
0.148





na: no analysis/measurement






Example 21
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS137
Plasmid pSPB130 (FIG. 7) was transferred into the dark red-violet rose variety “WKS137”, and 20 transformants were obtained. Accumulation of delphinidin was confirmed in all 17 of the pigment-analyzed plants (Table 21). The delphinidin content was 1.3% at maximum (average: 0.4%). No alteration in flower color was observed from RHS Color Chart 61b (Red-Purple group).
















TABLE 21







Del









Plant
Acylation
content
Del
Cya
Pel
Peo
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
























1
0.5
0.3
0.008
2.821
0.037
0.000
na
na
na


2
0.8
0.3
0.010
3.384
0.051
0.000
na
na
na


3
0.4
0.3
0.005
1.982
0.014
0.000
na
na
na


4
0.6
0.2
0.008
3.344
0.057
0.000
na
na
na


5
0.7
0.4
0.011
3.145
0.035
0.000
na
na
na


6
0.7
1.3
0.025
2.919
0.040
0.003
na
na
na


7
0.4
0.3
0.008
2.820
0.045
0.000
na
na
na


8
0.5
0.4
0.010
2.467
0.042
0.000
na
na
na


9
0.7
0.2
0.010
3.836
0.024
0.000
na
na
na


10
0.1
0.5
0.008
1.743
0.016
0.000
na
na
na


11
0.7
0.4
0.011
2.593
0.027
0.003
na
na
na


12
0.6
0.3
0.007
2.393
0.022
0.000
0.048
3.026
2.812


13
1.4
0.2
0.009
3.756
0.065
0.000
na
na
na


14
0.7
0.4
0.008
2.149
0.024
0.001
na
na
na


15
0.8
0.5
0.007
2.281
0.041
0.000
na
na
na


16
0.5
0.5
0.007
1.314
0.014
0.000
na
na
na


17
1.0
0.2
0.007
2.892
0.051
0.000
na
na
na





na: no analysis/measurement






Example 22
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-acyltransferase gene into WKS140
Plasmid pSPB130 (FIG. 7) was transferred into the pale violet rose variety “WKS140”, and 197 transformants were obtained. Accumulation of delphinidin was confirmed in 37 of the 45 pigment-analyzed plants (Tables 22 and 23). The delphinidin content was 94% at maximum (average: 47%). The flower color was altered from RHS Color Chart 186d (Greyed-Purple group) to 79d (Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.














TABLE 22







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















1
3.5
0.0
0.000
0.090
na
na
na


2
2.5
0.0
0.000
0.093
0.096
2.429
0.246


3
5.5
63.5
0.061
0.035
0.688
1.090
0.106


4
13.2
17.7
0.013
0.059
na
na
na


5
5.4
11.6
0.017
0.129
na
na
na


6
3.6
12.3
0.011
0.078
na
na
na


7
13.6
11.7
0.009
0.069
na
na
na


8
4.1
22.3
0.012
0.041
0.057
1.950
0.492


9
3.3
0.0
0.000
0.071
na
na
na


10
2.6
18.6
0.017
0.076
na
na
na


11
4.2
18.6
0.012
0.052
0.130
3.101
1.172


12
6.5
25.0
0.026
0.079
0.251
2.300
0.592


13
1.3
0.0
0.000
0.062
0.000
2.200
0.552


14
22.7
85.4
0.261
0.045
1.649
0.943
0.126


15
20.9
57.4
0.093
0.069
0.481
1.418
0.182


16
16.4
39.9
0.052
0.078
na
na
na


17
15.2
50.8
0.074
0.072
na
na
na


18
6.1
22.6
0.036
0.111
0.148
2.152
0.279


19
2.7
0.0
0.000
0.033
na
na
na


20
9.1
52.6
0.041
0.037
na
na
na


21
4.4
46.2
0.075
0.087
na
na
na


22
8.5
34.7
0.040
0.075
0.195
1.847
0.394


23
11.0
30.9
0.018
0.040
0.155
1.106
0.142


24
13.4
46.8
0.056
0.063
na
na
na


25
2.8
5.1
0.006
0.107
na
na
na





na: no analysis/measurement




















TABLE 23







Del




K


Plant
Acylation
content
Del
Cya
M
Q
(mg/


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
g)






















26
4.1
6.8
0.007
0.098
na
na
na


27
31.4
93.4
0.252
0.018
1.434
0.361
0.052


28
13.4
86.7
0.101
0.016
1.237
1.740
0.499


29
32.3
94.2
0.200
0.012
0.862
0.131
0.029


30
13.0
89.7
0.176
0.020
0.553
0.289
0.026


31
12.3
87.1
0.150
0.022
1.007
0.674
0.135


32
6.7
9.9
0.009
0.086
na
na
na


33
11.5
67.4
0.108
0.052
na
na
na


34
5.0
11.2
0.014
0.110
0.074
2.588
0.659


35
12.5
79.7
0.088
0.022
1.192
1.185
0.574


36
15.0
83.4
0.065
0.013
1.478
1.147
0.570


37
1.8
0.0
0.000
0.068
na
na
na


38
1.3
44.3
0.105
0.132
0.582
3.259
1.232


39
2.5
73.6
0.114
0.041
na
na
na


40
14.0
85.3
0.165
0.028
1.881
1.035
0.180


41
0.5
4.3
0.006
0.144
na
na
na


42
9.9
53.3
0.040
0.035
0.373
1.038
0.164


43
33.5
87.4
0.275
0.040
1.851
0.701
0.148


44
1.3
0.0
0.000
0.073
na
na
na


45
1.5
0.0
0.000
0.062
na
na
na





na: no analysis/measurement






Example 23
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS77
Plasmid pSPB130 (FIG. 7) was transferred into the dark red-purple rose variety “WKS77”, and 35 transformants were obtained. Accumulation of delphinidin was confirmed in all 17 of the pigment-analyzed plants (Table 24). The delphinidin content was 57% at maximum (average: 33%). The flower color was altered from RHS Color Chart 57a (Red-Purple group) to 71a (Red-Purple group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.















