# US 11,299,742 B2 · Plant having blue flower color and breeding method therefor

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,299,742 B2
- Document ID: US11299742B2
- Status: Granted
- Kind: B2
- Application number: 16/088,943
- Filed: 2017-03-13
- Published: 2020-10-15
- Granted: 2022-04-12
- Earliest claimed: 2016-03-31
- Assignees: Suntory Holdings Limited
- Applicants: Suntory Holdings Limited
- Inventors: Naonobu Noda; Masayoshi Nakayama; Mitsuru Douzono; Satoshi Hongo; Ryutaro Aida; Yukihisa Katsumoto
- Web page: https://patentgrep.com/patent/US11299742B2

## CPC

- A01H 3/00: Processes for modifying phenotypes {, e.g. symbiosis with bacteria} (A01H4/00 takes precedence)
- C12N 15/825: {involving pigment biosynthesis}
- A01H 5/02: Flowers
- A01H 6/1424: {Chrysanthemum}
- A01H 6/305: {Dianthus carnations}
- A01H 6/749: {Rosa, i.e. roses}
- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- C12N 5/10: Cells modified by introduction of foreign genetic material
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The purpose of the present invention is to provide a breeding method for a plant having a blue flower color with a simpler blue color development controlling technique, without requiring complex mechanisms for blue color development that have been previously presented and techniques reproducing such mechanisms. Delphinidin-based anthocyanins, in which the both 3′ and 5′-positions of the anthocyanin B-ring have been glycosylated, and flavone glycosides or flavonol glycosides as copigment are made to coexist in the cells of flower organ such as petals.

## Claims

### Claim 1

A method of creating a plant with a blue flower color, the method comprising the step of transforming the cells of the plant to express a Campanula-derived flavonoid 3′,5′-hydroxylase transgene (CamF3′5′H) and a Clitoria-derived anthocyanin 3′,5′-O-glucosyltransferase transgene (CtA3′5′GT) so that delphinidin-based anthocyanins having glycosyl-residues at both the 3′- and 5′-positions of the anthocyanin B-ring coexist with a flavone glycoside or flavonol glycoside in the cells of the plant,
wherein the transformation is carried out by:
introducing the Clitoria-derived anthocyanin 3′,5′-O-glucosyltransferase transgene into the cells of the plant;
further introducing the Campanula-derived flavonoid 3′,5′-hydroxylase transgene into the cells of the plant when the cells of the plant do not accumulate any delphinidin-based anthocyanins; and
further optionally introducing a flavone synthase transgene or a flavonol synthase transgene into the cells of the plant when the cells of the plant do not accumulate any flavone glycosides or flavonol glycosides,
wherein the plant is rose, lily, carnation, dahlia, Phalaenopsis aphrodite, or chrysanthemum,
wherein the delphinidin-based anthocyanins having glycosyl groups at both the 3′- and 5′-positions of the anthocyanin B-ring and the flavone glycoside or the flavonol glycoside coexist in a quantity ratio of 1:5 to 1:10, and wherein the intravacuolar pH in the cells of the plant is 5.2 to 6.4.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the flavone glycoside is selected from the group consisting of luteolin glycoside, tricetin glycoside, apigenin glycoside, acacetin glycoside, and their combinations.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the luteolin glycoside is luteolin 7-malonyl glucoside, luteolin 7-glucoside, luteolin 7,3′-diglucoside, luteolin 8-C-glucoside, luteolin 6-C-glucoside, or a derivative thereof.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the tricetin glycoside is tricetin 7-malonyl glucoside or a derivative thereof.

### Claim 5 (depends on 2)

The method according to claim 2, wherein the apigenin glycoside is apigenin 7-glucoside, apigenin 7-rutinoside, apigenin 8-C-glucoside, apigenin 6-C-glucoside, or a derivative thereof.