TABLE 24







Del








Plant
Acylation
content
Del
Cya
Pel
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























1
6.2
42.5
1.153
1.552
0.008
0.484
0.679
0.196


2
7.6
38.6
0.618
0.979
0.005
0.267
0.465
0.094


3
3.9
40.4
0.706
1.030
0.011
1.266
1.768
0.722


4
2.0
46.9
0.372
0.417
0.004
0.363
0.608
0.276


5
5.4
40.6
0.540
0.784
0.005
1.077
1.809
0.645


6
2.0
44.7
1.078
1.325
0.009
0.516
1.034
0.382


7
2.1
46.5
0.398
0.453
0.005
0.353
0.792
0.569


8
5.8
39.7
0.647
0.980
0.005
0.425
0.706
0.183


9
4.7
40.0
0.844
1.268
0.000
0.310
0.764
0.199


10
7.6
39.7
1.345
2.033
0.009
0.350
0.635
0.119


11
14.1
2.9
0.068
2.274
0.013
na
na
na


12
12.8
6.9
0.126
1.688
0.009
na
na
na


13
12.7
4.2
0.109
2.468
0.012
0.060
1.541
0.366


14
13.0
20.9
0.704
2.669
0.000
0.407
2.502
0.694


15
19.3
43.5
1.011
1.308
0.007
0.357
0.843
0.276


16
19.6
6.1
0.092
1.414
0.010
0.120
1.740
0.477


17
22.8
56.6
1.068
0.814
0.004
0.604
0.503
0.126





na: no analysis/measurement






Example 24
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS82
Plasmid pSPB130 (FIG. 7) was transferred into the pale violet rose variety “WKS82”, and 89 transformants were obtained. Accumulation of delphinidin was confirmed in all 44 of the pigment-analyzed plants (Tables 25 and 26). The delphinidin content was 91% at maximum (average: 49%). The flower color was altered from RHS Color Chart 186d (Greyed-Purple group) to 80c (Purple-Violet group). However, no color of the Violet group, Violet-Blue group or Blue group according to the RHSCC was achieved and the target blue rose could not be obtained.















TABLE 25







Del








Plant
Acylation
content
Del
Cya
Pel
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























1
10.5
52.3
0.055
0.050
0.000
0.430
0.883
0.083


2
15.9
62.5
0.091
0.054
0.000
0.570
0.549
0.030


3
15.9
36.6
0.044
0.076
0.000
0.622
2.221
0.102


4
6.8
40.0
0.023
0.034
0.000
0.247
0.986
0.172


5
15.0
82.9
0.087
0.018
0.000
5.451
0.403
0.042


6
na
89.7
0.072
0.008
0.000
0.853
0.163
0.062


7
9.5
89.5
0.101
0.012
0.000
0.719
0.144
0.019


8
14.7
11.4
0.012
0.090
0.000
na
na
na


9
11.6
29.3
0.024
0.059
0.000
na
na
na


10
8.7
15.2
0.010
0.053
0.000
na
na
na


11
7.9
59.0
0.046
0.032
0.000
0.580
0.619
0.022


12
8.5
55.6
0.060
0.048
0.000
1.318
1.615
0.165


13
13.9
42.3
0.026
0.035
0.000
0.603
1.094
0.052


14
10.1
10.3
0.008
0.073
0.000
na
na
na


15
10.6
18.8
0.018
0.079
0.000
na
na
na


16
9.3
11.7
0.009
0.066
0.000
na
na
na


17
14.3
76.2
0.112
0.035
0.000
3.741
1.587
0.377


18
12.7
76.7
0.101
0.031
0.000
1.608
0.656
0.075


19
9.8
71.7
0.057
0.022
0.000
1.403
0.455
0.041


20
5.3
14.1
0.011
0.068
0.000
0.132
2.999
0.720


21
3.5
18.5
0.008
0.035
0.000
na
na
na


22
7.7
23.1
0.017
0.055
0.000
0.141
0.929
0.034


23
5.4
19.0
0.015
0.065
0.000
0.297
4.128
1.350





na: no analysis/measurement





















TABLE 26







Del








Plant
Acylation
content
Del
Cya
Pel
M
Q
K


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























24
1.1
42.1
0.036
0.050
0.000
0.609
2.929
0.679


25
22.7
91.0
0.079
0.008
0.000
0.964
0.218
0.018


26
6.1
61.3
0.048
0.030
0.000
0.490
0.468
0.029


27
8.7
91.3
0.097
0.009
0.000
2.053
0.339
0.123


28
9.4
59.9
0.060
0.040
0.000
1.537
1.631
0.422


29
5.5
51.2
0.040
0.038
0.000
0.688
0.723
0.038


30
5.1
61.4
0.056
0.032
0.003
0.637
0.537
0.087


31
7.0
53.3
0.037
0.032
0.000
0.706
1.032
0.051


32
5.7
58.1
0.071
0.051
0.000
1.592
1.478
0.220


33
4.3
64.6
0.092
0.050
0.000
0.849
0.753
0.035


34
6.4
61.7
0.042
0.026
0.000
0.477
0.468
0.023


35
8.9
58.8
0.048
0.034
0.000
0.646
0.928
0.063


36
6.2
11.6
0.007
0.057
0.000
0.094
1.132
0.066


37
7.1
51.2
0.038
0.036
0.000
0.911
1.135
0.079


38
5.8
50.8
0.029
0.028
0.000
0.868
1.105
0.096


39
5.5
47.0
0.027
0.023
0.007
1.366
1.632
0.105


40
4.9
67.0
0.044
0.022
0.000
0.795
0.586
0.051


41
na
61.1
0.053
0.033
0.000
1.310
1.466
0.259


42
9.6
71.0
0.074
0.030
0.000
0.460
0.337
0.023


43
1.2
27.6
0.009
0.024
0.000
na
na
na


44
5.2
13.8
0.013
0.078
0.000
na
na
na





na: no analysis/measurement






Example 25
Transfer of Pansy F3′5′H Gene (#40) and Torenia Anthocyanin 5-Acyltransferase Gene into WKS91
Plasmid pSPB130 (FIG. 7) was transferred into the light orange rose variety “WKS91”, and 10 transformants were obtained. Accumulation of delphinidin was confirmed in only one of the two pigment-analyzed plants (Table 27). The delphinidin content was 2% at maximum. No alteration in flower color was observed from RHS Color Chart 43c (Red group).












TABLE 27







Del





Plant
Acylation
content
Del
Cya
Pel


No.
(%)
(%)
(mg/g)
(mg/g)
(mg/g)







1
0.7
0.0
0.000
0.090
0.307


2
0.0
1.8
0.006
0.040
0.295









Example 26
Expression of Pansy F3′5′H Gene (#40) and Iris DFR Gene and Suppression of Rose Endogenous DFR Gene in Lavande
RNA was obtained from blue iris petals of cut flowers, and polyA+RNA was prepared therefrom. A cDNA library was prepared from the polyA+RNA with λZAPII (Stratagene) as the vector, using a cDNA library preparation kit (Stratagene) according to the manufacturer's recommended protocol. An iris DFR gene fragment was prepared by the same method as reported for obtaining gentian DFR gene fragment (Tanaka et al. Plant Cell Physiol. 37, 711-716 1996).
The approximately 400 bp DNA fragment obtained was recovered with Gene Clean according to the manufacturer's recommended protocol, and was subcloned in pCR-TOPO. Determination of the nucleotide sequence revealed a sequence homologous to the rose DFR gene. The DNA fragment was used for screening of the iris cDNA library, and iris DFR cDNA including the full-length amino acid sequence was obtained. The total nucleotide sequence of the cDNA in the clone designated as pSPB906 was determined. The nucleotide sequence is listed as SEQ ID NO: 9, and the corresponding amino acid sequence is listed as SEQ ID NO: 10.
Next, an approximately 3.9 kb DNA fragment obtained by digestion of pSPB580 with BamHI and XhoI was linked with an approximately 1.5 kb DNA fragment obtained by digestion of pSPB906 with BamHI and XhoI, and the obtained plasmid was designated as pSPB909.
A vector for transcription of double-stranded RNA for the rose DFR cDNA in plants was prepared in the following manner. An approximately 3.5 kb DNA fragment (including Mac1 promoter, rose DFR cDNA and mas terminator) obtained by partial digestion of pCGP1364 (Tanaka et al., Plant Cell Physiol. (1995) 36, 1023-1031) with PstI was inserted at the PstI site of pUC19 (Yanisch-Perron C et al., Gene 33:103-119, 1985) to obtain plasmids, among which a plasmid having the HindIII site of pUC19 near the MacI promoter was designated as pCGP1394.
Next, an approximately 1.4 kb DNA fragment obtained by digestion of pCGP1394 with HindIII and SacII was ligated with an approximately 1.9 kb DNA fragment obtained by digestion of pCGP1394 with PstI, blunting of the ends and further digestion with SacII, and with a binary vector fragment obtained by digestion of pBinPLUS with SacI, blunting of the ends and further digestion with HindIII, to obtain pSPB185. Plasmid pSPB185 was digested with XbaI, blunted and ligated with a SalI linker to obtain pSPB521. An approximately 700 bp DNA fragment obtained by digestion of pUE6 with HindIII and BamHI was ligated with a binary vector DNA fragment obtained by digestion of pSPB521 with HindIII and SacI and with a GUS gene fragment obtained by digestion of pE2113 with BamHI and SacI, to obtain pSPB528.
Plasmid pSPB528 is a binary vector having a structural gene inserted between the enhancer-containing cauliflower mosaic virus 35S promoter and the manopine synthase terminator, which is expressible in plants. Also, in order to shorten the 5′-end non-translated sequence of rose DFR cDNA in pCGP645, plasmid pCGP645 was digested with SmaI and PvuI, blunted and re-ligated to obtain pCGP645s.
The 5′-end sequence of rose DFR cDNA was obtained by PCR amplification using pCGP645s as the template and a reverse primer and the synthetic primer RDF310 (5′-CCCTCGAGCCCTTGATGGCCTCGTCG-3′) (SEQ ID NO: 19) as the primers, and was cloned in pCRTOPO. The DNA nucleotide sequence was determined and absence of errors by PCR was confirmed. This plasmid was designated as pSPB569. Also, a rose DFR cDNA 5′-end sequence with a different length was obtained by amplification using pCGP645s as the template and a reverse primer and the synthetic primer RDF830 (5′-GGGTCGACGCGGCCCTCTGCTTTCGG-3′) (SEQ ID NO: 20) as the primers, and was cloned in pCRTOPO. The DNA nucleotide sequence was determined and absence of errors by PCR was confirmed.
This plasmid was designated as pSPB570. A binary vector DNA fragment obtained by digestion of pSPB528 with BamHI and SacI, and an approximately 0.3 kb DNA fragment obtained by digestion of pSPB569 with Sad and XhoI, were ligated with a DNA fragment obtained by digestion of pSPB570 with BamHI and SalI, to obtain pSPB572. This vector is designed for transcription of double-stranded RNA for rose DFR cDNA in plants.
Plasmid pUE6 was digested with Sad and blunted, and a SalI linker was inserted to obtain pUE8. A DNA fragment obtained by digesting pUE8 with HindIII and EcoRI was introduced at the HindIII and EcoRI sites of pBinPLUS to obtain plasmid pSPB189. An approximately 3.7 kb DNA fragment obtained by digestion of pSPB189 with BamHI and SalI was ligated with an approximately 1.8 kb DNA fragment obtained by complete digestion of pCGP1961 with BamHI followed by partial digestion with XhoI, to obtain plasmid pSPB567. After PacI digestion and dephosphorylation treatment of pSPB572, it was linked with an approximately 2.8 kb DNA fragment obtained by digestion of pSPB567 with PacI, and a plasmid with transcription of the nptII gene and pansy F3′5′H #40 in the same direction was selected and designated as pSPB905.
After AscI digestion and dephosphorylation treatment of pSPB905, it was linked with an approximately 2.5 kb DNA fragment obtained by digestion of pSPB909 with AscI, and a plasmid with transcription of the iris DFR gene in the same direction as the nptII gene was obtained and designated as pSPB919 (FIG. 8). This plasmid is expected to allow transcription of the iris DFR gene and pansy F3′S′H #40 gene in rose, while suppressing expression of the rose DFR gene due to transcription of double-stranded RNA. The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pSPB919 (FIG. 8) was transferred into the pale violet rose variety “Lavande”, and 87 transformants were obtained. Accumulation of delphinidin was confirmed in 31 of the 38 pigment-analyzed plants (Tables 28 and 29). The delphinidin content was 100% at maximum (average: 76%). The flower color was altered from RHS Color Chart 186c (Greyed-Purple group) to 85a,b (Violet group).
RNA was extracted from rose petals in the same manner as explained above, and after separating the RNA by agarose gel electrophoresis, it was transferred onto Hybond N (Amersham) (for example, Tanaka et al., 1995). The mRNA was detected using a DIG Northern Starter Kit (Roche) by the manufacturer's recommended protocol. The rose DFR mRNA was detected using pCGP645 (Tanaka et al., Plant Cell Physiol. 36, 1023-1031, 1995) as template and a T7 primer transcript as the probe.
Detection of pansy F3′5′H #40 mRNA was accomplished using pCGP1961 as template and a T7 primer transcript as the probe. Detection of iris DFR mRNA was accomplished using pSPB906 as template and a T7 primer transcript as the probe. Pansy F3′5′H #40 and iris DFR gene mRNA were detected in the altered-color roses. On the other hand, rose DFR mRNA was significantly reduced compared to the host and a band was detected at the low molecular weight position, indicating decomposition of the rose DFR mRNA.