### Claim 6 (depends on 2)

The method according to claim 2, wherein the acacetin glycoside is acacetin 7-rutinoside or a derivative thereof.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the flavonol glycoside is selected from the group consisting of kaempferol glycoside, quercetin glycoside, and their combinations.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the kaempferol glycoside is kaempferol 3-glucoside or a derivative thereof.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the quercetin glycoside is quercetin 3-glucoside, quercetin 3-(6″-malonyl) glucoside, quercetin 3-rutinoside, or a derivative thereof.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the delphinidin-based anthocyanins having glycosyl groups at both the 3′- and 5′-positions of the anthocyanin B-ring are selected from the group consisting of delphinidin 3-(6″-malonyl)glucoside-3′,5′-diglucoside (ternatin C5), delphinidin 3,3′,5′-triglucoside (preternatin C5), and their combinations.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the plant is rose.

### Claim 12

A transformed plant with a blue flower color, its inbred progeny, or its outbred progeny, wherein the plant is rose, lily, carnation, dahlia, Phalaenopsis aphrodite, or chrysanthemum,
wherein the cells of the transform plant, the inbred progeny, or the outbred progeny express a Campanula-derived flavonoid 3′,5′-hydroxylase transgene (CamF3′5′H) and a Clitoria-derived anthocyanin 3′,5′-O-glucosyltransferase transgene (CtA3′5′GT),
wherein delphinidin-based anthocyanins having glycosyl groups at both the 3′- and 5′-positions of the anthocyanin B-ring coexist with a copigment, in a quantity ratio of 1:5 to 1:10, in the cells of the transformed plant, the inbred progeny, or the outbred progeny,
wherein the copigment is a flavone glycoside or a flavonol glycoside, and
wherein the intravacuolar pH of the cells of the transformed plant is 5.2 to 6.4.

### Claim 13 (depends on 12)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 12, wherein the flavone glycoside is selected from the group consisting of luteolin glycoside, tricetin glycoside, apigenin glycoside, acacetin glycoside, and their combinations.

### Claim 14 (depends on 13)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 13, wherein the luteolin glycoside is luteolin 7-malonyl glucoside, luteolin 7-glucoside, luteolin 7,3′-diglucoside, luteolin 8-C-glucoside, luteolin 6-C-glucoside, or a derivative thereof.

### Claim 15 (depends on 13)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 13, wherein the tricetin glycoside is tricetin 7-malonyl glucoside or a derivative thereof.

### Claim 16 (depends on 13)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 13, wherein the apigenin glycoside is apigenin 7-glucoside, apigenin 7-rutinoside, apigenin 8-C-glucoside, apigenin 6-C-glucoside, or a derivative thereof.

### Claim 17 (depends on 13)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 13, wherein the acacetin glycoside is acacetin 7-rutinoside or a derivative thereof.

### Claim 18 (depends on 12)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 12, wherein the flavonol glycoside is selected from the group consisting of kaempferol glycoside, quercetin glycoside, and their combinations.

### Claim 19 (depends on 18)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 18, wherein the kaempferol glycoside is kaempferol 3-glucoside or a derivative thereof.

### Claim 20 (depends on 18)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 18, wherein the quercetin glycoside is quercetin 3-glucoside, quercetin 3-(6″-malonyl) glucoside, quercetin 3-rutinoside, or a derivative thereof.

### Claim 21 (depends on 12)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 12, wherein the delphinidin-based anthocyanins having glycosyl groups at both the 3′- and 5′-positions of the anthocyanin B-ring are selected from the group consisting of delphinidin 3-(6″-malonyl)glucoside-3′,5′-diglucoside (ternatin C5), delphinidin 3,3′,5′-triglucoside (preternatin C5), and their combinations.

### Claim 22 (depends on 12)

The transformed plant, its inbred progeny, or its outbred progeny according to claim 12, wherein the plant is rose.

### Claim 23 (depends on 12)

Propagules, partial plant bodies, tissue or cells of the transformed plant, its inbred progeny, or its outbred progeny according to claim 12.

### Claim 24 (depends on 12)

Cut flowers of the transformed plant, its inbred progeny, or its outbred progeny of claim 12, or a processed form created from the cut flowers.