TABLE 28






Del







Plant
content
Del
Cya
M
Q
K


No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
0.0
0.000
0.105
0.036
0.856
0.038


2
0.0
0.000
0.125
na
na
na


3
0.0
0.000
0.091
0.023
0.851
0.101


4
0.0
0.000
0.116
0.000
1.336
0.087


5
0.0
0.000
0.048
na
na
na


6
88.5
0.086
0.011
1.626
1.187
0.411


7
90.8
0.089
0.009
0.797
1.548
0.087


8
84.0
0.046
0.009
0.163
0.699
0.016


9
87.8
0.062
0.009
0.193
0.760
0.022


10
89.3
0.072
0.009
0.210
0.575
0.033


11
91.5
0.049
0.005
0.398
0.805
0.050


12
91.5
0.032
0.003
0.100
0.811
0.014


13
85.7
0.040
0.007
0.092
0.497
0.012


14
64.9
0.040
0.021
0.263
0.327
0.015


15
88.3
0.041
0.005
na
na
na


16
66.4
0.011
0.006
0.036
1.221
0.030


17
79.7
0.008
0.002
0.030
0.765
0.009


18
100.0
0.010
0.000
0.048
1.343
0.067


19
95.9
0.040
0.002
0.159
0.136
0.004


20
65.4
0.016
0.008
0.090
1.244
0.048


21
18.8
0.011
0.049
0.048
0.855
0.020


22
0.0
0.000
0.110
0.000
1.274
0.079


23
0.0
0.000
0.140
0.000
1.952
0.200





na: no analysis/measurement



















TABLE 29






Del







Plant
content
Del
Cya
M
Q
K


No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)







24
41.4
0.102
0.144
0.265
0.417
0.015


25
34.3
0.042
0.081
0.167
0.429
0.024


26
34.6
0.023
0.043
na
na
na


27
41.4
0.082
0.116
0.232
0.385
0.019


28
37.7
0.046
0.076
0.254
0.429
0.018


29
36.1
0.032
0.057
0.151
0.235
0.042


30
97.2
0.052
0.002
0.208
0.088
0.004


31
93.0
0.038
0.003
0.347
0.137
0.007


32
98.2
0.101
0.002
0.339
0.258
0.029


33
91.3
0.039
0.004
na
na
na


34
91.9
0.041
0.004
0.332
0.120
0.007


35
96.8
0.052
0.002
na
na
na


36
96.7
0.084
0.003
0.342
0.168
0.010


37
88.0
0.014
0.002
0.076
1.000
0.029


38
84.5
0.016
0.003
0.074
1.121
0.025





na: no analysis/measurement






Example 27
Expression of Pansy F3′5′H Gene (#40) and Nierembergia DFR gene, and Suppression of Rose Endogenous DFR gene in Lavande
RNA was obtained from petals of the Nierembergia hybrida cultivar Fairy Bell Patio Light Blue (Suntory Flowers Co., Ltd.), and polyA+RNA was prepared therefrom. A cDNA library was prepared from the polyA+RNA with λZAPII (Stratagene) as the vector, using a cDNA library synthesis kit (Stratagene) according to the manufacturer's recommended protocol. The cDNA library was screened using DIG-labeled petunia DFR cDNA (from pCGP1405).
The screening conditions were according to the plaque hybridization method using a DIG-labeling system, according to the manufacturer's recommended protocol. However, the formaldehyde concentration was 30% for the pre-hybridization and hybridization buffers, and hybridization was carried out overnight at 37° C. The membrane was rinsed at 55° C. in 5×SSC containing 1% SDS. Plasmids were recovered from 20 plaques among the numerous positive signals, and their nucleotide sequences were determined using Reverse Primer (Takara). These exhibited high homology with the DFR genes of other plants including petunia. The total nucleotide sequence of the cDNA in the clone designated as pSPB709 was determined. The nucleotide sequence is listed as SEQ ID NO: 11, and the corresponding amino acid sequence is listed as SEQ ID NO: 12
An approximately 3.9 kb DNA fragment obtained by digestion of pSPB580 with BamHI and XhoI was linked with an approximately 1.5 kb DNA fragment obtained by digestion of pSPB709 with BamHI and XhoI, to obtain plasmid pSPB910. After AscI digestion and dephosphorylation treatment of pSPB910, it was linked with an approximately 2.5 kb DNA fragment obtained by digestion of pSPB910 with AscI, and a plasmid with transcription of the Nierembergia DFR gene in the same direction as the nptII gene was obtained and designated as pSPB920 (FIG. 9). This plasmid is expected to allow transcription of the Nierembergia DFR gene and pansy F3′5′H #40 gene in rose, while suppressing expression of the rose DFR gene due to transcription of double-stranded RNA. The plasmid was transferred into Agrobacterium tumefaciens Ag10.
Plasmid pSPB920 (FIG. 9) was transferred into the pale violet rose variety “Lavande”, and 56 transformants were obtained. Accumulation of delphinidin was confirmed in 23 of the 24 pigment-analyzed plants (Table 30). The delphinidin content was 100% at maximum (average: 43%). The flower color was altered from RHS Color Chart 186c (Greyed-Purple group) to 85b (Violet group).













TABLE 30






Del







Plant
content
Del
Cya
M
Q
K


No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)





















1
69.5
0.025
0.002
0.081
2.265
0.066


2
85.4
0.024
0.004
0.114
1.355
0.032


3
71.8
0.006
0.002
0.043
0.781
0.027


4
100.0
0.012
0.000
0.414
0.283
0.030


5
88.2
0.015
0.002
0.506
0.126
0.030


6
100.0
0.013
0.000
0.430
0.123
0.008


7
33.3
0.019
0.038
na
na
na


8
37.3
0.012
0.020
na
na
na


9
48.2
0.012
0.013
na
na
na


10
18.9
0.011
0.049
0.053
1.023
0.022


11
39.7
0.037
0.056
0.120
1.157
0.035


12
9.4
0.010
0.095
na
na
na


13
11.0
0.008
0.062
na
na
na


14
24.4
0.017
0.054
0.128
1.852
0.181


15
12.4
0.015
0.102
na
na
na


16
89.7
0.089
0.010
0.530
1.424
0.165


17
15.4
0.006
0.035
na
na
na


18
22.3
0.006
0.019
0.018
1.286
0.038


19
10.4
0.007
0.058
0.039
1.673
0.045


20
28.3
0.006
0.015
0.028
0.932
0.025


21
35.2
0.015
0.028
0.105
0.743
0.028


22
16.0
0.010
0.052
na
na
na


23
0.0
0.000
0.018
0.013
1.764
0.027


24
13.7
0.007
0.042
0.033
1.469
0.041





na: no analysis/measurement






Example 28
Inheritance of Traits to Progeny
Cross-breeding was carried out using a transformant (LA/919-2-13) obtained by transfer of pSPB919 (FIG. 8) into the pale violet rose variety “Lavande” as the pollen parent and non-recombinant WKS77 or WKS133 as the maternal parent (Suzuki, S., “Bara, Hanazufu”, Shogakkann, p. 256-260, 1990). Fruit was collected on the 100th day after pollination. Seed production was accomplished by first peeling the fruit, harvesting the achene, peeling the achene, and then removing the germ and embedding it on moistened filter paper in a dish. The water used for seed production was sterilized water containing 1 ml/l PPMTM (Plant Preservative Mixture, Plant Cell Technology, Inc.) and 50 mg/l kanamycin, and seedlings were raised by potting only the normally budded plants.
Accumulation of delphinidin was confirmed in all 40 of the pigment-analyzed transformant progeny (Tables 31 and 32). The delphinidin content was 99% at maximum (average: 46%).














TABLE 31








Del







Plant
content
Del
Cya
Pel
Peo



No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























1
89.8
0.494
0.056
0.000
0.000



2
96.1
3.900
0.153
0.005
0.000



3
55.9
0.836
0.660
0.000
0.000



4
24.6
0.041
0.127
0.000
0.000



5
23.5
1.108
3.605
0.009
0.002



6
25.9
0.191
0.545
0.003
0.000



7
0.5
0.013
2.552
0.012
0.002



8
75.8
0.283
0.090
0.000
0.000



9
95.9
1.420
0.061
0.000
0.000



10
30.8
0.862
1.841
0.007
0.105



11
13.3
0.068
0.441
0.004
0.000



12
23.9
0.529
1.667
0.023
0.000



13
43.7
0.280
0.362
0.000
0.000



14
19.3
0.035
0.145
0.000
0.000



15
0.6
0.008
1.418
0.021
0.000



16
20.8
0.048
0.183
0.000
0.000



17
92.5
2.257
0.177
0.007
0.000



18
66.4
2.496
1.247
0.015
0.000



19
42.4
0.369
0.497
0.004
0.000



20
75.6
0.597
0.183
0.010
0.000



21
19.6
0.271
1.103
0.008
0.000



22
71.0
0.107
0.044
0.000
0.000



23
0.6
0.006
0.850
0.004
0.000
























TABLE 32








Del







Plant
content
Del
Cya
Pel
Peo



No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)























24
16.7
0.053
0.263
0.000
0.000



25
71.8
0.211
0.083
0.000
0.000



26
18.6
0.177
0.769
0.003
0.000



27
1.3
0.009
0.652
0.004
0.000



28
59.7
0.183
0.124
0.000
0.000



29
39.6
0.124
0.187
0.003
0.000



30
21.4
0.187
0.684
0.003
0.000



31
0.6
0.005
0.763
0.004
0.000



32
38.8
0.226
0.353
0.003
0.000



33
50.5
0.154
0.151
0.000
0.000



34
28.0
0.267
0.682
0.003
0.000



35
83.9
0.204
0.039
0.000
0.000



36
64.9
0.380
0.205
0.000
0.000



37
78.8
0.239
0.064
0.000
0.000



38
97.4
0.614
0.016
0.000
0.000



39
98.7
0.805
0.011
0.000
0.000



40
54.9
0.083
0.068
0.000
0.000










Example 29
Expression of Pansy F3′5′H #40 Gene and Iris DFR Gene and Suppression of Rose Endogenous DFR Gene in WKS140
Plasmid pSPB919 was transferred into the pale violet rose variety “WKS140”, and 89 transformants were obtained. Accumulation of delphinidin was confirmed in 74 of the 79 pigment-analyzed plants. The delphinidin content was 100% at maximum (average: 68%). The flower color was altered from RHS Color Chart 186d (Greyed-Purple group) to primarily 84c (Violet group).











TABLE 33





Plant
Del
Del
Cya
Pel


No.
(%)
(mg/g)
(mg/g)
(mg/g)



















1
0.0%
0.0000
0.0423
0.0000


2
89.9%
0.0242
0.0027
na


3
90.0%
0.0245
0.0027
na


4
88.6%
0.0093
0.0012
na


5
43.5%
0.0042
0.0054
na


6
91.2%
0.0118
0.0011
na


7
81.2%
0.0027
0.0006
na


8
81.0%
0.0173
0.0041
na


9
73.9%
0.0733
0.0259
na


10
62.9%
0.0321
0.0190
na


11
91.9%
0.0962
0.0084
na


12
99.1%
0.1606
0.0015
na


13
94.7%
0.0588
0.0033
na


14
100.0%
0.0839
0.0000
na


15
0.0%
0.0000
0.0005
na


16
98.4%
0.0296
0.0005
na


17
80.4%
0.1748
0.0451
na


18
94.6%
0.0190
0.0000
na


19
0.0%
0.0000
0.0714
na


20
34.3%
0.0099
0.0191
na


21
30.9%
0.0126
0.0282
na


22
65.6%
0.0294
0.0154
na


23
24.1%
0.0205
0.0646
na





na: no analysis/measurement






Example 30
Expression of Pansy F3′5′H #40 Gene and Iris DFR Gene and Suppression of Rose Endogenous DFR Gene in WKS77
Plasmid pSPB919 was transferred into the dark red-purple rose variety “WKS77”, and 50 transformants were obtained. Accumulation of delphinidin was confirmed in 21 of the 23 pigment-analyzed plants. The delphinidin content was 81% at maximum (average: 19%). The flower color was altered from RHS Color Chart 57a (Red-Purple group) to 77b (Purple group).











TABLE 34





Plant
Del
Del
Cya
Pel


No.
(%)
(mg/g)
(mg/g)
(mg/g)



















1
26.0%
1.2028
3.4033
0.0117


2
41.5%
0.6473
0.9093
0.0048


3
80.8%
0.2210
0.0526
na


4
68.0%
0.1865
0.0878
na


5
68.5%
0.2090
0.0951
0.0010


6
1.5%
0.0119
0.7731
0.0051


7
1.5%
0.0114
0.7304
0.0041


8
0.2%
0.0069
2.9266
0.0063


9
0.2%
0.0017
1.0791
0.0062


10
0.0%
0.0000
0.5013
0.0043


11
0.1%
0.0028
2.3418
0.0110


12
0.4%
0.0091
2.4603
0.0126


13
0.2%
0.0040
1.7766
0.0096


14
0.3%
0.0026
0.9046
0.0052


15
0.0%
0.0000
1.6063
0.0100


16
22.2%
0.3279
1.1392
0.0049


17
24.0%
0.2638
0.8288
0.0052


18
1.4%
0.0240
1.6777
0.0118


19
1.1%
0.0186
1.6352
0.0101


20
26.7%
0.2645
0.7230
0.0037


21
22.7%
0.2200
0.7460
0.0046


22
40.1%
0.8929
1.3374
0.0071





na: no analysis/measurement






Example 31
Expression of Pansy F3′5′H #40 Gene and Nierembergia DFR Gene and Suppression of Rose Endogenous DFR Gene in WKS77
Plasmid pSPB920 was transferred into the dark red-purple rose variety “WKS77”, and 30 transformants were obtained. Accumulation of delphinidin was confirmed in 26 of the 27 pigment-analyzed plants. The delphinidin content was 98% at maximum (average: 60%). The flower color was altered from RHS Color Chart 57a (Red-Purple group) to 77b (Purple group).











TABLE 35





Plant
Del
Del
Cya
Pel


No.
(%)
(mg/g)
(mg/g)
(mg/g)



















1
93.9%
0.1679
0.0110
0.0000


2
97.6%
0.2311
0.0058
na


3
96.3%
0.1684
0.0065
na


4
97.1%
0.1012
0.0017
na


5
9.6%
0.0946
0.7810
0.1104


6
21.9%
0.1462
0.5166
0.0034


7
12.7%
0.1097
0.7495
0.0049


8
97.9%
0.1942
0.0042
na


9
98.1%
0.1228
0.0024
na


10
3.2%
0.0360
1.0689
0.0035


11
3.1%
0.0267
0.9587
0.0032


12
4.8%
0.1138
2.2562
0.0049


13
6.2%
0.1066
1.5999
0.0080


14
96.5%
0.3541
0.0132
na


15
2.1%
0.0173
0.7852
0.0068


16
94.7%
0.2898
0.0160
0.0000


17
96.7%
0.0819
0.0020
0.0000


18
95.8%
0.6969
0.0309
na


19
96.4%
0.4868
0.0181
na


20
64.3%
0.3092
0.1724
na


21
26.9%
0.2740
0.7431
0.0025


22
19.9%
0.3760
1.5028
0.0071


23
88.2%
0.0316
0.0042
na


24
94.2%
0.0259
0.0016
na


25
90.4%
0.0481
0.0051
na





na: no analysis/measurement






Example 32
Expression of Pansy F3′5′H#40 Gene and Petunia DFR Gene and Suppression of Rose Endogenous DFR Gene in WKS77
Plasmid pSPB921 was transferred into the dark red-purple rose variety “WKS77”, and 15 transformants were obtained. Accumulation of delphinidin was confirmed in 12 of the 13 pigment-analyzed plants. The delphinidin content was 98% at maximum (average: 60%). The flower color was altered from RHS Color Chart 57a (Red-Purple group) to 72b (Red-Purple group).











TABLE 36





Plant
Del
Del
Cya
Pel


No.
(%)
(mg/g)
(mg/g)
(mg/g)



















1
90.0%
0.0549
0.0061
na


2
38.4%
0.3397
0.5402
0.0041


3
56.9%
0.7834
0.5824
0.0099


4
58.5%
0.0196
0.0139
na


5
90.3%
0.1336
0.0144
na


6
90.9%
0.1251
0.0126
na


7
86.7%
0.1771
0.0274
na


8
91.6%
0.0113
0.0010
na


9
97.5%
0.0864
0.0022
na


10
9.5%
0.2687
2.6591
0.0000


11
8.8%
0.1421
1.4598
0.0071


12
0.4%
0.0060
1.3554
0.0053





na: no analysis/measurement






Example 33
Inheritance of Traits to Progeny
Cross-breeding was carried out in the same manner as Example 28, using a transformant (LA/919-4-10) obtained by transfer of pSPB919 into the pale violet rose variety “Lavande” as the pollen parent and the non-recombinant rose variety “Black Baccara” as the maternal parent. Fruit was collected on the 100th day after pollination. Seed production was accomplished by first peeling the fruit, harvesting the achene, peeling the achene, and then removing the germ and embedding it on moistened filter paper in a dish. The water used for seed production was sterilized water containing 1 ml/l PPMTM (Plant Preservative Mixture, Plant Cell Technology, Inc.) and 50 mg/l kanamycin, and seedlings were raised by potting only the normally budded plants.
Accumulation of delphinidin was confirmed in all 18 of the pigment-analyzed transformant progeny. The delphinidin content was 99.8% at maximum (average: 98.7%).











TABLE 37





Plant
Del
Del
Cya
Pel


No.
(%)
(mg/g)
(mg/g)
(mg/g)



















1
97.8%
0.6633
0.0142
0.0009


2
99.0%
0.9002
0.0096
na


3
98.5%
0.5385
0.0080
na


4
99.5%
2.0561
0.0087
0.0016


5
99.8%
1.6556
0.0034
na


6
96.6%
0.5601
0.0200
na


7
99.0%
0.6148
0.0063
na


8
98.9%
1.6867
0.0193
na


9
95.0%
0.5740
0.0304
na


10
96.9%
0.1152
0.0036
na


11
99.3%
0.0683
0.0005
na


12
99.6%
0.1248
0.0005
na


13
99.5%
0.3574
0.0010
0.0000


14
99.6%
0.5500
0.0021
na


15
99.6%
1.2322
0.0049
na


16
99.7%
1.4384
0.0042
na


17
99.8%
0.5117
0.0010
na


18
98.3%
0.8073
0.0140
na





na: no analysis/measurement






Example 34
Expression of Pansy F3′5′H #40 Gene and Suppression of Rose Endogenous F3′H Gene in WKS77
Plasmid pSPB1106 (FIG. 10) was transferred into the dark red-purple rose variety “WKS77”, and 40 transformants were obtained. Accumulation of delphinidin was confirmed in all 26 of the pigment-analyzed plants. The delphinidin content was 80.0% at maximum (average: 30.5%). The flower color underwent a major alteration from RHS Color Chart 57a (Red-Purple group) to 83d (Violet group).














TABLE 38





Plant
Del
Del
Cya
Pel
M
Q
K


No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)






















1
68.7%
0.5497
0.2275
0.0241
na
na
na


2
78.8%
0.3449
0.0830
0.0096
na
na
na


3
80.0%
0.6949
0.1604
0.0144
na
na
na


4
71.2%
0.4377
0.1563
0.0214
na
na
na


5
72.7%
0.5260
0.1715
0.0266
0.3812
0.2275
1.7669


6
70.7%
0.3829
0.1449
0.0146
na
na
na


7
10.3%
0.0358
0.3031
0.0071
na
na
na


8
15.6%
0.1847
0.9530
0.0444
na
na
na


9
4.8%
0.0739
1.4586
0.0149
na
na
na


10
1.1%
0.0114
1.0411
0.0144
na
na
na


11
54.0%
1.3206
1.1166
0.0092
na
na
na


12
57.8%
0.8842
0.6410
0.0056
na
na
na


13
0.9%
0.0242
2.5500
0.0168
na
na
na


14
23.0%
0.2087
0.6909
0.0062
na
na
na


15
12.7%
0.1645
1.1271
0.0058
na
na
na


16
26.4%
0.5275
1.4645
0.0132
na
na
na


17
18.7%
0.3555
1.5310
0.0109
na
na
na


18
24.2%
0.4388
1.3687
0.0072
na
na
na


19
64.7%
0.4029
0.1945
0.0249
0.6368
0.3949
2.0567


20
0.1%
0.0021
1.8646
0.0077
na
na
na


21
0.0%
0.0000
0.9708
0.0062
na
na
na


22
0.1%
0.0022
2.6049
0.0127
na
na
na


23
0.4%
0.0066
1.8002
0.0066
na
na
na


24
0.5%
0.0079
1.4670
0.0056
0.0000
1.3096
0.2414


25
17.3%
0.1000
0.4671
0.0099
na
na
na


26
18.3%
0.1232
0.5418
0.0052
na
na
na





na: no analysis/measurement






Example 35
Expression of Pansy F3′5′H #40 Gene and Suppression of Rose Endogenous F3′H Gene in Lavande
Plasmid pSPB1106 was transferred into the pale violet rose variety “Lavande”, and 40 transformants were obtained. Accumulation of delphinidin was confirmed in 23 of the 25 pigment-analyzed plants. The delphinidin content was 98.3% at maximum (average: 46.9%).














TABLE 39





Plant
Del
Del
Cya
Pel
M
Q
K


No.
(%)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g)






















1
76.8%
0.0732
0.0188
0.0032
0.5705
0.1595
0.3073


2
80.1%
0.1441
0.0296
0.0061
0.5298
0.1881
4.3294


3
3.7%
0.0086
0.2174
0.0027
na
na
na


4
4.4%
0.0079
0.1691
0.0034
na
na
na


5
8.8%
0.0158
0.1557
0.0070
na
na
na


6
39.0%
0.0212
0.0128
0.0204
0.0000
0.0363
1.3107


7
44.4%
0.0089
0.0027
0.0084
0.0756
0.0573
1.3689


8
40.4%
0.0165
0.0071
0.0172
0.0365
0.0592
2.5211


9
42.0%
0.0087
0.0036
0.0084
0.0752
0.0596
1.2661


10
13.5%
0.0153
0.0939
0.0040
0.1288
1.0594
0.5440


11
81.6%
0.2252
0.0447
0.0061
0.3947
0.1401
0.3947


12
78.8%
0.1022
0.0239
0.0036
0.6700
0.2137
0.5847


13
81.7%
0.2125
0.0438
0.0036
1.3616
0.4621
0.7478


14
80.9%
0.1829
0.0388
0.0044
0.4100
0.2405
0.0567


15
70.9%
0.0664
0.0204
0.0069
0.4230
0.1221
0.1788


16
0.0%
0.0000
0.0844
0.0000
na
na
na


17
98.0%
0.2363
0.0048
0.0000
0.0000
1.0613
0.2698


18
98.3%
0.1398
0.0025
0.0000
0.0479
0.7060
0.1299


19
4.2%
0.0078
0.1724
0.0040
0.0000
0.8627
0.2075


20
0.0%
0.0000
0.1696
0.0043
na
na
na


21
60.0%
0.0333
0.0115
0.0107
0.0000
0.0740
1.8678


22
14.3%
0.0091
0.0454
0.0088
0.1096
0.5305
0.6453


23
15.1%
0.0082
0.0408
0.0053
na
na
na


24
17.6%
0.0082
0.0324
0.0059
na
na
na


25
24.4%
0.0147
0.0375
0.0080
0.0000
0.2147
0.9765





na: no analysis/measurement






These results demonstrate that the transferred exogenous gene was inherited and expressed by the progeny, and that the trait of delphinidin production which is not found in ordinary rose petals was successfully inherited by the rose progeny. Thus, this gene can be used for cross-breeding cultivation of roses with altered colors to create roses with new colors including blue and purple.
INDUSTRIAL APPLICABILITY
By artificially suppressing function of the endogenous metabolic pathway such as, for example, expression of dihydroflavonol reductase, in rose, and expressing the gene coding for pansy flavonoid 3′,5′-hydroxylase and a gene coding for dihydroflavonol reductase from species other than rose, it is possible to create blue to violet roses. These genes are inherited by subsequent generations, and the blue rose trait can be utilized for cross-breeding.

## Claims

1. A method for producing a rose, comprising:
artificially suppressing a rose flavonoid synthesis pathway by artificially suppressing expression of either a rose endogenous dihydroflavonol reductase (DFR) using a double-stranded RNA (dsRNA) of the DFR gene or a rose endogenous flavonoid 3′-hydroxylase (F3′H) using a dsRNA of the F3′H gene; and
expressing a pansy gene coding for flavonoid 3′,5′-hydroxylase (F3′5′H) having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

2. The method for producing a rose according to claim 1, further comprising expressing a gene coding for dihydroflavonol reductase derived from a plant other than rose.

3. The method for producing a rose according to claim 1, wherein the plant other than rose is selected from the group consisting of iris, Nierembergia, and petunia.

4. The rose obtained by the method according to any one of claim 1, 2, or 3, or a progeny or a tissue thereof, the progeny or the tissue comprising the dsRNA of the DFR gene or the dsRNA of the F3′H gene and the pansy gene coding for F3′5′H, having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

5. The rose, progeny, or tissue thereof, according to claim 4, wherein the petal color of the rose is violet.

6. The rose, progeny, or tissue thereof, according to claim 4, wherein the petal color of the rose belongs to the “Violet group”, according to the Royal Horticultural Society Colour Chart (RHSCC).

7. The rose, progeny, or tissue thereof, according to claim 4, wherein the petal color of the rose belongs to “Violet group” 85a or 85b according to the Royal Horticultural Society Colour Chart (RHSCC).

8. A method for producing a rose, comprising:
artificially suppressing a rose flavonoid synthesis pathway by artificially suppressing expression of a rose endogenous dihydroflavonol reductase (DFR) using a dsRNA of the DFR gene;
expressing a pansy gene coding for flavonoid 3′,5′-hydroxylase (F3′5′H) having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and
expressing a gene coding for a dihydroflavonol reductase derived from a plant other than rose.

9. The method for producing a rose according to claim 8, wherein the plant other than rose is selected from the group consisting of iris, Nierembergia, and petunia.

10. The rose obtained by the method according to claim 8, or a progeny or a tissue thereof, the progeny or the tissue comprising the dsRNA of the DFR gene and the pansy gene coding for F3′5′H having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

11. The rose, the progeny, or the tissue thereof, according to claim 10, wherein the petal color of the rose is violet.

12. The rose, the progeny, or the tissue according to claim 10, wherein the petal color of the rose belongs to the “Violet group”, according to the Royal Horticultural Society Colour Chart (RHSCC).

13. The rose, or progeny or tissue thereof, according to claim 10, wherein the petal color of the rose belongs to “Violet group” 85a or 85b according to the Royal Horticultural Society Colour Chart (RHSCC).